Syncytic oncolytic herpes simplex mutants as effective cancer therapeutics

Abstract

The present invention provides a non-natural herpes simplex virus ("HSV"), a composition comprising, consisting essentially of, or consisting of the HSV, and a method of preparing the HSV or infecting cells with the HSV. Also provided herein are methods of treating cancer or inhibiting the growth or metastasis of cancer cells in a subject in need thereof.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority under 35 USC §119(e) to U.S. Provisional Application Nos. 62 / 818,577 and 62 / 932,725, filed on March 14, 2019 and November 8, 2019, respectively, the contents of each of which are incorporated herein by reference in their entirety.

[0003] Government Support Statement

[0004] This invention was made with government support under Award No. CA223104 from the National Institutes of Health and National Cancer Institute (NIH / NCI). The government may have certain rights in this invention.

[0005] Sequence Listing

[0006] This application contains a sequence listing, which has been submitted electronically in ASCII format, the contents of which are incorporated herein by reference in their entirety. This ASCII copy was created on March 13, 2020, is named 106887-7660_ST25.txt, and is 245,014 bytes in size. Background Art

[0007] Oncolytic herpes simplex virus (oHSV) (such as Imlygic, recently approved by the FDA for the treatment of melanoma) TM ) have shown promising antitumor efficacy. These vectors have two main mechanisms of action: (1) a lytic phase, determined by direct infection and cell lysis; and (2) an immune phase, driven by stimulation of antitumor immunity. However, not all cancers respond similarly, as viral spread in some cancers is inherently slow. In culture, cells have varying degrees of tolerance to viruses. In animals, variations in tumor stroma and immune cell composition lead to variations in viral spread and immune response capacity. Therefore, strategies to improve the potency of the lytic phase to achieve optimal therapeutic effects remain needed. The present invention addresses these needs and provides related advantages. Summary of the Invention

[0008] The present invention provides a non-naturally occurring herpes simplex virus ("HSV"), wherein the virus comprises, consists essentially of, or consists of a mutation in one or more of the following: (a) a gene encoding glycoprotein E ("gE"), (b) a gene encoding infected cell protein 0 ("ICP0"), (c) a gene encoding DNA packaging terminase subunit 1, (d) a gene encoding ICP8, or (e) a gene encoding ICP34.5.

[0009] In another aspect, the present invention provides a composition or pharmaceutical composition comprising, consisting essentially of, or consisting of a non-naturally occurring HSV of the present invention. In another aspect, the present invention provides a method of infecting a cell comprising, consisting essentially of, or consisting of contacting the cell with a non-naturally occurring HSV or a composition or pharmaceutical composition comprising the non-naturally occurring HSV.

[0010] In one aspect, the present invention provides a method for preparing a non-natural HSV of the present invention, comprising, consisting essentially of, or consisting of the following steps: mutating a gene in a non-natural HSV virus particle or introducing a transgene into the non-natural HSV. In another aspect, a method for producing a non-natural HSV vector comprises, consisting essentially of, or consisting of the following steps: (a) introducing a 17TermA HSV vector and an rRp450 HSV vector into a host cell; (b) growing the host cell for at least three generations; and (c) isolating HSV particles produced by the host cell.

[0011] Also provided is a method for inhibiting the growth or metastasis of cancer cells or metastatic cancer cells, comprising, consisting essentially of, or consisting of the following steps: contacting the cells with an effective amount of a non-natural HSV vector or a composition or pharmaceutical composition containing a non-natural HSV vector as described herein. The contacting is in vitro or in vivo. In one aspect, the contacting is performed in vivo by administering a non-natural HSV or composition or pharmaceutical composition to a subject. In vitro, the method is performed by contacting the non-natural HSV with the cells. The in vitro method can be used to test new therapies, or as a personalized analysis to determine whether the therapy is suitable for the cancer to be treated. Additional cancer therapies can be combined with treatments that can be performed simultaneously or sequentially with the methods disclosed herein.

[0012] The cancer cells to be treated can be solid tumors or blood cancers, such as carcinomas or sarcomas, and non-limiting examples include pancreatic cancer, renal cancer, small cell lung cancer, brain cancer, neuroblastoma, neurological cancer, bone cancer, lymphoma, myeloma, colon cancer, uterine cancer, breast cancer, leukemia, liver cancer, prostate cancer, skin cancer, or melanoma. The cells belong to any species, such as mammals and humans, and when performed in vitro, they can be from cultured cell lines or primary cells, such as from tissue biopsies. The cells can be adult or juvenile cells or cancer stem cells (i.e., cancer cells with characteristics associated with normal stem cells, particularly the ability to produce all cell types found in a particular cancer sample) or cancer cells that do not have characteristics associated with normal stem cells. In one embodiment, the cells express the N-myc proto-oncogene protein (MYCN) and / or express MYCN at a higher level than non-cancerous cells.

[0013] On the other hand, the present invention also provides a method for treating cancer or metastatic cancer, or inhibiting the growth or metastasis of cancer cells in a subject in need thereof, the method comprising the following steps, or consisting essentially of the following steps, or consisting of the following steps: administering to the subject an effective amount of the non-natural HSV, composition or pharmaceutical composition of the present invention. The subject to be treated can be any species, such as mammals and humans, such as dogs, horses, cows, cats, monkeys, rats or mice. Administration can be used as a first-line therapy, a second-line therapy, a third-line therapy, a fourth-line therapy or a fifth-line therapy. Additional cancer therapies can be combined with treatments that can be performed simultaneously or sequentially with the methods disclosed herein. The cancer cells to be treated can be solid tumors or blood cancers, such as carcinomas or sarcomas, and non-limiting examples include pancreatic cancer, kidney cancer, small cell lung cancer, brain cancer, neuroblastoma, neurocarcinoma, bone cancer, lymphoma, myeloma, colon cancer, uterine cancer, breast cancer, leukemia, liver cancer, prostate cancer, skin cancer or melanoma.

[0014] The methods of the present invention can be combined with appropriate diagnostics to monitor the remission or progression of the disease. Several methods for such monitoring are known in the art.

[0015] In one aspect, the present invention provides a method for inducing cell lysis, comprising, consisting essentially of, or consisting of contacting a cell with an effective amount of a non-natural HSV, composition, or pharmaceutical composition of the present invention. The contacting is in vitro or in vivo. In one aspect, the contacting is performed in vivo by administering the non-natural HSV, composition, or pharmaceutical composition to a subject. In vitro, the method is performed by contacting the non-natural HSV with a cell. The in vitro method can be used to test new therapies or as a personalized analysis to determine whether the therapy is suitable for the subject to be treated. Additional cytolytic therapies can be combined with treatments that can be performed simultaneously or sequentially with the methods disclosed herein.

[0016] The cancer cells to be treated can be solid tumors or blood cancers, such as carcinomas or sarcomas, and non-limiting examples include pancreatic cancer, renal cancer, small cell lung cancer, brain cancer, neuroblastoma, neurocarcinoma, bone cancer, lymphoma, myeloma, colon cancer, uterine cancer, breast cancer, leukemia, liver cancer, prostate cancer, skin cancer, or melanoma. The cells are of any species, such as mammals and humans, and when performed in vitro, they can be from cultured cell lines or primary cells, such as from a tissue biopsy. The cells can be adult or juvenile cells, or cancer stem cells, or cancer cells that do not have characteristics associated with normal stem cells. The therapy can be combined with appropriate assays to test the effectiveness of the therapy, such as cancer remission or progression.

[0017] In another aspect, the present invention also provides a kit comprising, consisting essentially of, or consisting of the non-natural HSV, composition, and / or pharmaceutical composition of the present invention.

[0018] Description of the accompanying drawings.

[0019] Figures 1A-1B The generation of Mut-3 and Mut-3Δ34.5 ( Figure 1A ) and Mut-3 SNP ( Figure 1B Schematic diagram of HSV Mut-3. HSV Mut-3 was isolated from serial passages of mixed TermA and rRp450 in a non-permissive line ("directed evolution"); and the attenuated mutant Mut-3Δ34.5 ( Figure 1A ). Figure 1B A sequence alignment of Mut-3 and its parental viruses is shown. Non-synonymous mutations that differ from either parent in Mut-3 are shaded with backslashes, including UL15, UL29, US8, RL1, and RL2. Genomic sequences identical to 17TermA are shown as open boxes in the bottom panel; those identical to rRp450 are marked with forward slashes.

[0020] Figures 2A-2C The plaque size and receptor usage of Mut-3, Mut-3Δ34.5, rRp450, and 17TermA are shown. The applicant simultaneously performed plaque analysis on the four viruses and scanned and analyzed the plaque images using a Keyence HS multifunctional fluorescence microscope BZ-II analyzer 3 days later. Figure 2A ) Original (top) and masked (bottom, numbered plaque) images of four viruses. ( Figure 2B ) The average plaque size (left) and number of plaques (right) of the four viruses were calculated. The plaque sizes of Mut-3 and Mut-3Δ34.5 were significantly larger than those of the parental viruses rRp450 and 17TermA. ( Figure 2CIn vitro cytotoxicity / MTS assays were performed on a CHO cell panel. Applicants infected CHO-K1 (as labeled), CHO-Nectin-1 (as labeled), CHO-Nectin-2 (as labeled), and CHO-HVEM (as labeled) with four viruses at varying multiplicities of infection (MOI). Applicants measured cell viability using a colorimetric cell proliferation / MTS assay three days post-infection (PVI) relative to untreated controls. Only CHO-Nectin-1 and CHO-HVEM, but not CHO-K1 or CHO-Nectin-2 (primarily required for HSV-2 entry), were sensitive to treatment with the four viruses, indicating that Mut-3 and Mut-3Δ34.5 do not bypass the receptor barrier and can still rely on canonical HSV entry receptors to infect cells.

[0021] Figures 3A-3C Mut-3Δ34.5 is more effective than 17TermA (γ134.5-null) in killing human and mouse neuroblastoma cells, which is not due to an increase in the production of infectious viral particles in vitro. Figure 3A ) In vitro cytotoxicity / MTS assay of neuroblastoma cell lines. Applicants infected human (SK-N-AS) and mouse (975A2) neuroblastoma cells with Mut-3 (as labeled), Mut-3Δ34.5 (as labeled), or 17TermA (as labeled) at varying MOIs. Applicants measured cell viability by MTS assay 4 days post-virus infection (pvi) relative to untreated controls. N = 6, error bars represent SEM. ****p < 0.0001, two-way ANOVA. ( Figure 3B ) In vitro viral replication assay. Applicants infected neuroblastoma cell lines with 17TermA (as indicated) or Mut-3Δ34.5 (as indicated) at an MOI of 0.1 (upper panel, human line SK-N-as) or an MOI of 0.5 (lower panel, mouse line 975A2) and washed with PBS 2 hours post-infection (PVI). Applicants obtained cell lysates 2, 24, 48, and 72 hours post-infection (PVI) and determined viral yield by plaque assay. N = 3. Error bars represent SD. *p < 0.05, **p < 0.001, ****p < 0.0001, two-way ANOVA. ( Figure 3C ) compared the relative free release of virions from Mut-3Δ34.5 versus 17TermA infected neuroblastoma cultures. Figure 3B ) by collecting the supernatant and pellet and performing plaque analysis on two separate fractions over time, rather than as described in Figure 3BThe analysis was performed as a whole. Applicants calculated relative free virions as the ratio of the entire culture (supernatant + particles) to infectious particles in the supernatant. Mut-3Δ34.5 showed significantly more relative released virions than 17TermA at 48 hours. N = 3. Error bars represent SD. *p < 0.05.

[0022] Figures 4A-4H TEM analysis of Mut-3Δ34.5 and 17TermA uptake in neuroblastoma cells. Neuroblastoma cells SK-N-AS and 975A2 were infected with Mut-3Δ34.5 and 17TermA at an MOI of 50 for 20 minutes at 37°C. (Figures 4A, 4B, 4E, and 4F) Insets show that Mut-3Δ34.5 virions are primarily located in endocytic vesicles (arrowheads except the right arrow in Figure 4B), with few fusions with the plasma membrane (right arrow in Figure 4B). (Figures 4C, 4D, 4G, and 4H) Insets show that 17TermA virions are primarily located in endocytic vesicles (arrowheads except the bottom arrow in Figure 4G), with few fusions with the plasma membrane (bottom arrow in Figure 4G). TEM analysis was performed using a Hitachi H-7650 TEM. N: nucleus. Scale bar: 500 nm.

[0023] Figures 5A-5C : Attenuated 17Δ34.5 virions showed comparable potency to 17TermA in neuroblastoma cells. Applicants generated 17Δ34.5 by CRISPR-Cas9 gene editing technology to replace the wild-type strain 17 with an EGFP expression cassette. + The g134.5 gene in Figure 5A ) Image of plaques of 17Δ34.5 clone on Vero cells. After three rounds of plaque purification, both B4 and G1 clones produced 100% GFP-positive (left panel) and non-syncytial (right panel, phase contrast) plaques. Two days after viral infection (PVI), the cells were plated with EVOS. ® Images were taken with the FL imaging system. Scale bar: 400 μm. ( Figure 5B ) In vitro cytotoxicity / MTS assay of neuroblastoma cell lines. Applicants used wild-type strain 17 + Neuroblastoma cell lines SK-N-AS and 975A2 were infected with 17D34.5 clone B4 (as labeled), 17TermA (as labeled), 17D34.5 clone B4 (as labeled), or 17D34.5 clone G1 (as labeled) at different MOIs. Applicants measured cell viability 3 days post-virus infection (pvi) relative to untreated controls using an MTS assay. The potency of the two 17D34.5 clones was significantly lower than that of their wild-type counterparts, strain 17+, but comparable to that of 17TermA. The DNA sequences of the EGFP expression cassette and EGFP are shown in Figure 2. Figure 5C As shown (SEQ ID NO: 11).

[0024] Figures 6A-6C : Mut-3Δ34.5 showed faster viral gene transfer and cell killing compared to 17Δ34.5. Applicants treated (Figure 6A) Vero (Figure 6B) SK-N-AS (Figure 6C) 975A2 cells with mock CTL, Mut-3Δ34.5, or 17Δ34.5 at different MOIs and used IncuCyte ZOOM live cell imaging to monitor GFP-positive areas (top panels) and cell confluence (bottom panels) over time. N = 6 wells per condition, and n = 2 measurements per well per time point. Post-virus infection (pvi). The Mann-Whitney U test was used to compare the time to reach maximum GFP area between viruses in (Figure 6A). Error bars represent SD .

[0025] Figure 7 Mut-3Δ34.5 is more effective than 17TermA in controlling human neuroblastoma growth in vivo. Athymic nude mice bearing sub-q SK-N-AS tumors were intratumorally injected with phosphate-buffered saline (PBS) control (as indicated, n = 8), three doses of 1e7 pfu of 17TermA (as indicated, n = 8), or Mut-3Δ34.5 (as indicated, n = 9). Kaplan–Meier survival curves were plotted. Statistical significance between 17TermA and Mut-3Δ34.5 was scored using the log-rank test. *p < 0.05.

[0026] Figures 8A-8CMut-3ΔICP6, an attenuated version of Mut-3, induces higher cytotoxicity in the human neuroblastoma cell line CHP-134 compared to the oncolytic herpesvirus rRp450. (Figure 8A) A map of Mut-3ΔICP6, constructed using a CRISPR-Cas9 gene editing strategy, replaces the UL39 gene encoding ICP-6 with a CMV-driven GFP reporter gene cassette. An MTS cell viability assay compared the cytotoxicity of Mut-3, rRp450, and Mut3-ΔICP6 / D7-1 in the human neuroblastoma cell line. Cells were seeded at 4000 cells / well in 96-well culture dishes, cultured overnight at 37°C, and then infected with each listed virus at a multiplicity of infection (MOI) of 0.001, 0.01, 0.1, and 1 infectious virus particles per cell. Cell Titer96 AQuesous Non-Radioactive Cell Proliferation Assay / MTS (Promega, Madison, WI) was used for the assay on day 3 or 4 post-infection, following the manufacturer's instructions. Each sample was run in quadruplicate, and results are presented as percentage of cell viability relative to an uninfected control. Error bars represent standard deviation. Statistical significance was assessed using a t-test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. The results are presented graphically in Figure 8B for SK-N-AS and Figure 8C for CHP-134.

[0027] Figures 9A-9B : Compared with rRp450, Mut-3ΔICP6 induces higher cytotoxicity in mouse neuroblastoma cell lines Neuro-2a and 975A2. Figures 9A-9B Shown are the results of an MTS cell viability assay comparing the cytotoxicity of Mut-3, rRp450, and Mut3-ΔICP6 / D7-1 in a mouse neuroblastoma cell line. Cells were seeded at 4000 cells / well in 96-well culture dishes, cultured overnight at 37°C, and then infected with each listed virus at a multiplicity of infection (MOI) of 0.001, 0.01, 0.1, and 1 infectious virus particles per cell. Assays were performed on day 3 or 4 post-infection using the Cell Titer96 AQuesous Non-Radioactive Cell Proliferation Assay / MTS (Promega, Madison, WI), following the manufacturer's instructions. Each sample was run in quadruplicate, and results are shown as percentage of cell viability relative to an uninfected control. Statistical significance was assessed using a t-test. **p ≤ 0.01, ***p ≤ 0.001. Figure 9A: Neuro-2a and Figure 9B: 975A2.

[0028] Figures 10A-10BMut-3ΔICP6 produced significantly higher viral yields than rRp450 in the mouse neuroblastoma cell line 975A2 at 48 and 72 hours of infection. Mouse 975A2 (Figure 10A) and human SK-N-AS (Figure 10B) neuroblastoma cells were plated at 2 x 10 5 Cells were seeded in 12-well dishes and cultured overnight at 37°C. SK-N-AS cells (Figure 10B) or 975A2 cells (Figure 10A) were infected at an MOI of 0.01 in 200 µL of serum-free medium for 2 hours with gentle agitation every 20 minutes. Cells were washed once with PBS and overlaid with 1 mL of complete medium. Cells and supernatants were then harvested at 2, 24, 48, and 72 hours post-infection, frozen and thawed three times, and serially diluted and titrated on Vero cells to determine infectious virus yield using a standard plaque assay. Each sample was run in triplicate. Error bars represent standard deviation. Statistical significance was assessed using a t-test. *p ≤ 0.05, **p ≤ 0.01.

[0029] Figures 11A-11B Mut-3Δ34.5 and Mut-3ΔICP6 were significantly less active than the Mut-3 virus on differentiated human keratinocytes. Human keratinocytes (HFKs) were grown in EpiLife medium (Cascade Biologics) supplemented with human keratinocyte growth supplement according to the manufacturer's instructions. Undifferentiated HFKs (Figure 11A) were seeded into 96-well plates at a density of 2,000 cells per well and cultured overnight. Cultures were then infected with Mut-3, Mut-3Δ34.5 / C8G5, or Mut-3ΔICP6 D7-1 at MOIs of 0.0004, 0.004, 0.04, 0.4, and 4. Three days after viral infection, cell viability was determined using an MTS assay. Twenty-four hours after seeding, differentiated HFKs were generated by adding 10% FBS and 1 mmol / l CaCl2 to the culture medium and allowed to culture for an additional 48 hours before infection. Four days after viral infection, the viability of differentiated cells was determined using an MTS assay (Figure 11B). Each sample was run in quadruplicate. Error bars represent standard deviation. Statistical significance was assessed using a t-test. *p ≤ 0.05, **p ≤ 0.01.

[0030] Figure 12Intravenous injection of 1e6 pfu of the wild-type KOS virus strain is lethal to naive Balb / c mice. Description of the safety study of Mut-3Δ34.5 and Mut-3ΔICP6. The applicant initiated a viral biodistribution study by injecting naive, non-tumor-bearing Balb / c mice (20 males and 20 females) with 1e8 plaque-forming units (pfu) of each virus via the tail vein. The applicant then sacrificed the animals on days 1, 14, 28, 56, and 85 (n=4 per time point), and collected peripheral blood, testes (males), ovaries (females), spleen, lungs, kidneys, heart, lung, and brain for analysis. The applicant observed each mouse daily for the first two weeks after viral administration and then twice weekly until the scheduled sacrifice date. The body weight of each mouse was recorded before infection and weekly thereafter. Wild-type KOS virus was administered to each group of mice as a positive (non-safety) control (doses ranged from 1e5 to 1e7 pfu per mouse (n=3)). The applicants also performed pathological analysis of the harvested organs, comparing samples obtained from mice inoculated with the mutant viruses with those from mice inoculated with wild-type KOS virus. The applicants also used plaque assays to test the replication potential of these viruses. The results show survival curves for mice that received a single dose of 1e5, 1e6, or 1e7 pfu of wild-type KOS virus.

[0031] Figures 13A–13B: Naive Balb / c mice can tolerate intravenous injections of up to 1e8 pfu of Mut-3∆34.5 (C8G5) or Mut-3∆ICP6 (D7-1). The applicant completed safety and biodistribution studies and found that mice receiving up to 1e8 pfu of Mut-3∆34.5 (C8G5) or Mut-3∆ICP6 (D7-1) remained healthy until the designated sacrifice date (up to 85 days) by physical examination. In female mice, plaque assays revealed infectious virus in the heart, kidneys, liver, ovaries, and spleen only 24 hours after infection. Figures 13A and 13B present the results for female and male mice, respectively.

[0032] Figure 14 Replicated HSV could be detected in the brain, kidney, and ovaries of mice that received 1e6 pfu or more of wild-type KOS virus. Figure 14 This table summarizes the results of plaque analysis in tissues of mice injected with 1e6 or 1e7 pfu of wild-type KOS virus. Mice were sacrificed 5–6 days post-viral injection (PVI), after the onset of disease symptoms (kyphosis, lethargy, hind limb paralysis, etc.). "+" indicates detectable plaques, and "-" indicates their absence. Gray-shaded boxes indicate unavailable data.

[0033] Figure 15This table shows that replicated HSV was detectable in nearly all tissues (except the lungs) obtained from mice that received 1e8 pfu of C8G5 or D7-1 24 hours after viral infection (PVI). This table summarizes the results of plaque analysis of tissues from mice injected with Mut∆34.5 or Mut3∆ICP6 24 hours after viral infection. "+" indicates detectable plaques, and "-" indicates their absence. Gray-shaded boxes indicate that these data were not available.

[0034] Figure 16 The table shows that no replicated HSV was detected in any tissue obtained from mice that received 1e8 pfu of C8G5 or D7-1 14 days after viral infection (PVI). The table summarizes the results of plaque analysis of tissues from mice that were injected with Mut∆34.5 or Mut3∆ICP6 14 days after viral infection. "-" indicates no detectable plaques.

[0035] Figure 17 The table shows that no HSV replication was detected in any tissue obtained from mice that received 1e8 pfu of C8G5 or D7-1 at 28 days post-viral infection (PVI). This table summarizes the results of plaque analysis of tissues from mice that were injected with Mut∆34.5 or Mut3∆ICP6 at 28 days post-viral infection. "-" indicates no detectable plaques. Gray-shaded boxes indicate that these data were not available.

[0036] Figure 18 Table 1 summarizes the results of plaque analysis of tissues from mice injected with Mut∆34.5 or Mut3∆ICP6 at 56 days post-viral infection (PVI). A "-" indicates no detectable plaques.

[0037] Figure 19 The table shows that no replicated HSV was detected in any tissue obtained from mice that received 1e8 pfu of C8G5 or D7-1 at 85 days post-viral infection (pvi). The table summarizes the results of plaque analysis of tissues from mice that were injected with Mut∆34.5 or Mut3∆ICP6 at 85 days post-viral infection. "-" indicates no detectable plaques.

[0038] Figures 20A–20D show that the plaque size of Mut-3ΔgE is significantly smaller than that of Mut-3 or Mut-3D34.5 (C8G5). Figure 20A: Mut-3ΔgE was constructed using a CRISPR-Cas9 gene editing strategy, in which the Us8 gene encoding the glycoprotein gE was replaced with a CMV-driven GFP reporter gene cassette. Figure 20B: Non-syncytial GFP-positive plaque phenotype of Mut-3ΔgE clones 28D5-B4 and 28D5-H1. Vero cells were infected with serial dilutions of 28D5-B4 or 28D5-H1 for 2 hours and overlaid with culture medium. Plaques were photographed 3 days post-virus infection (pvi). Figure 20C: qPCR analysis of Mut-3ΔgE clones 28D5-B4 and 28D5-H1 demonstrates the loss of Us8 / gE. Baby hamster kidney (BHK) cells were infected with Mut-3∆gE clones 28D5-B4 or 28D5-H1, and lysates were collected when a cytopathic effect of ≥50% was observed. 50-100 ng of genomic DNA isolated from virus-infected BHK cell lysates was used for qPCR reactions. Results are presented as fold increase in gE / Us8 or GFP relative to the Us8a control HSV gene. Figure 20D: Comparison of plaque size between Mut-3∆gE clones 28D5-B4 and 28D5-H1 and Mut-3 or Mut-3∆34.5 / C8G5, as measured by a standard plaque assay performed in Vero cells. Images of plaques of Mut-3, Mut-3∆34.5, Mut-3∆gE clone 28D5-B4, and clone 28D5-H1 were taken 3 days post-infection (pvi).

[0039] Figures 21A–21D present results showing that 17ΔgE produces significantly smaller plaques than 17syn+ or 17Δ34.5. Figure 21A: 17ΔgE was constructed using a CRISPR-Cas9 gene editing strategy, in which the Us8 gene encoding the glycoprotein gE was replaced with a CMV-driven GFP reporter gene cassette. Figure 21B: Non-syncytial and small GFP-positive plaque phenotype of 17ΔgE clone 12G5. Vero cells were infected with serial dilutions of 17ΔgE 12G5 for 2 hours and overlaid with overlay medium. Plaques were photographed 3 days post-infection (pvi). Figure 21C: qPCR analysis of 17ΔgE clone 12G5 demonstrates the loss of Us8 / gE. Baby hamster kidney (BHK) cells were infected with 17ΔgE clone 12G5, and lysates were collected when a cytopathic effect of ≥50% was observed. 50–100 ng of genomic DNA isolated from virus-infected BHK cell lysates was used for qPCR reactions. Results are shown as gE / Us8 or GFP fold increase relative to the HSV gene in the ICP6 control group. Figure 21D: Comparison of plaque size between 17ΔgE clone 12G5 17syn+ or 17Δ34.5 clone B4, obtained from a standard plaque assay performed in Vero cells. Plaque images were taken 3 days post-infection (pvi).

[0040] Detailed description

[0041] The following will describe more fully embodiments according to the present invention. However, various aspects of the present invention may be embodied in different forms and should not be construed as limited to the embodiments described herein. Rather, these embodiments are provided to make the present invention thorough and complete and to fully convey the scope of the present invention to those skilled in the art. The terminology used in the description herein is for the purpose of describing specific embodiments only and is not intended to be limiting.

[0042] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of this application and the related art, and should not be interpreted in an idealized or overly formal sense unless expressly defined herein. Although not expressly defined below, such terms should be interpreted according to their common meaning.

[0043] The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present invention.All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0044] The practice of the present technology will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art.

[0045] Unless the context indicates otherwise, the various features of the invention described herein may be used in any combination. Furthermore, the present invention contemplates that in some embodiments, any feature or combination of features described herein may be excluded or omitted. For illustration, if the specification states that a compound includes components A, B, and C, it is specifically intended that any one or combination of A, B, or C may be omitted or disclaimed, individually or in any combination.

[0046] Unless expressly indicated otherwise, all specified embodiments, features, and terms are intended to include the stated embodiment, feature, or term and its biological equivalents.

[0047] All numerical designations, such as pH, temperature, time, concentration, and molecular weight, including ranges, are approximate and may vary by increments of (+) or (-) 1.0 or 0.1, as appropriate, or by + / - 15%, or by 10%, or by 5%, or by 2%. It will be understood that, although not always explicitly stated, all numerical designations are preceded by the term "about." It will also be understood that, although not always explicitly stated, the reagents described herein are merely exemplary reagents and that equivalents of such reagents are known in the art.

[0048] As used herein, the term "comparable" refers to having the same level as a reference level, or within a variation range of + / - 50%, or 45%, or 40%, or 35%, or 20%, or 25%, or 20%, or 15%, or 10%, or 5%, or 2% compared to a reference level.

[0049] Throughout this disclosure, various publications, patents, and published patent specifications are referenced by identifying references or numerals. Full citations for publications identified by numerals can be found immediately preceding the claims. The disclosures of these publications, patents, and published patent specifications are hereby incorporated by reference in their entireties to more fully describe the state of the art to which this invention pertains.

[0050] definition

[0051] Unless otherwise indicated, the practice of the present technology will employ conventional techniques of organic chemistry, pharmacology, immunology, molecular biology, microbiology, cell biology, and recombinant DNA within the skill of the art. For example, see Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd edition (1989); Current Protocols In Molecular Biology (FM Ausubel, et al. eds., (1987)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: APractical Approach (MJ MacPherson, BD Hames and GR Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, a Laboratory Manual, and Animal Cell Culture (RI Freshney, ed. (1987)).

[0052] As used in the description of the invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0053] As used herein, the term "comprising" is intended to indicate that compositions and methods include the recited elements, but not to exclude other elements. As used herein, the transition phrase consisting essentially of (and grammatical variations) will be interpreted as including the recited materials or steps, as well as those that do not materially affect the basic and novel characteristics of the described embodiment. These characteristics are described in the method embodiments. Therefore, the term "consisting essentially of" as used herein should not be interpreted as equivalent to "comprising." "Consisting of" should mean excluding trace elements in excess of other ingredients and the essential method steps for applying the compositions disclosed herein. Aspects defined by each of these transition terms are within the scope of the present invention.

[0054] As used herein, the term "about" when referring to a measurable value (eg, an amount or concentration, etc.) is intended to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the stated amount.

[0055] The terms "acceptable," "effective," or "sufficient" when used to describe the selection of any ingredient, range, dosage form, etc. disclosed herein means that the ingredient, range, dosage form, etc. is suitable for the disclosed purpose.

[0056] Also as used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of a combination when construed in the alternative ("or").

[0057] As used herein, the term "cell" may refer to a prokaryotic or eukaryotic cell, optionally obtained from a subject or a commercial source.

[0058] "Eukaryotic cells" refer to all living organisms except prokaryotes. They can be easily distinguished by the membrane-enclosed nucleus. Animals, plants, fungi, and protists are eukaryotic organisms or organisms, whose cells are composed of complex structures composed of an inner membrane and a cytoskeleton. The most typical membrane-enclosed structure is the nucleus. Unless otherwise specified, the term "host" includes eukaryotic hosts, including, for example, yeast, higher plants, insects, and mammalian cells. Non-limiting examples of eukaryotic cells or hosts include monkeys, cows, pigs, mice, rats, birds, reptiles, and humans, such as HEK293 cells and 293T cells.

[0059] "Prokaryotic cells" typically lack a nucleus or any other membrane-enclosed organelles and are divided into two domains: bacteria and archaea. In addition to chromosomal DNA, these cells can also contain genetic information in loops called episomes. Bacterial cells are very small, roughly the size of animal mitochondria (approximately 1-2 μm in diameter and 10 μm long). Prokaryotic cells have three main shapes: rod-shaped, spherical, and spiral. Rather than undergoing the complex replication process of eukaryotic cells, bacterial cells divide by binary fission. Examples include, but are not limited to, Bacillus, Escherichia coli, and Salmonella.

[0060] The term "encoding" as applied to a nucleic acid sequence refers to a polynucleotide that is described as "encoding" a polypeptide and that, in its native state or when manipulated by methods well known to those skilled in the art, can be transcribed and / or translated to produce mRNA for the polypeptide and / or fragments thereof. The antisense strand is the complementary sequence of such a nucleic acid, from which the coding sequence can be deduced.

[0061] The terms "equivalent" or "bioequivalent" are used interchangeably when referring to a particular molecule, organism, or cell material, and mean a material having minimal homology while maintaining a desired structure or function. Non-limiting examples of equivalent polypeptides include polypeptides having at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95% identity to it or a polypeptide sequence, or polypeptides encoded by polynucleotides or their complementary sequences that hybridize to the polynucleotides encoding the polypeptide sequence under highly stringent conditions. Highly stringent conditions are described herein and are incorporated herein by reference. Alternatively, an equivalent is a polypeptide encoded by a polynucleotide or its complementary sequence that has at least 70%, or at least 75%, or 80%, or at least 85%, or at least 90%, or at least 95% identity to a reference polynucleotide (e.g., a wild-type polynucleotide), or at least 97% sequence identity, or at least 99% identity. In one aspect, the equivalent polypeptide or polynucleotide has the same or substantially similar biological function as the reference polypeptide or polynucleotide, respectively, e.g., cytolytic function, anti-tumor, anti-metastatic or anti-cancer function, as determined by an appropriate cell assay or animal model as described herein.

[0062] Non-limiting examples of equivalent polypeptides include polynucleotides that are at least 60%, or at least 65%, or at least 70%, or at least 75%, or 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97% identical to the reference polynucleotide. Equivalents also include polynucleotides that hybridize to the reference polynucleotide under highly stringent conditions, or their complementary sequences.

[0063] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) having a certain percentage (e.g., 80%, 85%, 90% or 95%) of "sequence identity" to another sequence means that, when aligned, the bases (or amino acids) of that percentage are identical when comparing the two sequences. Comparisons and percent homology or sequence identity can be determined using software programs known in the art, such as those described in Current Protocols in Molecular Biology (Ausubel et al., eds. 1987) Supplement 30, section 7.7.18, Table 7.7.1. In certain embodiments, default parameters are used for alignment. Using default parameters, a non-limiting exemplary alignment program can be performed using BLAST. Specifically, exemplary programs include BLASTN and BLASTP using the following default parameters: genetic code = standard; filter = none; strand = two strands; cutoff = 60; expectation = 10; matrix = BLOSUM62; description = 50 sequences; sort by = high score; databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. Detailed information about these programs can be found at: ncbi.nlm.nih.gov / cgi-bin / BLAST. Sequence identity and percent identity can be determined by combining them in clustalW (available at: genome.jp / tools / clustalw / , last accessed January 13, 2017).

[0064] "Homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence, which may be aligned for comparison. When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An "unrelated" or "nonhomologous" sequence is one that has less than 40% identity, or less than 25% identity, to one of the sequences of the invention.

[0065] "Homology" or "identity" or "similarity" can also refer to two nucleic acid molecules that hybridize under stringent conditions.

[0066] "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized by hydrogen bonds between the bases of the nucleotide residues. Hydrogen bonds can occur by Watson-Crick base pairing, Hoogstein binding, or any other sequence-specific manner. The complex comprises, or consists essentially of, or consists of two chains that form a double-stranded structure, three or more chains that form a multi-chain complex, a single self-hybridizing chain, or any combination of these. A hybridization reaction can constitute a step in a broader process, such as the initiation of a PCR reaction, or the cleavage of a polynucleotide by a ribozyme.

[0067] Examples of stringent hybridization conditions include an incubation temperature of about 25°C to about 37°C; a hybridization buffer concentration of about 6×SSC to about 10×SSC; a formamide concentration of about 0% to 25%; and a wash solution of about 4×SSC to about 8×SSC. Examples of moderately stringent hybridization conditions include an incubation temperature of about 40°C to about 50°C; a buffer concentration of about 9×SSC to 2×SSC; a formamide concentration of about 30% to 50%; and a wash solution of about 5×SSC to about 2×SSC. Highly stringent hybridization refers to hybridization of the oligonucleotide to the target sequence without mismatches (or perfect complementarity). Examples of high stringency conditions include an incubation temperature of about 55°C to about 68°C; a buffer concentration of about 1×SSC to 0.1×SSC; a formamide concentration of about 55% to 75%; and a wash solution of about 1×SSC, 0.1×SSC, or deionized water. Typically, hybridization incubation times range from 5 minutes to 24 hours, with one, two, or more wash steps, and wash incubation times of about 1, 2, or 15 minutes. SSC is a 0.15 M NaCl and 15 mM sodium citrate buffer. It will be appreciated that SSC equivalents using other buffer systems may be used.

[0068] As used herein, "expression" refers to the process by which a polynucleotide is transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0069] "Gene" refers to a polynucleotide containing at least one open reading frame (ORF) that, after transcription and translation, encodes a specific polypeptide or protein. "Gene product" or, alternatively, "gene expression product" refers to the amino acids (e.g., peptides or polypeptides) produced when a gene is transcribed and translated.

[0070] "Under transcriptional control" is a term well known in the art, meaning that the transcription of a polynucleotide sequence (typically a DNA sequence) is dependent upon its operable linkage to elements that facilitate initiation or promotion of transcription. "Operably linked" means that the polynucleotides are arranged in a manner that allows them to function in the cell. In one aspect, the present invention provides a promoter that is operably linked to a downstream sequence (e.g., an HSV virulence gene or a mutant thereof).

[0071] The term "encoding" as applied to a nucleic acid sequence refers to a polynucleotide that is described as "encoding" a polypeptide and that, in its native state or when manipulated by methods well known to those skilled in the art, can be transcribed and / or translated to produce mRNA for the polypeptide and / or fragments thereof. The antisense strand is the complementary sequence of such a nucleic acid, from which the coding sequence can be deduced.

[0072] As used herein, the term "isolated" refers to a molecule, biological or cellular material that is substantially free of other material.

[0073] As used herein, the term "functional" may be applied to the modification of any molecule, biological product, or cellular material to achieve a particular specific effect.

[0074] As used herein, the terms "nucleic acid sequence" and "polynucleotide" are used interchangeably to refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derivatized nucleotide bases.

[0075] The term "wild type" refers to a gene or gene product that has the characteristics of the gene or gene product when isolated from a natural source. In some embodiments, a wild type gene or gene product comprises a slightly different sequence even for one viral strain.

[0076] The term "mutant" refers to a gene or gene product that exhibits an alteration in sequence and / or functional properties (i.e., an altered characteristic) compared to a wild-type gene or gene product or a gene or gene product from another mutant strain. In one embodiment, the other mutant strain comprises a 17TermA or rR450 strain.

[0077] The term "mutation" refers to a variation in the DNA sequence from a wild-type or other mutant strain. A mutation may or may not produce a functional property in an organism. There are many types of mutations, including but not limited to insertions, deletions, truncations, frameshifts, substitutions, and point mutations.

[0078] The term "point mutation" refers to a mutation having a single nucleotide base change, insertion, or deletion of genetic material, DNA or RNA.

[0079] A "deletion" refers to a mutation in which a portion of a chromosome or DNA sequence is missing.

[0080] "Frameshift" refers to a mutation in a DNA sequence caused by the insertion or deletion of multiple nucleotides that are not divisible by three.

[0081] "Substitution" refers to a mutation in which one or several nucleotides of a gene are replaced.

[0082] "Truncation" refers to a mutation that eliminates the N-terminal or C-terminal portion of a protein by proteolysis or manipulation of the structural gene, or premature termination of protein elongation due to the presence of a stop codon in the structural gene due to a nonsense mutation.

[0083] In some embodiments, the mutation is a non-synonymous mutation. The term "non-synonymous mutation" refers to a mutation that changes the amino acid sequence of a protein, in contrast to a synonymous mutation that does not change the amino acid sequence.

[0084] As used herein, the term "promoter" refers to any sequence that regulates the expression of a coding sequence (e.g., a gene). For example, a promoter can be constitutive, inducible, repressible, or tissue-specific. A "promoter" is a control sequence, which is a region of a polynucleotide sequence that controls the initiation and rate of transcription. It can contain genetic elements to which regulatory proteins and molecules can bind, such as RNA polymerase and other transcription factors. Non-limiting exemplary promoters include the ROS sarcoma virus (RSV) LTR promoter (optionally with an RSV enhancer), the cytomegalovirus (CMV) promoter, the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerate kinase (PGK) promoter, the U6 promoter, or the EF1 promoter. In some embodiments, the promoter is the chicken β-actin ("CBA") promoter.

[0085] Other non-limiting exemplary promoters with specific target specificities are provided below, including but not limited to CMV, EF1a, SV40, PGK1 (human or mouse), P5, Ubc, human β-actin, CAG, TRE, UAS, Ac5, polyhedrin, CaMKIIa, Gal1, TEF1, GDS, ADH1, CaMV35S, Ubi, H1, U6, α-1-antitrypsin. Synthetically derived promoters can be used for ubiquitous or tissue-specific expression. In addition, virally derived promoters, some of which are described above, can be used in the methods disclosed herein, such as CMV, HIV, adenovirus, and AAV promoters. In some embodiments, the promoter is coupled to an enhancer to increase transcription efficiency.

[0086] An enhancer is a regulatory element that increases the expression of a target sequence. A "promoter / enhancer" is a polynucleotide containing sequences that provide both promoter and enhancer functions. For example, the long terminal repeats of retroviruses contain both promoter and enhancer functions. Enhancers / promoters can be "endogenous," "exogenous," or "heterologous." An "endogenous" enhancer / promoter is one naturally associated with a given gene in the genome. An "exogenous" or "heterologous" enhancer / promoter is one that has been juxtaposed to a gene through genetic manipulation (i.e., molecular biology techniques) such that transcription of that gene is controlled by the associated enhancer / promoter.

[0087] As used herein, the term "tumor-specific promoter or tissue-specific promoter" refers to a promoter that allows expression of a gene under the control of the promoter, particularly in a desired tumor cell or tissue. Non-limiting examples of tissue-specific promoters that can be used in the present invention include prostate-specific antigen (PSA) promoter, prostate-specific membrane antigen (PSMA) promoter, casein promoter, IgG promoter, chorionic villus antigen promoter, elastase promoter, porphobilinogen deaminase promoter, insulin promoter, growth hormone factor promoter, acetylcholine receptor promoter, alcohol dehydrogenase promoter, and α or β globin promoter.

[0088] Non-limiting examples of tumor-specific promoters used in the present invention include telomerase reverse transcriptase promoter, glial fibrillary acidic protein promoter, E2F promoter, survivin promoter, COX-2 promoter, EGD-2 promoter, ELF-1 promoter, hypoxia-specific promoter, carcinoembryonic antigen promoter, and stromelysin 3 promoter.

[0089] The term "cryoprotectant" refers to a compound or material that is capable of protecting one or more tissues, viruses, or other biological agents from damage or injury. Examples of cryoprotectants include, but are not limited to, chondroitin sulfate, glycosaminoglycan dimethyl sulfoxide, cell-permeable organic solutes, polysaccharides, glycerol, Dulbecco's Minimum Essential Medium (DMEM), glutamine, D-glucose, sodium pyruvate, fetal bovine serum, papaverine, DMSO, glycerol, trehalose, KH2PO4, K2HPO4, KCl, mannitol, NaHCO3, sodium ascorbate, 1,2-propylene glycol, formamide, 2,3-butanediol, probucol, curcumin, and mixtures thereof.

[0090] The terms "protein," "peptide," and "polypeptide" are used interchangeably and in their broadest sense to refer to a compound composed of two or more subunits of amino acids, amino acid analogs, or peptidomimetics. The subunits can be linked by peptide bonds. In another aspect, the subunits can be linked by other bonds, such as esters, ethers, and the like. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that a protein's or peptide's sequence may contain, consist essentially of, consist essentially of, consist of, consist essentially of, or consist of. As used herein, the term "amino acid" refers to natural and / or unnatural or synthetic amino acids, including glycine and the D and L optical isomers, amino acid analogs, and peptidomimetics.

[0091] As used herein, the term "vector" refers to a non-chromosomal nucleic acid that contains, consists essentially of, or consists of an intact replicon, such that the vector can be replicated when placed inside a cell, for example, by a transformation process. A vector can be a viral vector or a non-viral vector. Viral vectors include retroviruses, adenoviruses, herpes simplex virus ("HSV"), baculoviruses, modified baculoviruses, pasteurian viruses, or other modified natural viruses. Exemplary non-viral vectors for delivery of nucleic acids include: naked DNA; DNA complexed with cationic liposomes, alone or in combination with cationic polymers; anionic and cationic liposomes; DNA-protein complexes and particles, including DNA condensed with cationic polymers (such as isomeric polylysine, oligopeptides of defined length, and polyethyleneimine), in some cases contained in liposomes, consisting essentially of, or consisting of; and the use of ternary complexes comprising, consisting essentially of, or consisting of a virus and polylysine DNA.

[0092] "Viral vector" is defined as a recombinantly produced virus or viral particle comprising, or consisting essentially of, or consisting of a polynucleotide that is delivered to a host cell in vivo, in vitro or in vitro. Examples of viral vectors include retroviral vectors, AAV vectors, lentiviral vectors, adenoviral vectors, alphaviral vectors, and the like. Alphaviral vectors, such as vectors based on Semliki Forest virus and vectors based on Sindbis virus, have also been developed for gene therapy and immunotherapy. See, Schlesinger and Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 and Ying, et al. (1999) Nat. Med. 5(7): 823-827.

[0093] In another embodiment, expression of an HSV virulence protein (e.g., wild-type or mutant) is regulated by a promoter that is an inducible promoter. In a specific related embodiment, the promoter is an inducible tetracycline promoter. Tet-Off and Tet-On gene expression systems provide researchers with ready access to regulated, high-level gene expression systems, described as Tet-Off and Tet-On. In the Tet-Off system, gene expression is turned on when tetracycline (Tc) or doxycycline (Dox; a Tc derivative) is removed from the culture medium. In contrast, in the Tet-On system, expression is turned on by the addition of Dox. Both systems allow gene expression to be tightly regulated by varying concentrations of Tc or Dox. The maximum expression levels in the Tet system are very high and compare well to those achieved with strong constitutive mammalian promoters (e.g., CMV). Unlike other inducible mammalian expression systems, gene regulation in the Tet system is highly specific, so interpretation of results is not complicated by pleiotropic effects or nonspecific induction. In Escherichia coli, the Tet repressor (TetR) negatively regulates genes within the tetracycline resistance operon on the Tn10 transposable element. In the absence of Tc, TetR blocks transcription of these genes by binding to the tet operator sequence (tetO). TetR and tetO provide the basis for regulation and induction in mammalian experimental systems. In the Tet-On system, the regulatory protein is based on the "reverse" Tet repressor (rTetR), which is generated by four amino acid changes in TetR (Hillen & Berens, Mechanisms underlying expression of Tn10 encoded tetracycline resistance. Annu Rev Microbiol. 1994;48:345-69; Gossen et al., Transcriptional activation by tetracyclines in mammalian cells. Science. 1995 Jun 23;268(5218):1766-9). The produced protein rtTA (reverse tTA also known as tetracycline activator protein) is encoded by the pTet-On regulatory plasmid.

[0094] In a related embodiment, the vector further comprises, consists essentially of, or consists of: a nucleic acid encoding a tetracycline activator protein; and a promoter that regulates expression of the tetracycline activator protein.

[0095] Other inducible systems that can be used with the vectors, isolated cells, viral packaging systems, and methods described herein include ecdysone, estrogen, progesterone, chemical inducers of dimerization, and regulation by isopropyl-β-D1-thiogalactopyranoside (IPTG).

[0096] As used herein, the term "recombinant expression system" or "recombinant vector" refers to one or more genetic constructs for expressing certain genetic materials formed by recombination.

[0097] A "gene delivery vector" is defined as any molecule capable of carrying a polynucleotide for insertion into a host cell. Examples of gene delivery vectors are: liposomes, micelles, biocompatible polymers, including natural and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; and bacteria or viruses, such as baculoviruses, adenoviruses, and retroviruses, phages, cosmids, plasmids, fungal vectors, and other recombinant vectors commonly used in the art, which have been described for expression in a variety of eukaryotic and prokaryotic hosts and can be used for gene therapy as well as simple protein expression.

[0098] The polynucleotides disclosed herein can be delivered to cells or tissues using gene delivery vectors. As used herein, "gene delivery," "gene transfer," "transduction," and the like refer to terms for introducing exogenous polynucleotides (sometimes referred to as "transgenes") into host cells, regardless of the method used to introduce them. Such methods include a variety of well-known techniques, such as vector-mediated gene transfer (such as by viral infection / transfection or various other protein- or lipid-based gene delivery complexes) and techniques for facilitating the delivery of "naked" polynucleotides (such as electroporation, "gene gun" delivery, and various other techniques for introducing polynucleotides). The introduced polynucleotides can be stably or temporarily maintained in the host cell. Stable maintenance generally requires that the introduced polynucleotides contain a replication origin that is compatible with the host cell, or are integrated into a replicon of the host cell, such as an extrachromosomal replicon (such as a plasmid) or a nuclear chromosome or mitochondrial chromosome. Many vectors are known to be able to mediate gene transfer to mammalian cells, as known in the art and described herein.

[0099] A "plasmid" is an extrachromosomal DNA molecule that is separate from chromosomal DNA and capable of replicating independently of it. In many cases, it is circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within microbial populations and often provide a selective advantage under given environmental conditions. Plasmids may carry genes that confer resistance to naturally occurring antibiotics in competitive environmental niches, or they may produce proteins that act as toxins under similar circumstances.

[0100] Plasmids used in genetic engineering are called plasmid vectors. Many commercially available plasmids are available for this purpose. The gene to be replicated is inserted into a copy of the plasmid, which contains the gene that confers resistance to a specific antibiotic and a multiple cloning site (MCS or polylinker), a short region containing several commonly used restriction sites that allows for easy insertion of DNA fragments. Another major use of plasmids is to produce large quantities of proteins. In this case, researchers grow bacteria containing a plasmid carrying the gene of interest. Just as the bacterium produces a protein to confer antibiotic resistance, it can also be coaxed into producing large quantities of protein from the inserted gene.

[0101] As used herein, the term "herpes simplex virus" or "HSV" refers to the herpes simplex virus that produces the effects of the present invention, including wild-type and mutant herpes simplex viruses. In one embodiment, the mutant non-natural HSV is obtained by mutating or modifying any gene of the wild-type HSV or by inserting any exogenous gene. HSV serotypes include, consist essentially of, or consist of HSV type 1 (or HSV-1) or HSV type 2 (or HSV-2). HSV-1 is an enveloped double-stranded DNA virus. In one embodiment, HSV-1 can infect human cells. In another embodiment, a sequence, gene, or multiple genes can be incorporated into HSV-1. The size of the combined sequence can be approximately 1 base, 5 bases, 10 bases, 100 bases, 1 kb, 10 kb, 100 kb, or 150 kb. HSV-1 can induce cell lysis at a relatively low multiplicity of infection (MOI), and its proliferation can be inhibited by antiviral drugs. In one embodiment, the HSV viral DNA remains extrachromosomal and is not incorporated into the host cell genome. HSV-1 can include multiple strains (e.g., KOS and McKrae). See Wang et al., (2013) Virus Res. 173(2):436–440. In one embodiment, HSV-1 is HSV-1 KOS strain. In another embodiment, HSV-1 is HSV-1 McKrae strain.

[0102] There are several HSV mutants, such as 17TermA HSV and rRp450 HSV. The term "17TermA HSV" refers to a mutant HSV-1 virus that contains the entire ICP34.5 gene, but with a stop codon inserted before 100 bp of the coding region, resulting in premature termination of protein expression and expression of a 30-amino acid truncated protein. The 17TermA HSV mutant exhibits growth defects due to the truncated ICP34.5 protein. See, Orvedahl et al., (2007) Cell Host & Microbe, 1:1, 23-25. The term "rRp450" refers to an attenuated herpes simplex 1 vector that lacks the virally encoded ribonucleotide reductase or ICP6. See, Aghi M et al., (1999) Cancer Res., 59(16):3861-5.

[0103] The HSV genome encodes multiple virulence proteins, including, but not limited to, glycoprotein E ("gE"), infected cell protein 0 ("ICP0"), infected cell protein 6 ("ICP6"), DNA packaging terminase subunit 1, infected cell protein 8 ("ICP8"), and infected cell protein 34.5 ("ICP34.5"). An exemplary HSV1 genome can be found in the NCBI reference sequence: NC_001806.2, last accessed on March 13, 2020.

[0104] The term "gE-encoding gene" refers to a gene or DNA fragment encoding the gE protein. An exemplary gE-encoding gene can be identified at positions 33-2555 of the HSV-1 genome sequence in the NCBI reference sequence: NC_001806.2. The term "ICP6 protein" refers to infected cell protein 6, encoded by the HSV genome. ICP6 is a subunit of ribonucleotide reductase (RR), a key enzyme for nucleotide metabolism and viral DNA synthesis in non-dividing cells.

[0105] A "dysfunctional" protein refers to a protein whose original protein function is impaired or non-functional. In one embodiment, the dysfunctional protein is caused by a deletion or substitution in the coding sequence. For example, if the ICP6 gene is dysfunctional, deleted or inactivated, HSV cannot replicate in normal non-dividing cells. However, in actively dividing cells with increased RR activity, the enzymatic activity that the virus lacks is compensated, allowing the virus to replicate. The DNA and amino acid sequences of ICP 34.5 are provided in SEQ ID Nos: 1, 2 and 5-10. The DNA and amino acid sequences of gE are provided in SEQ ID Nos: 12-19. The DNA and amino acid sequences of ICP 0 are provided in SEQ ID Nos: 20-26. The DNA and amino acid sequences of DNA packaging terminase subunit 1 are provided in SEQ ID Nos: 35-42. The DNA and amino acid sequences of ICP 8 are provided in SEQ ID Nos: 27-34. The DNA and amino acid sequences of ICP 6 are provided in SEQ ID Nos: 43-50.

[0106] The term "ICP0 encoding gene" refers to a gene encoding an ICPO protein, or a DNA fragment thereof. Exemplary DNA and amino acid sequences of ICP 0 are provided in SEQ ID Nos: 20-26. The term "DNA packaging terminase subunit 1 encoding gene" refers to a gene encoding a DNA packaging terminase subunit 1 protein or peptide, or a DNA fragment thereof. Exemplary DNA and amino acid sequences of DNA packaging terminase subunit 1 are provided in SEQ ID Nos: 35-42. The term "ICP 8 encoding gene" refers to a gene encoding an ICP 8 protein, or a DNA fragment thereof. Exemplary DNA and amino acid sequences of ICP 8 are provided in SEQ ID Nos: 27-34. The term "ICP 34.5 encoding gene" refers to a gene encoding an ICP 34.5 protein, or a DNA fragment thereof. Exemplary DNA and amino acid sequences of ICP 34.5 are provided in SEQ ID Nos: 1, 2, and 5-10. The term "glycoprotein E ("gE") encoding gene" refers to a gene encoding a gE protein, or a DNA fragment thereof. Exemplary DNA and amino acid sequences of gE are provided in SEQ ID Nos: 12-19.

[0107] The term "gene deletion or inactivation" refers to the deletion of all or part of a gene or the inhibition of gene expression by replacing certain bases, modifying, inserting unnecessary sequences, etc. Deletion or inactivation of HSV genes (e.g., gE, ICP0, and ICP8) can be performed by those skilled in the art using known methods or methods based on such methods. For example, a method using homologous recombination can be employed. For example, a DNA fragment containing a portion of the HSV gene and sequences unrelated to the HSV gene can be cloned into a suitable plasmid vector and then introduced into HSV, thereby inducing homologous recombination in certain regions of the HSV gene, thereby isolating and inactivating the HSV gene. Alternatively, mutation or deletion of the HSV gene can be caused by spontaneous mutation during viral passage.

[0108] In the context of gene transfer mediated by a DNA viral vector (e.g., herpes simplex virus), a vector construct refers to a polynucleotide comprising, consisting essentially of, or consisting of the viral genome or a portion thereof and a transgene. Thus, in one aspect, the non-natural HSV further comprises a transgene encoding a therapeutic polynucleotide or protein.

[0109] Vectors containing a promoter and a cloning site operably linked to a polynucleotide are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo and are commercially available from sources such as Agilent Technologies (Santa Clara, Calif.) and Promega Biotech (Madison, Wis.). To optimize expression and / or in vitro transcription, it may be necessary to remove, add, or alter the 5′ and / or 3′ untranslated portion of the clone to eliminate additional, potentially inappropriate, alternative translation start codons or other sequences that may interfere with or reduce expression, either at the transcriptional or translational level. Alternatively, a consensus ribosome binding site can be inserted immediately 5′ of the start codon to enhance expression.

[0110] Gene delivery vectors also include DNA / liposome complexes, micelles, and targeted viral protein DNA complexes. Liposomes that further comprise, consist essentially of, consist essentially of, consist essentially of, or consist of targeting antibodies or fragments thereof can be used in the methods disclosed herein. In addition to delivering polynucleotides to cells or cell populations, proteins described herein can be directly introduced into cells or cell populations by non-limiting protein transfection techniques, or culture conditions that enhance expression and / or promote activity of the proteins disclosed herein are other non-limiting techniques.

[0111] As used herein, the term "signal peptide" or "signal polypeptide" refers to an amino acid sequence that is typically present at the N-terminus of a newly synthesized secretory or membrane polypeptide or protein. Its function is to direct the polypeptide to a specific cellular location, for example, across the cell membrane, into the cell membrane, or into the cell nucleus. In some embodiments, the signal peptide is removed after localization. Examples of signal peptides are well known in the art. Non-limiting examples are those described in U.S. Patent Nos. 8,853,381, 5,958,736, and 8,795,965.

[0112] In one aspect, HSV is detectably labeled. As used herein, the term "label" refers to a directly or indirectly detectable compound or composition, such as a polynucleotide or protein, such as an antibody, that is directly or indirectly coupled to a composition to be detected to produce a "labeled" composition. The term also includes sequences coupled to a polynucleotide that provide a signal when expressed from the inserted sequence, such as green fluorescent protein (GFP). The label itself can be detectable (e.g., radioisotope labels or fluorescent labels) or, in the case of enzymatic labels, can catalyze a chemical alteration of a detectable substrate compound or composition. Labels can be suitable for small-scale detection or more suitable for high-throughput screening. Suitable labels include, but are not limited to, radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes, and proteins, including enzymes. Labels can be simply detected or quantified. A simple detection response typically includes a response that merely confirms the presence of, consists essentially of, or consists of, while a quantified response typically includes a response with a quantifiable (e.g., digitally reportable) value, such as intensity, polarization, and / or other characteristic. In luminescent or fluorescent assays, the detectable response can be produced directly using a luminophore or fluorophore associated with the assay component that actually participates in binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component.

[0113] The luminescent label example that produces signal includes but is not limited to bioluminescence and chemiluminescence.Detectable luminescent response generally includes the variation or the occurrence of luminescent signal, or is basically composed of it, or is composed of it. Suitable method and the luminophore that are used for luminescent label analysis component are known in the art, for example, described in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th ed.).The example of luminescent probe includes but is not limited to aequorin and luciferase.

[0114] Examples of suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, red pigment, coumarin, methylcoumarin, pyrene, apple green, stilbene, Lucifer Yellow, Cascade Blue™ and Texas Red. Other suitable optical dyes are described in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th ed.).

[0115] On the other hand, fluorescent labels are functionalized to facilitate covalent attachment to cellular components present in or on the surface of a cell or tissue, such as cell surface markers. Suitable functional groups include, but are not limited to, isothiocyanate, amino, haloacetyl, maleimide, succinimidyl ester, and sulfonyl halide, all of which can be used to attach the fluorescent label to a second molecule. The choice of fluorescent label functional group depends on the attachment site of the linker, reagent, label, or second label.

[0116] Attachment of the fluorescent label can be directly attached to the cellular component or compound, or can be attached through a linker. Suitable binding pairs for indirectly linking the fluorescent label to the intermediate include, but are not limited to, antigen / antibody, such as rhodamine / anti-rhodamine, biotin / avidin, and biotin / streptavidin.

[0117] The phrase "solid support" refers to a non-aqueous surface, such as a "culture plate," "gene chip," or "microarray." Such gene chips or microarrays can be used for diagnostic and therapeutic purposes by many techniques known to those skilled in the art. In one technique, oligonucleotides are linked and arrayed on a gene chip for determining DNA sequences by hybridization methods, such as those outlined in U.S. Patent Nos. 6,025,136 and 6,018,041. The polynucleotides of the present invention can be modified into probes, which can then be used to detect genetic sequences. For example, such techniques are described in U.S. Patent Nos. 5,968,740 and 5,858,659. Probes can also be attached or fixed to electrode surfaces for electrochemical detection of nucleic acid sequences, as described in Kayem et al. U.S. Patent No. 952,172 and Kelley et al. (1999) Nucleic Acids Res. 27: 4830-4837.

[0118] A "composition" refers to a combination of an active polypeptide, polynucleotide, or antibody and another inert (eg, a detectable label) or active (eg, a gene delivery vector) compound or composition.

[0119] A "pharmaceutical composition" refers to a composition comprising an active polypeptide, polynucleotide or antibody in combination with a carrier (inert or active, such as a solid support) such that the composition is suitable for diagnostic or therapeutic use in vitro, in vivo or in vitro.

[0120] As used herein, the term "pharmaceutically acceptable carrier" includes any standard pharmaceutical carrier, such as phosphate-buffered saline, water, and emulsions, such as oil / water or water / oil emulsions, and various types of wetting agents. The composition may also include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see Martin (1975) Remington's Pharm. Sci., 15th Ed. (Mack Publ. Co., Easton).

[0121] A "subject" for diagnosis or treatment is a cell or animal, such as a mammal or human. Subjects are not limited to a particular species and include non-human animals being diagnosed or treated, as well as animals affected by infections or animal models, for example, simians, murines (e.g., rats, mice, chinchillas), canines (e.g., dogs), lagomorphs (e.g., rabbits), livestock, sports animals, and pets. Human patients are also included within this term.

[0122] As used herein, the term "tissue" refers to tissue of a living or dead organism, or any tissue derived from or designed to simulate a living or dead organism. Tissue may be healthy, diseased, and / or have genetic mutations. Biological tissue may include any single tissue (e.g., a collection of cells that can be interconnected) or tissue that makes up an organ or body part or region of an organism. The tissue may comprise, consist essentially of, or be composed of homogenous cellular material, or may be a composite structure, such as found in body regions including the chest, which may include, for example, lung tissue, bone tissue, and / or muscle tissue. Exemplary tissues include, but are not limited to, tissue derived from the liver, lung, thyroid, skin, pancreas, blood vessels, bladder, kidney, brain, bile duct, duodenum, abdominal aorta, iliac vein, heart, and intestine, including any combination thereof.

[0123] As used herein, "treating" a disease in a subject means (1) preventing the onset of symptoms or disease in a subject who is susceptible to or who does not yet display symptoms of the disease; (2) inhibiting the disease or arresting its development; or (3) ameliorating or causing regression of the disease or disease symptoms. As understood in the art, "treatment" is an approach used to obtain a beneficial or desired result, including a clinical result. For the purposes of the present technology, a beneficial or desired result may include one or more of, but is not limited to: alleviation or amelioration of one or more symptoms; reduction in the extent of a condition (including a disease); stabilization (i.e., not worsening) of the condition (including a disease); delay or alleviation of a condition (including a disease); progression, improvement, or remission of a condition (including a disease); and remission (whether partial or complete), whether or not detectable.

[0124] As used herein, the term "effective amount" refers to an amount sufficient to achieve the desired effect. In the context of therapeutic or prophylactic applications, the effective amount will depend on the type and severity of the disease in question, as well as the characteristics of the individual subject, such as general health, age, sex, weight, and tolerance to the pharmaceutical composition. In the context of gene therapy, in some embodiments, an effective amount is an amount sufficient to restore partial or full function of the gene deficient in the subject. In one aspect, an effective amount is an amount that provides a multiplicity of infection (MOI) of 0.001 to 1 infectious viral particles per cell, with ranges therebetween. Non-limiting examples include an MOI of at least 0.001, or at least 0.01, or at least 0.1, or at least 1, or 0.01 to 1, or 0.1 to 1, or about 0.01 to 0.1, or less than 1, or less than 0.1, or less than 0.01 infectious viral particles per cell. In other embodiments, an effective amount of HSV viral particles is an amount sufficient to cause lysis of the subject's cells. In some embodiments, an effective amount is the amount required to increase galactose metabolism in a subject in need thereof. One skilled in the art will be able to determine an appropriate amount based on these and other factors.

[0125] In some embodiments, the effective amount will depend on the size and nature of the application. It will also depend on the nature and sensitivity of the target object and the method of use. Those skilled in the art will be able to determine the effective amount based on these and other considerations. According to embodiments, the effective amount may include one or more administrations of the composition, or consist essentially of, or consist essentially of, or consist of.

[0126] As used herein, the term "administration" or "administering" refers to the delivery of a substance to a subject (such as an animal or human). Administration can be a single administration, continuous administration, or intermittent administration, for example, intratumoral or intravenous administration, throughout the course of treatment. Methods for determining the most effective mode of administration and dosage are known to those skilled in the art and will vary with the composition used for treatment, the purpose of treatment, and the age, health, or sex of the subject being treated. Single or multiple administrations can be selected by the treating physician at the dosage level and pattern, and for pets and animals, by the treating veterinarian at the dosage level and pattern. Formulations and methods for administration of appropriate dosages are known in the art. The route of administration can be determined, and methods for determining the most effective route of administration are known to those skilled in the art and will vary with the composition used for treatment, the purpose of treatment, the health status or disease stage of the subject being treated, and the target cells or tissues. Non-limiting examples of routes of administration include direct and systemic, e.g., intravenous, intraarterial, intramuscular, intracardiac, intrathecal, subventricular, epidural, intracerebral, intratumoral, intracranial, intraventricular, subretinal, intravitreal, intraarticular, intraocular, intraperitoneal, intrauterine, intradermal, subcutaneous, transdermal, transmucosal, and inhalation.

[0127] Oncolytic herpes simplex virus (oHSV)

[0128] The anti-tumor efficacy of oHSV stems from direct cell killing (lytic phase) and enhanced anti-cancer immunity (immune phase). These viruses have been engineered in various ways to selectively target cancer cells. To achieve cancer selectivity, the most common mutation is deletion of the neurovirulence gene γ134.5 / RL1. Expression of ICP34.5, encoded by γ134.5 / RL1, is essential for HSV-1 to antagonize the host cell antiviral protein kinase RNA-activated (PKR) pathway, which normally phosphorylates e-IF2α in response to viral infection and halts protein translation. ICP34.5 redirects cellular protein phosphatase-1 (PP1) to dephosphorylate e-IF2α, allowing efficient viral replication. Many cancer cells, including γ134.5-null mutants, are defective in the PKR response and thus support HSV vector replication. Some vectors have been constructed by mutating key metabolic viral genes, such as ribonucleotide reductase (RR), the large subunit of which is encoded by the ICP6 / UL39 gene. Because many malignant cells have increased expression and activity of RRs, ICP6-null mutants replicate selectively in highly proliferative cancer cells due to the presence of abundant ribonucleotide pools.

[0129] HSV entry, cell-to-cell spread, and syncytium formation

[0130] HSV-1 entry involves a series of steps involving the interaction of viral glycoproteins with cell surface molecules. First, glycoproteins B and C (gB and gC) attach to cellular heparan sulfate proteoglycans, followed by gD binding to viral entry receptors (i.e., nectin-1, herpesvirus entry mediator (HVEM), or 3-O-heparan sulfate (3-OS-HS)). This process further allows gH / gL to interact with gB and trigger fusion of the viral envelope with the target cell membrane, leading to the release of virion capsid and intercalating proteins into the cell. Several reports suggest that membrane fusion is also crucial for the subsequent lateral spread of the virus between adjacent cells. Viral cell-to-cell spread occurs when the virus spreads from an infected cell to a neighboring uninfected cell through cell-cell contacts, even in the presence of virus-neutralizing antibodies. The presence of gE / gI enhances cell-to-cell spread, while gE subtype mutants reduce cell-to-cell fusion and plaque size. Syncytia form when multiple adjacent cells fuse to form multinucleated giant cells. Syncytia-triggering mutations have been found in at least four HSV viral genes (i.e., gB, gK, UL20, and UL24).

[0131] The present invention provides a strategy for developing clinically relevant viral vectors by combining "directed evolution" and CRISPR / Cas9 technology. This potent combination represents a substantial departure from prior art approaches. Without being bound by theory, it is anticipated that unexpected mechanisms may lead to increased viral potency in killing tumors. This is expected to reveal previously unknown mutations that contribute to the hyperfusogenic phenotype and enhanced potency, as well as a stepping stone for developing the next generation of oHSVs. The resulting oHSVs exhibit enhanced lytic phases, longer sustainability, and maximized therapeutic efficacy, consistent with the goals of the National Cancer Institute's clinical and translational exploratory / developmental research.

[0132] While the focus is on the "lytic phase" of viral therapy, a longer-lasting viral response promotes inflammatory responses in the body, thus facilitating the subsequent "immune phase" of viral therapy for optimal anti-tumor efficacy. Efficacy studies in pediatric cancer models can address the challenges of using non-natural HSVs for pediatric cancer treatment. The resulting modified viruses are also expected to be effective in a variety of adult cancers.

[0133] Mode for Carrying Out the Invention

[0134] Although HSV can target or infect a wide range of cells to induce lysis, viral infection itself has been shown to cause cytotoxicity and other pathologies, such as encephalitis, esophagitis, and pneumonia. Numerous HSV genes influence pathogenicity. For example, γ34.5 (RL1) can cause neuropathology. ICP6 (UL39), ribonucleotide reductase, thymidine kinase (UL23), uracil-DNA glycosylase (UL2), dUTPase (UL50), and DNA polymerase (UL30) are involved in HSV nucleotide metabolism and virulence. Therefore, there is a need to generate attenuated but replication-competent HSV particles to achieve their tumor suppressive function while minimizing side effects.

[0135] This article provides a novel virus, named Mut-3, which exhibits a giant syncytial plaque phenotype. Applicants isolated Mut-3 from serial passages of a mixture of 17TermA and rRp450 in a non-permissive line ("directed evolution") and constructed an attenuated mutant, Mut-3Δ34.5 ( Figure 1A ). Whole genome sequence analysis showed that Mut-3 acquired complete copies of γ134.5 / RL1 and UL39 (which encodes ICP6), making its genotype similar to that of the wild-type (WT) virus. Without being bound by theory, the applicant found that the lytic activity of Mut-3 was even higher than that of many WT viruses, suggesting that its enhanced potency may be related to other genomic changes (in addition to its complete viral genome). Five non-synonymous mutations were found in Mut-3 that were different from either parental virus, including an alanine to threonine mutation (A151T) at position 151 in the gene encoding gE. An attenuated version of the Mut-3 virus was provided, in which γ134.5 / RL1 was replaced with green fluorescent protein (GFP) by CRISPR / Cas9 gene editing, and was designated Mut-3Δ34.5 ( Figure 1A , bottom, labeled "CRISPR / Cas9" step). See, Figure 1B A summary of sequence comparisons of Mut-3 and its parental viruses is shown. Nonsynonymous mutations in Mut-3 that differ from either parent are shaded with backslashes and include UL15, UL29, US8, RL1, and RL2. Genomic sequences identical to 17TermA are shown as open boxes; sections identical to rRp450 are shaded with forward slashes.

[0136] Thus, the present invention provides a non-natural herpes simplex virus ("HSV"), wherein the virus comprises, consists essentially of, or consists of a mutation in a virulence gene from one or more of the following groups: (a) glycoprotein E ("gE") encoding gene, (b) infected cell protein 0 ("ICP0") encoding gene, (c) DNA packaging terminase subunit 1 encoding gene, (d) ICP8 encoding gene, or (e) ICP34.5 encoding gene. In one embodiment, the HSV further comprises, consists essentially of, or consists of a gene encoding a dysfunctional ICP34.5 protein and / or a gene encoding a dysfunctional ICP6 protein. In another embodiment, the gene encoding a dysfunctional ICP34.5 protein comprises, consists essentially of, or consists of a polynucleotide having a sequence at least 95% identical to SEQ ID No. 1, 5, 7, 9, or 51, and equivalents thereof, provided that the equivalent retains the mutated or altered amino acid or nucleotide. In another embodiment, the gene encoding the dysfunctional ICP6 protein comprises, consists essentially of, consists essentially of, consists of, consists of, or consists of a polynucleotide having a sequence at least 95% identical to SEQ ID No. 43, 45, 47 or 49, and equivalents thereof, provided that the equivalent retains the mutated or altered amino acid or nucleotide compared to the wt sequence.

[0137] In certain embodiments, the gE encoding gene of non-natural herpes simplex virus comprises the following sequence, or is essentially composed of the following sequence, or is composed of the following sequence: there is a polynucleotide and its equivalent with SEQ ID NO: 12,14,16,18 at least 80%, 85%, 90%, 95%, 97% or 99% identical sequence, provided that the equivalent keeps the amino acid or nucleotide that mutates or changes compared with the wt sequence. In another embodiment, the HSV comprising such equivalent enters cells and / or spreads and / or replicates DNA between cells at a level suitable with the non-natural HSV comprising the mutation gE with the amino acid sequence of SEQ ID NO: 13. Non-limiting examples of assessing HSV entry into cells, intercellular propagation and replication of DNA can be found in the embodiments. In another embodiment, the polynucleotide encoding of the gE encoding gene has a polypeptide selected from the amino acid sequence of SEQ ID NO: 13,15,17 and 19.

[0138] In certain embodiments, the ICPO encoding gene of the non-natural herpes simplex virus comprises, consists essentially of, or consists of a sequence selected from SEQ ID NOs: 20, 22, 24, 25, and 53, a sequence of any one of SEQ ID NOs: 20, 22, 24, 25, and 53 without one or two introns, a polynucleotide having a sequence at least 80%, 85%, 90%, 95%, 97%, or 99% identical to SEQ ID NOs: 20, 22, 24, 25, and 53, and equivalents thereof, provided that the equivalent retains the mutated or altered amino acid or nucleotide compared to the wt sequence. In one embodiment, the introns are indicated in the sequence listing and as follows: nucleotides (nt) 58 to nt 861 of SEQ ID NO: 20, nt 1529 to nt 1663 of SEQ ID NO: 20, nt 58 to nt 822 of SEQ ID NO: 22, nt 1490 to nt 1625 of SEQ ID NO: 22, 58 to nt 862 of SEQ ID NO: 24, nt 1530 to nt 1668 of SEQ ID NO: 24, nt 58 to nt 861 of SEQ ID NO: 25, nt 1529 to nt 1663 of SEQ ID NO: 25, nt 58 to nt 822 of SEQ ID NO: 53, nt 1490 to nt 1625 of SEQ ID NO: 53. In another embodiment, the equivalent encodes an ICPO polypeptide having a level of function comparable to that of wild-type ICPO or a mutant ICPO having the amino acid sequence of SEQ ID NO: 21 (e.g., promoting transcription of viral genes, disrupting nuclear structures called nuclear dots or promyelocytic leukemia (PML) nuclear bodies, and binding to neuron-specific proteins to alter expression of host and viral genes).Examples of assessing such functions can be found Lee HR, Kim DJ, Lee JM, et al. (June 2004). "Ability of the human cytomegalovirus IE1protein to modulate sumoylation of PML correlates with its functional activities in transcriptional regulation and infectivity in cultured fibroblast cells". J. Virol. 78 (12): 6527–42; Gu H, Liang Y, Mandel G, Roizman B (May 2005). "Components of the REST / CoREST / histone deacetylaserepressor complex are disrupted, modified, and translocated in HSV-1-infectedcells". Proc. Natl. Acad. Sci. USA 102 (21): 7571–6; and Pinnoji RC, Bedadala GR, George B, Holland TC, Hill JM, Hsia SC (2007). "Repressorelement-1 silencing transcription factor / neuronal restrictive silencer factor (REST / NRSF) can regulate HSV-1 immediate-early transcription via histonemodification". Virol. J. 4: 56. Additionally or alternatively, HSV comprising such equivalents enters cells and / or spreads between cells and / or replicates DNA at levels comparable to those of non-natural HSV comprising a mutant ICPO having an amino acid sequence of SEQ ID NO: 21. Non-limiting examples of assessing HSV cell entry, cell-to-cell spread, and DNA replication can be found in the Examples. In another embodiment, the polynucleotide encoding the ICPO gene encodes a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 23, and 26.

[0139] In certain embodiments, the non-natural herpes simplex virus ICP8 encoding gene comprises, consists essentially of, or consists of a sequence selected from SEQ ID NOs: 27, 29, 31, 33, a polynucleotide having a sequence at least 80%, 85%, 90%, 95%, 97%, or 99% identical to SEQ ID NOs: 27, 29, 31, and 33, and equivalents thereof, provided that the equivalent retains the mutated or altered amino acid or nucleotide sequence compared to the wt sequence. In another embodiment, the equivalent encodes an ICP8 polypeptide having a level of function comparable to that of wild-type ICP8 or a mutant ICP8 having the amino acid sequence of SEQ ID NO: 28 (e.g., annealing to single-stranded DNA (ssDNA), unwinding small fragments of double-stranded DNA, or destabilizing double-stranded DNA during the initiation of replication). Such functions can be assessed by methods available in the art, for example, Boehmer, PE; Lehman, IR (1993). "Herpes simplex virus type 1 ICP8: Helix-destabilizing properties". Journal of Virology. 67 (2): 711–5. Additionally or alternatively, an HSV comprising such an equivalent enters cells and / or spreads between cells and / or replicates DNA at a level comparable to that of a non-natural HSV comprising a mutant ICP8 having the amino acid sequence of SEQ ID NO: 28. Non-limiting examples of assessing HSV entry into cells, spread between cells, and replication of DNA can be found in the Examples. In another embodiment, the polynucleotide encoding the ICP8 gene encodes a polypeptide having an amino acid sequence selected from SEQ ID NOs: 28, 30, 32, and 34.

[0140] In certain embodiments, the DNA packaging terminase subunit 1 encoding gene of the non-natural herpes simplex virus comprises the following sequence, or is essentially composed of the following sequence, or is composed of the following sequence: a sequence selected from SEQ ID NO: 35, 37, 39, 41, a polynucleotide having a sequence at least 80%, 85%, 90%, 95%, 97% or 99% identical to SEQ ID NO: 35, 37, 39 and 41 and its equivalent, provided that the equivalent maintains a mutation or altered amino acid or nucleotide compared to the wt sequence. In another embodiment, the HSV comprising such equivalents enters cells and / or spreads between cells and / or replicates DNA at a level comparable to that of the non-natural HSV comprising a mutant DNA packaging terminase subunit 1 with the amino acid sequence of SEQ ID NO: 36. Non-limiting examples of evaluating HSV entry into cells, cell-to-cell spread and DNA replication can be found in the embodiments. In another embodiment, the polynucleotide encoding gene for the DNA packaging terminase subunit 1 has a polypeptide selected from the group consisting of SEQ ID NO: 36, 38, 40 and 42 amino acid sequences.

[0141] In certain embodiments, the HSV comprises, consists essentially of, or consists of a polynucleotide having a sequence selected from SEQ ID NOs: 12, 20, 27, and 35. In another embodiment, the HSV does not have functional ICP34.5 (i.e., encodes a dysfunctional ICP34.5 or does not encode ICP34.5). Additionally or alternatively, the HSV does not have functional ICP6 (i.e., encodes a dysfunctional ICP6 or does not encode ICP6). In another embodiment, the mutation in the virulence gene comprises, consists essentially of, or consists of an insertion, deletion, truncation, frameshift, substitution, or point mutation, for example, of the ICP34.5 gene and / or ICP6 gene. In another embodiment, the HSV lacks a gene encoding a functional ICP34.5 protein and / or a functional ICP6 protein. In another embodiment, the mutation is a non-synonymous mutation in a virulence gene.

[0142] In one embodiment, the mutations in the non-natural HSV of the invention encode one or more of the following: (a) an alanine to threonine mutation at position 151 of the gE protein, (b) an arginine to histidine mutation at position 258 of the ICPO protein, (c) an alanine to threonine mutation at position 376 of the DNA packaging terminase subunit 1 protein, (d) a threonine to methionine mutation at position 1155 of the ICP8 protein, or (e) a proline to histidine mutation at position 119 of the ICP34.5 protein. In another embodiment, the non-natural HSV comprises, consists essentially of, or consists of one or more of SEQ ID No. 2, SEQ ID No. 13, SEQ ID No. 21, SEQ ID No. 28, or SEQ ID No. 36, and equivalents thereof, provided that the equivalent retains the mutated or altered amino acid or nucleotide compared to the wt sequence.

[0143] In certain embodiments, a non-naturally occurring herpes simplex virus ("HSV") is provided, wherein the virus comprises, consists essentially of, or consists of a mutation in one or more of: (a) gE, (b) ICP0, (c) DNA packaging terminase subunit 1, (d) ICP8, or (e) ICP34.5. In certain embodiments, the HSV does not comprise a functional ICP34.5 protein (e.g., ICP34.5 from a 17TermA strain or an rRp450 strain). In another embodiment, the HSV does not comprise any ICP34.5 protein. Additionally or alternatively, the HSV does not comprise a functional ICP6 protein (e.g., ICP6 from a 17TermA strain or an rRp450 strain). In another embodiment, the HSV does not comprise any ICP6 protein.

[0144] In one embodiment, the mutation on the non-natural HSV of the present invention is one or more of the following: (a) an alanine to threonine mutation at position 151 of the gE protein, (b) an arginine to histidine mutation at position 258 of the ICP0 protein, (c) an alanine to threonine mutation at position 376 of the DNA packaging terminase subunit 1 protein, (d) a threonine to methionine mutation at position 1155 of the ICP8 protein, or (e) a proline to histidine mutation at position 119 of the ICP34.5 protein.

[0145] In certain embodiments, the gE of the non-natural HSV comprises, consists essentially of, or consists of an amino acid sequence selected from SEQ ID NOs: 13, 15, 17, and 19. In another embodiment, the non-natural HSV further comprises a polynucleotide encoding a gE amino acid sequence, for example, a polynucleotide having a sequence selected from SEQ ID NOs: 12, 14, 16, 18, and equivalents thereof. In certain embodiments, the ICPO of the non-natural HSV comprises, consists essentially of, or consists of an amino acid sequence selected from SEQ ID NOs: 21, 23, and 26. In another embodiment, the non-natural HSV further comprises a polynucleotide encoding an amino acid sequence of ICPO, for example, a polynucleotide having a sequence selected from SEQ ID NOs: 20, 22, 24, 25, 53, and equivalents thereof. In certain embodiments, the ICP8 of the non-natural HSV comprises, consists essentially of, or consists of an amino acid sequence selected from SEQ ID NOs: 28, 30, 32, and 34. In another embodiment, the non-natural HSV further comprises a polynucleotide encoding an amino acid sequence of ICP8, for example, a polynucleotide having a sequence selected from SEQ ID NOs: 27, 29, 31, 33, and their equivalents. In certain embodiments, the DNA packaging terminase subunit 1 of the non-natural HSV comprises, consists essentially of, or consists of an amino acid sequence selected from SEQ ID NOs: 36, 38, 40, and 42. In another embodiment, the non-natural HSV further comprises a polynucleotide encoding an amino acid sequence of DNA packaging terminase subunit 1, for example, a polynucleotide having a sequence selected from SEQ ID NOs: 35, 37, 39, 41, or their equivalents.

[0146] In certain embodiments, the non-naturally occurring HSV comprises, consists essentially of, or consists of one or more polypeptides having an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 21, 28, and 36. In another embodiment, the non-naturally occurring HSV comprises, consists essentially of, or consists of one or more polynucleotides encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 13, 21, 28, and 36.

[0147] In certain embodiments, the non-natural HSV comprises, consists essentially of, or consists of: (a) a polynucleotide encoding an amino acid sequence selected from the group consisting of SEQ ID Nos. 2, 6, 8, 10, and 52, and / or a polynucleotide having a sequence selected from the group consisting of SEQ ID Nos. 1, 5, 7, 9, and 51; (b) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID Nos. 2, 6, 8, 10, and 52; (c) a polynucleotide encoding an amino acid sequence selected from the group consisting of SEQ ID Nos. 13, 15, 17, and 19, and / or a polynucleotide having a sequence selected from the group consisting of SEQ ID Nos. 12, 14, 16, and 18; (d) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID Nos. 13, 15, 17, and 19; (e) a polynucleotide encoding an amino acid sequence selected from the group consisting of SEQ ID Nos. 21, 23, and 26, and / or a polynucleotide having a sequence selected from the group consisting of SEQ ID Nos. 20, 22, 24, 25 and 53 or a sequence thereof without one or two or more introns; (f) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs. 21, 23 and 26; (g) a polynucleotide encoding an amino acid sequence selected from the group consisting of SEQ ID NOs. 28, 30, 32 and 34, and / or a polynucleotide having a sequence selected from the group consisting of SEQ ID NOs. 27, 29, 31 and 33; (h) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs. 28, 30, 32 and 34; (i) a polynucleotide encoding an amino acid sequence selected from the group consisting of SEQ ID NOs. 36, 38, 40 and 42, and / or a polynucleotide having a sequence selected from the group consisting of SEQ ID NOs. 35, 37, 39 and 41; (j) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs. 36, 38, 40 and 42; (k) a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs. 44, 46, 48 and 50, and / or a polynucleotide having a sequence selected from SEQ ID NOs. 43, 45, 47 and 49; (l) a polypeptide having an amino acid sequence selected from SEQ ID NOs. 44, 46, 48 and 50.

[0148] In another embodiment, the non-natural HSV comprises, consists essentially of, or consists of a polynucleotide having a sequence identical to at least one segment of a virulence gene from 17TermA HSV and its equivalents. In another embodiment, the non-natural HSV comprises, consists essentially of, or consists of a polynucleotide having a sequence identical to at least one segment of a virulence gene from rRp450 HSV and its equivalents. In certain embodiments, the non-natural HSV is derived from an HSV type 1 ("HSV-1") or HSV type 2 ("HSV-2") strain. In one embodiment, the non-natural HSV is derived from an HSV-1 KOS strain. In another embodiment, the non-natural HSV further comprises, consists essentially of, or consists of a transgene.

[0149] Since the HSV disclosed herein retains its lytic function, on the other hand, the present invention also provides a method for treating cancer or inhibiting the growth or metastasis of cancer cells in a subject in need thereof, the method comprising the following steps, or consisting essentially of the following steps, or consisting of the following steps: administering to the subject an effective amount of the non-natural HSV of the present invention, or a composition comprising or consisting essentially of a non-natural HSV or consisting of a non-natural HSV. In one aspect, cancers include pancreatic cancer, kidney cancer, small cell lung cancer, brain cancer, neurological cancer, neuroblastoma, bone cancer, lymphoma, myeloma, colon cancer, uterine cancer, breast cancer, leukemia, liver cancer, prostate cancer, skin cancer or melanoma. The subject receiving treatment can be an adult or pediatric patient, such as a mammal or a human patient. In another embodiment, the non-natural HSV vector or composition or pharmaceutical composition is administered locally or systemically by injection, infusion, instillation and / or inhalation. In another embodiment, the subject is a mammal. In certain embodiments, the mammal is a mouse, rat, guinea pig, non-human primate, dog, cat, horse, cow, pig, goat or sheep. In another embodiment, the subject is a human.

[0150] In another aspect, the present invention provides a method for inducing cell lysis, comprising, consisting essentially of, or consisting of contacting a cell with a non-naturally occurring HSV or a composition comprising, consisting essentially of, or consisting of a non-naturally occurring HSV. In one embodiment, the cell is a cancer cell. In another aspect, the cell is a cultured cell (for use as a preclinical model or preclinical analysis) or a cell isolated from a subject. The cell can be cultured or in isolated tissue. Non-limiting examples of such cells include cells from pancreatic cancer, renal cancer, small cell lung cancer, brain cancer, neuroblastoma, neural cancer, bone cancer, lymphoma, myeloma, colon cancer, uterine cancer, breast cancer, leukemia, liver cancer, prostate cancer, skin cancer, or melanoma. These cells can be isolated from a mammal, for example, a human, which can be an adult or a juvenile (child).

[0151] In another aspect, the present invention provides a method for infecting a cell, comprising, consisting essentially of, or consisting of contacting the cell with a non-natural HSV. In one embodiment, the cell is a eukaryotic cell. In another embodiment, the cell is a lymphocyte. In one embodiment, the cell is a cancer cell, such as a blood cancer or a solid tumor cell, such as a carcinoma or sarcoma. In another aspect, the cell is a cultured cell (for use as a preclinical model or preclinical analysis) or a cell isolated from a subject. The cell can be cultured or in isolated tissue. Non-limiting examples of such cells include cells from pancreatic cancer, renal cancer, small cell lung cancer, brain cancer, neuroblastoma, neural cancer, bone cancer, lymphoma, myeloma, colon cancer, uterine cancer, breast cancer, leukemia, liver cancer, prostate cancer, skin cancer, or melanoma. These cells can be isolated from a mammal, for example, a human, such as an adult or a juvenile (child).

[0152] Applicants have discovered that conventional Epstein-Barr virus ("EBV")-infected lymphocytes are resistant to conventional oncolytic HSV ("oHSV"). Without being bound by theory, this resistance is at least in part due to low levels of HSV entry receptor expression on EBV-infected lymphocytes. Surprisingly, the HSV claimed in the present invention is able to induce lysis in EBV-infected lymphocytes that are resistant to wild-type strain 17, KOS, and McKrae HSV viruses. Thus, in one embodiment, the cell comprises, consists essentially of, or consists of an EBV-infected cell or, alternatively, a cell that is resistant to EBV. In another embodiment, the cell comprises, consists essentially of, or consists of virulence elements of Epstein-Barr virus ("EBV"). In another embodiment, the cell comprises, consists essentially of, or consists of an EBV-infected lymphocyte. In another embodiment, the HSV claimed herein has a higher infection rate in EBV-infected cells compared to a control group. In one embodiment, the control group comprises, consists essentially of, or consists of a conventional oHSV. In another embodiment, the conventional oHSV comprises wild-type strain 17 HSV, KOS HSV, or McKrae HSV.

[0153] Preparation of HSV mutants or derivatives

[0154] The preparation of HSV mutants or derivatives involves altering or mutating a gene or combination of genes encoded by the parent HSV strain. For example, a derivative may have an HSV-1 or HSV-2 genomic sequence modified by nucleotide substitutions (e.g., 1, 2, or 3 to 10, 25, 50, or 100 substitutions). The HSV-1 or HSV-2 genome can be selectively or alternatively modified by one or more insertions and / or deletions and / or extensions at one or both ends. Genetic modification methods are known in the art, such as CRISPR, recombinant construction, or point mutations. One of ordinary skill in the art knows how to prepare HSV mutants as needed.

[0155] In addition to targeted gene modification methods, HSV mutants can also be generated spontaneously. For example, the cultivation of viruses such as HSV involves a technique called continuous passaging. In order to culture and maintain the virus, suitable cells are infected with the virus, the virus replicates within the cells, and then the virus is harvested; fresh cells are then infected again, and this process constitutes a cycle of continuous passaging. For example, for HSV, each cycle may take several days. As described above, this continuous passaging may lead to changes in the characteristics or gene sequence of the viral strain, in which case characteristics that are beneficial to the clinical application of HSV are selected. For example, enhanced characteristics may include rapid replication, or the ability to spread along axons to infect human cells. In addition, spontaneous mutations can be prepared by infecting cells with one or more HSVs.

[0156] Thus, the present invention provides a method for preparing HSV or a mutant or derivative thereof by mutating a gene in HSV. In another embodiment, the method comprises, consists essentially of, or consists of the following steps: inducing a transgene into a non-natural HSV.

[0157] In another aspect, provided herein is a method for producing HSV viral particles, comprising, consisting essentially of, or consisting of the following steps: (a) introducing a 17TermA HSV vector and an rRp450 HSV vector into a host cell; (b) growing the host cell for at least three passages; and (c) isolating the HSV particles produced by the host cell. In one embodiment, HSV is introduced into the host cell by transfection, infection, transformation, electroporation, injection, microinjection, or a combination thereof. In another embodiment, the host cell is grown for at least three, 10, 20, 30, 40, or 50 passages. In some embodiments, the host cell comprises, consists essentially of, or consists of a complementary gene product to support replication of the introduced HSV vector. In another embodiment, the complementary gene encodes an ICP6 protein and / or an ICP34.5 protein. In another embodiment, the HSV particles produced in this manner comprise, consist essentially of, or consist of an HSV vector of the present invention.

[0158] In certain embodiments, provided herein is a method for preparing a non-natural HSV viral particle of the present invention. The method comprises, consists essentially of, or consists of the following steps: (a) introducing a non-natural HSV vector into a host cell; (b) growing the host cell; and (c) isolating HSV particles produced by the host cell.

[0159] In certain embodiments, provided herein is a method for producing a non-naturally occurring HSV viral particle of the present invention, comprising, consisting essentially of, or consisting of the following steps: (a) introducing a polynucleotide encoding the viral genome of a non-naturally occurring HSV vector into a host cell; (b) growing the host cell; and (c) collecting and isolating the HSV particles produced by the host cell. In one embodiment, the nucleic acid sequence encoding the viral genome is introduced into the host cell by transfection, infection, transformation, electroporation, injection, microinjection, or a combination thereof. In one embodiment, the nucleic acid sequence encoding the viral genome is introduced into the host cell in a vector. In another embodiment, the vector is a viral vector (e.g., HSV) or a non-viral vector (e.g., a plasmid or nanoparticle). In another embodiment, the vector is HSV. In certain embodiments, the host cell comprises, consists essentially of, or consists of a complementary gene product to support replication of the introduced HSV vector. In one embodiment, the complementary gene product is provided in the host cell by a helper virus. In another embodiment, the complementary gene encodes ICP6 protein and / or ICP34.5 protein. In another embodiment, the HSV particles so produced comprise, consist essentially of, or consist of an HSV vector of the invention.

[0160] In one embodiment, the isolating step refers to a process of substantially separating the HSV from other materials (e.g., host cells, cell debris, culture medium, or any other reagents used to culture the host cells) by, for example, centrifugation, filtration, chromatography, or any combination thereof. Non-limiting examples can be found in Sia et al, Optimal purification method for Herpes-based viral vectors that confers minimal cytotoxicity for systemic route of vector administration. J Virol Methods. 2007 Feb;139(2):166-74.

[0161] Composition

[0162] In another aspect, the present invention provides a composition comprising, consisting essentially of, or consisting of a non-naturally occurring HSV as described herein. Compositions (including pharmaceutical compositions) comprising, consisting essentially of, or consisting of an agent or viral particle as described herein can be prepared by conventional mixing, dissolving, granulating, grinding, emulsifying, encapsulating, embedding, or lyophilizing techniques. Such compositions can be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, adjuvants, or adjuvants to facilitate processing of the viral particles provided herein into pharmaceutically acceptable formulations.

[0163] The agents and viral particles of the present technology can be administered parenterally (e.g., intramuscularly, intraperitoneally, intravenously, intracerebroventricularly ("ICV"), intracerebral injection or infusion, subcutaneous injection or implant), orally, by nasal inhalation spray, vaginally, rectally, sublingually, urethrally (e.g., urethral suppositories), or by topical administration (e.g., gels, ointments, creams, aerosols, etc.), and can be formulated, alone or together, into suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, adjuvants, and solvents suitable for each route of administration.

[0164] In one embodiment, the present invention relates to a composition comprising, consisting essentially of, or consisting of a non-natural HSV viral particle as described herein and a vector.

[0165] In another embodiment, the present invention relates to a pharmaceutical composition comprising, consisting essentially of, or consisting of a non-natural viral particle as described herein and a pharmaceutically acceptable carrier. In another embodiment, the composition comprises, consists essentially of, or consists of a cryoprotectant that facilitates freezing and thawing of the non-natural HSV without significant loss of toxicity.

[0166] In another embodiment, the present invention relates to a pharmaceutical composition comprising, consisting essentially of, or consisting of a therapeutically effective amount of a non-natural HSV viral particle as described herein and a pharmaceutically acceptable carrier.

[0167] The pharmaceutical composition for administering HSV viral particles can be conveniently presented in the form of dosage units and can be prepared by any method well known in the pharmaceutical field. For example, the HSV viral particles provided herein can be uniformly and tightly combined with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, the product is formed into the desired dosage form to prepare a pharmaceutical composition. In the pharmaceutical composition, the compound provided herein is included in an amount sufficient to produce the desired therapeutic effect. For example, the pharmaceutical composition of the present invention can be in a form suitable for almost any mode of administration, including, for example, topical, ocular, oral, mucosal, systemic, nasal, injection, infusion, transdermal, rectal and vaginal administration, or a form suitable for administration by inhalation or insufflation.

[0168] For topical administration, the non-natural HSV viral particles can be formulated into solutions, gels, ointments, creams, suspensions, etc., as is well known in the art.

[0169] Systemic formulations include those designed for administration by injection (e.g., subcutaneous, intravenous, infusion, intramuscular, intradural, or intraperitoneal injection), as well as those designed for transdermal, transmucosal, oral, or pulmonary administration.

[0170] Effective injectable formulations include sterile suspensions, solutions or emulsions of HSV virus particles provided herein in aqueous or oily solvents. The compositions may further comprise preparatons, such as suspending agents, stabilizers and / or dispersants. Injectable formulations may be presented in unit dosage form, such as ampoules or multi-dose containers, and may contain added preservatives.

[0171] Alternatively, the injectable formulation can be provided in powder form so that it can be reconstituted with a suitable solvent before use, including but not limited to sterile, heat-free water, buffer, and glucose solution. To this end, the HSV virus particles provided herein can be dried by any technique known in the art (e.g., lyophilized) and reconstituted before use.

[0172] For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art.

[0173] For oral administration, the pharmaceutical composition can be in the form of, for example, lozenges, tablets, or capsules prepared by conventional methods using pharmaceutically acceptable excipients such as binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, or silicon dioxide); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Tablets can be coated, for example, with sugar, film, or enteric coatings by methods known in the art.

[0174] Compositions for oral administration can be prepared according to any method known in the art for manufacturing pharmaceutical compositions, and such compositions may include agents selected from sweeteners, flavorings, colorants, and preservatives to provide pharmaceutically elegant and palatable formulations. Tablets contain the HSV viral particles provided herein, mixed with non-toxic, pharmaceutically acceptable excipients suitable for tablet manufacture. These excipients may include, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate; granulating and disintegrant agents (such as corn starch or alginic acid); binders (such as starch, gelatin, or gum arabic); and lubricants (such as magnesium stearate, stearic acid, or talc). Tablets may be uncoated or coated using known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained effect over a longer period. For example, time-delay materials such as glyceryl monostearate or glyceryl distearate may be used. These may also be coated using techniques well known to those skilled in the art. Pharmaceutical compositions of this technology may also be in the form of oil-in-water emulsions.

[0175] Liquid preparations for oral administration can take the form of, for example, elixirs, solutions, syrups, or suspensions, or can be presented as a dry product before use with water or other suitable solvents. Such liquid preparations can be prepared by conventional methods using pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifiers (e.g., lecithin or gum arabic); non-aqueous solvents (e.g., almond oil, oily esters, ethanol, cremophore™, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl paraben or sorbic acid). The preparations may also optionally contain buffer salts, preservatives, flavorings, colorants, and sweeteners.

[0176] In one embodiment, a method for preparing a non-natural HSV of the present invention is provided, the method comprising, consisting essentially of, or consisting of mutating a gene in a non-natural HSV virus particle or introducing a transgene into a non-natural HSV. In another aspect, a method for preparing a non-natural HSV vector comprises, consisting essentially of, or consisting of: (a) introducing a 17TermA HSV vector and an rRp450 HSV vector into a host cell; (b) growing the host cell for at least three generations; and (c) isolating HSV particles produced by the host cell.

[0177] Also provided is a method for inhibiting the growth or metastasis of cancer cells or metastatic cancer cells, the method comprising, consisting essentially of, or consisting of the following steps: contacting the cells with an effective amount of a non-natural HSV vector or a composition or pharmaceutical composition containing a non-natural HSV vector as described herein. The contact is in vitro or in vivo. In one aspect, the contact is performed in vivo by administering a non-natural HSV or composition or pharmaceutical composition to a subject. In vitro, the method is performed by contacting the non-natural HSV with the cells. The in vitro method can be used to test new therapies, or as a personalized analysis to determine whether the therapy is suitable for the cancer to be treated. Additional cancer therapies can be combined with treatments that can be performed simultaneously or sequentially with the methods disclosed herein.

[0178] The cancer cells to be treated can be solid tumors or blood cancers, such as carcinomas or sarcomas, and non-limiting examples include pancreatic cancer, renal cancer, small cell lung cancer, brain cancer, neuroblastoma, neurological cancer, bone cancer, lymphoma, myeloma, colon cancer, uterine cancer, breast cancer, leukemia, liver cancer, prostate cancer, skin cancer, or melanoma. The cells belong to any species, such as mammals and humans, and when performed in vitro, they can be from cultured cell lines or primary cells, such as from tissue biopsies. The cells can be adult or juvenile cells or cancer stem cells (i.e., cancer cells with characteristics associated with normal stem cells, particularly the ability to produce all cell types found in a particular cancer sample) or cancer cells that do not have characteristics associated with normal stem cells. In one embodiment, the cells express the N-myc proto-oncogene protein (MYCN) and / or express MYCN at a higher level than non-cancerous cells.

[0179] In addition, the present invention also provides a method for treating cancer or inhibiting the growth or metastasis of cancer cells in a subject in need thereof, the method comprising the following steps, or consisting essentially of the following steps, or consisting of the following steps: administering to the subject an effective amount of the non-natural HSV, composition or pharmaceutical composition of the present invention. The subject to be treated can be any species, such as mammals and humans, such as dogs, horses, cows, cats, monkeys, rats or mice. Administration can be used as a first-line therapy, a second-line therapy, a third-line therapy, a fourth-line therapy or a fifth-line therapy. Additional cancer therapies can be combined with treatments that can be performed simultaneously or sequentially with the methods disclosed herein. The cancer cells to be treated can be solid tumors or blood cancers, such as carcinomas or sarcomas, and non-limiting examples include pancreatic cancer, kidney cancer, small cell lung cancer, brain cancer, neuroblastoma, neurocarcinoma, bone cancer, lymphoma, myeloma, colon cancer, uterine cancer, breast cancer, leukemia, liver cancer, prostate cancer, skin cancer or melanoma.

[0180] The methods of the present invention can be combined with appropriate diagnostics to monitor the remission or progression of the disease. Several methods for such monitoring are known in the art.

[0181] Further provided is a method of inducing cell lysis, comprising, consisting essentially of, or consisting of contacting cells with an effective amount of a non-natural HSV, composition, or pharmaceutical composition of the present invention. The contacting is in vitro or in vivo. In one aspect, the contacting is performed in vivo by administering the non-natural HSV, composition, or pharmaceutical composition to a subject. In vitro, the method is performed by contacting the non-natural HSV with cells. The in vitro method can be used to test new therapies or as a personalized analysis to determine whether the therapy is suitable for the subject to be treated. Additional cytolytic therapies can be combined with treatments that can be performed simultaneously or sequentially with the methods disclosed herein.

[0182] The cells to be treated can be solid tumors or blood cancers, such as carcinomas or sarcomas, and non-limiting examples include pancreatic cancer, renal cancer, small cell lung cancer, brain cancer, neuroblastoma, neurocarcinoma, bone cancer, lymphoma, myeloma, colon cancer, uterine cancer, breast cancer, leukemia, liver cancer, prostate cancer, skin cancer, or melanoma. The cells are of any species, such as mammals and humans, and when performed in vitro, they can be from cultured cell lines or primary cells, such as from a tissue biopsy. The cells can be adult or juvenile cells, or cancer stem cells, or cancer cells that do not have characteristics associated with normal stem cells. The therapy can be combined with appropriate assays to test the effectiveness of the therapy, such as cancer remission or progression.

[0183] Application of HSV virus particles in the preparation of medicines

[0184] The HSV and compositions of the present invention can also be used to prepare medicaments for treating various pathologies described herein. Methods and techniques for preparing medicaments for the compositions are known in the art. For illustrative purposes only, the pharmaceutical formulations and delivery routes are described in detail herein.

[0185] Therefore, it will be readily understood by those skilled in the art that any one or more of the compositions described above, including many specific embodiments, can be prepared by applying standard pharmaceutical manufacturing procedures to prepare drugs for treating the various diseases described herein. Such drugs can be delivered to a subject using delivery methods known in the pharmaceutical field.

[0186] Administration of additional therapeutic agents

[0187] The methods disclosed herein may comprise, consist essentially of, or consist of administering an effective amount of an additional therapeutic agent to enhance or improve the therapeutic effect of the methods disclosed herein. In one embodiment, the additional therapeutic agent comprises, consists essentially of, or consists of surgical resection of the tumor, an anti-tumor agent (e.g., a small molecule or immunotherapy or cytolytic therapy).

[0188] Administration of the therapy or substance of the present invention to a patient will follow the general dosing regimen for a particular primary or secondary treatment, taking into account the toxicity of the treatment, if any. It is anticipated that treatment cycles will be repeated as necessary. It is also contemplated that various standard therapies as well as surgical interventions may be used in combination with the therapy.

[0189] As will be apparent to those skilled in the art, combination therapy may take the form of simultaneous or sequential administration of the combination therapy.

[0190] Reagent test kit

[0191] In some embodiments, the reagents or non-natural HSV described herein can be assembled into pharmaceutical or diagnostic or research kits to facilitate their use in therapeutic, diagnostic or research applications. The kit may include one or more reagents described herein, as well as instructions describing the intended application and proper use of these reagents. Specifically, the kit may include instructions for mixing one or more components of the kit and / or separating and mixing samples and applying them to a subject. In certain embodiments, the reagents in the kit are pharmaceutical formulations and dosages suitable for specific applications and methods of administration. Kits for research purposes may contain components in appropriate concentrations or quantities for conducting various experiments.

[0192] The design of the kit can facilitate the use of the methods described herein and can take a variety of forms. Where applicable, each composition of the kit can be provided in liquid form (e.g., a solution) or solid form (e.g., a dry powder). In some cases, some compositions can be composable or otherwise processable (e.g., to an active form), such as by adding a suitable solvent or other species (e.g., water or cell culture medium), which can be provided with or without the kit. In some embodiments, the composition can be provided in a preservation solution (e.g., a cryogenic preservation solution). Non-limiting examples of preservation solutions include DMSO, paraformaldehyde, and CryoStor® (Stem Cell Technologies, Vancouver, Canada). In some embodiments, the preservation solution contains an amount of a metalloproteinase inhibitor.

[0193] As used herein, "instructions" may define the components of the instructions and / or promotional material and generally refer to written instructions for or associated with the packaging of the method, recombinant vector, or composition. Instructions may also include any oral or electronic instructions provided in any manner so that the user will clearly recognize that the instructions are associated with the kit, such as audiovisual (e.g., videotape, DVD, etc.), Internet and / or web-based communication, etc. The written instructions may be in a format prescribed by a governmental agency regulating the manufacture, use, or sale of drugs or biological products. The instructions may also reflect approval for manufacture, use, or sale by an animal regulatory agency.

[0194] In some embodiments, the kit comprises any one or more of the components described herein in one or more containers. For example, in one embodiment, the kit may include instructions for mixing one or more components of the kit and / or separating and mixing a sample and applying it to a subject. The kit may include containers for holding the reagents described herein. The HSV may be in liquid, gel, or solid (powder) form. The HSV may be aseptically prepared, packaged in a syringe, and shipped refrigerated. Alternatively, it may be placed in a vial or other container for storage. A second container may contain other sterile prepared reagents. Alternatively, the kit may include premixed active agents and shipped in a syringe, vial, test tube, or other container. The kit may include one or more or all of the components required to administer the agent to a subject, such as a syringe, a topical application device, or an IV needle and bag.

[0195] Screening test

[0196] The present invention also provides screening assays to identify known and novel compounds and combinations as potential therapeutic agents. For example, one skilled in the art can also determine whether HSV provides therapeutic benefit in vitro by contacting HSV with a sample cell or tissue to be treated. The cell or tissue can be from any species, such as monkey, dog, cow, sheep, rat, mouse, or human.

[0197] Contacting can also be performed in an appropriate animal model or in vivo in a human patient. When performed in vitro, HSV can be added directly to the cell culture medium. When performed in vitro, the method can be used to screen new combination therapies, formulations, or treatment regimens prior to administration to an animal or human patient.

[0198] In another aspect, the assay entails contacting a first sample comprising appropriate cells or tissue (a "control sample") with an effective amount of an HSV disclosed herein, and contacting a second sample of appropriate cells or tissue (a "test sample") with the HSV, agent, or combination to be analyzed. In one aspect, in the case of cancer, growth inhibition is determined for the first and second cell samples. If the growth inhibition of the second sample is substantially the same as or greater than that of the first sample, the agent is a potential therapeutic drug. In one aspect, substantially the same or greater cell growth inhibition is a difference of less than about 1%, or less than about 5%, or less than about 10%, or greater than about 10%, or greater than about 20%, or greater than about 50%, or greater than about 90%. Contact can be performed in vitro or in vivo. Methods for determining cell growth inhibition are well known in the art.

[0199] In another aspect, the test agent is contacted with a third sample of cells or tissues comprising a control and normal counterpart of the test sample, and an agent is selected that treats the second cell or tissue sample without adversely affecting the third sample. For the purposes of the assays described herein, suitable cells or tissues are described herein, such as cancer or other diseases described herein. Examples of such methods include, but are not limited to, cancer cells or tissues obtained by biopsy or blood.

[0200] The efficacy of the test composition is determined using methods known in the art, including but not limited to cell viability assays.

[0201] On the other hand, the assay requires at least two cell types, the first of which is a suitable control cell.

[0202] These assays can also be used to predict whether a subject will respond appropriately to treatment according to the present invention by delivering HSV to a sample containing cells to be treated and analyzing the treatment (which will vary with pathology), or to screen for new drugs and combinations. In one aspect, cells or tissue are obtained from a subject or patient by biopsy. The present invention also provides kits for determining whether pathological cells or patients will respond appropriately to treatment according to the present invention by providing at least one composition of the present invention and instructions for use.

[0203] Test cells can be grown in small multiwell plates and used to measure the biological activity of test compounds. For purposes of the present invention, a successful HSV or other agent will prevent the growth of or kill cancer cells, but leave control cell types unharmed.

[0204] The following examples are provided to illustrate but not to limit the present invention. Example

[0205] Generation of HSV mutants

[0206] Non-permissive cell lines were infected with 17TermA and rRp450 (“directed evolution”) and cultured after serial passage to isolate HSV Mut-3 mutants containing a WT-like genotype. The HSV Mut-3 mutant was then used to construct the attenuated mutant Mut-3∆34.5 ( Figure 1A ). Figure 1B A sequence comparison of Mut-3 and its parental viruses is shown. Nonsynonymous mutations in Mut-3 that differ from either parent are indicated by backslashes and include UL15, UL29, US8, RL1, and RL2. Genomic sequences identical to 17TermA are shown as open boxes; sequences identical to rRp450 are indicated by forward slashes.

[0207] A potent oHSV mutant, Mut-3, was isolated, harboring a WT-like genotype. Both Mut-3 and its attenuated version, Mut-3∆34.5, rely on the canonical HSV entry protein, nectin-1, or the herpesvirus entry mediator (HVEM) for successful infection (not shown). Compared to 17∆34.5 (a ∆34.5-null virus derived from wild-type strain 17 via the same CRISPR / Cas9 gene-editing strategy), the Mut-3∆34.5 virus exhibited much earlier gene transfer kinetics, as measured by the onset of detectable GFP-positive areas. Furthermore, infection with Mut-3∆34.5 resulted in increased cell death, as measured by its smaller area of ​​cell confluence. However, Mut-3∆34.5 appeared to be less replicative than either 17∆34.5 or the parental virus, 17TermA, of Mut-3∆34.5. These results suggest that even in the attenuated Mut-3∆34.5 version, unique genomic alterations result in a syncytial phenotype (not shown) and enhanced potency. In a study of epidermal growth factor receptor (EGFR)-repurposed HSV, it was reported that the introduction of syncytial mutations did not compromise entry and spread specificity. Without being bound by theory, the applicants believe that the underlying cause of the syncytial phenotype in Mut-3∆34.5 may result from altered kinetics of viral fusion to the cell membrane, thereby affecting entry and / or virus-mediated cell-to-cell fusion, leading to faster cell killing and reduced viral particle production.

[0208] No mutations were found in the canonical HSV-1 entry glycoproteins gB, gD, gH, and gL. However, unlike either parental strain, Mut-3 harbored nonsynonymous (NS) mutations in five genes: RL1, RL2, UL15, UL29, and Us8 / gE. Both Mut-3 and its attenuated version, Mut-3∆34.5, displayed a fusion phenotype, indicating that this phenomenon was unrelated to RL1. Genome-wide analysis also demonstrated that this phenotype was unrelated to previously reported mutations, as the amino acid sequence of Mut-3 was identical to either the reference strain 17 genome (non-syncytial virus) in rRp450 and gK / UL53 and UL20, or to 17TermA (non-syncytial virus) in gB / UL27 and UL24. The A151T mutation in gE / Us8 was the only glycoprotein (virion surface protein) in Mut-3 to have a NS change compared to its parental strains. Although gE is not implicated in viral attachment or entry, gE / gI dimerization mediates cell-to-cell spread of the virus, as mutations in gE are associated with smaller plaque size. **Applicants hypothesize that A151T in gE of Mut-3∆34.5 may underlie its syncytial and potentiated phenotypes. Table 1 lists all five NS mutations identified in the five genes of Mut-3, along with the corresponding function of each gene, its potential role in the Mut-3 potentiated phenotype, and the proposed studies, according to applicants' research priorities (from top to bottom). The other three mutations (except RL1) can also be identified, as was done for gE A151T.

[0209] Table 1 HSV protein mutations and their functions, nt: nucleotide; aa: amino acid.

[0210]

[0211]

[0212] Comparison of HSV mutants

[0213] At the same time Figure 1A The four viruses shown were subjected to plaque assays, and the plaque images were scanned and analyzed 3 days later using a Keyence HS multifunctional fluorescence microscope BZ-II analyzer. Figure 1A and 1BAs shown, the plaque size of Mut-3 and Mut-3Δ34.5 was significantly larger than that of the parental viruses rRp450 and 17TermA. In the in vitro cytotoxicity / MTS assay of CHO cell lines, CHO-K1, CHO-Nectin-1, CHO-Nectin-2, and CHO-HVEM were infected with the four viruses at different multiplicities of infection (MOI). Cell viability was measured by colorimetric cell proliferation and MTS assays 3 days after virus infection (pvi) relative to untreated controls. Only CHO-Nectin-1 and CHO-HVEM, but not CHO-K1 or CHO-Nectin-2 (primarily for HSV-2 entry), were sensitive to treatment with the four viruses ( Figure 2C Without being bound by theory, the results suggest that Mut-3 and Mut-3Δ34.5 do not bypass the receptor barrier and still rely on the canonical HSV entry receptor to infect cells.

[0214] The increased potency of Mut-3Δ34.5 in killing human and mouse neuroblastoma cells compared to 17TermA was not due to an increase in infectious virus production (Figure 3). Transmission electron microscopy (TEM) analysis also showed that after infection of neuroblastoma cells, Mut-3Δ34.5 virions were primarily located in endocytic vesicles, whereas 17TermA virions were primarily located in endocytic vesicles (Figure 4).

[0215] The attenuated 17Δ34.5 mutant was generated by CRISPR-Cas9 gene editing technology to replace the g134.5 gene in the wild-type strain 17+ with an EGFP expression cassette. The 17Δ34.5 mutant was less potent than its wild-type counterpart 17+ but comparable to 17TermA (Figure 5). The attenuation of 17Δ34.5 was further confirmed when Mut-3Δ34.5 showed faster viral gene transfer and cell killing compared to 17Δ34.5 (Figure 6). In addition, Mut-3Δ34.5 was more effective than 17TermA in controlling the growth of human neuroblastoma cells in vivo ( Figure 7 ).

[0216] Us8 / gE revertant in Mut-3Δ34.5 for loss-of-function studies

[0217] Introducing single nucleotide changes in the HSV genome using CRISPR / Cas9 technology alone is difficult because multiple copies of the genome are produced during viral replication and a selectable marker cannot be added to accompany the gene editing. A construct can be the result of a two-step process combining CRISPR / Cas9 technology: replacing the entire gE coding region with a reporter gene such as mCherry or red fluorescent protein (RFP) to construct gE-null. Mut-3∆34.5; then 2) replacing the reporter gene with the WT-gE coding region to generate a gE-WT Mut-3∆34.5 revertant. As described above, the extent to which the gE-null intermediate and the gE revertant Mut-3∆34.5 lose their phenotype can be observed. This construct is also provided by the present invention.

[0218] Us8 / gE A151T mutation in Mut-3 parent 17TermA for gain-of-function

[0219] As described above, the gE mutant 17TermA can be constructed in two steps: 1) completely knocking out the gE coding region and replacing it with a reporter gene (e.g., GFP); then 2) replacing the reporter gene by knocking in a gE coding region containing the A151T mutation. The phenotype of Mut-3∆34.5 will be determined. This construct is also provided herein.

[0220] If gE A151T is responsible for only some or no observed phenotypes, or even if it is, the present invention also provides additional mutations in Mut-3. Mutants are constructed by systematically employing similar gain-of-function and loss-of-function approaches, either alone or in combination, as described herein and shown in Table 1. The safety and efficacy of the attenuated Mut-3∆34.5 can be tested in mouse models. Therefore, the present invention provides animal models and testing methods for testing mutants.

[0221] The Mut-3 strain is a potent WT-like HSV mutant constructed by recombinantly constructing 17TermA and rRp450. By deleting the viral virulence protein γ134.5 / RL1, the attenuated version Mut-3Δ34.5 was created to ensure its safety for clinical use. In the applicant's pilot toxicology study, no adverse clinical symptoms or significant changes in body weight were observed in Balb / c mice after intravenous injection of up to 1e8 plaque-forming units (pfu) for more than 85 days (not shown). In addition, compared with 17TermA, Mut-3Δ34.5 showed no adverse clinical symptoms or significant changes in body weight in vitro ( Figure 3A ) and in vivo ( Figure 7 ) also showed antitumor efficacy in a highly aggressive neuroblastoma model.

[0222] Biodistribution of Mut-3Δ34.5 in naive non-tumor-bearing mice

[0223] The applicant's own studies have shown that naive Balb / c mice can tolerate up to 1e8 pfu of Mut-3∆34.5 virus delivered intravenously (iv) without any signs of disease for over 85 days. Biodistribution studies began with intravenous injections of the highest dose previously tested (1e8 pfu of Mut-3∆34.5 virus per mouse) in naive, non-tumor-bearing Balb / c mice of both sexes (30 mice per sex). Peripheral blood was collected, and the mice were sacrificed. Testes, ovaries, spleen, lungs, kidneys, heart, lung, and brain were harvested at 24 hours, 14 days, 28 days, 56 days, and 85 days pvi (n=6 per time point). Half of the organs were embedded in formalin for pathological analysis, and the other half were homogenized for qPCR analysis of the HSV genome and plaque analysis to determine the viral load in each organ. Mice were observed daily for two weeks after dosing and then twice weekly until the scheduled sacrifice. The body weight of each mouse was measured before and weekly after viral infection. Mice were sacrificed for adverse clinical signs or weight loss >20%, and viral activity in organs was analyzed as described above. 5 Up to 1 x 10 7 pfu (n=3)] were administered to each group of mice as a positive (non-safety) control. 6 A KOS virus dose of 1 pfu (100 μg) was uniformly lethal in FVBN mice within 2 to 3 days. qPCR was used to analyze HSV genome copies, plaque assays were used to assess viral activity, and pathological analysis was performed on organs from positive control mice showing signs of disease. These results served as a positive indicator / threshold for evaluating data collected from mice treated with Mut-3∆34.5. Tissue / organ pathological changes in the Mut-3∆34.5-treated group were further evaluated to demonstrate viral loads comparable to those in the positive control group. Six mice per group of each sex were used to assess biodistribution and safety and tolerability over time. Mut-3∆34.5 biodistribution was measured as HSV genome copies per nanogram of genomic DNA in various organs over time, using descriptive statistics and compared with univariate analysis where applicable.

[0224] Mut-3∆34.5 and other γ 1 34.5 / RL1 virus (17TermA and T-VEC) in various pediatric cancer cell lines In vitro cytotoxicity

[0225] Compared with 17TermA in human and mouse neuroblastoma cells, a superior killing effect of Mut-3∆34.5 was observed ( Figure 3A ).use Figure 3AThe same MTS used in the in vitro assay shown will be used in other pediatric cancer cell lines, such as sarcomas, malignant peripheral nerve sheath tumors (MPNSTs), and brain tumors (multiple models are available to the applicant) to determine whether the increased efficacy phenotype applies to different tumor types. The most potent cell line in each cell type will be used for in vivo efficacy studies.

[0226] Testing the efficacy of Mut-3∆34.5 in human pediatric tumor models compared to other oHSV therapies 17TermA and T-VEC Efficacy

[0227] One of three high-efficacy models for each tumor type was selected for efficacy studies in xenografts using 5- to 6-week-old female athymic nude mice. 3 At the time of the study, mice were pooled and randomly divided into three groups (n = 11 per group): i) phosphate-buffered saline (PBS) control, ii) Mut-3∆34.5 virus, or iii) 17TermA or T-VEC virus. To determine the efficacy between the viruses, at least 11 mice were used per group to detect significant differences in survival and tumor growth (20% vs 80% on day 20) with a minimum power of 80%. Based on the applicant's previous studies, each mouse was treated every other day with 1 x 10 mice in 100 μl of PBS. 7 pfu of virus or PBS alone (control group) were injected intratumorally three times. The tumor volume (twice a week) and body weight (once a week) of the mice were monitored for 80 days after virus injection. Endpoint indicators included tumor volume exceeding 2500 mm 3 , tumor diameter reached 2 cm, or body weight loss >20%. Animal survival rates were shown by Kaplan-Meier curves and compared between groups using the log-rank test.

[0228] Compared with other oHSV therapies (17TermA and T-VEC), Mut-3Δ34.5 is more effective in mouse pediatric tumor models. Efficacy

[0229] One of the two high-efficacy models for each tumor type was selected for the applicant's efficacy study in sex-matched C57BL6 mice (5 to 6 weeks old). Similarly, mice were pooled and randomized into three groups, each with n=11: i) PBS, ii) Mut-3∆34.5, iii) 17TermA or T-VEC. Each mouse was treated with 1 x 10 8 pfu of virus or PBS alone were injected intratumorally three times as in the applicant's previous study. Endpoints included tumor volume exceeding 2500 mm 3 , tumor diameter reached 2 cm, or body weight loss >20%. Animal survival rates were shown by Kaplan-Meier curves and compared between groups using the log-rank test.

[0230] Pathological analysis summary

[0231] Tissues from four wild-type KOS-injected mice (1e6 and 1e7 pfu, two mice at each dose) were submitted for pathological analysis through the OSU Comparative Pathology & Mouse Phenotyping Shared Resource. Tissues from Mut-3D34.5 / C8G5- and Mut-3DICP6 / D7-1-injected mice (24 hours and 14 days pvi time points, two mice at each time point) were subsequently submitted for pathological analysis.

[0232] Pathology report (female): KOS-injected mice

[0233] The presence of lymphoplasmacytic encephalitis in the brainstem is consistent with published reports of central nervous system pathology in Balb / c mice injected with HSV-1. All mice examined in this submission had this lesion, although one submission included only a small portion of the brainstem, resulting in a less severe lesion than in the other three mice. Adrenal gland sections were preserved along with kidney sections from two mice. Necrosis was evident in both the adrenal cortex and medulla of these mice.

[0234] Pathology report (female): 24 hours and 14 days after injection of Mut-3∆34.5 / C8G5 and Mut-3∆ICP6 / D7-1 mouse

[0235] Samples from mice sacrificed 24 hours after HSV infection all showed the following lesions of moderate to marked severity: periportal to mid-hepatic zone necrosis, including single hepatocytes and focal diffuse cord necrosis; marked splenic red pulp necrosis and moderate white pulp necrosis, with follicles smaller than expected. Several mice exhibited increased apoptosis / necrosis of luteal cells in follicles and ovarian tissue at 24 hours. This excess had not been noted in control mice evaluated several months prior but was observed in the mice described here.

[0236] In 14-day-old mice, oval cell hyperplasia was a common chronic sign of liver damage, likely in response to infection. A few lung samples showed necrotic and inflammatory lesions not seen in 24-hour-old animals. Overall, 14-day-old mice had fewer and milder lesions than 24-hour-old mice.

[0237] The laboratory-created mutant virus can cause necrosis in multiple cell types, especially within 24 hours, including liver cells, spleen cells in the red and white pulp, and cells in the ovaries.

[0238] Equivalent

[0239] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0240] The embodiments illustratively described herein may suitably be practiced in the absence of any elements, limitations or restrictions not specifically disclosed herein. Thus, for example, the terms "comprising," "containing," "including," and the like are to be interpreted broadly and not restrictively. Furthermore, the terms and expressions used herein have been used as terms of description and not of limitation, and in the use of such terms and expressions, there is no intention to exclude any equivalents of the features shown and described or portions thereof, but it is to be understood that various modifications may be made within the scope of the present disclosure.

[0241] Therefore, it should be understood that although the present disclosure has been specifically disclosed through specific embodiments and optional features, those skilled in the art may resort to modifications, improvements and variations of the embodiments disclosed herein, and such modifications, improvements and variations are considered to be within the scope of the present disclosure. The materials, methods and examples provided herein are representative of specific embodiments and are exemplary and are not intended to limit the scope of the present disclosure.

[0242] The scope of the present disclosure has been described broadly and generically herein. Each narrower species and subgeneric grouping falling within the generic disclosure also forms part of this disclosure. This includes the generic description with a proviso or negative limitation removing any subject matter from the genus, whether or not the excised material is specifically cited herein.

[0243] All publications, patent applications, patents, and other references mentioned herein, including all formulae and figures, are incorporated by reference in their entirety to the same extent as if individually incorporated by reference. In the event of conflict, the present specification, including definitions, will control.

[0244] Other embodiments are set forth in the following claims.

[0245] SEQ ID NO. 1 Figure 3: DNA sequences of ICP34.5 in the Mut 3 and Mut-3∆ICP6 strains. The C356A mutation is shown in bold and italics in the sequence below.

[0246] ATGGCCCGCCCGCCGCCATCGCGGCCCCCGCCGCCCCCGGCCGCCCGG

[0247] GCCCACGGGCGCGGTCCCAACCGCACAGTCCCAGGTAACCTCCACGCCCA

[0248] ACTCGGAACCCGTGGTCAGGAGCGCGCCCGCGGCCGCCCCGCCGCCGCCC

[0249] CCCGCCAGTGGGCCCCCGCCTTCTTGTTCGCTGCTGCTGCGCCAGTGGCT

[0250] CCACGTTCCCGAGTCCGCGTCCGACGACGACGACGACGACTGGCCGGACA

[0251] GCCCCCCGCCCGAGCCGGCGCCAGAGGCCCGGCCCACCGCCGCCGCCCCC

[0252] CGCCCCCGGTCCCCACCGCCCGGCGCGGGCCCGGGGGGCGGGGCTAACCC

[0253] CTCCC A CCCCCCCTCACGCCCCTTCCGCCTTCCGCCGCGCCTCGCCCTCC

[0254] GCCTGCGCGTCACCGCAGAGCACCTGGCGCGCCTGCGCCTGCGACGCGCG

[0255] GGCGGGGAGGGGGCGCCGAAGCCCCCCGCGACCCCCGCGACCCCCGCGAC

[0256] CCCCACGCGGGTGCGCTTCTCGCCCCACGTCCGGGTGCGCCACCTGGTGG

[0257] TCTGGGCCTCGGCCGCCCGCCTGGCGCGCCGCGGCTCGTGGGCCCGCGAG

[0258] CGGGCCGACCGGGCTCGGTTCCGGCGCCGGGTGGCGGAGGCCGAGGCGGT

[0259] CATCGGGCCGTGCCTGGGGCCCGAGGCCCGTGCCCGGGCCCTGGCCCGCG

[0260] GAGCCGGCCCGGCGAACTCGGTCTAA

[0261] SEQ ID NO. 2: Amino acid sequence of ICP34.5 in Mut3 and Mut-3∆ICP6. The P119H mutation is shown in bold and italics in the sequence below.

[0262] MARRRRHRGPRRRPPGPTGAVPTAQSQVTSTPNSEPVVRSAPAAAPPPP

[0263] PASGPPPSCSLLLRQWLHVPESASDDDDDWPDSPPPEPAPEARPTAAAP

[0264] RPRSPPPGAGPGGGANPS H PPSRPFRLPPRLALRLRVTAEHLARLRLRRA

[0265] GGEGAPKPPATPATPATPTRVRFSPHVRVRHLVVWASAARLARRGSWARE

[0266] RADRARFRRRVAEAEAVIGPCLGPEARARALARGAGPANSV

[0267] SEQ ID NO. 3: DNA coding sequence of EGFP. The ICP34.5 coding region was replaced by the EGFP expression cassette in Mut3∆IC34.5 ( Figure 5C , SEQ ID NO. 11).

[0268] ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGT

[0269] CGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGG

[0270] GCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACC

[0271] ACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTA

[0272] CGGCGTGCAGTGCTTCAGCCGCTACCCGACCACATGAAGCAGCACGACT

[0273] TCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTC

[0274] TTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGG

[0275] CGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGG

[0276] ACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAAC

[0277] GTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAA

[0278] GATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACC

[0279] AGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCAC

[0280] TACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGA

[0281] TCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCA

[0282] TGGACGAGCTGTACAAGAAGCTTAGCCATGGCTTCCCGCCGGAGGTGGAG

[0283] GAGCAGGATGATGGCACGCTGCCCATGTCTTGTGCCCAGGAGAGCGGGAT

[0284] GGACCGTCACCCTGCAGCCTGTGCTTCTGCTAGGATCAATGTGTAG

[0285] SEQ ID NO. 4: Amino acid coding sequence of EGFP.

[0286] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICT

[0287] TGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIF

[0288] FKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN

[0289] VYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH

[0290] YLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKKLSHGFPPEVE

[0291] EQDDGTLPMSCAQESGMDRHPAACASARINV

[0292] SEQ ID NO. 5: DNA sequence of ICP34.5 in strain 17TermA.

[0293] ATGGCCCGCCGCCGCCGCCATCGCGGCCCCCGCCGCCCCCGGCCGCCCGG

[0294] GCCCACGGGCGCCGTCCCAACCGCACAGTCCCAGGTAACCTAGACTAGTC

[0295] TAGCGTAACCTCCACGCCCAACTCGGAACCCGCGGTCAGGAGCGCGCCCG

[0296] CGGCCGCCCCGCCGCCGCCCCCCGCCGGTGGGCCCCCGCCTTCTTGTTCG

[0297] CTGCTGCTGCGCCAGTGGCTCCACGTTCCCGAGTCCGCGTCCGACAACGA

[0298] CGATGACGACGACTGGCCGGACAGCCCCCCGCCCGAGCCGGCGCCAGAGG

[0299] CCCGGCCCACCGCCGCCGCCCCCCGGCCCCGGCCCCCACCGCCCGGCGTG

[0300] GGCCCGGGGGGCGGGGCTGACCCCTCCCACCCCCCCTCGCGCCCCTTCCG

[0301] CCTTCCGCCGCGCCTCGCCCTCCGCCTGCGCGTCACCGCGGAGCACCTGG

[0302] CGCGCCTGCGCCTGCGACGCGCGGGCGGGGAGGGGGCGCCGGAGCCCCCC

[0303] GCGACCCCCGCGACCCCCGCGACCCCCGCGACCCCCGCGACCCCCGCGCG

[0304] GGTGCGCTTCTCGCCCCACGTCCGGGTGCGCCACCTGGTGGTCTGGGCCT

[0305] CGGCCGCCCGCCTGGCGCGCCGCGGCTCGTGGGCCCGCGAGCGGGCCGAC

[0306] CGGGCTCGGTTCCGGCGCCGGGTGGCGGAGGCCGAGGCGGTCATCGGGCC

[0307] GTGCCTGGGGCCCGAGGCCCGTGCCCGGGCCCTGGCCCGCGGAGCCGGCC

[0308] CGGCGAACTCGGTCTAA

[0309] SEQ ID NO. 6: Amino acid sequence of ICP34.5 in strain 17TermA.

[0310] MARRRRHRGPRRPRPPGPTGAVPTAQSQVT*

[0311] (*Stop codon - the predicted* sequence after this is not expressed)

[0312] SEQ ID NO. 7: DNA sequence of ICP34.5 in strain rRp450.

[0313] ATGGCCCGCCGCCGCCATCGCGGCCCCCGCCGCCCCCGGCCGCCCGGGCC

[0314] CACGGGCGCGGTCCCAACCGCACAGTCCCAGGTAACCTCCACGCCCAACT

[0315] CGGAACCCGTGGTCAGGAGCGCGCCCGCGGCCGCCCCGCCGCCGCCCCCC

[0316] GCCAGTGGGCCCCCGCCTTCTTGTTCGCTGCTGCTGCGCCAGTGGCTCCA

[0317] CGTTCCCGAGTCCGCGTCCGACGACGACGACGACGACTGGCCGGACAGCC

[0318] CCCCGCCCGAGCCGGCGCCAGAGGCCCGGCCCACCGCCGCCGCCCCCCGC

[0319] CCCCGGTCCCCACCGCCCGGCGCGGGCCCGGGGGGCGGGGCTAACCCCTC

[0320] CCCCCCCCCCTCACGCCCCTTCCGCCTTCCGCCGCGCCTCGCCCTCCGCC

[0321] TGCGCGTCACCGCAGAGCACCTGGCGCGCCTGCGCCTGCGACGCGCGGGC

[0322] GGGGAGGGGGCGCCGAAGCCCCCCGCGACCCCCGCGACCCCCGCGACCCC

[0323] CACGCGGGTGCGCTTCTCGCCCCACGTCCGGGTGCGCCACCTGGTGGTCT

[0324] GGGCCTCGGCCGCCCGCCTGGCGCGCCGCGGCTCGTGGGCCCGCGAGCGG

[0325] GCCGACCGGGCTCGGTTCCGGCGCCGGGTGGCGGAGGCCGAGGCGGTCAT

[0326] CGGGCCGTGCCTGGGGCCCGAGGCCCGTGCCCGGGCCCTGGCCCGCGGAG

[0327] CCGGCCCGGCGAACTCGGTCTAA

[0328] SEQ ID NO. 8: Amino acid sequence of ICP34.5 in rRp450 strain.

[0329] MARRRHRGPRRPRPPGPTGAVPTAQSQVTSTPNSEPVVRSAPAAAPPPPP

[0330] ASGPPPSCSLLLRQWLHVPESASDDDDDDWPDSPPPEPAPEARPTAAAPR

[0331] PRSPPPGAGPGGGANPSPPPSRPFRLPPRLALRLRVTAEHLARLRLRRAG

[0332] GEGAPKPPATPATPATPTRVRFSPHVRVRHLVVWASAARLARRGSWARER

[0333] ADRARFRRRVAEAEAVIGPCLGPEARARALARGAGPANSV

[0334] SEQ ID NO. 9: DNA sequence of ICP34.5 in wild - type strain 17.

[0335] ATGGCCCGCCGCCGCCGCCATCGCGGCCCCCGCCGCCCCCGGCCGCCCGG

[0336] GCCCACGGGCGCGGTCCCAACCGCACAGTCCCAGGTAACCTCCACGCCCA

[0337] ACTCGGAACCCGTGGTCAGGAGCGCGCCCGCGGCCGCCCCGCCGCCGCCC

[0338] CCCGCCAGTGGGCCCCCGCCTTCTTGTTCGCTGCTGCTGCGCCAGTGGCT

[0339] CCACGTTCCCGAGTCCGCGTCCGACGACGACGACGACGACTGGCCGGACA

[0340] GCCCCCCGCCCGAGCCGGCGCCAGAGGCCCGGCCCACCGCCGCCGCCCCC

[0341] CGCCCCCGGTCCCCACCGCCCGGCGCGGGCCCGGGGGGCGGGGCTAACCC

[0342] CTCCCACCCCCCCTCACGCCCCTTCCGCCTTCCGCCGCGCCTCGCCCTCC

[0343] GCCTGCGCGTCACCGCAGAGCACCTGGCGCGCCTGCGCCTGCGACGCGCG

[0344] GGCGGGGAGGGGGCGCCGAAGCCCCCCGCGACCCCCGCGACCCCCGCGAC

[0345] CCCCACGCGGGTGCGCTTCTCGCCCCACGTCCGGGTGCGCCACCTGGTGG

[0346] TCTGGGCCTCGGCCGCCCGCCTGGCGCGCCGCGGCTCGTGGGCCCGCGAG

[0347] CGGGCCGACCGGGCTCGGTTCCGGCGCCGGGTGGCGGAGGCCGAGGCGGT

[0348] CATCGGGCCGTGCCTGGGGCCCGAGGCCCGTGCCCGGGCCCTGGCCCGCG

[0349] GAGCCGGCCCGGCGAACTCGGTCTAA

[0350] SEQ ID NO. 51: DNA sequence of ICP34.5 in wild-type strain 17.

[0351] ATGGCCCGCCGCCGCCGCCATCGCGGCCCCCGCCGCCCCCGGCCGCCCGGGCCCACGGGCGCCGTCCCAACCGCACAGTCCCAGGTAACCTCCACGCCCAACTCGGAACCCGCGGTCAGGAGCGCGCCCGCGGCCGCCCCGCCGCCGCCCCCCGCCGGTGGGCCCCCGCCTTCTTGTTCGCTGCTGCTGCGCCAGTGGCTCCACGTTCCCGAGTCCGCGTCCGACGACGACGATGACGACGACTGGCCGGACAGCCCCCCGCCCGAGCCGGCGCCAGAGGCCCGGCCCACCGCCGCCGCCCCCCGGCCCCGGCCCCCACCGCCCGGCGTGGGCCCGGGGGGCGGGGCTGACCCCTCCCACCCCCCCTCGCGCCCCTTCCGCCTTCCGCCGCGCCTCGCCCTCCGCCTGCGCGTCACCGCGGAGCACCTGGCGCGCCTGCGCCTGCGACGCGCGGGCGGGGAGGGGGCGCCGGAGCCCCCCGCGACCCCCGCGACCCCCGCGACCCCCGCGACCCCCGCGACCCCCGCGCGGGTGCGCTTCTCGCCCCACGTCCGGGTGCGCCACCTGGTGGTCTGGGCCTCGGCCGCCCGCCTGGCGCGCCGCGGCTCGTGGGCCCGCGAGCGGGCCGACCGGGCTCGGTTCCGGCGCCGGGTGGCGGAGGCCGAGGCGGTCATCGGGCCGTGCCTGGGGCCCGAGGCCCGTGCCCGGGCCCTGGCCCGCGGAGCCGGCCCGGCGAACTCGGTCTAA

[0352] SEQ ID NO. 10: Amino acid sequence of ICP34.5 in wild-type strain 17.

[0353] MARRRRHRGPRRPRPPGPTGAVPTAQSQVTSTPNSEPVVRSAPAAAPPPP

[0354] PASGPPPSCSLLLRQWLHVPESASDDDDDDWPDSPPPEPAPEARPTAAAP

[0355] RPRSPPPGAGPGGGANPSHPPSRPFRLPPRLALRLRVTAEHLARLRLRRA

[0356] GGEGAPKPPATPATPATPTRVRFSPHVRVRHLVVWASAARLARRGSWARE

[0357] RADRARFRRRVAEAEAVIGPCLGPEARARALARGAGPANSV

[0358] SEQ ID NO. 52: Amino acid sequence of ICP34.5 in wild-type strain 17.

[0359] MARRRRHRGPRRPRPPGPTGAVPTAQSQVTSTPNSEPAVRSAPA

[0360] AAPPPPPAGGPPPSCSLLLRQWLHVPESASDDDDDDDWPDSPPPEPAPEARPTAAAPR

[0361] PRPPPPGVGPGGGADPSHPPSRPFRLPPRLALRLRVTAEHLARLRLRRAGGEGAPEPP

[0362] [[ID=2l]]ATPATPATPATPATPARVRFSPHVRVRHLVVWASAARLARRGSWARERADRARFRRRV

[0363] AEAEAVIGPCLGPEARARALARGAGPANSV

[0364] SEQ ID NO: 11: DNA sequence of the EGFP expression cassette.

[0365] AACACATTAATTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATA

[0366] AAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGT

[0367] TACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTC

[0368] ACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATG

[0369] TCTGCTCGAAGCGGCCGGCCGCCCCGACTCTAGACTACACATTGATCCTA

[0370] GCAGAAGCACAGGCTGCAGGGTGACGGTCCATCCCGCTCTCCTGGGCACA

[0371] AGACATGGGCAGCGTGCCATCATCCTGCTCCTCCACCTCCGGCGGGAAGC

[0372] CATGGCTAAGCTTCTTGTACAGCTCGTCCATGCCGAGAGTGATCCCGGCG

[0373] GCGGTCACGAACTCCAGCAGGACCATGTGATCGCGCTTCTCGTTGGGGTC

[0374] TTTGCTCAGGGCGGACTGGGTGCTCAGGTAGTGGTTGTCGGGCAGCAGCA

[0375] CGGGGCCGTCGCCGATGGGGGTGTTCTGCTGGTAGTGGTCGGCGAGCTGC

[0376] ACGCTGCCGTCCTCGATGTTGTGGCGGATCTTGAAGTTCACCTTGATGCC

[0377] GTTCTTCTGCTTGTCGGCCATGATATAGACGTTGTGGCTGTTGTAGTTGT

[0378] ACTCCAGCTTGTGCCCCAGGATGTTGCCGTCCTCCTTGAAGTCGATGCCC

[0379] TTCAGCTCGATGCGGTTCACCAGGGTGTCGCCCTCGAACTTCACCTCGGC

[0380] GCGGGTCTTGTAGTTGCCGTCGTCCTTGAAGAAGATGGTGCGCTCCTGGA

[0381] CGTAGCCTTCGGGCATGGCGGACTTGAAGAAGTCGTGCTGCTTCATGTGG

[0382] TCGGGGTAGCGGCTGAAGCACTGCACGCCGTAGGTCAGGGTGGTCACGAG

[0383] GGTGGGCCAGGGCACGGGCAGCTTGCCGGTGGTGCAGATGAACTTCAGGG

[0384] TCAGCTTGCCGTAGGTGGCATCGCCCTCGCCCTCGCCGGACACGCTGAAC

[0385] TTGTGGCCGTTTACGTCGCCGTCCAGCTCGACCAGGATGGGCACCACCCC

[0386] GGTGAACAGCTCCTCGCCCTTGCTCACCATCCGGGAATTGCGGCCGCGGG

[0387] TACAATTCCGCAGCTTTTAGAGCAGAAGTAACACTTCCGTACAGGCCTAG

[0388] AAGTAAAGGCAACATCCACTGAGGAGCAGTTCTTTGATTTGCACCACCAC

[0389] CGGATCCGGGACCTGAAATAAAAAGACAAAAAGACTAAACTTACCAGTTAA

[0390] CTTTCTGGTTTTTCAGTTCCTCGAGTACCGGATCCTCTAGAGTCCGGAGG

[0391] CTGGATCGGTCCCGGTGTCTTCTATGGAGGTCAAAACAGCGTGGATGGCG

[0392] TCTCCAGGCGATCTGACGGTTCACTAAACGAGCTCTGCTTATATAGACCT

[0393] CCCACCGTACACGCCTACCGCCCATTTGCGTCAATGGGGCGGAGTTGTTA

[0394] CGACATTTTGGAAAGTCCCGTTGATTTTGGTGCCAAAACAAACTCCCATT

[0395] GACGTCAATGGGGTGGAGACTTGGAAATCCCCGTGAGTCAAACCGCTATC

[0396] CACGCCCATTGATGTACTGCCAAAACCGCATCACCATGGTAATAGCGATG

[0397] ACTAATACGTAGATGTACTGCCAAGTAGGAAAGTCCCATAAGGTCATGTA

[0398] CTGGGCATAATGCCAGGCGGGCCATTTACCGTCATTGACGTCAATAGGGG

[0399] GCGTACTTGGCATATGATACACTTGATGTACTGCCAAGTGGGCAGTTTAC

[0400] CGTAAATACTCCACCCATTGACGTCAATGGAAAGTCCCTATTGGCGTTAC

[0401] TATGGGAACATACGTCATTATTGACGTCAATGGGCGGGGGTCGTTGGGCG

[0402] GTCAGCCAGGCGGGCCATTTACCGTAAGTTATGTAACGACCTGCAGGCAT

[0403] GCAAGCTCGAATTCGAACACGCAGATGCAGTCGGGGCGGCAGATCTTAAT

[0404] TAATGGCTGGTTGTTTGTTGT

[0405] SEQ ID NO: 12: DNA sequence of gE (glycoprotein E) in the Mut-3, Mut-3∆34.5 or Mut-3∆ICP6 virus strains. The g451a mutation is shown in bold and italic in the following sequence.

[0406] ATGGATCGCGGGGCGGTGGTGGGGTTTCTTCTCGGTGTTTGTGTTGTATC

[0407] GTGCTTGGCGGGAACGCCCAAAACGTCCTGGAGACGGGTGAGTGTCGGCG

[0408] AGGACGTTTCGTTGCTTCCAGCTCCGGGGCCTACGGGGCGCGGCCCGACC

[0409] CAGAAACTACTATGGGCCGTGGAACCCCTGGATGGGTGCGGCCCCTTACA

[0410] CCCGTCGTGGGTCTCGCTGATGCCCCCCAAGCAGGTGCCCGAGACGGTCG

[0411] TGGATGCGGCGTGCATGCGCGCTCCGGTCCCGCTGGCGATGGCGTACGCC

[0412] CCCCCGGCCCCATCTGCGACCGGGGGTCTACGAACGGACTTCGTGTGGCA

[0413] GGAGCGCGCGGCCGTGGTTAACCGGAGTCTGGTTATTCACGGGGTCCGAG

[0414] AGACGGACAGCGGCCTGTATACCCTGTCCGTGGGCGACATAAAGGACCCG

[0415] A CTCGCCAAGTGGCCTCGGTGGTCCTGGTGGTGCAACCGGCCCCAGTTCC

[0416] GACCCCACCCCCGACCCCAGCCGATTACGACGAGGATGACAATGACGAGG

[0417] GCGAGGACGAAAGTCTCGCCGGCACTCCCGCCAGCGGGACCCCCCGGCTC

[0418] CCGCCTCCCCCCGCCCCCCCGAGGTCTTGGCCCAGCGCCCCCGAAGTCTC

[0419] ACATGTGCGTGGGGTGACCGTGCGTATGGAGACTCCGGAAGCTATCCTGT

[0420] TTTCCCCCGGGGAGACGTTCAGCACGAACGTCTCCATCCATGCCATCGCC

[0421] CACGACGACCAGACCTACTCCATGGACGTCGTCTGGTTGAGGTTCGACGT

[0422] GCCGACCTCGTGTGCCGAGATGCGAATATACGAATCGTGTCTGTATCACC

[0423] CGCAGCTCCCAGAATGTCTGTCCCCGGCCGACGCGCCGTGCGCCGCGAGT

[0424] ACGTGGACGTCTCGCCTGGCCGTCCGCAGCTACGCGGGGTGTTCCAGAAC

[0425] AAACCCCCCACCGCGCTGTTCGGCCGAGGCTCACATGGAGCCCGTCCCGG

[0426] GGCTGGCGTGGCAGGCGGCCTCCGTCAATCTGGAGTTCCGGGACGCGTCC

[0427] CCACAACACTCCGGCCTGTATCTGTGTGTGGTGTACGTCAACGACCATAT

[0428] TCACGCCTGGGGCCACATTACCATCAGCACCGCGGCGCAGTACCGGAACG

[0429] CGGTGGTGGAACAGCCCCTCCCACAGCGCGGCGCGGATTTGGCCGAGCCC

[0430] ACCCACCCGCACGTCGGGGCCCCTCCCCACGCGCCCCCAACCCACGGCGC

[0431] CCTGCGGTTAGGGGCGGTGATGGGGGCCGCCCTGCTGCTGTCTGCGCTGG

[0432] GGTTGTCGGTGTGGGCGTGTATGACCTGTTGGCGCAGGCGTGCCTGGCGG

[0433] GCGGTTAAAAGCAGGGCCTCGGGTAAGGGGCCCACGTACATTCGCGTGGC

[0434] CGACAGCGAGCTGTACGCGGACTGGAGCTCGGACAGCGAGGGAGAACGCG

[0435] ACCAGGTCCCGTGGCTGGCCCCCCCGGAGAGACCCGACTCTCCCTCCACC

[0436] AATGGATCCGGCTTTGAGATCTTATCACCAACGGGCTCCGTCTGTATACCC

[0437] CCGTAGCGACGGGCATCAATCTCGCCGCCAGCTCACAACCTTTGGATCCG

[0438] GAAGGCCCGATCGCCGTTACTCCCAGGCCTCGATTCGTCCGTCTTCTGG

[0439] TAA

[0440] SEQ ID NO: 13: Amino acid sequence of gE (glycoprotein E) from Mut-3, Mut-3∆34.5, or Mut-3∆ICP6 strains. The A151T mutation is shown in bold and italics in the sequence below.

[0441] MDRGAVVGFLLGVCVVSCLAGTPKTSWRRVSVGEDVSLLPAPGPTGRGPT

[0442] QKLLWAVEPLDGCGPLHPSWVSLMPPKQVPETVVDAACMRAPVPLAMAYA

[0443] PPAPSATGGLRTDFVWQERAAVVNRSLVIHGVRETDSGLYTLSVGDIKDP

[0444] T RQVASVVLVVQPAPVPTPPPTPADYDEDDNDEGEDESLAGTPASGTPRL

[0445] PPPPAPPRSWPSAPEVSHVRGVTVRMETPEAILFSPGETFSTNVSIHAIA

[0446] HDDQTYSMDVVWLRFDVPTSCAEMRIYESCLYHPQLPECLSPADAPCAAS

[0447] TWTSRLAVRSYAGCSRTNPPPRCSAEAHMEPVPGLAWQAASVNLEFRDAS

[0448] PQHSGLYLCVVYVNDHIHAWGHITISTAAQYRNAVVEQPLPQRGADLAEP

[0449] THPHVGAPPHAPPTHGALRLGAVMGAALLLSALGLSVWACMTCWRRRAWR

[0450] AVKSRASGKGPTYIRVADSELYADWSSDSEGERDQVPWLAPPERPDSPST

[0451] NGSGFEILSPTAPSVYPRSDGHQSRRQLTTFGSGRPDRRYSQASDSSVFW

[0452] SEQ ID NO: 14: DNA sequence of gE in strain 17TermA

[0453] ATGGATCGCGGGGCGGTGGTGGGGTTTCTTCTCGGTGTTTGTGTTGTATC

[0454] GTGCTTGGCGGGAACGCCCAAAACGTCCTGGAGACGGGTGAGTGTCGGCG

[0455] AGGACGTTTCGTTGCTTCCAGCTCCGGGGCCTACGGGGCGCGGCCCGACC

[0456] CAGAAACTACTATGGGCCGTGGAACCCCTGGATGGGTGCGGCCCCTTACA

[0457] CCCGTCGTGGGTCTCGCTGATGCCCCCCAAGCAGGTGCCCGAGACGGTCG

[0458] TGGATGCGGCGTGCATGCGCGCTCCGGTCCCGCTGGCGATGGCGTACGCC

[0459] CCCCCGGCCCCATCTGCGACCGGGGGTCTACGAACGGACTTCGTGTGGCA

[0460] GGAGCGCGCGGCCGTGGTTAACCGGAGTCTGGTTATTCACGGGGTCCGAG

[0461] AGACGGACAGCGGCCTGTATACCCTGTCCGTGGGCGACATAAAGGACCCG

[0462] GCTCGCCAAGTGGCCTCGGTGGTCCTGGTGGTGCAACCGGCCCCAGTTCC

[0463] GACCCCACCCCCGACCCCAGCCGATTACGACGAGGATGACAATGACGAGG

[0464] GCGAGGACGAAAGTCTCGCCGGCACTCCCGCCAGCGGGACCCCCCGGCTC

[0465] CCGCCTCCCCCCGCCCCCCCGAGGTCTTGGCCCAGCGCCCCCGAAGTCTC

[0466] ACATGTGCGTGGGGTGACCGTGCGTATGGAGACTCCGGAAGCTATCCTGT

[0467] TTTCCCCCGGGGAGACGTTCAGCACGAACGTCTCCATCCATGCCATCGCC

[0468] CACGACGACCAGACCTACTCCATGGACGTCGTCTGGTTGAGGTTCGACGT

[0469] GCCGACCTCGTGTGCCGAGATGCGAATATACGAATCGTGTCTGTATCACC

[0470] CGCAGCTCCCAGAATGTCTGTCCCCGGCCGACGCGCCGTGCGCCGCGAGT

[0471] ACGTGGACGTCTCGCCTGGCCGTCCGCAGCTACGCGGGGTGTTCCAGAAC

[0472] AAACCCCCCACCGCGCTGTTCGGCCGAGGCTCACATGGAGCCCGTCCCGG

[0473] GGCTGGCGTGGCAGGCGGCCTCCGTCAATCTGGAGTTCCGGGACGCGTCC

[0474] CCACAACACTCCGGCCTGTATCTGTGTGTGGTGTACGTCAACGACCATAT

[0475] TCACGCCTGGGGCCACATTACCATCAGCACCGCGGCGCAGTACCGGAACG

[0476] CGGTGGTGGAACAGCCCCTCCCACAGCGCGGCGCGGATTTGGCCGAGCCC

[0477] ACCCACCCGCACGTCGGGGCCCCTCCCCACGCGCCCCCAACCCACGGCGC

[0478] CCTGCGGTTAGGGGCGGTGATGGGGGCCGCCCTGCTGCTGTCTGCACTGG

[0479] GGTTGTCGGTGTGGGCGTGTATGACCTGTTGGCGCAGGCGTGCCTGGCGG

[0480] GCGGTTAAAAGCAGGGCCTCGGGTAAGGGGCCCACGTACATTCGCGTGGC

[0481] CGACAGCGAGCTGTACGCGGACTGGAGCTCGGACAGCGAGGGAGAACGCG

[0482] ACCAGGTCCCGTGGCTGGCCCCCCCGGAGAGACCCGACTCTCCCTCCACC

[0483] AATGGATCCGGCTTTGAGATCTTATCACCAACGGCTCCGTCTGTATACCC

[0484] CCGTAGCGATGGGCATCAATCTCGCCGCCAGCTCACAACCTTTGGATCCG

[0485] GAAGGCCCGATCGCCGTTACTCCCAGGCCTCCGATTCGTCCGTCTTCTGG

[0486] TAA

[0487] SEQ ID NO: 15: Amino acid sequence of gE in strain 17TermA.

[0488] MDRGAVVGFLLGVCVVSCLAGTPKTSWRRVSVGEDVSLLPAPGPTGRGPT

[0489] QKLLWAVEPLDGCGPLHPSWVSLMPPKQVPETVVDAACMRAPVPLAMAYA

[0490] PPAPSATGGLRTDFVWQERAAVVNRSLVIHGVRETDSGLYTLSVGDIKDP

[0491] ARQVASVVLVVQPAPVPTPPPTPADYDEDDNDEGEDESLAGTPASGTPRL

[0492] PPPPAPPRSWPSAPEVSHVRGVTVRMETPEAILFSPGETFSTNVSIHAIA

[0493] HDDQTYSMDVVWLRFDVPTSCAEMRIYESCLYHPQLPECLSPADAPCAAS

[0494] TWTSRLAVRSYAGCSRTNPPPRCSAEAHMEPVPGLAWQAASVNLEFRDAS

[0495] PQHSGLYLCVVYVNDHIHAWGHITISTAAQYRNAVVEQPLPQRGADLAEP

[0496] THPHVGAPPHAPPTHGALRLGAVMGAALLLSALGLSVWACMTCWRRRAWR

[0497] AVKSRASGKGPTYIRVADSELYADWSSDSEGERDQVPWLAPPERPDSPST

[0498] NGSGFEILSPTAPSVYPRSDGHQSRRQLTTFGSGRPDRRYSQASDSSVFW

[0499] SEQ ID NO. 16: DNA sequence of gE in strain rRp450.

[0500] ATGGATCGCGGGGCGGTGGTGGGGTTTCTTCTCGGTGTTTGTGTTGTATC

[0501] GTGCTTGGCGGGAACGCCCAAAACGTCCTGGAGACGGGTGAGTGTCGGCG

[0502] AGGACGTTTCGTTGCTTCCAGCTCCGGGGCCTACGGGGCGCGGCCCGACC

[0503] CAGAAACTACTATGGGCCGTGGAACCCCTGGATGGGTGCGGCCCCTTACA

[0504] CCCGTCGTGGGTCTCGCTGATGCCCCCCAAGCAGGTGCCCGAGACGGTCG

[0505] TGGATGCGGCGTGCATGCGCGCTCCGGTCCCGCTGGCGATGGCGTACGCC

[0506] CCCCCGGCCCCATCTGCGACCGGGGGTCTACGAACGGACTTCGTGTGGCA

[0507] GGAGCGCGCGGCCGTGGTTAACCGGAGTCTGGTTATTCACGGGGTCCGAG

[0508] AGACGGACAGCGGCCTGTATACCCTGTCCGTGGGCGACATAAAGGACCCG

[0509] GCTCGCCAAGTGGCCTCGGTGGTCCTGGTGGTGCAACCGGCCCCAGTTCC

[0510] GACCCCACCCCCGACCCCAGCCGATTACGACGAGGATGACAATGACGAGG

[0511] GCGAGGACGAAAGTCTCGCCGGCACTCCCGCCAGCGGGACCCCCCGGCTC

[0512] CCGCCTCCCCCCGCCCCCCCGAGGTCTTGGCCCAGCGCCCCCGAAGTCTC

[0513] ACATGTGCGTGGGGTGACCGTGCGTATGGAGACTCCGGAAGCTATCCTGT

[0514] TTTCCCCCGGGGAGACGTTCAGCACGAACGTCTCCATCCATGCCATCGCC

[0515] CACGACGACCAGACCTACTCCATGGACGTCGTCTGGTTGAGGTTCGACGT

[0516] GCCGACCTCGTGTGCCGAGATGCGAATATACGAATCGTGTCTGTATCACC

[0517] CGCAGCTCCCAGAATGTCTGTCCCCGGCCGACGCGCCGTGCGCCGCGAGT

[0518] ACGTGGACGTCTCGCCTGGCCGTCCGCAGCTACGCGGGGTGTTCCAGAAC

[0519] AAACCCCCCACCGCGCTGTTCGGCCGAGGCTCACATGGAGCCCGTCCCGG

[0520] GGCTGGCGTGGCAGGCGGCCTCCGTCAATCTGGAGTTCCGGGACGCGTCC

[0521] CCACAACACTCCGGCCTGTATCTGTGTGTGGTGTACGTCAACGACCATAT

[0522] TCACGCCTGGGGCCACATTACCATCAGCACCGCGGCGCAGTACCGGAACG

[0523] CGGTGGTGGAACAGCCCCTCCCACAGCGCGGCGCGGATTTGGCCGAGCCC

[0524] ACCCACCCGCACGTCGGGGCCCCTCCCCACGCGCCCCCAACCCACGGCGC

[0525] CCTGCGGTTAGGGGCGGTGATGGGGGCCGCCCTGCTGCTGTCTGCGCTGG

[0526] GGTTGTCGGTGTGGGCGTGTATGACCTGTTGGCGCAGGCGTGCCTGGCGG

[0527] GCGGTTAAAAGCAGGGCCTCGGGTAAGGGGCCCACGTACATTCGCGTGGC

[0528] CGACAGCGAGCTGTACGCGGACTGGAGCTCGGACAGCGAGGGAGAACGCG

[0529] ACCAGGTCCCGTGGCTGGCCCCCCCGGAGAGACCCGACTCTCCCTCCACC

[0530] AATGGATCCGGCTTTGAGATCTTATCACCAACGGCTCCGTCTGTATACCC

[0531] CCGTAGCGACGGGCATCAATCTCGCCGCCAGCTCACAACCTTTGGATCCG

[0532] GAAGGCCCGATCGCCGTTACTCCCAGGCCTCCGATTCGTCCGTCTTCTGG

[0533] TAA

[0534] SEQ ID NO. 17: Amino acid sequence of gE in rRp450 strain.

[0535] MDRGAVVGFLLGVCVVSCLAGTPKTSWRRVSVGEDVSLLPAPGPTGRGPT

[0536] QKLLWAVEPLDGCGPLHPSWVSLMPPKQVPETVVDAACMRAPVPLAMAYA

[0537] PPAPSATGGLRTDFVWQERAAVVNRSLVIHGVRETDSGLYTLSVGDIKDP

[0538] ARQVASVVLVVQPAPVPTPPPTPADYDEDDNDEGEDESLAGTPASGTPRL

[0539] PPPPAPPRSWPSAPEVSHVRGVTVRMETPEAILFSPGETFSTNVSIHAIA

[0540] HDDQTYSMDVVWLRFDVPTSCAEMRIYESCLYHPQLPECLSPADAPCAAS

[0541] TWTSRLAVRSYAGCSRTNPPPRCSAEAHMEPVPGLAWQAASVNLEFRDAS

[0542] PQHSGLYLCVVYVNDHIHAWGHITISTAAQYRNAVVEQPLPQRGADLAEP

[0543] THPHVGAPPHAPPTHGALRLGAVMGAALLLSALGLSVWACMTCWRRRAWR

[0544] AVKSRASGKGPTYIRVADSELYADWSSDSEGERDQVPWLAPPERPDSPST

[0545] NGSGFEILSPTAPSVYPRSDGHQSRRQLTTFGSGRPDRRYSQASDSSVFW

[0546] SEQ ID NO. 18: DNA sequence of gE in wild - type strain 17.

[0547] ATGGATCGCGGGGCGGTGGTGGGGTTTCTTCTCGGTGTTTGTGTTGTATC

[0548] GTGCTTGGCGGGAACGCCCAAAACGTCCTGGAGACGGGTGAGTGTCGGCG

[0549] AGGACGTTTCGTTGCTTCCAGCTCCGGGGCCTACGGGGCGCGGCCCGACC

[0550] CAGAAACTACTATGGGCCGTGGAACCCCTGGATGGGTGCGGCCCCTTACA

[0551] CCCGTCGTGGGTCTCGCTGATGCCCCCCAAGCAGGTGCCCGAGACGGTCG

[0552] TGGATGCGGCGTGCATGCGCGCTCCGGTCCCGCTGGCGATGGCGTACGCC

[0553] CCCCCGGCCCCATCTGCGACCGGGGGTCTACGAACGGACTTCGTGTGGCA

[0554] GGAGCGCGCGGCCGTGGTTAACCGGAGTCTGGTTATTCACGGGGTCCGAG

[0555] AGACGGACAGCGGCCTGTATACCCTGTCCGTGGGCGACATAAAGGACCCG

[0556] GCTCGCCAAGTGGCCTCGGTGGTCCTGGTGGTGCAACCGGCCCCAGTTCC

[0557] GACCCCACCCCCGACCCCAGCCGATTACGACGAGGATGACAATGACGAGG

[0558] GCGAGGACGAAAGTCTCGCCGGCACTCCCGCCAGCGGGACCCCCCGGCTC

[0559] CCGCCTCCCCCCGCCCCCCCGAGGTCTTGGCCCAGCGCCCCCGAAGTCTC

[0560] ACATGTGCGTGGGGTGACCGTGCGTATGGAGACTCCGGAAGCTATCCTGT

[0561] TTTCCCCCGGGGAGACGTTCAGCACGAACGTCTCCATCCATGCCATCGCC

[0562] CACGACGACCAGACCTACTCCATGGACGTCGTCTGGTTGAGGTTCGACGT

[0563] GCCGACCTCGTGTGCCGAGATGCGAATATACGAATCGTGTCTGTATCACC

[0564] CGCAGCTCCCAGAATGTCTGTCCCCGGCCGACGCGCCGTGCGCCGCGAGT

[0565] ACGTGGACGTCTCGCCTGGCCGTCCGCAGCTACGCGGGGTGTTCCAGAAC

[0566] AAACCCCCCACCGCGCTGTTCGGCCGAGGCTCACATGGAGCCCGTCCCGG

[0567] GGCTGGCGTGGCAGGCGGCCTCCGTCAATCTGGAGTTCCGGGACGCGTCC

[0568] CCACAACACTCCGGCCTGTATCTGTGTGTGGTGTACGTCAACGACCATAT

[0569] TCACGCCTGGGGCCACATTACCATCAGCACCGCGGCGCAGTACCGGAACG

[0570] CGGTGGTGGAACAGCCCCTCCCACAGCGCGGCGCGGATTTGGCCGAGCCC

[0571] ACCCACCCGCACGTCGGGGCCCCTCCCCACGCGCCCCCAACCCACGGCGC

[0572] CCTGCGGTTAGGGGCGGTGATGGGGGCCGCCCTGCTGCTGTCTGCGCTGG

[0573] GGTTGTCGGTGTGGGCGTGTATGACCTGTTGGCGCAGGCGTGCCTGGCGG

[0574] GCGGTTAAAAGCAGGGCCTCGGGTAAGGGGCCCACGTACATTCGCGTGGC

[0575] CGACAGCGAGCTGTACGCGGACTGGAGCTCGGACAGCGAGGGAGAACGCG

[0576] ACCAGGTCCCGTGGCTGGCCCCCCCGGAGAGACCCGACTCTCCCTCCACC

[0577] AATGGATCCGGCTTTGAGATCTTATCACCAACGGCTCCGTCTGTATACCC

[0578] CCGTAGCGACGGGCATCAATCTCGCCGCCAGCTCACAACCTTTGGATCCG

[0579] GAAGGCCCGATCGCCGTTACTCCCAGGCCTCCGATTCGTCCGTCTTCTGG

[0580] TAA

[0581] SEQ ID NO. 19: Amino acid sequence of gE in wild-type strain 17.

[0582] MDRGAVVGFLLGVCVVSCLAGTPKTSWRRVSVGEDVSLLPAPGPTGRGPT

[0583] QKLLWAVEPLDGCGPLHPSWVSLMPPKQVPETVVDAACMRAPVPLAMAYA

[0584] PPAPSATGGLRTDFVWQERAAVVNRSLVIHGVRETDSGLYTLSVGDIKDP

[0585] ARQVASVVLVVQPAPVPTPPPTPADYDEDDNDEGEDESLAGTPASGTPRL

[0586] PPPPAPPRSWPSAPEVSHVRGVTVRMETPEAILFSPGETFSTNVSIHAIA

[0587] HDDQTYSMDVVWLRFDVPTSCAEMRIYESCLYHPQLPECLSPADAPCAAS

[0588] TWTSRLAVRSYAGCSRTNPPPRCSAEAHMEPVPGLAWQAASVNLEFRDAS

[0589] PQHSGLYLCVVYVNDHIHAWGHITISTAAQYRNAVVEQPLPQRGADLAEP

[0590] THPHVGAPPHAPPTHGALRLGAVMGAALLLSALGLSVWACMTCWRRRAWR

[0591] AVKSRASGKGPTYIRVADSELYADWSSDSEGERDQVPWLAPPERPDSPST

[0592] NGSGFEILSPTAPSVYPRSDGHQSRRQLTTFGSGRPDRRYSQASDSSVFW

[0593] SEQ ID NO. 20: DNA sequence of ICP0 from Mut 3, Mut-3∆34.5, and Mut-3∆ICP6 strains. The a848c (intronic) and g1712a (for R258H) mutations are indicated in bold and italics in the following sequences. Both introns are included (i.e., nucleotides (nt) 58 to nt 861 and nt 1529 to nt 1663 of the following sequence; see also the sequence in parentheses).

[0594] ATGGAGCCCCGCCCCGGAGCGAGTACCCGCCGGCCTGAGGGCCGCCCCCA

[0595] GCGCGAG(GTGAGGGGCCGGGCGCCATGTCTGGGGCGCCATGTCTGGGGCG

[0596] CCATGTCTGGGGCGCCATGTCTGGGGCGCCATGTTGGGGGGCGCCATGTT

[0597] GGGGGGCGCCATGTTGGGGGACCCCCGACCCTTACACTGGAACCGGCCGC

[0598] CATGTTGGGGGACCCCCACTCATACACGGGAGCCGGGCGCCATGTTGGGG

[0599] CGCCATGTTAGGGGGCGTGGAACCCCGTGACACTATATATACAGGGACCG

[0600] GGGGCGCCATGTTAGGGGGCGCGGAACCCCCTGACCCTATATATACAGGG

[0601] ACCGGGGTCGCCCTGTTAGGGGTCGCCATGTGACCCCCTGACTTTATATA

[0602] TACAGACCCCCACACCTACACATGGCCCCTTTGACTCAGACGCAGGGCC

[0603] CGGGGTCGCCGTGGGACCCCCCTGACTCATACACAGAGACACGCCCCCAC

[0604] AACAAACACACAGGGACCGGGGTCCGCCGTGTTAGGGGGCGTGGTCCCCAC

[0605] TGACTCATACGCAGGGCCCCCTTACTCACACGCATCTAGGGGGGTGGGGA

[0606] GGAGCCGCCCGCCATATTTGGGGGACGCCGTGGGACCCCCGACTCCGGTG

[0607] CGTCTGGAGGGCGGGAGAAGAGGGAAGAAGAGGGGTCGGGATCCAAAGGA

[0608] CGGACCCAGACCACCTTTGGTTGCAGACCCCTTTCTCCCCCCTCTTCCGA

[0609] GGCCAGCAGGGGGGCAGGACTTTGTGAGGCGGGGGGGGAGGGGGAACTCG

[0610] TGGGCGCTGATTGACGCGGGAAATCCCCCCATTCTTACCCGCCCCCC C TT

[0611] TTTCCCCTCAG)CCCGCCCCGGATGTCTGGGTGTTTCCCTGCGACCGAGAC

[0612] CTGCCGGACAGCAGCGACTCGGAGGCGGAGACCGAAGTGGGGGGGCGGGG

[0613] GGACGCCGACCACCATGACGACGACTCCGCCTCCGAGGCGGACAGCACGG

[0614] ACACGGAACTGTTCGAGACGGGGCTGCTGGGGCCGCAGGGCGTGGATGGG

[0615] GGGGCGGTCTCGGGGGGGAGCCCCCCCCGCGAGGAAGACCCCGGCAGTTG

[0616] CGGGGGCGCCCCCCCTCGAGAGGACGGGGGGAGCGACGAGGGCGACGTGT

[0617] GCGCCGTGTGCACGGATGAGATCGCGCCCCACCTGCGCTGCGACACCTTC

[0618] CCGTGCATGCACCGCTTCTGCATCCCGTGCATGAAAACCTGGATGCAATT

[0619] GCGCAACACCTGCCCGCTGTGCAACGCCAAGCTGGTGTACCTGATAGTGG

[0620] GCGTGACGCCCAGCGGGTCGTTCAGCACCATCCCGATCGTGAACGACCCC

[0621] CAGACCCGCATGGAGGCCGAGGAGGCCGTCAGGGCGGGCACGGCCGTGGA

[0622] CTTTATCTGGACGGGCAATCAGCGGTTCGCCCCGCGGTACCTGACCCTGG

[0623] GGGGGCACACGGTGAGGGCCCTGTCGCCCACCCACCCGGAACCCACCACG

[0624] GACGAGGATGACGACGACCTGGACGACG(GTGAGGCGGGGGGCGGCAAGGA

[0625] CCCTGGGGGAGGAGGAGGAGGAGGGGGGGGAGGGAGGAATAGGCGGGCGG

[0626] GCGAGGAAAGGGCGGGCCGGGGAGGGGGCGTAACCTGATCGCGCCCCCCG

[0627] TTGTCTCTTGCAG)CAGACTACGTACCGCCCGCCCCCCGCCGGACGCCCCG

[0628] CGCCCCCCCAC A CAGAGGCGCCGCCGCGCCCCCCGTGACGGGCGGGGCGT

[0629] CTCACGCAGCCCCCCAGCCGGCCGCGGCTCGGACAGCGCCCCCCTCGGCG

[0630] CCCATCGGGCCACACGGCAGCAGTAACACCAACACCACCACCAACAGCAG

[0631] CGGCGGCGGCGGCTCCCGCCAGTCGCGAGCCGCGGCGCCGCGGGGGGCGT

[0632] CTGGCCCCTCCGGGGGGGTTGGGGTTGGGGTTGGGGTTGTTGAAGCGGAG

[0633] GCGGGGCGGCCGAGGGGCCGGACGGGCCCCCTTGTCAACAGACCCGCCCC

[0634] CCTTGCAAACAACAGAGACCCCATAGTGATCAGCGACTCCCCCCCGGCCT

[0635] CTCCCCACAGGCCCCCCGCGGCGCCCATGCCAGGCTCCGCCCCCCGCCCC

[0636] GGGCCCCCCGCGTCCGCGGCCGCGTCGGGACCCGCGCGCCCCCGCGCGGC

[0637] CGTGGCCCCGTGCGTGCGAGCGCCGCCTCCGGGGCCCGGCCCCCGCGCCC

[0638] CGGCCCCCGGGGCGGAGCCGGCCGCCCGCCCCGCGGACGCGCGCCGTGTG

[0639] CCCCAGTCGCACTCGTCCCTGGCTCAGGCCGCGAACCAAGAACAGAGTCT

[0640] GTGCCGGGCGCGTGCGACGGTGGCGCGCGGCTCGGGGGGGCCGGGCGTGG

[0641] AGGGTGGGCACGGGCCCTCCCGCGGCGCCGCCCCCTCCGGCGCCGCCCCG

[0642] CTCCCCTCCGCCGCCTCTGTCGAGCAGGAGGCGGCGGTGCGTCCGAGGAA

[0643] GAGGCGCGGGTCGGGCCAGGAAAACCCCTCCCCCCAGTCCACGCGTCCCC

[0644] CCCTCGCGCCGGCAGGGGCCAAGAGGGCGGCGACGCACCCCCCCTCCGAC

[0645] TCAGGGCCGGGGGGGCGCGGCCAGGGTGGGCCCGGGACCCCCCTGACGTC

[0646] CTCGGCGGCCTCCGCCTCTTCCTCCTCTGCCTCTTCCTCCTCGGCCCCGA

[0647] CCCCCGCGGGGGCCGCCTCTTCCGCCGCCGGGGCCGCGTCCTCCTCCGCT

[0648] TCCGCCTCCTCGGGCGGGGCCGTCGGTGCCCTGGGAGGGAGACAAGAGGA

[0649] AACCTCCCTCGGCCCCCGCGCTGCTTCTGGGCCGCGGGGGCCGAGGAAGT

[0650] GTGCCCGGAAGACGCGCCACGCGGAGACTTCCGGGGCCGTCCCCGCGGGC

[0651] GGCCTCACGCGCTACCTGCCCATCTCGGGGGTCTCTAGCGTGGTCGCCCT

[0652] GTCGCCTTACGTGAACAAGACGATCACGGGGGACTGCCTGCCCATCCTGG

[0653] ACATGGAGACGGGGAACATCGGGGCGTACGTGGTCCTGGTGGACCAGACG

[0654] GGAAACATGGCGACCCGGCTGCGGGCCGCGGTCCCCGGCTGGAGCCGCCG

[0655] CACCCTGCTCCCCGAGACCGCGGGTAACCACGTGATGCCCCCCGAGTACC

[0656] CGACGGCCCCCGCGTCGGAGTGGAACAGCCTCTGGATGACCCCCGTGGGG

[0657] AACATGCTGTTCGACCAGGGCACCCTAGTGGGCGCCCTGGACTTCCGCAG

[0658] CCTGCGGTCTCGGCACCCGTGGTCCGGGGAGCAGGGGGCGTCGACCCGGG

[0659] ACGAGGGAAAACAATAA

[0660] SEQ ID NO. 21: Amino acid sequence of ICP0 in Mut 3, Mut-3∆34.5, and Mut-3∆ICP6. The R258H mutation is shown in italics and bold in the sequence below.

[0661]

[0662]

[0663] SEQ ID NO. 22:17 DNA sequence of ICPO in TermA strain, including two introns (i.e., nucleotides (nt) 58 to nt 822 and nt 1490 to nt 1625 of the sequence below, see also the sequence in parentheses).

[0664] ATGGAGCCCCGCCCCGGAGCGAGTACCCGCCGGCCTGAGGGCCGCCCCCA

[0665] GCGCGAG(GTGAGGGGCCGGGCGCCATGTCTGGGGCGCCATATTGGGGGGC

[0666] GCCATATTGGGGGGCGCCATGTTGGGGGACCCCCGACCCTTACACTGGAA

[0667] CCGGCCGCCATGTTGGGGGACCCCCACTCATACACGGGAGCCGGGCGCCA

[0668] TGTTGGGGCGCCATGTTAGGGGGCGTGGAACCCCGTGACACTATATATAC

[0669] AGGGACCGGGGGCGCCATGTTAGGGGGTGCGGAACCCCCTGACCCTATAT

[0670] ATACAGGGACCGGGGTCGCCCTGTTGGGGGTCGCCATGTGACCCCCTGAC

[0671] TTTATATATACAGACCCCCAACACATACACATGGCCCCTTTGACTCAGAC

[0672] GCAGGGCCCGGGGTCGCCGTGGGACCCCCTGACTCATACACAGAGACACG

[0673] CCCCCACAACAAACACACAGGGACCGGGGTCGCCGTGTTGGGGGCGTGGT

[0674] CCCCACTGACTCATACGCAGGCCCCCCTTACTCACACGCATCTAGGGGGG

[0675] TGGGGAGGAGCCGCCCGCCATATTTGGGGGACGCCGTGGGACCCCCGACT

[0676] CCGGTGCGTCTGGAGGGCGGGAGAAGAGGGAAGAAGAGGGGTCGGGATCC

[0677] AAAGGACGGACCCAGACCACCTTTGGTTGCAGACCCCTTTCTCCCCCCTC

[0678] TTCCGAGGCCAGCAGGGGGGCAGGACTTTGTGAGGCGGGGGGGGGAGAGG

[0679] GGGAACTCGTGGGTGCTGATTGACGCGGGAAATCCCCCCCCATTCTTACC

[0680] CGCCCCCCTTTTTTCCCCTTAG)CCCGCCCCGGATGTCTGGGTGTTTCCCT

[0681] GCGACCGAGACCTGCCGGACAGCAGCGACTCTGAGGCGGAGACCGAAGTG

[0682] GGGGGGCGGGGGGACGCCGACCACCATGACGACGACTCCGCCTCCGAGGC

[0683] GGACAGCACGGACACGGAACTGTTCGAGACGGGGCTGCTGGGGCCGCAGG

[0684] GCGTGGATGGGGGGGCGGTCTCGGGGGGGAGCCCCCCCCGCGAGGAAGAC

[0685] CCCGGCAGTTGCGGGGGCGCCCCCCCTCGAGAGGACGGGGGGAGCGACGA

[0686] GGGTGACGTGTGCGCCGTGTGCACGGATGAGATCGCGCCCCACCTGCGCT

[0687] GCGACACCTTCCCGTGCATGCACCGCTTCTGCATCCCGTGCATGAAAACC

[0688] TGGATGCAATTGCGCAACACCTGCCCGCTGTGCAACGCCAAGCTGGTGTA

[0689] CCTGATAGTGGGCGTGACGCCCAGCGGGTCGTTCAGCACCATCCCGATCG

[0690] TGAACGACCCCCAGACCCGCATGGAGGCCGAGGAGGCCGTCAGGGCGGGC

[0691] ACGGCCGTGGACTTTATCTGGACGGGCAATCAGCGGTTCGCCCCGCGGTA

[0692] CCTGACCCTGGGGGGGCACACGGTGAGGGCCCTGTCGCCCACCCACCCGG

[0693] AACCCACCACGGACGAGGATGACGACGACCTGGACGACG(GTGAGGCGGGG

[0694] GGCGGCAAGGACCCTGGGGGAGGAGGAGGAGGAGGGGGGGGGAGGGAGGA

[0695] ATAGGCGGGCGGGCGAGGAAAGGGCGGGCCGGGGAGGGGGCGTAACCTGA

[0696] TCGCGCCCCCCGTTGTCTCTTGCAG)CAGACTACGTACCGCCCGCCCCCCG

[0697] CCGGACGCCCCGCGCCCCCCCACGCAGAGGCGCCGCCGCGCCCCCCGTGA

[0698] CGGGCGGGGCGTCTCACGCAGCCCCCCAGCCGGCCGCGGCTCGGACAGCG

[0699] CCCCCCTCGGCGCCCATCGGGCCACACGGCAGCAGTAACACCAACACCAC

[0700] CACCAACAGCAGCGGCGGCGGCGGCTCCCGCCAGTCGCGAGCCGCGGCGC

[0701] CGCGGGGGGCGTCTGGCCCCTCCGGGGGGGTTGGGGTTGGGGTTGGGGTT

[0702] GTTGAAGCGGAGGCGGGGCGGCCGAGGGGCCGGACGGGCCCCCTTGTCAA

[0703] CAGACCCGCCCCCCTTGCAAACAACAGAGACCCCATAGTGATCAGCGACT

[0704] CCCCCCCGGCCTCTCCCCACAGGCCCCCCGCGGCGCCCATGCCAGGCTCC

[0705] GCCCCCCGCCCCGGGCCCCCCGCGTCCGCGGCCGCGTCGGGACCCGCGCG

[0706] CCCCCGCGCGGCCGTGGCCCCGTGCGTGCGAGCGCCGCCTCCGGGGCCCG

[0707] GCCCCCGCGCCCCGGCCCCCGGGGCGGAGCCGGCCGCCCGCCCCGCGGAC

[0708] GCGCGCCGTGTGCCCCAGTCGCACTCGTCCCTGGCTCAGGCCGCGAACCA

[0709] AGAACAGAGTCTGTGCCGGGCGCGTGCGACGGTGGCGCGCGGCTCGGGGG

[0710] GGCCGGGCGTGGAGGGTGGGCACGGGCCCTCCCGCGGCGCCGCCCCCTCC

[0711] GGCGCCGCCCCGCTCCCCTCCGCCGCCTCTGTCGAGCAGGAGGCGGCGGT

[0712] GCGTCCGAGGAAGAGGCGCGGGTCGGGCCAGGAAAACCCCTCCCCCCAGT

[0713] CCACGCGTCCCCCCCTCGCGCCGGCAGGGGCCAAGAGGGCGGCGACGCAC

[0714] CCCCCCTCCGACTCAGGGCCGGGGGGGCGCGGCCAGGGTGGGCCCGGGAC

[0715] CCCCCTGACGTCCTCGGCGGCCTCCGCCTCTTCCTCCTCTGCCTCTTCCT

[0716] CCTCGGCCCCGACCCCCGCGGGGGCCGCCTCTTCCGCCGCCGGGGCCGCG

[0717] TCCTCCTCCGCTTCCGCCTCCTCGGGCGGGGCCGTCGGTGCCCTGGGAGG

[0718] GAGACAAGAGGAAACCTCCCTCGGCCCCCGCGCTGCTTCTGGGCCGCGGG

[0719] GGCCGAGGAAGTGTGCCCGGAAGACGCGCCACGCGGAGACTTCCGGGGCC

[0720] GTCCCCGCGGGCGGCCTCACGCGCTACCTGCCCATCTCGGGGGTCTCTAG

[0721] CGTGGTCGCCCTGTCGCCTTACGTGAACAAGACTATCACGGGGGACTGCC

[0722] TGCCCATCCTGGACATGGAGACGGGGAACATCGGGGCGTACGTGGTCCTG

[0723] GTGGACCAGACGGGAAACATGGCGACCCGGCTGCGGGCCGCGGTCCCCGG

[0724] CTGGAGCCGCCGCACCCTGCTCCCCGAGACCGCGGGTAACCACGTGATGC

[0725] CCCCCGAGTACCCGACGGCCCCCGCGTCGGAGTGGAACAGCCTCTGGATG

[0726] ACCCCCGTGGGGAACATGCTGTTCGACCAGGGCACCCTAGTGGGCGCCCT

[0727] GGACTTCCGCAGCCTGCGGTCTCGGCACCCGTGGTCCGGGGAGCAGGGGG

[0728] CGTCGACCCGGGACGAGGGAAAACAATAA

[0729] SEQ ID NO. 23:17 Amino acid sequence of ICPO in TermA strain.

[0730]

[0731]

[0732] SEQ ID NO. 24: DNA sequence of ICPO in rRp450 strain, including two introns (i.e., nucleotides (nt) 58 to nt 862 and nt 1530 to nt 1668 of the sequence below, see also the sequence in parentheses).

[0733] ATGGAGCCCCGCCCCGGAGCGAGTACCCGCCGGCCTGAGGGCCGCCCCCA

[0734] GCGCGAG(GTGAGGGGCCGGGCGCCATGTCTGGGGCGCCATGTCTGGGGCG

[0735] CCATGTCTGGGGCGCCATGTCTGGGGCGCCATGTTGGGGGGCGCCATGTT

[0736] GGGGGGCGCCATGTTGGGGGACCCCCGACCCTTACACTGGAACCGGCCGC

[0737] CATGTTGGGGGACCCCCACTCATACACGGGAGCCGGGCGCCATGTTGGGG

[0738] CGCCATGTTAGGGGGCGTGGAACCCCGTGACACTATATATACAGGGACCG

[0739] GGGGCGCCATGTTAGGGGGCGCGGAACCCCCTGACCCTATATATACAGGG

[0740] ACCGGGGTCGCCCTGTTAGGGGTCGCCATGTGACCCCCTGACTTTATATA

[0741] TACAGACCCCCAACACCTACACATGGCCCCTTTGACTCAGACGCAGGGCC

[0742] CGGGGTCGCCGTGGGACCCCCCTGACTCATACACAGAGACACGCCCCCAC

[0743] AACAAACACACAGGGACCGGGGTCGCCGTGTTAGGGGGCGTGGTCCCCAC

[0744] TGACTCATACGCAGGGCCCCCTTACTCACACGCATCTAGGGGGGTGGGGA

[0745] GGAGCCGCCCGCCATATTTGGGGGACGCCGTGGGACCCCCGACTCCGGTG

[0746] CGTCTGGAGGGCGGGAGAAGAGGGAAGAAGAGGGGTCGGGATCCAAAGGA

[0747] CGGACCCAGACCACCTTTGGTTGCAGACCCCTTTCTCCCCCCTCTTCCGA

[0748] GGCCAGCAGGGGGGCAGGACTTTGTGAGGCGGGGGGGGAGGGGGAACTCG

[0749] TGGGCGCTGATTGACGCGGGAAATCCCCCCATTCTTACCCGCCCCCCCTT

[0750] TTTTCCCCTCAG)CCCGCCCCGGATGTCTGGGTGTTTCCCTGCGACCGAGA

[0751] CCTGCCGGACAGCAGCGACTCGGAGGCGGAGACCGAAGTGGGGGGGCGGG

[0752] GGGACGCCGACCACCATGACGACGACTCCGCCTCCGAGGCGGACAGCACG

[0753] GACACGGAACTGTTCGAGACGGGGCTGCTGGGGCCGCAGGGCGTGGATGG

[0754] GGGGGCGGTCTCGGGGGGGAGCCCCCCCCGCGAGGAAGACCCCGGCAGTT

[0755] GCGGGGGCGCCCCCCCTCGAGAGGACGGGGGGAGCGACGAGGGCGACGTG

[0756] TGCGCCGTGTGCACGGATGAGATCGCGCCCCACCTGCGCTGCGACACCTT

[0757] CCCGTGCATGCACCGCTTCTGCATCCCGTGCATGAAAACCTGGATGCAAT

[0758] TGCGCAACACCTGCCCGCTGTGCAACGCCAAGCTGGTGTACCTGATAGTG

[0759] GGCGTGACGCCCAGCGGGTCGTTCAGCACCATCCCGATCGTGAACGACCC

[0760] CCAGACCCGCATGGAGGCCGAGGAGGCCGTCAGGGCGGGCACGGCCGTGG

[0761] ACTTTATCTGGACGGGCAATCAGCGGTTCGCCCCGCGGTACCTGACCCTG

[0762] GGGGGGCACACGGTGAGGGCCCTGTCGCCCACCCACCCTGAGCCCACCAC

[0763] GGACGAGGATGACGACGACCTGGACGACG(GTGAGGCGGGGGGGCGGCGAG

[0764] GACCCTGGGGGAGGAGGAGGAGGGGGGGGGGGGGGAGGAATAGGCGGGCG

[0765] GGCGGGCGAGGAAAGGGCGGGCCGGGGAGGGGGCGTAACCTGATCGCGCC

[0766] CCCCGTTGTCTCTTGCAG)CAGACTACGTACCGCCCGCCCCCCGCCGGACG

[0767] CCCCGCGCCCCCCCACGCAGAGGCGCCGCCGCGCCCCCCGTGACGGGCGG

[0768] GGCGTCTCACGCAGCCCCCCAGCCGGCCGCGGCTCGGACAGCGCCCCCCT

[0769] CGGCGCCCATCGGGCCACACGGCAGCAGTAACACTAACACCACCACCAAC

[0770] AGCAGCGGCGGCGGCGGCTCCCGCCAGTCGCGAGCCGCGGTGCCGCGGGG

[0771] GGCGTCTGGCCCCTCCGGGGGGGTTGGGGTTGTTGAAGCGGAGGCGGGGC

[0772] GGCCGAGGGGCCGGACGGGCCCCCTTGTCAACAGACCCGCCCCCCTTGCA

[0773] AACAACAGAGACCCCATAGTGATCAGCGACTCCCCCCCGGCCTCTCCCCA

[0774] CAGGCCCCCCGCGGCGCCCATGCCAGGCTCCGCCCCCCGCCCCGGTCCCC

[0775] CCGCGTCCGCGGCCGCGTCGGGCCCCGCGCGCCCCCGCGCGGCCGTGGCC

[0776] CCGTGTGTGCGGGCGCCGCCTCCGGGGCCCGGCCCCCGCGCCCCGGCCCC

[0777] CGGGGCGGAGCCGGCCGCCCGCCCCGCGGACGCGCGCCGTGTGCCCCAGT

[0778] CGCACTCGTCCCTGGCTCAGGCCGCGAACCAAGAACAGAGTCTGTGCCGG

[0779] GCGCGTGCGACGGTGGCGCGCGGCTCGGGGGGGCCGGGCGTGGAGGGTGG

[0780] ACACGGGCCCTCCCGCGGCGCCGCCCCCTCCGGCGCCGCCCCCTCCGGCG

[0781] CCCCCCCGCTCCCCTCCGCCGCCTCTGTCGAGCAGGAGGCGGCGGTGCGT

[0782] CCGAGGAAGAGGCGCGGGTCGGGCCAGGAAAACCCCTCCCCCCAGTCCAC

[0783] GCGTCCCCCCCTCGCGCCGGCAGGGGCCAAGAGGGCGGCGACGCACCCCC

[0784] CCTCCGACTCAGGGCCGGGGGGGCGCGGCCAGGGAGGGCCCGGGACCCCC

[0785] CTGACGTCCTCGGCGGCCTCCGCCTCTTCCTCCTCCGCCTCTTCCTCCTC

[0786] GGCCCCGACTCCCGCGGGGGCCACCTCTTCCGCCACCGGGGCCGCGTCCT

[0787] CCTCCGCTTCCGCCTCCTCGGGCGGGGCCGTCGGTGCCCTGGGAGGGAGA

[0788] CAAGAGGAAACCTCCCTCGGCCCCCGCGCTGCTTCTGGGCCGCGGGGGCC

[0789] GAGGAAGTGTGCCCGGAAGACGCGCCACGCGGAGACTTCCGGGGCCGTCC

[0790] CCGCGGGCGGCCTCACGCGCTACCTGCCCATCTCGGGGGTCTCTAGCGTG

[0791] GTCGCCCTGTCGCCTTACGTGAACAAGACGATCACGGGGGACTGCCTGCC

[0792] CATCCTGGACATGGAGACGGGGAACATCGGGGCGTACGTGGTCCTGGTGG

[0793] ACCAGACGGGAAACATGGCGACCCGGCTGCGGGCCGCGGTCCCCGGCTGG

[0794] AGCCGCCGCACCCTGCTCCCCGAGACCGCGGGTAACCACGTGACGCCCCC

[0795] CGAGTACCCGACGGCCCCCGCGTCGGAGTGGAACAGCCTCTGGATGACCC

[0796] CCGTGGGGAACATGCTGTTCGACCAGGGCACCCTAGTGGGCGCCCTGGAC

[0797] TTCCGCAGCCTGCGGTCTCGGCACCCGTGGTCCGGGGAGCAGGGGGCGTC

[0798] GACCCGGGACGAGGGAAAACAATAA

[0799] SEQ ID NO. 25: DNA sequence of ICP0 in wild-type strain 17. It contains two introns (i.e., nucleotides (nt) 58 to nt 861 and nt 1529 to nt 1663 of the following sequence, see also the sequence in parentheses).

[0800] ATGGAGCCCCGCCCCGGAGCGAGTACCCGCCGGCCTGAGGGCCGCCCCCA

[0801] GCGCGAG(GTGAGGGGCCGGGCGCCATGTCTGGGGCGCCATGTCTGGGGCG

[0802] CCATGTCTGGGGCGCCATGTCTGGGGCGCCATGTTGGGGGGCGCCATGTT

[0803] GGGGGGCGCCATGTTGGGGGACCCCCGACCCTTACACTGGAACCGGCCGC

[0804] CATGTTGGGGGACCCCCACTCATACACGGGAGCCGGGCGCCATGTTGGGG

[0805] CGCCATGTTAGGGGGCGTGGAACCCCGTGACACTATATATACAGGGACCG

[0806] GGGGCGCCATGTTAGGGGGCGCGGAACCCCCTGACCCTATATATACAGGG

[0807] ACCGGGGTCGCCCTGTTAGGGGTCGCCATGTGACCCCCTGACTTTATATA

[0808] TACAGACCCCCAACACCTACACATGGCCCCTTTGACTCAGACGCAGGGCC

[0809] CGGGGTCGCCGTGGGACCCCCCTGACTCATACACAGAGACACGCCCCCAC

[0810] AACAAACACACAGGGACCGGGGTCGCCGTGTTAGGGGGCGTGGTCCCCAC

[0811] TGACTCATACGCAGGGCCCCCTTACTCACACGCATCTAGGGGGGTGGGGA

[0812] GGAGCCGCCCGCCATATTTGGGGGACGCCGTGGGACCCCCGACTCCGGTG

[0813] CGTCTGGAGGGCGGGAGAAGAGGGAAGAAGAGGGGTCGGGATCCAAAGGA

[0814] CGGACCCAGACCACCTTTGGTTGCAGACCCCTTTCTCCCCCCTCTTCCGA

[0815] GGCCAGCAGGGGGGCAGGACTTTGTGAGGCGGGGGGGGAGGGGGAACTCG

[0816] TGGGCGCTGATTGACGCGGGAAATCCCCCCATTCTTACCCGCCCCCCTTT

[0817] TTTCCCCTCAG)CCCGCCCCGGATGTCTGGGTGTTTCCCTGCGACCGAGAC

[0818] CTGCCGGACAGCAGCGACTCGGAGGCGGAGACCGAAGTGGGGGGGCGGGG

[0819] GGACGCCGACCACCATGACGACGACTCCGCCTCCGAGGCGGACAGCACGG

[0820] ACACGGAACTGTTCGAGACGGGGCTGCTGGGGCCGCAGGGCGTGGATGGG

[0821] GGGGCGGTCTCGGGGGGGAGCCCCCCCCGCGAGGAAGACCCCGGCAGTTG

[0822] CGGGGGCGCCCCCCCTCGAGAGGACGGGGGGAGCGACGAGGGCGACGTGT

[0823] GCGCCGTGTGCACGGATGAGATCGCGCCCCACCTGCGCTGCGACACCTTC

[0824] CCGTGCATGCACCGCTTCTGCATCCCGTGCATGAAAACCTGGATGCAATT

[0825] GCGCAACACCTGCCCGCTGTGCAACGCCAAGCTGGTGTACCTGATAGTGG

[0826] GCGTGACGCCCAGCGGGTCGTTCAGCACCATCCCGATCGTGAACGACCCC

[0827] CAGACCCGCATGGAGGCCGAGGAGGCCGTCAGGGCGGGCACGGCCGTGGA

[0828] CTTTATCTGGACGGGCAATCAGCGGTTCGCCCCGCGGTACCTGACCCTGG

[0829] GGGGGCACACGGTGAGGGCCCTGTCGCCCACCCACCCGGAACCCACCACG

[0830] GACGAGGATGACGACGACCTGGACGACG(GTGAGGCGGGGGGCGGCAAGGA

[0831] CCCTGGGGGAGGAGGAGGAGGAGGGGGGGGAGGGAGGAATAGGCGGGCGG

[0832] GCGAGGAAAGGGCGGGCCGGGGAGGGGGCGTAACCTGATCGCGCCCCCCG

[0833] TTGTCTCTTGCAG)CAGACTACGTACCGCCCGCCCCCCGCCGGACGCCCCG

[0834] CGCCCCCCCACGCAGAGGCGCCGCCGCGCCCCCCGTGACGGGCGGGGCGT

[0835] CTCACGCAGCCCCCCAGCCGGCCGCGGCTCGGACAGCGCCCCCCTCGGCG

[0836] CCCATCGGGCCACACGGCAGCAGTAACACCAACACCACCACCAACAGCAG

[0837] CGGCGGCGGCGGCTCCCGCCAGTCGCGAGCCGCGGCGCCGCGGGGGGCGT

[0838] CTGGCCCCTCCGGGGGGGTTGGGGTTGGGGTTGGGGTTGTTGAAGCGGAG

[0839] GCGGGGCGGCCGAGGGGCCGGACGGGCCCCCTTGTCAACAGACCCGCCCC

[0840] CCTTGCAAACAACAGAGACCCCATAGTGATCAGCGACTCCCCCCCGGCCT

[0841] CTCCCCACAGGCCCCCCGCGGCGCCCATGCCAGGCTCCGCCCCCCGCCCC

[0842] GGGCCCCCCGCGTCCGCGGCCGCGTCGGGACCCGCGCGCCCCCGCGCGGC

[0843] CGTGGCCCCGTGCGTGCGAGCGCCGCCTCCGGGGCCCGGCCCCCGCGCCC

[0844] CGGCCCCCGGGGCGGAGCCGGCCGCCCGCCCCGCGGACGCGCGCCGTGTG

[0845] CCCCAGTCGCACTCGTCCCTGGCTCAGGCCGCGAACCAAGAACAGAGTCT

[0846] GTGCCGGGCGCGTGCGACGGTGGCGCGCGGCTCGGGGGGGCCGGGCGTGG

[0847] AGGGTGGGCACGGGCCCTCCCGCGGCGCCGCCCCCTCCGGCGCCGCCCCG

[0848] CTCCCCTCCGCCGCCTCTGTCGAGCAGGAGGCGGCGGTGCGTCCGAGGAA

[0849] GAGGCGCGGGTCGGGCCAGGAAAACCCCTCCCCCCAGTCCACGCGTCCCC

[0850] CCCTCGCGCCGGCAGGGGCCAAGAGGGCGGCGACGCACCCCCCCTCCGAC

[0851] TCAGGGCCGGGGGGGCGCGGCCAGGGTGGGCCCGGGACCCCCCTGACGTC

[0852] CTCGGCGGCCTCCGCCTCTTCCTCCTCTGCCTCTTCCTCCTCGGCCCCGA

[0853] CCCCCGCGGGGGCCGCCTCTTCCGCCGCCGGGGCCGCGTCCTCCTCCGCT

[0854] TCCGCCTCCTCGGGCGGGGCCGTCGGTGCCCTGGGAGGGAGACAAGAGGA

[0855] AACCTCCCTCGGCCCCCGCGCTGCTTCTGGGCCGCGGGGGCCGAGGAAGT

[0856] GTGCCCGGAAGACGCGCCACGCGGAGACTTCCGGGGCCGTCCCCGCGGGC

[0857] GGCCTCACGCGCTACCTGCCCATCTCGGGGGTCTCTAGCGTGGTCGCCCT

[0858] GTCGCCTTACGTGAACAAGACGATCACGGGGGACTGCCTGCCCATCCTGG

[0859] ACATGGAGACGGGGAACATCGGGGCGTACGTGGTCCTGGTGGACCAGACG

[0860] GGAAACATGGCGACCCGGCTGCGGGCCGCGGTCCCCGGCTGGAGCCGCCG

[0861] CACCCTGCTCCCCGAGACCGCGGGTAACCACGTGATGCCCCCCGAGTACC

[0862] CGACGGCCCCCGCGTCGGAGTGGAACAGCCTCTGGATGACCCCCGTGGGG

[0863] AACATGCTGTTCGACCAGGGCACCCTAGTGGGCGCCCTGGACTTCCGCAG

[0864] CCTGCGGTCTCGGCACCCGTGGTCCGGGGAGCAGGGGGCGTCGACCCGGG

[0865] ACGAGGGAAAACAATAA

[0866] SEQ ID NO. 53: DNA sequence of ICP0 in wild-type strain 17. It contains two introns (i.e., nucleotides (nt) 58 to nt 822 and nt 1490 to nt 1625 of the following sequence, see also the sequences in parentheses).

[0867] ATGGAGCCCCGCCCCGGAGCGAGTACCCGCCGGCCTGAGGGCCGCCCCCA

[0868] GCGCGAG(GTGAGGGGCCGGGCGCCATGTCTGGGGCGCCATATTGGGGGGC

[0869] GCCATATTGGGGGGCGCCATGTTGGGGGACCCCCGACCCTTACACTGGAA

[0870] CCGGCCGCCATGTTGGGGGACCCCCACTCATACACGGGAGCCGGGCGCCA

[0871] TGTTGGGGCGCCATGTTAGGGGGCGTGGAACCCCGTGACACTATATATAC

[0872] [[ID=IS]]AGGGACCGGGGGCGCCATGTTAGGGGGTGCGGAACCCCCTGACCCTATAT

[0873] ATACAGGGACCGGGGTCGCCCTGTTGGGGGTCGCCATGTGACCCCCTGAC

[0874] TTTATATATACAGACCCCCAACACATACACATGGCCCCTTTGACTCAGAC

[0875] GCAGGGCCCGGGGTCGCCGTGGGACCCCCTGACTCATACACAGAGACACG

[0876] CCCCCACAACAAACACACAGGGACCGGGGTCGCCGTGTTGGGGGCGTGGT

[0877] CCCCACTGACTCATACGCAGGCCCCCCTTACTCACACGCATCTAGGGGGG

[0878] It should be noted that there seems to be a typo in line where "IS" should probably be "18". This has been left as is in the translation to maintain consistency with the original text.TGGGGAGGAGCCGCCCGCCATATTTGGGGGACGCCGTGGGACCCCCGACT

[0879] CCGGTGCGTCTGGAGGGCGGGAGAAGAGGGAAGAAGAGGGGTCGGGATCC

[0880] AAAGGACGGACCCAGACCACCTTTGGTTGCAGACCCCTTTCTCCCCCCTC

[0881] TTCCGAGGCCAGCAGGGGGGCAGGACTTTGTGAGGCGGGGGGGGGAGAGG

[0882] GGGAACTCGTGGGTGCTGATTGACGCGGGAAATCCCCCCCCATTCTTACC

[0883] CGCCCCCCTTTTTTCCCCTTAG)CCCGCCCCGGATGTCTGGGTGTTTCCCT

[0884] GCGACCGAGACCTGCCGGACAGCAGCGACTCTGAGGCGGAGACCGAAGTG

[0885] GGGGGGCGGGGGGACGCCGACCACCATGACGACGACTCCGCCTCCGAGGC

[0886] GGACAGCACGGACACGGAACTGTTCGAGACGGGGCTGCTGGGGCCGCAGG

[0887] GCGTGGATGGGGGGGCGGTCTCGGGGGGGAGCCCCCCCCGCGAGGAAGAC

[0888] CCCGGCAGTTGCGGGGGCGCCCCCCCTCGAGAGGACGGGGGGAGCGACGA

[0889] GGGCGACGTGTGCGCCGTGTGCACGGATGAGATCGCGCCCCACCTGCGCT

[0890] GCGACACCTTCCCGTGCATGCACCGCTTCTGCATCCCGTGCATGAAAACC

[0891] TGGATGCAATTGCGCAACACCTGCCCGCTGTGCAACGCCAAGCTGGTGTA

[0892] CCTGATAGTGGGCGTGACGCCCAGCGGGTCGTTCAGCACCATCCCGATCG

[0893] TGAACGACCCCCAGACCCGCATGGAGGCCGAGGAGGCCGTCAGGGCGGGC

[0894] ACGGCCGTGGACTTTATCTGGACGGGCAATCAGCGGTTCGCCCCGCGGTA

[0895] CCTGACCCTGGGGGGGCACACGGTGAGGGCCCTGTCGCCCACCCACCCGG

[0896] AGCCCACCACGGACGAGGATGACGACGACCTGGACGACG(GTGAGGCGGGG

[0897] GGCGGCAAGGACCCTGGGGGAGGAGGAGGAGGAGGGGGGGGGAGGGAGGA

[0898] ATAGGCGGGCGGGCGAGGAAAGGGCGGGCCGGGGAGGGGGCGTAACCTGA

[0899] TCGCGCCCCCCGTTGTCTCTTGCAG)CAGACTACGTACCGCCCGCCCCCCG

[0900] CCGGACGCCCCGCGCCCCCCCACGCAGAGGCGCCGCCGCGCCCCCCGTGA

[0901] CGGGCGGGGCGTCTCACGCAGCCCCCCAGCCGGCCGCGGCTCGGACAGCG

[0902] CCCCCCTCGGCGCCCATCGGGCCACACGGCAGCAGTAACACCAACACCAC

[0903] CACCAACAGCAGCGGCGGCGGCGGCTCCCGCCAGTCGCGAGCCGCGGCGC

[0904] CGCGGGGGGCGTCTGGCCCCTCCGGGGGGGTTGGGGTTGGGGTTGGGGTT

[0905] GTTGAAGCGGAGGCGGGGCGGCCGAGGGGCCGGACGGGCCCCCTTGTCAA

[0906] CAGACCCGCCCCCCTTGCAAACAACAGAGACCCCATAGTGATCAGCGACT

[0907] CCCCCCCGGCCTCTCCCCACAGGCCCCCCGCGGCGCCCATGCCAGGCTCC

[0908] GCCCCCCGCCCCGGGCCCCCCGCGTCCGCGGCCGCGTCGGGACCCGCGCG

[0909] CCCCCGCGCGGCCGTGGCCCCGTGCGTGCGAGCGCCGCCTCCGGGGCCCG

[0910] GCCCCCGCGCCCCGGCCCCCGGGGCGGAGCCGGCCGCCCGCCCCGCGGAC

[0911] GCGCGCCGTGTGCCCCAGTCGCACTCGTCCCTGGCTCAGGCCGCGAACCA

[0912] AGAACAGAGTCTGTGCCGGGCGCGTGCGACGGTGGCGCGCGGCTCGGGGG

[0913] GGCCGGGCGTGGAGGGTGGGCACGGGCCCTCCCGCGGCGCCGCCCCCTCC

[0914] GGCGCCGCCCCGCTCCCCTCCGCCGCCTCTGTCGAGCAGGAGGCGGCGGT

[0915] GCGTCCGAGGAAGAGGCGCGGGTCGGGCCAGGAAAACCCCTCCCCCCAGT

[0916] CCACGCGTCCCCCCCTCGCGCCGGCAGGGGCCAAGAGGGCGGCGACGCAC

[0917] CCCCCCTCCGACTCAGGGCCGGGGGGGCGCGGCCAGGGTGGGCCCGGGAC

[0918] CCCCCTGACGTCCTCGGCGGCCTCCGCCTCTTCCTCCTCTGCCTCTTCCT

[0919] CCTCGGCCCCGACCCCCGCGGGGGCCGCCTCTTCCGCCGCCGGGGCCGCG

[0920] TCCTCCTCCGCTTCCGCCTCCTCGGGCGGGGCCGTCGGTGCCCTGGGAGG

[0921] GAGACAAGAGGAAACCTCCCTCGGCCCCCGCGCTGCTTCTGGGCCGCGGG

[0922] GGCCGAGGAAGTGTGCCCGGAAGACGCGCCACGCGGAGACTTCCGGGGCC

[0923] GTCCCCGCGGGCGGCCTCACGCGCTACCTGCCCATCTCGGGGGTCTCTAG

[0924] CGTGGTCGCCCTGTCGCCTTACGTGAACAAGACTATCACGGGGGACTGCC

[0925] TGCCCATCCTGGACATGGAGACGGGGAACATCGGGGCGTACGTGGTCCTG

[0926] GTGGACCAGACGGGAAACATGGCGACCCGGCTGCGGGCCGCGGTCCCCGG

[0927] CTGGAGCCGCCGCACCCTGCTCCCCGAGACCGCGGGTAACCACGTGATGC

[0928] CCCCCGAGTACCCGACGGCCCCCGCGTCGGAGTGGAACAGCCTCTGGATG

[0929] ACCCCCGTGGGGAACATGCTGTTCGACCAGGGCACCCTAGTGGGCGCCCT

[0930] GGACTTCCGCAGCCTGCGGTCTCGGCACCCGTGGTCCGGGGAGCAGGGGG

[0931] CGTCGACCCGGGACGAGGGAAAACAATAA

[0932] SEQ ID NO. 26: Amino acid sequence of ICPO in wild-type 17 strain.

[0933]

[0934]

[0935] SEQ ID NO. 27: DNA sequence of ICP8 from Mut-3, Mut-3∆34.5, and Mut-3∆ICP6 strains. The c3464t mutation is shown in bold and italics in the sequence below.

[0936] ATGGAGACAAAGCCCAAGACGGCAACCACCATCAAGGTCCCCCCCGGGCC

[0937] CCTGGGATACGTGTACGCTCGCGCGTGTCCGTCCGAAGGCATCGAGCTTC

[0938] TGGCGTTACTGTCGGCACGCAGCGGCGATTCCGACGTCGCCGTGGCGCCC

[0939] CTGGTCGTGGGCCTGACCGTGGAGAGCGGCTTTGAGGCCAACGTGGCCGT

[0940] GGTCGTGGGTTCTCGCACGACGGGGCTCGGGGGTACCGCGGTGTCCCTGA

[0941] AACTGACGCCCTCGCACTACAGCTCGTCCGTGTACGTCTTTCACGGCGGC

[0942] CGGCACCTGGACCCCAGCACCCAGGCCCCGAACCTGACGCGACTTTGCGA

[0943] GCGGGCACGCCGCCATTTTGGCTTTTCGGACTACACCCCCCGGCCCGGCG

[0944] ACCTCAAACACGAGACGACGGGGGAGGCGCTGTGTGAGCGCCTCGGCCTG

[0945] GACCCGGACCGCGCCCTCCTGTATCTGGTCGTTACCGAGGGCTTCAAGGA

[0946] GGCCGTGTGCATCAACAACACCTTTCTGCACCTGGGAGGCTCGGACAAGG

[0947] TAACCATAGGCGGGGCGGAGGTGCACCGCATACCCGTGTACCCGTTGCAG

[0948] CTGTTCATGCCGGATTTTAGCCGTGTCATCGCAGAGCCGTTCAACGCCAA

[0949] CCACCGATCGATCGGGGAGAATTTTACCTACCCGCTTCCGTTTTTTAACC

[0950] GCCCCCTCAACCGCCTCCTGTTCGAGGCGGTCGTGGGACCCGCCGCCGTG

[0951] GCACTGCGATGCCGAAACGTGGACGCCGTGGCCCGCGCGGCCGCCCACCT

[0952] GGCGTTTGACGAAAACCACGAGGGCGCCGCCCTCCCCGCCGACATTACGT

[0953] TCACGGCCTTCGAAGCCAGCCAGGGTAAGACCCCGCGGGGCGGGCGCGAC

[0954] GGCGGCGGCAAGGGCCCGGCGGGCGGGTTCGAACAGCGCCTGGCCTCCGT

[0955] CATGGCCGGAGACGCCGCCCTGGCCCTCGATTCTATCGTGTCGATGGCCG

[0956] TCTTTGACGAGCCGCCCACCGACATCTCCGCGTGGCCGCTGTTCGAGGGC

[0957] CAGGACACGGCCGCGGCCCGCGCCAACGCCGTCGGGGCGTACCTGGCGCG

[0958] CGCCGCGGGACTCGTGGGGGCCATGGTATTTAGCACCAACTCGGCCCTCC

[0959] ATCTCACCGAGGTGGACGACGCCGGCCCGGCGGACCCAAAGGACCACAGC

[0960] AAACCCTCCTTTTACCGCTTCTTCCTCGTGCCCGGGACCCACGTGGCGGC

[0961] CAACCCACAGGTGGACCGCGAGGGACACGTGGTGCCCGGGTTCGAGGGTC

[0962] GGCCCACCGCGCCCCTCGTCGGCGGAACCCAGGAATTTGCCGGCGAGCAC

[0963] CTGGCCATGCTGTGTGGGTTTTCCCCGGCGCTGCTGGCCAAGATGCTGTT

[0964] TTACCTGGAGCGCTGCGACGGCGGCGTGATCGTCGGGCGCCAGGAGATGG

[0965] ACGTGTTTCGATACGTCGCGGACTCCAACCAGACCGACGTGCCCTGTAAC

[0966] CTATGCACCTTCGACACGCGCCACGCCTGCGTACACACGACGCTCATGCG

[0967] CCTCCGGGCGCGCCATCCAAAGTTCGCCAGCGCCGCCCGCGGAGCCATCG

[0968] GCGTCTTCGGGACCATGAACAGCATGTACAGCGACTGCGACGTGCTGGGA

[0969] AACTACGCCGCCTTCTCGGCCCTGAAGCGCGCGGACGGATCCGAGACCGC

[0970] CCGGACCATCATGCAGGAGACGTACCGCGCGGCGACCGAGCGCGTCATGG

[0971] CCGAACTCGAGACCCTGCAGTACGTGGACCAGGCGGTCCCCACGGCCATG

[0972] GGGCGGCTGGAGACCATCATCACCAACCGCGAGGCCCTGCATACGGTGGT

[0973] GAACAACGTCAGGCAGGTCGTGGACCGCGAGGTGGAGCAGCTGATGCGCA

[0974] ACCTGGTGGAGGGGAGGAACTTCAAGTTTCGCGACGGTCTGGGCGAGGCC

[0975] AACCACGCCATGTCCCTGACGCTGGACCCGTACGCGTGCGGGCCGTGCCC

[0976] CCTGCTTCAGCTTCTCGGGCGGCGATCCAACCTCGCCGTGTACCAGGACC

[0977] TGGCCCTGAGTCAGTGCCACGGGGTGTTCGCCGGGCAGTCGGTCGAGGGG

[0978] CGCAACTTTCGCAATCAATTCCAACCGGTGCTGCGGCGGCGCGTGATGGA

[0979] CATGTTTAACAACGGGTTTCTGTCGGCCAAAACGCTGACGGTCGCGCTCT

[0980] CGGAGGGGGCGGCTATCTGCGCCCCCAGCCTAACGGCCGGCCAGACGGCC

[0981] CCCGCCGAGAGCAGCTTCGAGGGCGACGTTGCCCGCGTGACCCTGGGGTT

[0982] TCCCAAGGAGCTGCGCGTCAAGAGCCGCGTGTTGTTCGCGGGCGCGAGCG

[0983] CCAACGCGTCCGAGGCCGCCAAGGCGCGGGTCGCCAGCCTCCAGAGCGCC

[0984] TACCAGAAGCCCGACAAGCGCGTGGACATCCTCCTCGGACCGCTGGGCTT

[0985] TCTGCTGAAGCAGTTCCACGCGGCCATCTTCCCCAACGGCAAGCCCCCGG

[0986] GGTCCAACCAGCCGAACCCGCAGTGGTTCTGGACGGCCCTCCAACGCAAC

[0987] CAGCTTCCCGCCCGGCTCCTGTCGCGCGAGGACATCGAGACCATCGCGTT

[0988] CATTAAAAAGTTTTCCCTGGACTACGGCGCGATAAACTTTATTAACCTGG

[0989] CCCCCAACAACGTGAGCGAGCTGGCGATGTACTACATGGCAAACCAGATT

[0990] CTGCGGTACTGCGATCACTCGACATACTTCATCAACACCCTTACGGCCAT

[0991] CATCGCGGGGTCCCGCCGTCCCCCCAGCGTGCAGGCTGCGGCCGCGTGGT

[0992] CCGCGCAGGGCGGGGCGGGCCTGGAGGCCGGGGCCCGCGCGCTGATGGAC

[0993] GCCGTGGACGCGCATCCGGGCGCGTGGACGTCCATGTTCGCCAGCTGCAA

[0994] CCTGCTGCGGCCCGTCATGGCGGCGCGCCCCATGGTCGTGTTGGGGTTGA

[0995] GCATCAGCAAGTACTACGGCATGGCCGGCAACGACCGTGTGTTTCAGGCC

[0996] GGGAACTGGGCCAGCCTGATGGGCGGCAAAAACGCGTGCCCGCTCCTTAT

[0997] TTTTGACCGCACCCGCAAGTTCGTCCTGGCCTGTCCCCGGGCCGGGTTTG

[0998] TGTGCGCGGCCTCAAGCCTCGGCGGCGGAGCGCACGAAAGCTCGCTGTGC

[0999] GAGCAGCTCCGGGGCATTATCTCCGAGGGCGGGGCGGCCGTCGCCAGTAG

[1000] CGTGTTCGTGGCGACCGTGAAAAGCCTGGGGCCCCGCACCCAGCAGCTGC <重要的专利文本内容,应准确翻译]]

[1001] AGATCGAGGACTGGCTGGCGCTCCTGGAGGACGAGTACCTAAGCGAGGAG

[1002] ATGATGGAGCTGACCGCGCGTGCCCTGGAGCGCGGCAACGGCGAGTGGTC

[1003] GACGGACGCGGCCCTGGAGGTGGCGCACGAGGCCGAGGCCCTAGTCAGCC

[1004] AACTCGGCAACGCCGGGGAGGTGTTTAACTTTGGGGATTTTGGCTGCGAG

[1005] GACGACAACGCGA T GCCGTTCGGCGGCCCGGGGGCCCCGGGACCGGCATT

[1006] TGCCGGCCGCAAACGGGCGTTCCACGGGGATGACCCGTTTGGGGAGGGGC

[1007] CCCCCGACAAAAAGGGAGACCTGACGTTGGATATGCTGTGA

[1008] SEQ ID NO. 28: Amino acid sequences of ICP8 of Mut-3, Mut-3∆34.5 and Mut-3∆ICP6 virus strains. The T1155M mutation is shown in bold and italic in the following sequence. 请注意,对于专利文本的翻译,准确性至关重要,建议在实际应用中结合专业知识和背景进行进一步审核和确认。如果还有其他疑问或需要进一步的帮助,请随时提问。

[1009] METKPKTATTIKVPPGPLGYVYARACPSEGIELLALLSARSGDSDVAVAP

[1010] LVVGLTVESGFEANVAVVVGSRTTGGLGGTAVSLKLTPSHYSSSVYVFHGG

[1011] RHLDPSTQAPNLTRLCERARRHFGFSDYTPRPGDLKHETTGEALCERLGL

[1012] DPDRALLYLVVTEGFKEAVCINNTFLHLGGSDKVTIGGAEVHRIPVYPLQ

[1013] LFMPDFSRVIAEPFNANHRSIGENFTYPLPFFNRPLNRLLFEAVVGPAAV

[1014] ALRCRNVDAVARAAAHLAFDENHEGAALPADITFTAFEASQGKTPRGGRD

[1015] GGGKGPAGGFEQRLASVMAGDAALALDSIVSMAVFDEPPTDISAWPLFEG

[1016] QDTAAARANAVGAYLARAAGLVGAMVFSTNSALHLTEVDDAGPADPKDHS

[1017] KPSFYRFLFLVPGTHVAANPQVDREGHVVPGFEGRPTAPLVGGTQEFAGEH

[1018] LAMLCGFSPALLAKMLFYLERCDGGVIVGRQEMDVFRYVADSNQTDVPCN

[1019] LCTFDTRHACVHTTLMRLRARHPKFASAARGAIGVFGTMNSMYSDCDVLG

[1020] NYAAFSALKRADGSETARTIMQETYRAATERVMAELETLQYVDQAVPTAM

[1021] GRLETIITNREALHTVVNNVRQVVDREVEQLMRNLVEGRNFKFRDGGLGEA

[1022] NHAMSLTLDPYACGPCPLLQLLGRRSNLAVYQDLALSQCHGVFAGQSVEG

[1023] RNFRNQFQPVLRRRVMDMFNNGFLSAKTLTVALSEGAAICAPSLTAGQTA

[1024] PAESSFEGDVARVTLGFPKELRVKSRVLFAGASANASEAAKARVASLQSA

[1025] YQKPDKRVDILLGPLGFLLKQFHAAIFPNGKPPGSNQPNPQWFWTALQRN

[1026] QLPARLLSREDIETIAFIKKFSLDYGAINFINLAPNNVSELAMYYMANQI

[1027] LRYCDHSTYFINTLTAIIAGSRRPPSVQAAAAWSAQGGAGLEAGARALMD

[1028] AVDAHPGAWTSMFASCNLLRPVMAARPMVVLGLSISKYYGMAGNDRVFQA

[1029] GNWASLMGGKNACPLLIFDRTRKFVLACPRAGFVCAASSLGGGAHESSLC

[1030] EQLRGIISEGGAAVASSVFVATVKSLGPRTQQLQIEDWLALLEDEYLSEE

[1031] MMELTARALERGNGEWSTDAALEVAHEAEALVSQLGNAGEVFNFGDFGCE

[1032] DDNA M PFGGPGAPGPAFAGRKRAFHGDDPFGEGPPDKKGDLTLDML

[1033] SEQ ID NO. 29: DNA sequence of ICP8 in strain 17TermA.

[1034] ATGGAGACAAAGCCCAAGACGGCAACCACCATCAAGGTCCCCCCCGGGCC

[1035] CCTGGGATACGTGTACGCTCGCGCGTGTCCGTCCGAAGGCATCGAGCTTC

[1036] TGGCGTTACTGTCGGCACGCAGCGGCGATTCCGACGTCGCCGTGGCGCCC

[1037] CTGGTCGTGGGCCTGACCGTGGAGAGCGGCTTTGAGGCCAACGTGGCCGT

[1038] GGTCGTGGGTTCTCGCACGACGGGGCTCGGGGGTACCGCGGTGTCCCTGA

[1039] AACTGACGCCCTCGCACTACAGCTCGTCCGTGTACGTCTTTCACGGCGGC

[1040] CGGCACCTGGACCCCAGCACCCAGGCCCCGAACCTGACGCGACTTTGCGA

[1041] GCGGGCACGCCGCCATTTTGGCTTTTCGGACTACACCCCCCGGCCCGGCG

[1042] ACCTCAAACACGAGACGACGGGGGAGGCGCTGTGTGAGCGCCTCGGCCTG

[1043] GACCCGGACCGCGCCCTCCTGTATCTGGTCGTTACCGAGGGCTTCAAGGA

[1044] GGCCGTGTGCATCAACAACACCTTTCTGCACCTGGGAGGCTCGGACAAGG

[1045] TAACCATAGGCGGGGCGGAGGTGCACCGCATACCCGTGTACCCGTTGCAG

[1046] CTGTTCATGCCGGATTTTAGCCGTGTCATCGCAGAGCCGTTCAACGCCAA

[1047] CCACCGATCGATCGGGGAGAATTTTACCTACCCGCTTCCGTTTTTTAACC

[1048] GCCCCCTCAACCGCCTCCTGTTCGAGGCGGTCGTGGGACCCGCCGCCGTG

[1049] GCACTGCGATGCCGAAACGTGGACGCCGTGGCCCGCGCGGCCGCCCACCT

[1050] GGCGTTTGACGAAAACCACGAGGGCGCCGCCCTCCCCGCCGACATTACGT

[1051] TCACGGCCTTCGAAGCCAGCCAGGGTAAGACCCCGCGGGGCGGGCGCGAC

[1052] GGCGGCGGCAAGGGCCCGGCGGGCGGGTTCGAACAGCGCCTGGCCTCCGT

[1053] CATGGCCGGAGACGCCGCCCTGGCCCTCGATTCTATCGTGTCGATGGCCG

[1054] TCTTTGACGAGCCGCCCACCGACATCTCCGCGTGGCCGCTGTTCGAGGGC

[1055] CAGGACACGGCCGCGGCCCGCGCCAACGCCGTCGGGGCGTACCTGGCGCG

[1056] CGCCGCGGGACTCGTGGGGGCCATGGTATTTAGCACCAACTCGGCCCTCC

[1057] ATCTCACCGAGGTGGACGACGCCGGCCCGGCGGACCCAAAGGACCACAGC

[1058] AAACCCTCCTTTTACCGCTTCTTCCTCGTGCCCGGGACCCACGTGGCGGC

[1059] CAACCCACAGGTGGACCGCGAGGGACACGTGGTGCCCGGGTTCGAGGGTC

[1060] GGCCCACCGCGCCCCTCGTCGGCGGAACCCAGGAATTTGCCGGCGAGCAC

[1061] CTGGCCATGCTGTGTGGGTTTTCCCCGGCGCTGCTGGCCAAGATGCTGTT

[1062] TTACCTGGAGCGCTGCGACGGCGGCGTGATCGTCGGGCGCCAGGAGATGG

[1063] ACGTGTTTCGATACGTCGCGGACTCCAACCAGACCGACGTGCCCTGTAAC

[1064] CTATGCACCTTCGACACGCGCCACGCCTGCGTACACACGACGCTCATGCG

[1065] CCTCCGGGCGCGCCATCCAAAGTTCGCCAGCGCCGCCCGCGGAGCCATCG

[1066] GCGTCTTCGGGACCATGAACAGCATGTACAGCGACTGCGACGTGCTGGGA

[1067] AACTACGCCGCCTTCTCGGCCCTGAAGCGCGCGGACGGATCCGAGACCGC

[1068] CCGGACCATCATGCAGGAGACGTACCGCGCGGCGACCGAGCGCGTCATGG

[1069] CCGAACTCGAGACCCTGCAGTACGTGGACCAGGCGGTCCCCACGGCCATG

[1070] GGGCGGCTGGAGACCATCATCACCAACCGCGAGGCCCTGCATACGGTGGT

[1071] GAACAACGTCAGGCAGGTCGTGGACCGCGAGGTGGAGCAGCTGATGCGCA

[1072] ACCTGGTGGAGGGGAGGAACTTCAAGTTTCGCGACGGTCTGGGCGAGGCC

[1073] AACCACGCCATGTCCCTGACGCTGGACCCGTACGCGTGCGGGCCGTGCCC

[1074] CCTGCTTCAGCTTCTCGGGCGGCGATCCAACCTCGCCGTGTACCAGGACC

[1075] TGGCCCTGAGTCAGTGCCACGGGGTGTTCGCCGGGCAGTCGGTCGAGGGG

[1076] CGCAACTTTCGCAATCAATTCCAACCGGTGCTGCGGCGGCGCGTGATGGA

[1077] CATGTTTAACAACGGGTTTCTGTCGGCCAAAACGCTGACGGTCGCGCTCT

[1078] CGGAGGGGGCGGCTATCTGCGCCCCCAGCCTAACGGCCGGCCAGACGGCC

[1079] CCCGCCGAGAGCAGCTTCGAGGGCGACGTTGCCCGCGTGACCCTGGGGTT

[1080] TCCCAAGGAGCTGCGCGTCAAGAGCCGCGTGTTGTTCGCGGGCGCGAGCG

[1081] CCAACGCGTCCGAGGCCGCCAAGGCGCGGGTCGCCAGCCTCCAGAGCGCC

[1082] TACCAGAAGCCCGACAAGCGCGTGGACATCCTCCTCGGACCGCTGGGCTT

[1083] TCTGCTGAAGCAGTTCCACGCGGCCATCTTCCCCAACGGCAAGCCCCCGG

[1084] GGTCCAACCAGCCGAACCCGCAGTGGTTCTGGACGGCCCTCCAACGCAAC

[1085] CAGCTTCCCGCCCGGCTCCTGTCGCGCGAGGACATCGAGACCATCGCGTT

[1086] CATTAAAAAGTTTTCCCTGGACTACGGCGCGATAAACTTTATTAACCTGG

[1087] CCCCCAACAACGTGAGCGAGCTGGCGATGTACTACATGGCAAACCAGATT

[1088] CTGCGGTACTGCGATCACTCGACATACTTCATCAACACCCTTACGGCCAT

[1089] CATCGCGGGGTCCCGCCGTCCCCCCAGCGTGCAGGCTGCGGCCGCGTGGT

[1090] CCGCGCAGGGCGGGGCGGGCCTGGAGGCCGGGGCCCGCGCGCTGATGGAC

[1091] GCCGTGGACGCGCATCCGGGCGCGTGGACGTCCATGTTCGCCAGCTGCAA

[1092] CCTGCTGCGGCCCGTCATGGCGGCGCGCCCCATGGTCGTGTTGGGGTTGA

[1093] GCATCAGCAAGTACTACGGCATGGCCGGCAACGACCGTGTGTTTCAGGCC

[1094] GGGAACTGGGCCAGCCTGATGGGCGGCAAAAACGCGTGCCCGCTCCTTAT

[1095] TTTTGACCGCACCCGCAAGTTCGTCCTGGCCTGTCCCCGGGCCGGGTTTG

[1096] TGTGCGCGGCCTCAAGCCTCGGCGGCGGAGCGCACGAAAGCTCGCTGTGC

[1097] GAGCAGCTCCGGGGCATTATCTCCGAGGGCGGGGCGGCCGTCGCCAGTAG

[1098] CGTGTTCGTGGCGACCGTGAAAAGCCTGGGGCCCCGCACCCAGCAGCTGC

[1099] AGATCGAGGACTGGCTGGCGCTCCTGGAGGACGAGTACCTAAGCGAGGAG

[1100] ATGATGGAGCTGACCGCGCGTGCCCTGGAGCGCGGCAACGGCGAGTGGTC

[1101] GACGGACGCGGCCCTGGAGGTGGCGCACGAGGCCGAGGCCCTAGTCAGCC

[1102] AACTCGGCAACGCCGGGGAGGTGTTTAACTTTGGGGATTTTGGCTGCGAG

[1103] GACGACAACGCGACGCCGTTCGGCGGCCCGGGGGCCCCGGGACCGGCATT

[1104] TGCCGGCCGCAAACGGGCGTTCCACGGGGATGACCCGTTTGGGGAGGGGC

[1105] CCCCCGACAAAAAGGGAGACCTGACGTTGGATATGCTGTGA

[1106] SEQ ID NO. 30: Amino acid sequence of ICP8 in strain 17TermA.

[1107] METKPKTATTIKVPPGPLGYVYARACPSEGIELLALLSARSGDSDVAVAP

[1108] LVVGLTVESGFEANVAVVVGSRTTGLGGTAVSLKLTPSHYSSSVYVFHGG

[1109] RHLDPSTQAPNLTRLCERARRHFGFSDYTPRPGDLKHETTGEALCERLGL

[1110] DPDRALLYLVVTEGFKEAVCINNTFLHLGGSDKVTIGGAEVHRIPVYPLQ

[1111] LFMPDFSRVIAEPFNANHRSIGENFTYPLPFFNRPLNRLLFEAVVGPAAV

[1112] ALRCRNVDAVARAAAHLAFDENHEGAALPADITFTAFEASQGKTPRGGRD

[1113] GGGKGPAGGFEQRLASVMAGDAALALDSIVSMAVFDEPPTDISAWPLFEG

[1114] QDTAAARANAVGAYLARAAGLVGAMVFSTNSALHLTEVDDAGPADPKDHS

[1115] KPSFYRFFLVPGTHVAANPQVDREGHVVPGFEGRPTAPLVGGTQEFAGEH

[1116] LAMLCGFSPALLAKMLFYLERCDGGVIVGRQEMDVFRYVADSNQTDVPCN

[1117] LCTFDTRHACVHTTLMRLRARHPKFASAARGAIGVFGTMNSMYSDCDVLG

[1118] NYAAFSALKRADGSETARTIMQETYRAATERVMAELETLQYVDQAVPTAM

[1119] GRLETIITNREALHTVVNNVRQVVDREVEQLMRNLVEGRNFKFRDGLGEA

[1120] NHAMSLTLDPYACGPCPLLQLLGRRSNLAVYQDLALSQCHGVFAGQSVEG

[1121] RNFRNQFQPVLRRRVMDMFNNGFLSAKTLTVALSEGAAICAPSLTAGQTA

[1122] PAESSFEGDVARVTLGFPKELRVKSRVLFAGASANASEAAKARVASLQSA

[1123] YQKPDKRVDILLGPLGFLLKQFHAAIFPNGKPPGSNQPNPQWFWTALQRN

[1124] QLPARLLSREDIETIAFIKKFSLDYGAINFINLAPNNVSELAMYYMANQI

[1125] LRYCDHSTYFINTLTAIIAGSRRPPSVQAAAAWSAQGGAGLEAGARALMD

[1126] AVDAHPGAWTSMFASCNLLRPVMAARPMVVLGLSISKYYGMAGNDRVFQA

[1127] GNWASLMGGKNACPLLIFDRTRKFVLACPRAGFVCAASSLGGGAHESSLC

[1128] EQLRGIISEGGAAVASSVFVATVKSLGPRTQQLQIEDWLALLEDEYLSEE

[1129] MMELTARALERGNGEWSTDAALEVAHEAEALVSQLGNAGEVFNFGDFGCE

[1130] DDNATPFGGPGAPGPAFAGRKRAFHGDDPFGEGPPDKKGDLTLDML

[1131] SEQ ID NO. 31: DNA sequence of ICP8 in strain rRp450.

[1132] ATGGAGACAAAGCCCAAGACGGCAACCACCATCAAGGTCCCCCCCGGGCC

[1133] CCTGGGATACGTGTACGCTCGCGCGTGTCCGTCCGAAGGCATCGAGCTTC

[1134] TGGCGTTACTGTCGGCGCGCAGCGGCGATGCCGACGTCGCCGTGGCGCCC <00023​​​​​​​​​​​​GCGGGCACGCCGCCATTTTGGCTTTTCGGACTACACCCCCCGGCCCGGCG

[1140] ACCTCAAACACGAGACGACGGGGGAGGCGCTGTGTGAGCGCCTCGGCCTG

[1141] GACCCGGACCGCGCCCTCCTGTATCTGGTCGTTACCGAGGGCTTCAAGGA

[1142] GGCCGTGTGCATCAACAACACCTTTCTGCACCTGGGAGGCTCGGACAAGG

[1143] TAACCATAGGCGGGGCGGAGGTGCACCGCATACCCGTGTATCCGTTGCAG

[1144] CTGTTCATGCCGGATTTTAGCCGGGTCATCGCCGAGCCGTTCAACGCCAA

[1145] CCACCGATCGATCGGGGAGAATTTTACCTACCCGCTTCCGTTTTTTAACC

[1146] GCCCCCTCAACCGCCTCCTGTTCGAGGCGGTCGTGGGACCCGCCGCCGTG

[1147] GCACTGCGATGCCGAAACGTGGACGCCGTGGCCCGCGCGGCCGCCCACCT

[1148] GGCGTTTGACGAAAACCACGAGGGCGCCGCCCTCCCCGCCGACATTACGT

[1149] TCACGGCCTTCGAAGCCAGCCAGGGTAAGACCCCGCGGGGTGGGCGCGAC

[1150] GGCGGCGGCAAGGGCCCGGCGGGCGGGTTCGAACAGCGCCTGGCCTCCGT

[1151] CATGGCCGGAGACGCCGCCCTGGCCCTCGAGTCTATCGTGTCGATGGCCG

[1152] TCTTCGACGAGCCGCCCACCGACATCTCCGCGTGGCCGCTGTGCGAGGGC

[1153] CAGGACACGGCCGCGGCCCGCGACAACGCCGTCGGGGCGTACCTGGCGCG

[1154] CGCCGCGGGACTCGTGGGGGCCATGGTATTTAGCACCAACTCGGCCCTCC

[1155] ATCTCACCGAGGTGGACGACGCCGGTCCGGCGGACCCAAAGGACCACAGC

[1156] AAACCCTCCTTTTACCGCTTCTTCCTCGTGCCCGGGACCCACGTGGCGGC

[1157] CAACCCACAGGTGGACCGCGAGGGACACGTGGTGCCCGGGTTCGAGGGTC

[1158] GGCCCACCGCGCCCCTCGTCGGCGGAACCCAGGAATTTGCCGGCGAGCAC

[1159] CTGGCCATGCTGTGTGGGTTTTCCCCGGCGCTGCTGGCCAAGATGCTGTT

[1160] TTACCTGGAGCGCTGCGACGGCGGCGTGATCGTCGGGCGCCAGGAGATGG

[1161] ACGTGTTTCGATACGTCGCGGACTCCAACCAGACCGACGTGCCCTGCAAC

[1162] CTGTGCACCTTCGACACGCGCCACGCCTGCGTACACACGACGCTCATGCG

[1163] CCTCCGGGCGCGCCATCCCAAGTTCGCCAGCGCCGCCCGCGGAGCCATCG

[1164] GCGTCTTCGGGACCATGAACAGCATGTACAGCGACTGCGACGTGCTGGGA

[1165] AACTACGCCGCCTTCTCGGCCCTGAAGCGCGCGGACGGATCCGAGACCGC

[1166] CCGGACCATCATGCAGGAGACGTACCGCGCGGCGACCGAGCGCGTCATGG

[1167] CCGAACTCGAGACCCTGCAGTACGTGGACCAGGCGGTCCCCACGGCCATG

[1168] GGGCGGCTGGAGACCATCATCACCAACCGCGAGGCCCTGCATACGGTGGT

[1169] GAACAACGTCAGGCAGGTCGTGGACCGCGAGGTGGAGCAGCTGATGCGCA

[1170] ACCTGGTGGAGGGGAGGAACTTCAAGTTTCGCGACGGTCTGGGCGAGGCC

[1171] AACCACGCCATGTCCCTGACGCTGGACCCGTACGCGTGCGGGCCATGCCC

[1172] CCTGCTTCAGCTTCTCGGGCGGCGATCCAACCTCGCCGTGTATCAGGACC

[1173] TGGCCCTGAGCCAGTGCCACGGGGTGTTCGCCGGGCAGTCGGTCGAGGGG

[1174] CGCAACTTTCGCAATCAATTCCAACCGGTGCTGCGGCGGCGCGTGATGGA

[1175] CATGTTTAACAACGGGTTTCTGTCGGCCAAAACGCTGACGGTCGCGCTCT

[1176] CGGAGGGGGCGGCTATCTGCGCCCCCAGCCTAACGGCCGGCCAGACGGCC

[1177] CCCGCCGAGAGCAGCTTCGAGGGCGACGTTGCCCGCGTGACCCTGGGGTT

[1178] TCCCAAGGAGCTGCGCGTCAAGAGCCGCGTGTTGTTCGCGGGCGCGAGCG

[1179] CCAACGCGTCCGAGGCCGCCAAGGCGCGGGTCGCCAGCCTCCAGAGCGCC

[1180] TACCAGAAGCCCGACAAGCGCGTGGACATCCTCCTCGGACCGCTGGGCTT

[1181] TCTGCTGAAGCAGTTCCACGCGGCCATCTTCCCCAACGGCAAGCCCCCGG

[1182] GGTCCAACCAGCCGAACCCGCAGTGGTTCTGGACGGCCCTCCAACGCAAC

[1183] CAGCTTCCCGCCCGGCTCCTGTCGCGCGAGGACATCGAGACCATCGCGTT

[1184] CATTAAAAAGTTTTCCCTGGACTACGGCGCGATAAACTTTATTAACCTGG

[1185] CCCCCAACAACGTGAGCGAGCTGGCGATGTACTACATGGCAAACCAGATT

[1186] CTGCGGTACTGCGATCACTCGACATACTTCATCAACACCCTCACGGCCAT

[1187] CATCGCGGGGTCCCGCCGTCCCCCCAGCGTGCAGGCGGCGGCCGCGTGGT

[1188] CCGCGCAGGGCGGGGCGGGCCTGGAGGCCGGGGCCCGCGCGCTGATGGAC

[1189] GCCGTGGACGCGCATCCGGGCGCGTGGACGTCCATGTTCGCCAGCTGCAA

[1190] CCTGCTGCGGCCCGTCATGGCGGCGCGCCCCATGGTCGTGTTGGGGTTGA

[1191] GCATCAGCAAATACTACGGCATGGCCGGCAACGACCGTGTGTTTCAGGCC

[1192] GGGAACTGGGCCAGCCTGATGGGCGGCAAAAACGCGTGCCCGCTCCTTAT

[1193] TTTTGACCGCACCCGCAAGTTCGTCCTGGCCTGTCCCCGGGCCGGGTTTG

[1194] TGTGCGCGGCCTCGAACCTCGGCGGCGGAGCGCACGAAAGCTCGCTGTGC

[1195] GAGCAGCTCCGGGGCATTATCTCCGAGGGCGGGGCGGCCGTCGCCAGTAG

[1196] CGTGTTCGTGGCGACCGTGAAAAGCCTGGGGCCCCGCACCCAGCAGCTGC

[1197] AGATCGAGGACTGGCTGGCGCTCCTGGAGGACGAGTACCTAAGCGAGGAG

[1198] ATGATGGAGCTGACCGCGCGTGCCCTGGAGCGCGGCAACGGCGAGTGGTC

[1199] GACGGACGCGGCCCTGGAGGTGGCGCACGAGGCCGAGGCCCTAGTCAGCC

[1200] AACTCGGCAACGCCGGGGAGGTGTTTAACTTTGGGGATTTTGGCTGCGAG

[1201] GACGACAACGCGACGCCGTTCGGCGGCCCGGGGGCCCCGGGACCGGCATT

[1202] TGCCGGCCGCAAACGGGCGTTCCACGGGGATGACCCGTTTGGGGAGGGGC

[1203] CCCCCGACAAAAAGGGAGACCTGACGTTGGATATGCTGTGA

[1204] SEQ ID NO. 32: Amino acid sequence of ICP8 in strain rRp450.

[1205] METKPKTATTIKVPPGPLGYVYARACPSEGIELLALLSARSGDADVAVAP

[1206] LVVGLTVESGFEANVAVVVGSRTTGLGGTAVSLKLTPSHYSSSVYVFHGG

[1207] RHLDPSTQAPNLTRLCERARRHFGFSDYTPRPGDLKHETTGEALCERLGL

[1208] DPDRALLYLVVTEGFKEAVCINNTFLHLGGSDKVTIGGAEVHRIPVYPLQ

[1209] LFMPDFSRVIAEPFNANHRSIGENFTYPLPFFNRPLNRLLFEAVVGPAAV

[1210] ALRCRNVDAVARAAAHLAFDENHEGAALPADITFTAFEASQGKTPRGGRD

[1211] GGGKGPAGGFEQRLASVMAGDAALALESIVSMAVFDEPPTDISAWPLCEG

[1212] QDTAAARDNAVGAYLARAAGLVGAMVFSTNSALHLTEVDDAGPADPKDHS

[1213] KPSFYRFFLVPGTHVAANPQVDREGHVVPGFEGRPTAPLVGGTQEFAGEH

[1214] LAMLCGFSPALLAKMLFYLERCDGGVIVGRQEMDVFRYVADSNQTDVPCN

[1215] LCTFDTRHACVHTTLMRLRARHPKFASAARGAIGVFGTMNSMYSDCDVLG

[1216] NYAAFSALKRADGSETARTIMQETYRAATERVMAELETLQYVDQAVPTAM

[1217] GRLETIITNREALHTVVNNVRQVVDREVEQLMRNLVEGRNFKFRDGLGEA

[1218] NHAMSLTLDPYACGPCPLLQLLGRRSNLAVYQDLALSQCHGVFAGQSVEG

[1219] RNFRNQFQPVLRRRVMDMFNNGFLSAKTLTVALSEGAAICAPSLTAGQTA

[1220] PAESSFEGDVARVTLGFPKELRVKSRVLFAGASANASEAAKARVASLQSA

[1221] YQKPDKRVDILLGPLGFLLKQFHAAIFPNGKPPGSNQPNPQWFWTALQRN

[1222] QLPARLLSREDIETIAFIKKFSLDYGAINFINLAPNNVSELAMYYMANQI

[1223] LRYCDHSTYFINTLTAIIAGSRRPPSVQAAAAWSAQGGAGLEAGARALMD

[1224] AVDAHPGAWTSMFASCNLLRPVMAARPMVVLGLSISKYYGMAGNDRVFQA

[1225] GNWASLMGGKNACPLLIFDRTRKFVLACPRAGFVCAASNLGGGAHESSLC

[1226] EQLRGIISEGGAAVASSVFVATVKSLGPRTQQLQIEDWLALLEDEYLSEE

[1227] MMELTARALERGNGEWSTDAALEVAHEAEALVSQLGNAGEVFNFGDFGCE

[1228] DDNATPFGGPGAPGPAFAGRKRAFHGDDPFGEGPPDKKGDLTLDML

[1229] SEQ ID NO. 33: DNA sequence of ICP8 in wild-type strain 17.

[1230] ATGGAGACAAAGCCCAAGACGGCAACCACCATCAAGGTCCCCCCCGGGCC

[1231] CCTGGGATACGTGTACGCTCGCGCGTGTCCGTCCGAAGGCATCGAGCTTC

[1232] TGGCGTTACTGTCGGCACGCAGCGGCGATTCCGACGTCGCCGTGGCGCCC

[1233] CTGGTCGTGGGCCTGACCGTGGAGAGCGGCTTTGAGGCCAACGTGGCCGT

[1234] GGTCGTGGGTTCTCGCACGACGGGGCTCGGGGGTACCGCGGTGTCCCTGA

[1235] AACTGACGCCCTCGCACTACAGCTCGTCCGTGTACGTCTTTCACGGCGGC

[1236] CGGCACCTGGACCCCAGCACCCAGGCCCCGAACCTGACGCGACTTTGCGA

[1237] GCGGGCACGCCGCCATTTTGGCTTTTCGGACTACACCCCCCGGCCCGGCG

[1238] ACCTCAAACACGAGACGACGGGGGAGGCGCTGTGTGAGCGCCTCGGCCTG

[1239] GACCCGGACCGCGCCCTCCTGTATCTGGTCGTTACCGAGGGCTTCAAGGA

[1240] GGCCGTGTGCATCAACAACACCTTTCTGCACCTGGGAGGCTCGGACAAGG

[1241] TAACCATAGGCGGGGCGGAGGTGCACCGCATACCCGTGTACCCGTTGCAG

[1242] CTGTTCATGCCGGATTTTAGCCGTGTCATCGCAGAGCCGTTCAACGCCAA

[1243] CCACCGATCGATCGGGGAGAATTTTACCTACCCGCTTCCGTTTTTTAACC

[1244] GCCCCCTCAACCGCCTCCTGTTCGAGGCGGTCGTGGGACCCGCCGCCGTG

[1245] GCACTGCGATGCCGAAACGTGGACGCCGTGGCCCGCGCGGCCGCCCACCT

[1246] GGCGTTTGACGAAAACCACGAGGGCGCCGCCCTCCCCGCCGACATTACGT

[1247] TCACGGCCTTCGAAGCCAGCCAGGGTAAGACCCCGCGGGGCGGGCGCGAC

[1248] GGCGGCGGCAAGGGCCCGGCGGGCGGGTTCGAACAGCGCCTGGCCTCCGT

[1249] CATGGCCGGAGACGCCGCCCTGGCCCTCGATTCTATCGTGTCGATGGCCG

[1250] TCTTTGACGAGCCGCCCACCGACATCTCCGCGTGGCCGCTGTTCGAGGGC

[1251] CAGGACACGGCCGCGGCCCGCGCCAACGCCGTCGGGGCGTACCTGGCGCG

[1252] CGCCGCGGGACTCGTGGGGGCCATGGTATTTAGCACCAACTCGGCCCTCC

[1253] ATCTCACCGAGGTGGACGACGCCGGCCCGGCGGACCCAAAGGACCACAGC

[1254] AAACCCTCCTTTTACCGCTTCTTCCTCGTGCCCGGGACCCACGTGGCGGC

[1255] CAACCCACAGGTGGACCGCGAGGGACACGTGGTGCCCGGGTTCGAGGGTC

[1256] GGCCCACCGCGCCCCTCGTCGGCGGAACCCAGGAATTTGCCGGCGAGCAC

[1257] CTGGCCATGCTGTGTGGGTTTTCCCCGGCGCTGCTGGCCAAGATGCTGTT

[1258] TTACCTGGAGCGCTGCGACGGCGGCGTGATCGTCGGGCGCCAGGAGATGG

[1259] ACGTGTTTCGATACGTCGCGGACTCCAACCAGACCGACGTGCCCTGTAAC

[1260] CTATGCACCTTCGACACGCGCCACGCCTGCGTACACACGACGCTCATGCG

[1261] CCTCCGGGCGCGCCATCCAAAGTTCGCCAGCGCCGCCCGCGGAGCCATCG

[1262] GCGTCTTCGGGACCATGAACAGCATGTACAGCGACTGCGACGTGCTGGGA

[1263] AACTACGCCGCCTTCTCGGCCCTGAAGCGCGCGGACGGATCCGAGACCGC

[1264] CCGGACCATCATGCAGGAGACGTACCGCGCGGCGACCGAGCGCGTCATGG

[1265] CCGAACTCGAGACCCTGCAGTACGTGGACCAGGCGGTCCCCACGGCCATG

[1266] GGGCGGCTGGAGACCATCATCACCAACCGCGAGGCCCTGCATACGGTGGT

[1267] GAACAACGTCAGGCAGGTCGTGGACCGCGAGGTGGAGCAGCTGATGCGCA

[1268] ACCTGGTGGAGGGGAGGAACTTCAAGTTTCGCGACGGTCTGGGCGAGGCC

[1269] AACCACGCCATGTCCCTGACGCTGGACCCGTACGCGTGCGGGCCGTGCCC

[1270] CCTGCTTCAGCTTCTCGGGCGGCGATCCAACCTCGCCGTGTACCAGGACC

[1271] TGGCCCTGAGTCAGTGCCACGGGGTGTTCGCCGGGCAGTCGGTCGAGGGG

[1272] CGCAACTTTCGCAATCAATTCCAACCGGTGCTGCGGCGGCGCGTGATGGA

[1273] CATGTTTAACAACGGGTTTCTGTCGGCCAAAACGCTGACGGTCGCGCTCT

[1274] CGGAGGGGGCGGCTATCTGCGCCCCCAGCCTAACGGCCGGCCAGACGGCC

[1275] CCCGCCGAGAGCAGCTTCGAGGGCGACGTTGCCCGCGTGACCCTGGGGTT

[1276] TCCCAAGGAGCTGCGCGTCAAGAGCCGCGTGTTGTTCGCGGGCGCGAGCG

[1277] CCAACGCGTCCGAGGCCGCCAAGGCGCGGGTCGCCAGCCTCCAGAGCGCC

[1278] TACCAGAAGCCCGACAAGCGCGTGGACATCCTCCTCGGACCGCTGGGCTT

[1279] TCTGCTGAAGCAGTTCCACGCGGCCATCTTCCCCAACGGCAAGCCCCCGG

[1280] GGTCCAACCAGCCGAACCCGCAGTGGTTCTGGACGGCCCTCCAACGCAAC

[1281] CAGCTTCCCGCCCGGCTCCTGTCGCGCGAGGACATCGAGACCATCGCGTT

[1282] CATTAAAAAGTTTTCCCTGGACTACGGCGCGATAAACTTTATTAACCTGG

[1283] CCCCCAACAACGTGAGCGAGCTGGCGATGTACTACATGGCAAACCAGATT

[1284] CTGCGGTACTGCGATCACTCGACATACTTCATCAACACCCTTACGGCCAT

[1285] CATCGCGGGGTCCCGCCGTCCCCCCAGCGTGCAGGCTGCGGCCGCGTGGT

[1286] CCGCGCAGGGCGGGGCGGGCCTGGAGGCCGGGGCCCGCGCGCTGATGGAC

[1287] GCCGTGGACGCGCATCCGGGCGCGTGGACGTCCATGTTCGCCAGCTGCAA

[1288] CCTGCTGCGGCCCGTCATGGCGGCGCGCCCCATGGTCGTGTTGGGGTTGA

[1289] GCATCAGCAAGTACTACGGCATGGCCGGCAACGACCGTGTGTTTCAGGCC

[1290] GGGAACTGGGCCAGCCTGATGGGCGGCAAAAACGCGTGCCCGCTCCTTAT

[1291] TTTTGACCGCACCCGCAAGTTCGTCCTGGCCTGTCCCCGGGCCGGGTTTG

[1292] TGTGCGCGGCCTCAAGCCTCGGCGGCGGAGCGCACGAAAGCTCGCTGTGC

[1293] GAGCAGCTCCGGGGCATTATCTCCGAGGGCGGGGCGGCCGTCGCCAGTAG

[1294] CGTGTTCGTGGCGACCGTGAAAAGCCTGGGGCCCCGCACCCAGCAGCTGC

[1295] AGATCGAGGACTGGCTGGCGCTCCTGGAGGACGAGTACCTAAGCGAGGAG

[1296] ATGATGGAGCTGACCGCGCGTGCCCTGGAGCGCGGCAACGGCGAGTGGTC

[1297] GACGGACGCGGCCCTGGAGGTGGCGCACGAGGCCGAGGCCCTAGTCAGCC

[1298] AACTCGGCAACGCCGGGGAGGTGTTTAACTTTGGGGATTTTGGCTGCGAG

[1299] GACGACAACGCGACGCCGTTCGGCGGCCCGGGGGCCCCGGGACCGGCATT

[1300] TGCCGGCCGCAAACGGGCGTTCCACGGGGATGACCCGTTTGGGGAGGGGC

[1301] CCCCCGACAAAAAGGGAGACCTGACGTTGGATATGCTGTGA

[1302] SEQ ID NO. 34: Amino acid sequence of ICP8 in wild-type strain 17.

[1303] METKPKTATTIKVPPGPLGYVYARACPSEGIELLALLSARSGDSDVAVAP

[1304] LVVGLTVESGFEANVAVVVGSRTTGLGGTAVSLKLTPSHYSSSVYVFHGG

[1305] RHLDPSTQAPNLTRLCERARRHFGFSDYTPRPGDLKHETTGEALCERLGL

[1306] DPDRALLYLVVTEGFKEAVCINNTFLHLGGSDKVTIGGAEVHRIPVYPLQ

[1307] LFMPDFSRVIAEPFNANHRSIGENFTYPLPFFNRPLNRLLFEAVVGPAAV

[1308] ALRCRNVDAVARAAAHLAFDENHEGAALPADITFTAFEASQGKTPRGGRD

[1309] GGGKGPAGGFEQRLASVMAGDAALALDSIVSMAVFDEPPTDISAWPLFEG

[1310] QDTAAARANAVGAYLARAAGLVGAMVFSTNSALHLTEVDDAGPADPKDHS

[1311] KPSFYRFLFLVPGTHVAANPQVDREGHVVPGFEGRPTAPLVGGTQEFAGEH

[1312] LAMLCGFSPALLAKMLFYLERCDGGVIVGRQEMDVFRYVADSNQTDVPCN

[1313] LCTFDTRHACVHTTLMRLRARHPKFASAARGAIGVFGTMNSMYSDCDVLG

[1314] NYAAFSALKRADGSETARTIMQETYRAATERVMAELETLQYVDQAVPTAM

[1315] GRLETIITNREALHTVVNNVRQVVDREVEQLMRNLVEGRNFKFRDGGLGEA

[1316] NHAMSLTLDPYACGPCPLLQLLGRRSNLAVYQDLALSQCHGVFAGQSVEG

[1317] RNFRNQFQPVLRRRVMDMFNNGFLSAKTLTVALSEGAAAICAPSLTAGQTA

[1318] PAESSFEGDVARVTLGFPKELRVKSRVLFAGASANASEAAKARVASLQSA

[1319] YQKPDKRVDILLGPLGFLLKQFHAAIFPNGKPPGSNQPNPQFWWTALQRN

[1320] QLPARLLSREDIETIAFIKFSLDYGAINFINLAPNNVSELAMYYMANQI

[1321] LRYCDHSTYFINTLTAIIAGSRRPPSVQAAAAWSAQGGAGLEAGARALMD

[1322] AVDAHPGAWTSMFASCNLLRPVMAARPMVVLGLSISKYYGMAGNDRVFQA

[1323] GNWASLMGGKNACPLLIFDRTRKFVLACPRAGFVCAASSLGGGAHESSLC

[1324] EQLRGIISEGGAAVASSVFVATVKSLGPRTQQLQIEDWLALLEDEYLSEE

[1325] MMELTARALERGNGEWSTDAALEVAHEAEALVSQLGNAGEVFNFGDFGCE

[1326] DDNATPFGGPGAPGPAFAGRKRAFHGDDPFGEGPPDKKGDLTLDML

[1327] SEQ ID NO. 35: DNA sequence of DNA packaging terminase subunit 1 of Mut-3, Mut-3∆34.5, and Mut-3∆ICP6 virus strains. The g1126a mutation is shown in bold and italics in the sequence below.

[1328] ATGTTTGGTCAGCAGCTGGCGTCCGACGTCCAGCAGTACCTGGAGCGCCT

[1329] CGAGAAACAGAGGCAACTTAAGGTGGGCGCGGACGAGGCGTCGGCGGGCC

[1330] TCACCATGGGCGGGCGATGCCCTACGAGTGCCCTTTTTAGATTTCGCGACC

[1331] GCGACCCCCAAGCGCCACCAGACCGTGGTCCCTGGCGTCGGGACGCTCCA

[1332] CGACTGCTGCGAGCACTCGCCGCTCTTCTCGGCCGTGGCGCGGCGGCTGC

[1333] TGTTTAATAGCCTGGTGCCGGCGCAACTAAAGGGGCGTGATTTCGGGGGC

[1334] GACCACACGGCCAAGCTGGAATTCCTGGCCCCCGAGTTGGTACGGGCGGT

[1335] GGCGCGACTGCGGTTTAAGGAGTGCGCGCCGGCGGACGTGGTGCCTCAGC

[1336] GTAACGCCTACTATAGCGTTCTGAATACGTTTCAGGCCCTCCACCGCTCC

[1337] GAAGCCTTTCGCCAGCTGGTGCACTTTGTGCGGGACTTTGCCCAGCTGCT

[1338] CAAAACCTCCTTCCGGGCCTCCAGCCTCACGGAGACCACGGGCCCCCCCA

[1339] AAAAACGGGCCAAGGTGGACGTGGCCACCCACGGCCGGACGTACGGCACG

[1340] CTGGAGCTGTTCCAAAAAATGATCCTTATGCACGCCACCTACTTTCTGGC

[1341] CGCCGTGCTCCTCGGGGACCACGCGGAGCAGGTCAACACGTTCCTGCGTC

[1342] TCGTGTTTGAGATCCCCCTGTTTAGCGACGCGGCCGTGCGCCACTTCCGC

[1343] CAGCGCGCCACCGTGTTTCTCGTCCCCCGGCGCCACGGCAAGACCTGGTT

[1344] TCTGGTGCCCCTCATCGCGCTGTCGCTGGCCTCCTTTCGGGGGATCAAGA

[1345] TCGGCTACACGGCGCACATCCGCAAGGCGACCGAGCCGGTGTTTGAGGAG

[1346] ATCGACGCCTGCCTGCGGGGCTGGTTCGGTTCGGCCCGAGTGGACCACGT

[1347] TAAAGGGGAAACCATCTCCTTCTCGTTTCCGGACGGGTCGCGCAGTACCA

[1348] TCGTGTTTGCCTCCAGCCACAACACAAACGGAATCCGAGGCCAGGACTTT

[1349] AACCTGCTCTTTGTCGACGAGGCCAACTTTATTCGCCCGGATGCGGTCCA

[1350] GACGATTATGGGCTTTCTCAACCAG A CCAACTGCAAGATTATCTTCGTGT

[1351] CGTCCACCAACACCGGGAAGGCCAGTACGAGCTTTTTGTACAACCTCCGC

[1352] GGGGCCGCAGACGAGCTTCTCAACGTGGTGACCTATATATGCGATGATCA

[1353] CATGCCGAGGGTGGTGACGCACACAAACGCCACGGCCTGTTCTTGTTATA

[1354] TCCTCAACAAGCCCGTTTTCATCACGATGGACGGGGCGGTTCGCCGGACC

[1355] GCCGATTTGTTTCTGGCCGATTCCTTCATGCAGGAGATCATCGGGGGCCA

[1356] GGCCAGGGAGACCGGCGACGACCGGCCCGTTCTGACCAAGTCTGCGGGGG

[1357] AGCGGTTTCTGTTGTACCGCCCCTCGACCACCACCAACAGCGGCCTCATG

[1358] GCCCCCGATTTGTACGTGTACGTGGATCCCGCGTTCACGGCCAACACCCG

[1359] AGCCTCCGGGACCGGCGTCGCTGTCGTCGGGCGGTACCGCGACGATTATA

[1360] TCATCTTCGCCCTGGAGCACTTTTTTCTCCGCGCGCTCACGGGCTCGGCC

[1361] CCCGCCGACATCGCCCGCTGCGTCGTCCACAGTCTGACGCAGGTCCTGGC

[1362] CCTGCATCCCGGGGCGTTTCGCGGCGTCCGGGTGGCGGTCGAGGGAAATA

[1363] GCAGCCAGGACTCGGCCGTCGCCATCGCCACGCACGTGCACACAGAGATG

[1364] CACCGCCTACTGGCCTCGGAGGGGGCCGACGCGGGCTCGGGCCCCGAGCT

[1365] TCTCTTCTACCACTGCGAGCCTCCCGGGAGCGCGGTGCTGTACCCCTTTT

[1366] TCCTGCTCAACAAACAGAAGACGCCCGCCTTTGAACACTTTATTAAAAAG

[1367] TTTAACTCCGGGGGCGTCATGGCCTCCCAGGAGATCGTTTCCGCGACGGT

[1368] GCGCCTGCAGACCGACCCGGTCGAGTATCTGCTCGAGCAGCTAAATAACC

[1369] TCACCGAAACCGTCTCCCCCAACACTGACGTCCGTACGTATTCCGGAAAA

[1370] CGGAACGGCGCCTCGGATGACCTTATGGTCGCCGTCATTATGGCCATCTA

[1371] CCTCGCGGCCCAGGCCGGACCTCCGCACACATTCGCTCCTATCATACGCG

[1372] TCTCGTGA

[1373] SEQ ID NO. 36: Amino acid sequence of DNA packaging terminase subunit 1 of Mut-3, Mut-3∆34.5, and Mut-3∆ICP6 virus strains. The A376T mutation is shown in bold and italics in the sequence below.

[1374] MFGQQLASDVQQYLERLEKQRQLKVGADEASAGLTMGGDALRVPFLDFAT

[1375] ATPKRHQTVVPGVGTLHDCCEHSPLFSAVARRLLFNSLVPAQLKGRDFGG

[1376] DHTAKLEFLAPELVRAVARLRFKECAPADVVPQRNAYYSVLNTFQALHRS

[1377] EAFRQLVHFVRDFAQLLKTSFRASSLTETTGPPKKRAKVDVATHGRTYGT

[1378] LELFQKMILMHATYFLAAVLLGDHAEQVNTFLRLVFEIPLFSDAAVRHFR

[1379] QRATVFLVPRRHGKTWFLVPLIALSLASFRGIKIGYTAHIRKATEPVFEE

[1380] IDACLRGWFGSARVDHVKGETISFSFPDGRSTIVFASSHNTNGIRGQDF

[1381] NLLFVDEANFIRPDAVQTIMGFLNQ T NCKIIFVSSTNTGKASTSFLYNLR

[1382] GAADELLNVVTYICDDHMPRVVTHTNATACSCYILNKPVFITMDGAVRRT

[1383] ADLFLADSFMQEIIGGQARETGDDRPVLTKSAGERFLLYRPSTTTNSGLM

[1384] APDLYVYVDPAFTANTRASGTGVAVVGRYRDDYIIFALEHFFLRALTGSA

[1385] PADIARCVVHSLTQVLALHPGAFRGVRVAVEGNSSQDSAVAIATHVHTEM

[1386] HRLLASEGADAGSGPELLFYHCEPPGSAVLYPFFLLNKQKTPAFEHFIKK

[1387] FNSGGVMASQEIVSATVRLQTDPVEYLLEQLNNLTETVSPNTDVRTYSGK

[1388] RNGASDDLMVAVIMAIYLAAQAGPPHTFAPIIRVS

[1389] SEQ ID NO. 37: DNA sequence of the DNA packaging terminase subunit 1 of the 17TermA virus strain.

[1390] ATGTTTGGTCAGCAGCTGGCGTCCGACGTCCAGCAGTACCTGGAGCGCCT

[1391] CGAGAAACAGAGGCAACTTAAGGTGGGCGCGGACGAGGCGTCGGCGGGCC

[1392] TCACCATGGGCGGCGATGCCCTACGAGTGCCCTTTTTAGATTTCGCGACC

[1393] GCGACCCCCAAGCGCCACCAGACCGTGGTCCCTGGCGTCGGGACGCTCCA

[1394] CGACTGCTGCGAGCACTCGCCGCTCTTCTCGGCCGTGGCGCGGCGGCTGC

[1395] TGTTTAATAGCCTGGTGCCGGCGCAACTAAAGGGGCGTGATTTCGGGGGC

[1396] GACCACACGGCCAAGCTGGAATTCCTGGCCCCCGAGTTGGTACGGGCGGT

[1397] GGCGCGACTGCGGTTTAAGGAGTGCGCGCCGGCGGACGTGGTGCCTCAGC

[1398] GTAACGCCTACTATAGCGTTCTGAATACGTTTCAGGCCCTCCACCGCTCC

[1399] GAAGCCTTTCGCCAGCTGGTGCACTTTGTGCGGGACTTTGCCCAGCTGCT

[1400] CAAAACCTCCTTCCGGGCCTCCAGCCTCACGGAGACCACGGGCCCCCCCA

[1401] AAAAACGGGCCAAGGTGGACGTGGCCACCCACGGCCGGACGTACGGCACG

[1402] CTGGAGCTGTTCCAAAAAATGATCCTTATGCACGCCACCTACTTTCTGGC

[1403] CGCCGTGCTCCTCGGGGACCACGCGGAGCAGGTCAACACGTTCCTGCGTC

[1404] TCGTGTTTGAGATCCCCCTGTTTAGCGACGCGGCCGTGCGCCACTTCCGC

[1405] CAGCGCGCCACCGTGTTTCTCGTCCCCCGGCGCCACGGCAAGACCTGGTT

[1406] TCTGGTGCCCCTCATCGCGCTGTCGCTGGCCTCCTTTCGGGGGATCAAGA

[1407] TCGGCTACACGGCGCACATCCGCAAGGCGACCGAGCCGGTGTTTGAGGAG

[1408] ATCGACGCCTGCCTGCGGGGCTGGTTCGGTTCGGCCCGAGTGGACCACGT

[1409] TAAAGGGGAAACCATCTCCTTCTCGTTTCCGGACGGGTCGCGCAGTACCA

[1410] TCGTGTTTGCCTCCAGCCACAACACAAACGGAATCCGAGGCCAGGACTTT

[1411] AACCTGCTCTTTGTCGACGAGGCCAACTTTATTCGCCCGGATGCGGTCCA

[1412] GACGATTATGGGCTTTCTCAACCAGGCCAACTGCAAGATTATCTTCGTGT

[1413] CGTCCACCAACACCGGGAAGGCCAGTACGAGCTTTTTGTACAACCTCCGC

[1414] GGGGCCGCAGACGAGCTTCTCAACGTGGTGACCTATATATGCGATGATCA

[1415] CATGCCGAGGGTGGTGACGCACACAAACGCCACGGCCTGTTCTTGTTATA

[1416] TCCTCAACAAGCCCGTTTTCATCACGATGGACGGGGCGGTTCGCCGGACC

[1417] GCCGATTTGTTTCTGGCCGATTCCTTCATGCAGGAGATCATCGGGGGCCA

[1418] GGCCAGGGAGACCGGCGACGACCGGCCCGTTCTGACCAAGTCTGCGGGGG

[1419] AGCGGTTTCTGTTGTACCGCCCCTCGACCACCACCAACAGCGGCCTCATG

[1420] GCCCCCGATTTGTACGTGTACGTGGATCCCGCGTTCACGGCCAACACCCG

[1421] AGCCTCCGGGACCGGCGTCGCTGTCGTCGGGCGGTACCGCGACGATTATA

[1422] TCATCTTCGCCCTGGAGCACTTTTTTCTCCGCGCGCTCACGGGCTCGGCC

[1423] CCCGCCGACATCGCCCGCTGCGTCGTCCACAGTCTGACGCAGGTCCTGGC

[1424] CCTGCATCCCGGGGCGTTTCGCGGCGTCCGGGTGGCGGTCGAGGGAAATA

[1425] GCAGCCAGGACTCGGCCGTCGCCATCGCCACGCACGTGCACACAGAGATG

[1426] CACCGCCTACTGGCCTCGGAGGGGGCCGACGCGGGCTCGGGCCCCGAGCT

[1427] TCTCTTCTACCACTGCGAGCCTCCCGGGAGCGCGGTGCTGTACCCCTTTT

[1428] TCCTGCTCAACAAACAGAAGACGCCCGCCTTTGAACACTTTATTAAAAAG

[1429] TTTAACTCCGGGGGCGTCATGGCCTCCCAGGAGATCGTTTCCGCGACGGT

[1430] GCGCCTGCAGACCGACCCGGTCGAGTATCTGCTCGAGCAGCTAAATAACC

[1431] TCACCGAAACCGTCTCCCCCAACACTGACGTCCGTACGTATTCCGGAAAA

[1432] CGGAACGGCGCCTCGGATGACCTTATGGTCGCCGTCATTATGGCCATCTA

[1433] CCTCGCGGCCCAGGCCGGACCTCCGCACACATTCGCTCCTATCATACGCG

[1434] TCTCGTGA [[ID=3,2]]

[1435] SEQ ID NO. 38: Amino acid sequence of the DNA packaging terminase subunit 1 of the 17TermA virus strain.

[1436] MFGQQLASDVQQYLERLEKQRQLKVGADEASAGLTMGGDALRVPFLDFAT

[1437] ATPKRHQTVVPGVGTLHDCCEHSPLFSAVARRLLFNSLVPAQLKGRDFGG

[1438] DHTAKLEFLAPELVRAVARLRFKECAPADVVPQRNAYYSVLNTFQALHRS

[1439] EAFRQLVHFVRDFAQLLKTSFRASSLTETTGPPKKRAKVDVATHGRTYGT

[1440] LELFQKMILMHATYFLAAVLLGDHAEQVNTFLRLVFEIPLFSDAAVRHFR

[1441] QRATVFLVPRRHGKTWFLVPLIALSLASFRGIKIGYTAHIRKATEPVFEE

[1442] IDACLRGWFGSARVDHVKGETISFSFPDGSRSTIVFASSHNTNGIRGQDF

[1443] NLLFVDEANFIRPDAVQTIMGFLNQANCKIIIFVSSTNTGKASTSFLYNLR

[1444] GAADELLNVVTYICDDHMPRVVTHTNATACSCYILNKPVFITMDGAVRRT

[1445] ADLFLADSFMQEIIGGQARETGDDRPVLTKSAGERFLLYRPSTTTNSGLM

[1446] APDLYVYVDPAFTANTRASGTGVAVVGRYRDDYIIFALEHFFLRALTGSA

[1447] PADIARCVVHSLTQVLALHPPGAFRGVRVAVEGNSSQDSAVAIATHVHTEM

[1448] HRLLASEGADAGSGPELLFYHCEPPGSAVLYPFFLLNKQKTPAFEHFIKK

[1449] FNSGGVMASQEIVSATVRLQTDPVEYLLEQLNNLTETVSPNTDVRTYSGK

[1450] RNGASDDLMVAVIMAIYLAAQAGPPHTFAPIIRVS

[1451] SEQ ID NO. 39: DNA sequence of the DNA packaging terminase subunit 1 of the rRp450 virus strain.

[1452] ATGTTTGGTCAGCAGCTGGCGTCCGACGTCCAGCAGTACCTGGAGCGCCT

[1453] CGAGAAACAGAGGCAACTTAAGGTGGGCGCGGACGAGGCGTCGGCGGGCC

[1454] TCACAATGGGCGGCGATGCCCTACGAGTGCCCTTTTTAGATTTCGCGACC

[1455] GCGACCCCCAAGCGCCACCAGACCGTGGTCCCGGGCGTCGGGACGCTCCA

[1456] CGACTGCTGCGAGCACTCGCCGCTCTTCTCGGCCGTGGCGCGGCGGCTGC

[1457] TGTTTAATAGCCTGGTGCCGGCGCAACTAAAGGGGCGTGATTTCGGGGGC

[1458] GACCACACGGCCAAGCTGGAATTCCTGGCCCCCGAGTTGGTACGGGCGGT

[1459] GGCGCGACTGCGGTTTAAGGAGTGCGCGCCGGCGGACGTGGTGCCTCAGC

[1460] GTAACGCCTACTATAGCGTTCTGAACACGTTTCAGGCCCTCCACCGCTCC

[1461] GAAGCCTTTCGCCAGCTGGTGCACTTTGTGCGGGACTTTGCCCAGCTGCT

[1462] TAAAACCTCCTTCCGGGCCTCCAGCCTCACGGAGACCACGGGCCCCCCAA

[1463] AAAAACGGGCCAAGGTGGACGTGGCCACCCACGGCCGGACGTACGGCACG

[1464] CTGGAGCTGTTCCAAAAAATGATCCTTATGCACGCCACCTACTTTCTGGC

[1465] CGCCGTGCTCCTCGGGGACCACGCGGAGCAGGTCAACACGTTCCTGCGTC

[1466] TCGTGTTTGAGATCCCCCTGTTTAGCGACGCGGCCGTGCGCCACTTCCGC

[1467] CAGCGCGCCACCGTGTTTCTCGTCCCCCGGCGCCACGGCAAGACCTGGTT

[1468] TCTAGTGCCCCTCATCGCGCTGTCGCTGGCCTCCTTTCGGGGGATCAAGA

[1469] TCGGCTACACGGCGCACATCCGCAAGGCGACCGAGCCGGTGTTTGAGGAG

[1470] ATCGACGCCTGCCTGCGGGGCTGGTTCGGTTCGGCCCGAGTGGACCACGT

[1471] TAAAGGGGAAACCATCTCCTTCTCGTTTCCGGACGGGTCGCGCAGTACCA

[1472] TCGTGTTTGCCTCCAGCCACAACACAAACGGAATCCGAGGCCAGGACTTT

[1473] AACCTGCTCTTTGTCGACGAGGCCAACTTTATTCGCCCGGATGCGGTCCA

[1474] GACGATTATGGGCTTTCTCAACCAGGCCAACTGCAAGATTATCTTCGTGT

[1475] CGTCCACCAACACCGGGAAGGCCAGTACGAGCTTTTTGTACAACCTCCGC

[1476] GGGGCCGCCGACGAGCTTCTCAACGTGGTGACCTATATATGCGATGATCA

[1477] CATGCCGCGGGTGGTGACGCACACAAACGCCACGGCCTGTTCTTGTTATA

[1478] TCCTCAACAAGCCCGTTTTCATCACGATGGACGGGGCGGTTCGCCGGACC

[1479] GCCGATTTGTTTCTGGCCGATTCCTTCATGCAGGAGATCATCGGGGGCCA

[1480] GGCCAGGGAGACCGGCGACGACCGGCCCGTTCTGACCAAGTCTGCGGGGG

[1481] AGCGGTTTCTGTTGTACCGCCCCTCGACCACCACCAACAGCGGCCTCATG

[1482] GCCCCCGATTTGTACGTGTACGTGGATCCCGCGTTCACGGCCAACACCCG

[1483] AGCCTCCGGGACCGGCGTCGCTGTCGTCGGGCGGTACCGCGACGATTATA

[1484] TCATCTTCGCCCTGGAGCACTTTTTTCTCCGCGCGCTCACGGGCTCGGCC

[1485] CCCGCCGACATCGCCCGCTGCGTCGTCCACAGTCTGACGCAGGTCCTGGC

[1486] CCTGCATCCCGGGGCGTTTCGCGGCGTCCGGGTGGCGGTCGAGGGAAATA

[1487] GCAGCCAGGACTCGGCCGTCGCCATCGCCACGCACGTGCACACAGAGATG

[1488] CACCGCCTACTGGCCTCGGAGGGGGCCGACGCGGGCTCGGGCCCCGAGCT

[1489] TCTCTTCTACCACTGCGAGCCTCCCGGGAGCGCGGTGCTGTACCCCTTTT

[1490] TCCTGCTCAACAAACAGAAGACGCCCGCCTTTGAACACTTTATTAAAAAG

[1491] TTTAACTCCGGGGGCGTCATGGCCTCCCAGGAGATCGTTTCCGCGACGGT

[1492] GCGCCTGCAGACCGACCCGGTCGAGTATCTGCTCGAGCAGCTGAATAACC

[1493] TCACCGAAACCGTCTCCCCCAACACGGACGTCCGTACGTATTCCGGAAAA

[1494] CGGAACGGCGCCTCGGATGACCTTATGGTCGCCGTCATTATGGCCATCTA

[1495] CCTTGCGGCCCAGGCCGGACCTCCGCACACATTCGCTCCCATCACACGCG

[1496] TTTCGTGA

[1497] SEQ ID NO. 40: Amino acid sequence of the DNA packaging terminase subunit 1 of the rRp450 virus strain.

[1498] MFGQQLASDVQQYLERLEKQRQLKVGADEASAGLTMGGDALRVPFLDFAT

[1499] ATPKRHQTVVPGVGTLHDCCEHSPLFSAVARRLLFNSLVPAQLKGRDFGG

[1500] DHTAKLEFLAPELVRAVARLRFKECAPADVVPQRNAYYSVLNTFQALHRS

[1501] EAFRQLVHFVRDFAQLLKTSFRASSLTETTGPPKKRAKVDVATHGRTYGT

[1502] LELFQKMILMHATYFLAAVLLGDHAEQVNTFLRLVFEIPLFSDAAVRHFR

[1503] QRATVFLVPRRHGKTWFLVPLIALSLASFRGIKIGYTAHIRKATEPVFEE

[1504] IDACLRGWFGSARVDHVKGETISFSFPDGSRSTIVFASSHNTNGIRGQDF

[1505] NLLFVDEANFIRPDAVQTIMGFLNQANCKIIFVSSTNTGKASTSFLYNLR

[1509]

[1506] GAADELLNVVTYICDDHMPRVVTHTNATACSCYILNKPVFITMDGAVRRT

[1507] ADLFLADSFMQEIIGGQARETGDDRPVLTKSAGERFLLYRPSTTTNSGLM

[1508] APDLYVYVDPAFTANTRASGTGVAVVGRYRDDYIIFALEHFFLRALTGSA

[1509] PADIARCVVHSLTQVLALHPGAFRGVRVAVEGNSSQDSAVAIATHVHTEM

[1510] HRLLASEGADAGSGPELLFYHCEPPGSAVLYPFFLLNKQKTPAFEHFIKK<00031F2>

[1511] FNSGGVMASQEIVSATVRLQTDPVEYLLEQLNNLTETVSPNTDVRTYSGK <C

[1512] RNGASDDLMVAVIMAIYLAAQAGPPHTFAPITRVS

[1513] SEQ ID NO. 41: DNA sequence of the DNA packaging terminase subunit 1 of wild-type strain 17.

[1514] ATGTTTGGTCAGCAGCTGGCGTCCGACGTCCAGCAGTACCTGGAGCGCCT

[1515] CGAGAAACAGAGGCAACTTAAGGTGGGCGCGGACGAGGCGTCGGCGGGCC

[1516] TCACCATGGGCGGCGATGCCCTACGAGTGCCCTTTTTAGATTTCGCGACC

[1517] GCGACCCCCAAGCGCCACCAGACCGTGGTCCCTGGCGTCGGGACGCTCCA

[1518] CGACTGCTGCGAGCACTCGCCGCTCTTCTCGGCCGTGGCGCGGCGGCTGC

[1519] TGTTTAATAGCCTGGTGCCGGCGCAACTAAAGGGGCGTGATTTCGGGGGC

[1520] GACCACACGGCCAAGCTGGAATTCCTGGCCCCCGAGTTGGTACGGGCGGT

[1521] GGCGCGACTGCGGTTTAAGGAGTGCGCGCCGGCGGACGTGGTGCCTCAGC

[1522] GTAACGCCTACTATAGCGTTCTGAATACGTTTCAGGCCCTCCACCGCTCC

[1523] GAAGCCTTTCGCCAGCTGGTGCACTTTGTGCGGGACTTTGCCCAGCTGCT

[1524] CAAAACCTCCTTCCGGGCCTCCAGCCTCACGGAGACCACGGGCCCCCCCA

[1525] AAAAACGGGCCAAGGTGGACGTGGCCACCCACGGCCGGACGTACGGCACG

[1526] CTGGAGCTGTTCCAAAAAATGATCCTTATGCACGCCACCTACTTTCTGGC

[1527] CGCCGTGCTCCTCGGGGACCACGCGGAGCAGGTCAACACGTTCCTGCGTC

[1528] TCGTGTTTGAGATCCCCCTGTTTAGCGACGCGGCCGTGCGCCACTTCCGC

[1529] CAGCGCGCCACCGTGTTTCTCGTCCCCCGGCGCCACGGCAAGACCTGGTT

[1530] TCTGGTGCCCCTCATCGCGCTGTCGCTGGCCTCCTTTCGGGGGATCAAGA

[1531] TCGGCTACACGGCGCACATCCGCAAGGCGACCGAGCCGGTGTTTGAGGAG

[1532] ATCGACGCCTGCCTGCGGGGCTGGTTCGGTTCGGCCCGAGTGGACCACGT

[1533] TAAAGGGGAAACCATCTCCTTCTCGTTTCCGGACGGGTCGCGCAGTACCA

[1534] TCGTGTTTGCCTCCAGCCACAACACAAACGGAATCCGAGGCCAGGACTTT

[1535] AACCTGCTCTTTGTCGACGAGGCCAACTTTATTCGCCCGGATGCGGTCCA

[1536] GACGATTATGGGCTTTCTCAACCAGGCCAACTGCAAGATTATCTTCGTGT

[1537] CGTCCACCAACACCGGGAAGGCCAGTACGAGCTTTTTGTACAACCTCCGC

[1538] GGGGCCGCAGACGAGCTTCTCAACGTGGTGACCTATATATGCGATGATCA

[1539] CATGCCGAGGGTGGTGACGCACACAAACGCCACGGCCTGTTCTTGTTATA

[1540] TCCTCAACAAGCCCGTTTTCATCACGATGGACGGGGCGGTTCGCCGGACC

[1541] GCCGATTTGTTTCTGGCCGATTCCTTCATGCAGGAGATCATCGGGGGCCA

[1542] GGCCAGGGAGACCGGCGACGACCGGCCCGTTCTGACCAAGTCTGCGGGGG

[1543] AGCGGTTTCTGTTGTACCGCCCCTCGACCACCACCAACAGCGGCCTCATG

[1544] GCCCCCGATTTGTACGTGTACGTGGATCCCGCGTTCACGGCCAACACCCG

[1545] AGCCTCCGGGACCGGCGTCGCTGTCGTCGGGCGGTACCGCGACGATTATA

[1546] TCATCTTCGCCCTGGAGCACTTTTTTCTCCGCGCGCTCACGGGCTCGGCC

[1547] CCCGCCGACATCGCCCGCTGCGTCGTCCACAGTCTGACGCAGGTCCTGGC

[1548] CCTGCATCCCGGGGCGTTTCGCGGCGTCCGGGTGGCGGTCGAGGGAAATA

[1549] GCAGCCAGGACTCGGCCGTCGCCATCGCCACGCACGTGCACACAGAGATG

[1550] CACCGCCTACTGGCCTCGGAGGGGGCCGACGCGGGCTCGGGCCCCGAGCT

[1551] TCTCTTCTACCACTGCGAGCCTCCCGGGAGCGCGGTGCTGTACCCCTTTT

[1552] TCCTGCTCAACAAACAGAAGACGCCCGCCTTTGAACACTTTATTAAAAAG

[1553] TTTAACTCCGGGGGCGTCATGGCCTCCCAGGAGATCGTTTCCGCGACGGT

[1554] GCGCCTGCAGACCGACCCGGTCGAGTATCTGCTCGAGCAGCTAAATAACC

[1555] TCACCGAAACCGTCTCCCCCAACACTGACGTCCGTACGTATTCCGGAAAA

[1556] CGGAACGGCGCCTCGGATGACCTTATGGTCGCCGTCATTATGGCCATCTA

[1557] CCTCGCGGCCCAGGCCGGACCTCCGCACACATTCGCTCCTATCATACGCG <>

[1558] TCTCGTGA

[1559] SEQ ID NO. 42: Amino acid sequence of the DNA packaging terminase subunit 1 of wild-type strain 17.

[1560] MFGQQLASDVQQYLERLEKQRQLKVGADEASAGLTMGGDALRVPFLDFAT

[1561] ATPKRHQTVVPGVGTLHDCCEHSPLFSAVARRLLFNSLVPAQLKGRDFGG

[1562] DHTAKLEFLAPELVRAVARLRFKECAPADVVPQRNAYYSVLNTFQALHRS <>

[1563] EAFRQLVHFVRDFAQLLKTSFRASSLTETTGPPKKRAKVDVATHGRTYGT

[1564] LELFQKMILMHATYFLAAVLLGDHAEQVNTFLRLVFEIPLFSDAAVRHFR

[1565] QRATVFLVPRRHGKTWFLVPLIALSLASFRGIKIGYTAHIRKATEPVFEE

[1566] IDACLRGWFGSARVDHVKGETISFSFPDGSRSTIVFASSHNTNGIRGQDF

[1567] NLLFVDEANFIRPDAVQTIMGFLNQANCKIIFVSSTNTGKASTSFLYNLR

[1568] GAADELLNVVTYICDDHMPRVVTHTNATACSCYILNKPVFITMDGAVRRT

[1569] ADLFLADSFMQEIIGGQARETGDDRPVLTKSAGERFLLYRPSTTTNSGLM

[1570] APDLYVYVDPAFTANTRASGTGVAVVGRYRDDYIIFALEHFFLRALTGSA

[1571] PADIARCVVHSLTQVLALHPGAFRGVRVAVEGNSSQDSAVAIATHVHTEM

[1572] HRLLASEGADAGSGPELLFYHCEPPGSAVLYPFFLLNKQKTPAFEHFIKK

[1573] FNSGGVMASQEIVSATVRLQTDPVEYLLEQLNNLTETVSPNTDVRTYSGK<00032​​​​​​​​​​​​​​​​​​​​​​​​​​​​GACCGCCGTCGTGGCATTCGGGGGAACCCCACGTCGCTCGGCGGGGACGT

[1584] CTACCGGTACCCAGACGGCCGACGTCCCCACCGAGGCCCTTGGGGGCCCC

[1585] CCTCCTCCTCCCCGCTTCACCCTGGGTGGCGGCTGTTGTTCCTGTCGCGA

[1586] CACACGGCGCCGCTCTGCGGTATTCGGGGGGGAGGGGGATCCAGTCGGCC

[1587] CCGCGGAGTTCGTCTCGGACGACCGGTCGTCCGATTCCGACTCGGATGAC

[1588] TCGGAGGACACGGACTCGGAGACGCTGTCACACGCCTCCTCGGACGTGTC

[1589] CGGCGGGGCCACGTACGACGACGCCCTTGACTCCGATTCGTCATCGGATG

[1590] ACTCCCTGCAGATAGATGGCCCCGTGTGTCGCCCGTGGAGCAATGACACC

[1591] GCGCCCCTGGATGTTTGCCCCGGGACCCCCGGCCCGGGCGCCGACGCCGG

[1592] TGGTCCCTCAGCGGTAGACCCACACGCGCCGACGCCAGAGGCCGGCGCTG

[1593] GTCTTGCGGCCGATCCCGCCGTGGCCCGGGACGACGCGGAGGGGCTTTCG

[1594] GACCCCCGGCCACGTCTGGGAACGGGCACGGCCTACCCCGTCCCCCTGGA

[1595] ACTCACGCCCGAGAACGCGGAGGCCGTGGCGCGCTTTCTGGGAGATGCCG

[1596] TGAACCGCGAACCCGCGCTCATGCTGGAGTACTTTTGCCGGTGCGCCCGC

[1597] GAGGAAACCAAGCGTGTCCCCCCCAGGACATTCGGCAGCCCCCCTCGCCT

[1598] CACGGAGGACGACTTTGGGCTTCTCAACTACGCGCTCGTGGAGATGCAGC

[1599] GCCTGTGTCTGGACGTTCCTCCGGTCCCGCCGAACGCATACATGCCCTAT

[1600] TATCTCAGGGAGTATGTGACGCGGCTGGTCAACGGGTTCAAGCCGCTGGT

[1601] GAGCCGGTCCGCTCGCCTTTACCGCATCCTGGGGGTTCTGGTGCACCTGC

[1602] GGATCCGGACCCGGGAGGCCTCCTTTGAGGAGTGGCTGCGATCCAAGGAA

[1603] GTGGCCCTGGATTTTGGCCTGACGGAAAGGCTTCGCGAGCACGAAGCCCA

[1604] GCTGGTGATCCTGGCCCAGGCTCTGGACCATTACGACTGTCTGATCCACA

[1605] GCACACCGCACACGCTGGTCGAGCGGGGGCTGCAATCGGCCCTGAAGTAT

[1606] GAGGAGTTTTACCTAAAGCGTTTTGGCGGGCACTACATGGAGTCCGTCTT

[1607] CCAGATGTACACCCGCATCGCCGGCTTTTTGGCCTGCCGGGCCACGCGCG

[1608] GCATGCGCCACATCGCCCTGGGGCGAGAGGGGTCGTGGTGGGAAATGTTC

[1609] AAGTTCTTTTTCCACCGCCTCTACGACCACCAGATCGTACCGTCGACCCC

[1610] CGCCATGCTGAACCTGGGGACCCGCAACTACTACACCTCCAGCTGCTACC

[1611] TGGTAAACCCCCAGGCCACCACAAACAAGGCGACCCTGCGGGCCATCACC

[1612] AGCAACGTCAGTGCCATCCTCGCCCGCAACGGGGGCATCGGGCTATGCGT

[1613] GCAGGCGTTTAACGACTCCGGCCCCGGGACCGCCAGCGTCATGCCCGCCC

[1614] TCAAGGTCCTTGACTCGCTGGTGGCGGCGCACAACAAAGAGAGCGCGCGT

[1615] CCGACCGGCGCGTGCGTGTACCTGGAGCCGTGGCACACCGACGTGCGGGC

[1616] CGTGCTCCGGATGAAGGGGGTCCTCGCCGGCGAAGAGGCCCAGCGCTGCG

[1617] ACAATATCTTCAGCGCCCTCTGGATGCCAGACCTGTTTTTCAAGCGCCTG

[1618] ATTCGCCACCTGGACGGCGAGAAGAACGTCACATGGACCCTGTTCGACCG

[1619] GGACACCAGCATGTCGCTCGCCGACTTTCACGGGGAGGAGTTCGAGAAGC

[1620] TCTACCAGCACCTCGAGGTCATGGGGTTCGGCGAGCAGATACCCATCCAG

[1621] GAGCTGGCCTATGGCATTGTGCGCAGTGCGGCCACGACCGGGAGCCCCTT

[1622] CGTCATGTTCAAAGACGCGGTGAACCGCCACTACATCTACGACACCCAGG

[1623] GGGCGGCCATCGCCGGCTCCAACCTCTGCACCGAGATCGTCCATCCGGCC

[1624] TCCAAGCGATCCAGTGGGGTCTGCAACCTGGGAAGCGTGAATCTGGCCCG

[1625] ATGCGTCTCCAGGCAGACGTTTGACTTTGGGCGGCTCCGCGACGCCGTGC

[1626] AGGCGTGCGTGCTGATGGTGAACATCATGATCGACAGCACGCTACAACCC

[1627] ACGCCCCAGTGCACCCGCGGCAACGACAACCTGCGGTCCATGGGAATCGG

[1628] CATGCAGGGCCTGCACACGGCCTGCCTGAAGCTGGGGCTGGATCTGGAGT

[1629] CTGTCGAATTTCAGGACCTGAACAAACACATCGCCGAGGTGATGCTGCTG

[1630] TCGGCGATGAAGACCAGCAACGCGCTGTGCGTTCGCGGGGCCCGTCCCTT

[1631] CAACCACTTTAAGCGCAGCATGTATCGCGCCGGCCGCTTTCACTGGGAGC

[1632] GCTTTCCGGACGCCCGGCCGCGGTACGAGGGCGAGTGGGAGATGCTACGC

[1633] CAGAGCATGATGAAACACGGCCTGCGCAACAGCCAGTTTGTCGCGCTGAT

[1634] GCCCACCGCCGCCTCGGCGCAGATCTCGGACGTCAGCGAGGGCTTTGCCC

[1635] CCCTGTTCACCAACCTGTTCAGCAAGGTGACCCGGGACGGCGAGACGCTG

[1636] CGCCCCAACACGCTCCTGCTAAAGGAACTGGAACGCACGTTTAGCGGGAA

[1637] GCGCCTCCTGGAGGTGATGGACAGTCTCGACGCCAAGCAGTGGTCCGTGG

[1638] CGCAGGCGCTCCCGTGCCTGGAGCCCACCCACCCCCTCCGGCGATTCAAG

[1639] ACCGCGTTTGACTACGACCAGAAGTTGCTGATCGACCTGTGTGCGGACCG

[1640] CGCCCCCTACGTCGACCATAGCCAATCCATGACCCTGTATGTCACGGAGA

[1641] AGGCGGACGGGACCCTCCCAGCCTCCACCCTGGTCCGCCTTCTGGTCCAC

[1642] GCATATAAGCGCGGACTAAAAACAGGGATGTACTACTGCAAGGTTCGCAA

[1643] GGCGACCAACAGCGGGGTCTTTGGCGGCGACGACAACATTGTCTGCACGA

[1644] GCTGCGCGCTGTGA

[1645] SEQ ID NO. 44: Amino acid sequence of ICP6 in Mut 3 and Mut-3∆34.5 virus strains.

[1646] MASRPAASSPVEARAPVGGQEAGGPSAATQGEAAGAPLAHGHHVYCQRVN

[1647] GVMVLSDKTPGSASYRISDSNFVQCGSNCTMIIDGDVVRGRPQDPGAAAS

[1648] PAPFVAVTNIGAGSDGGTAVVAFGGTPRRSAGTSTGTQTADVPTEALGGP

[1649] PPPPRFTLGGGCCSCRDTRRRSAVFGGEGDPVGPAEFVSDDRSSDSDSDD

[1650] SEDTDSETLSHASSDVSGGATYDDALDSDSSSDDSLQIDGPVCRPWSNDT

[1651] APLDVCPGTPGPGADAGGPSAVDPHAPTPEAGAGLAADPAVARDDAEGLS

[1652] DPRPRLGTGTAYPVPLELTPENAEAVARFLGDAVNREPALMLEYFCRCAR

[1653] EETKRVPPRTFGSPPRLTEDDFGLLNYALVEMQRLCLDVPPVPPNAYMPY

[1654] YLREYVTRLVNGFKPLVSRSARLYRILGVLVHLRIRTREASFEEWLRSKE

[1655] VALDFGLTERLREHEAQLVILAQALDHYDCLIHSTPHTLVERGLQSALKY

[1656] EEFYLKRFGGHYMESVFQMYTRIAGFLACRATRGMRHIALGREGSWWEMF

[1657] KFFFHRLYDHQIVPSTPAMLNLGTRNYYTSSCYLVNPQATTNKATLRAIT

[1658] SNVSAILARNGGIGLCVQAFNDSGPGTASVMPALKVLDSLVAAHNKESAR

[1659] PTGACVYLEPWHTDVRAVLRMKGVLAGEEAQRCDNIFSALWMPDLFFKRL

[1660] IRHLDGEKNVTWTLFDRDTSMSLADFHGEEFEKLYQHLEVMGFGEQIPIQ

[1661] ELAYGIVRSAATTGSPFVMFKDAVNRHYIYDTQGAAIAGSNLCTEIVHPA

[1662] SKRSSGVCNLGSVNLARCVSRQTFDFGRLRDAVQACVLMVNIMIDSTLQP

[1663] TPQCTRGNDNLRSMGIGMQGLHTACLKLGLDLESVEFQDLNKHIAEVMLL<0​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ACGGAGACGTGGTGCGCGGGCGCCCCCAGGACCCGGGGGCCGCGGCATCC

[1676] CCCGCTCCCTTCGTTGCGGTGACAAACATCGGAGCCGGCAGCGACGGCGG

[1677] GACCGCCGTCGTGGCATTCGGGGGAACCCCACGTCGCTCGGCGGGGACGT

[1678] CTACCGGTACCCAGACGGCCGACGTCCCCACCGAGGCCCTTGGGGGCCCC

[1679] CCTCCTCCTCCCCGCTTCACCCTGGGTGGCGGCTGTTGTTCCTGTCGCGA

[1680] CACACGGCGCCGCTCTGCGGTATTCGGGGGGGAGGGGGATCCAGTCGGCC

[1681] CCGCGGAGTTCGTCTCGGACGACCGGTCGTCCGATTCCGACTCGGATGAC

[1682] TCGGAGGACACGGACTCGGAGACGCTGTCACACGCCTCCTCGGACGTGTC

[1683] CGGCGGGGCCACGTACGACGACGCCCTTGACTCCGATTCGTCATCGGATG

[1684] ACTCCCTGCAGATAGATGGCCCCGTGTGTCGCCCGTGGAGCAATGACACC

[1685] GCGCCCCTGGATGTTTGCCCCGGGACCCCCGGCCCGGGCGCCGACGCCGG

[1686] TGGTCCCTCAGCGGTAGACCCACACGCGCCGACGCCAGAGGCCGGCGCTG

[1687] GTCTTGCGGCCGATCCCGCCGTGGCCCGGGACGACGCGGAGGGGCTTTCG

[1688] GACCCCCGGCCACGTCTGGGAACGGGCACGGCCTACCCCGTCCCCCTGGA

[1689] ACTCACGCCCGAGAACGCGGAGGCCGTGGCGCGCTTTCTGGGAGATGCCG

[1690] TGAACCGCGAACCCGCGCTCATGCTGGAGTACTTTTGCCGGTGCGCCCGC

[1691] GAGGAAACCAAGCGTGTCCCCCCCAGGACATTCGGCAGCCCCCCTCGCCT

[1692] CACGGAGGACGACTTTGGGCTTCTCAACTACGCGCTCGTGGAGATGCAGC

[1693] GCCTGTGTCTGGACGTTCCTCCGGTCCCGCCGAACGCATACATGCCCTAT

[1694] TATCTCAGGGAGTATGTGACGCGGCTGGTCAACGGGTTCAAGCCGCTGGT

[1695] GAGCCGGTCCGCTCGCCTTTACCGCATCCTGGGGGTTCTGGTGCACCTGC

[1696] GGATCCGGACCCGGGAGGCCTCCTTTGAGGAGTGGCTGCGATCCAAGGAA

[1697] GTGGCCCTGGATTTTGGCCTGACGGAAAGGCTTCGCGAGCACGAAGCCCA

[1698] GCTGGTGATCCTGGCCCAGGCTCTGGACCATTACGACTGTCTGATCCACA

[1699] GCACACCGCACACGCTGGTCGAGCGGGGGCTGCAATCGGCCCTGAAGTAT

[1700] GAGGAGTTTTACCTAAAGCGTTTTGGCGGGCACTACATGGAGTCCGTCTT

[1701] CCAGATGTACACCCGCATCGCCGGCTTTTTGGCCTGCCGGGCCACGCGCG

[1702] GCATGCGCCACATCGCCCTGGGGCGAGAGGGGTCGTGGTGGGAAATGTTC

[1703] AAGTTCTTTTTCCACCGCCTCTACGACCACCAGATCGTACCGTCGACCCC

[1704] CGCCATGCTGAACCTGGGGACCCGCAACTACTACACCTCCAGCTGCTACC

[1705] TGGTAAACCCCCAGGCCACCACAAACAAGGCGACCCTGCGGGCCATCACC

[1706] AGCAACGTCAGTGCCATCCTCGCCCGCAACGGGGGCATCGGGCTATGCGT

[1707] GCAGGCGTTTAACGACTCCGGCCCCGGGACCGCCAGCGTCATGCCCGCCC

[1708] TCAAGGTCCTTGACTCGCTGGTGGCGGCGCACAACAAAGAGAGCGCGCGT

[1709] CCGACCGGCGCGTGCGTGTACCTGGAGCCGTGGCACACCGACGTGCGGGC

[1710] CGTGCTCCGGATGAAGGGGGTCCTCGCCGGCGAAGAGGCCCAGCGCTGCG

[1711] ACAATATCTTCAGCGCCCTCTGGATGCCAGACCTGTTTTTCAAGCGCCTG

[1712] ATTCGCCACCTGGACGGCGAGAAGAACGTCACATGGACCCTGTTCGACCG

[1713] GGACACCAGCATGTCGCTCGCCGACTTTCACGGGGAGGAGTTCGAGAAGC

[1714] TCTACCAGCACCTCGAGGTCATGGGGTTCGGCGAGCAGATACCCATCCAG

[1715] GAGCTGGCCTATGGCATTGTGCGCAGTGCGGCCACGACCGGGAGCCCCTT

[1716] CGTCATGTTCAAAGACGCGGTGAACCGCCACTACATCTACGACACCCAGG

[1717] GGGCGGCCATCGCCGGCTCCAACCTCTGCACCGAGATCGTCCATCCGGCC

[1718] TCCAAGCGATCCAGTGGGGTCTGCAACCTGGGAAGCGTGAATCTGGCCCG

[1719] ATGCGTCTCCAGGCAGACGTTTGACTTTGGGCGGCTCCGCGACGCCGTGC

[1720] AGGCGTGCGTGCTGATGGTGAACATCATGATCGACAGCACGCTACAACCC

[1721] ACGCCCCAGTGCACCCGCGGCAACGACAACCTGCGGTCCATGGGAATCGG

[1722] CATGCAGGGCCTGCACACGGCCTGCCTGAAGCTGGGGCTGGATCTGGAGT

[1723] CTGCCGAATTTCAGGACCTGAACAAACACATCGCCGAGGTGATGCTGCTG

[1724] TCGGCGATGAAGACCAGCAACGCGCTGTGCGTTCGCGGGGCCCGTCCCTT

[1725] CAACCACTTTAAGCGCAGCATGTATCGCGCCGGCCGCTTTCACTGGGAGC

[1726] GCTTTCCGGACGCCCGGCCGCGGTACGAGGGCGAGTGGGAGATGCTACGC

[1727] CAGAGCATGATGAAACACGGCCTGCGCAACAGCCAGTTTGTCGCGCTGAT

[1728] GCCCACCGCCGCCTCGGCGCAGATCTCGGACGTCAGCGAGGGCTTTGCCC

[1729] CCCTGTTCACCAACCTGTTCAGCAAGGTGACCCGGGACGGCGAGACGCTG

[1730] CGCCCCAACACGCTCCTGCTAAAGGAACTGGAACGCACGTTTAGCGGGAA

[1731] GCGCCTCCTGGAGGTGATGGACAGTCTCGACGCCAAGCAGTGGTCCGTGG

[1732] CGCAGGCGCTCCCGTGCCTGGAGCCCACCCACCCCCTCCGGCGATTCAAG

[1733] ACCGCGTTTGACTACGACCAGAAGTTGCTGATCGACCTGTGTGCGGACCG

[1734] CGCCCCCTACGTCGACCATAGCCAATCCATGACCCTGTATGTCACGGAGA

[1735] AGGCGGACGGGACCCTCCCAGCCTCCACCCTGGTCCGCCTTCTGGTCCAC

[1736] GCATATAAGCGCGGACTAAAAACAGGGATGTACTACTGCAAGGTTCGCAA

[1737] GGCGACCAACAGCGGGGTCTTTGGCGGCGACGACAACATTGTCTGCATGA

[1738] GCTGCGCGCTGTGA

[1739] SEQ ID NO. 46: Amino acid sequence of ICP6 in strain 17TermA.

[1740] MASRPAASSPVEARAPVGGQEAGGPSAATQGEAAGAPLAHGHHVYCQRVN

[1741] GVMVLSDKTPGSASYRISDSNFVQCGSNCTMIIDGDVVRGRPQDPGAAAS

[1742] PAPFVAVTNIGAGSDDGGTAVVAFGGTPRRSAGTSTGTQTADVPTEALGGP

[1743] PPPPRFTLGGGCSCRDTRRRSAVFGGEGDPVGPAEFVSDDRSSDSDSDD

[1744] SEDTDSETLSHASSDVSGGATYDDALDSDSSSDDSLQIDGPVCRPWSNDT

[1745] APLDVCPGTPGPGADAGGPSAVDPHAPTPEAGLAADPAVARDDAEGLS

[1746] DPRPRLGTGTAYPVPLELTPENAEAVARFLGDAVNREPALMLEYFCRCAR

[1747] EETKRVPPRTFGSPPRLTEDDFGLLNYALVEMQRLCLDVPPPPPNAYMPY

[1748] YLREYVTRLVNGFKPLVSRSARLYRILGVLVHLRIRTREASFEEWLRSKE

[1749] VALDFGLTERLREHEAQLVILAQALDHYDCLIHSTPTHLVERGLQSALKY

[1750] EEFYLKRFGGHYMESVFQMYTRIAGFLACRATRGMRHIALGREGSWWEMF

[1751] KFFFHRLYDHQIVPSTPAMLNLGTRNYYTSSCYLVNPQATTNKATLRAIT

[1752] SNVSAILARNGGIGLCVQAFNDSGPGTASVMPALKVLDSLVAAHNKESAR

[1753] PTGACVYLEPWHTDVRAVLRMKGVLAGEEAQRCDNIFSALWMPDLFFKRL

[1754] IRHLDGEKNVTWTLFDRDTSMSLADFHGEEFEKLYQHLEVMGFGEQIPIQ

[1755] ELAYGIVRSAATTGSPFVMFKDAVNRHYIYDTQGAAIAGSNLCTEIVHPA

[1756] SKRSSGVCNLGSVNLARCVSRQTFDFGRLRDAVQACVLMVNIMIDSTLQP

[1757] TPQCTRGNDNLRSMGIGMQGLHTACLKLGLDLESAEFQDLNKHIAEVMLL

[1758] SAMKTSNALCVRGARPFNHFKRSMYRAGRFHWERFPDARPRYEGEWEMLR

[1759] QSMMKHGLRNSQFVALMPTAASAQISDVSEGFAPLFTNLFSKVTRDGETL

[1760] RPNTLLLKELERTFSGKRLLEVMDSLDAKQWSVAQALPCLEPTHPLRRFK

[1761] TAFDYDQKLLIDLCADRAPYVDHSQSMTLYVTEKADGTLPASTLVRLLVH

[1762] AYKRGLKTGMYYCKVRKATNSGVFGGDDNIVCMSCAL

[1763] SEQ ID NO. 47: DNA sequence of rat cytochrome P450 2B1 in strain rRp450.

[1764] GAACCCCTTCGCC ATG GAGCCCAGTATCTTGCTCCTCCTTGCTCTCCTTG

[1765] TGGGCTTCTTGTTACTCTTAGTCAGGGGACACCCAAAGTCCCGTGGCAAC

[1766] TTCCCACCAGGACCTCGTCCCCTTCCCCTCTTGGGGAACCTCCTGCAGTT

[1767] GGACAGAGGGGGCCTCCTCAATTCCTTCATGCAGCTTCGAGAAAAATATG

[1768] GAGATGTGTTCACAGTACACCTGGGACCAAGGCCTGTGGTCATGCTATGT

[1769] GGGACAGACACCATAAAGGAGGCTCTGGTGGGCCAAGCTGAGGATTTCTC

[1770] TGGTCGGGGAACAATCGCTGTGATTGAGCCAATCTTCAAGGAATATGGTG

[1771] TGATCTTTGCCAATGGGGAACGCTGGAAGGCCCTTCGGCGATTCTCTCTG

[1772] GCTACCATGAGAGACTTTGGGATGGGAAAGAGGAGTGTGGAAGAACGGAT

[1773] TCAGGAGGAAGCCCAATGTTTGGTGGAGGAACTGCGGAAATCCCAGGGAG

[1774] CCCCACTGGATCCCACCTTCCTCTTCCAGTGCATCACAGCCAACATCATC

[1775] TGCTCCATTGTGTTTGGAGAGCGCTTTGACTACACAGACCGCCAGTTCCT

[1776] GCGCCTGTTGGAGCTGTTCTACCGGACCTTTTCCCTCCTAAGTTCATTCT

[1777] CCAGCCAGGTGTTTGAGTTCTTCTCTGGGTTCCTGAAATACTTTCCTGGT

[1778] GCCCACAGACAAATCTCCAAAAACCTCCAGGAAATCCTCGATTACATTGG

[1779] CCATATTGTGGAGAAGCACAGGGCCACCTTAGACCCAAGCGCTCCACGAG

[1780] ACTTCATCGACACTTACCTTCTGCGCATGGAGAAGGAGAAGTCGAACCAC

[1781] CACACAGAGTTCCATCATGAGAACCTCATGATCTCCCTGCTCTCTCTCTT

[1782] CTTTGCTGGCACTGAGACCAGCAGCACCACACTCCGCTATGGTTTCCTGC

[1783] TGATGCTCAAGTACCCCCATGTCGCAGAGAAAGTCCAAAAGGAGATTGAT

[1784] CAGGTGATCGGCTCACACCGGCTACCAACCCTTGATGACCGCAGTAAAAT

[1785] GCCATACACTGATGCAGTTATCCATGAGATTCAGAGGTTTTCAGATCTTG

[1786] TCCCTATTGGAGTACCACACAGAGTCACCAAAGACACCATGTTCCGAGGG

[1787] TACCTGCTTCCCAAGAACACTGAAGTGTACCCCATCCTGAGTTCAGCTCT

[1788] CCATGACCCACAGTACTTTGACCACCCAGACAGCTTCAATCCTGAACACT

[1789] TCCTGGATGCCAATGGGGCACTGAAAAAGAGTGAAGCTTTCATGCCCTTC

[1790] TCCACAGGAAAGCGCATTTGTCTTGGCGAAGGCATTGCCCGAAATGAATT

[1791] GTTCCTCTTCTTCACCACCATCCTCCAGAACTTCTCTGTGTCAAGCCATT

[1792] TGGCTCCCAAGGACATTGACCTCACGCCCAAGGAGAGTGGCATTGGAAAA

[1793] ATACCTCCAACGTACCAGATCTGCTTCTCAGCTCGGTGATCCGGCTGAGG

[1794] CAGCCATGTGCCCCAGTTCTGTTGGGAATGGAACTTGTTTATTGCAGCTT

[1795] ATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCA

[1796] TTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATC

[1797] TTATCATGTCTGGATCCCCGGGCGAGCTCGAATTCCTCCTTTGAGGAGTG

[1798] GCTGCGATCCAAGGAAGTGGCCCTGGACTTTGGCCTGACGGAAAGGCTTC

[1799] GCGAGCACGAAGCCCAGCTGGTGATCCTGGCCCAGGCTCTGGACCATTAC

[1800] GACTGTCTGATCCACAGCACACCGCACACGCTGGTCGAGCGGGGGCTGCA

[1801] ATCGGCCCTGAAGTATGAGGAGTTTTACCTAAAGCGCTTTGGCGGGCACT

[1802] ACATGGAGTCCGTCTTCCAGATGTACACCCGCATCGCCGGCTTTTTGGCC

[1803] TGCCGGGCCACGCGCGGCATGCGCCACATCGCCCTGGGGCGAGAGGGGTC

[1804] GTGGTGGGAAATGTTCAAGTTCTTTTTCCACCGCCTCTACGACCACCAGA

[1805] TCGTACCGTCGACCCCCGCCATGCTGAACCTGGGGACCCGCAACTACTAC

[1806] ACCTCCAGCTGCTACCTGGTAAACCCCCAGGCCACCACAAACAAGGCGAC

[1807] CCTGCGGGCCATCACCAGCAACGTCAGCGCCATCCTCGCCCGCAACGGGG

[1808] GCATCGGGCTATGCGTGCAGGCGTTTAACGACTCCGGCCCCGGGACCGCT

[1809] AGCGTCATACCCGCCCTCAAGGTCCTCGACTCGCTGGTGGCGGCGCACAA

[1810] CAAAGAGAGCGCGCGTCCAACCGGCGCGTGCGTGTACCTGGAGCCGTGGC

[1811] ACACCGACGTGCGGGCCGTGCTCCGGATGAAGGGGGTCCTCGCCGGCGAA

[1812] GAGGCCCAGCGCTGCGACAATATCTTCAGCGCCCTCTGGATGCCAGACCT

[1813] GTTTTTCAAGCGCCTGATTCGCCACCTGGACGGCGAGAAGAACGTCACAT

[1814] GGACCCTGTTCGACCGGGACACCAGCATGTCGCTCGCCGACTTTCACGGG

[1815] GAGGAGTTCGAGAAGCTCTACCAGCACCTCGAGGTCATGGGGTTCGGCGA

[1816] GCAGATACCCATCCAGGAGCTGGCCTATGGCATTGTGCGCAGTGCGGCCA

[1817] CGACCGGGAGCCCCTTCGTCATGTTCAAAGACGCGGTGAACCGCCACTAC

[1818] ATCTACGACACCCAGGGGGCGGCCATCGCCGGCTCCAACCTCTGCACCGA

[1819] GATCGTCCATCCGGCCTCCAAGCGATCCAGTGGGGTCTGCAATCTGGGAA

[1820] GCGTGAATCTGGCCCGATGCGTCTCCAGGCAGACGTTTGACTTTGGGCGG

[1821] CTCCGCGACGCCGTGCAGGCGTGCGTGCTGATGGTGAACATCATGATCGA

[1822] CAGCACGCTACAACCCACGCCCCAGTGCACCCGCGGCAACGACAACCTGC

[1823] GGTCCATGGGAATCGGCATGCAGGGCCTGCACACGGCCTGCCTGAAGCTG

[1824] GGGCTGGATCTGGAGTCTGTCGAATTTCAGGACCTGAACAAACACATCGC

[1825] CGAGGTGATGCTGCTGTCGGCGATGAAGACCAGCAACGCGCTGTGCGTTC

[1826] GCGGGGCCCGTCCCTTCAACCACTTTAAGCGCAGCATGTATCGCGCCGGC

[1827] CGCTTTCACTGGGAGCGCTTTCCGGACGCCCGGCCGCGGTACGAGGGCGA

[1828] GTGGGAGATGCTACGCCAGAGCATGATGAAACACGGCCTGCGCAACAGCC

[1829] AGTTTGTCGCGCTGATGCCCACCGCCGCCTCGGCGCAGATCTCGGACGTC

[1830] AGCGAGGGCTTTGCCCCCCTGTTCACCAACCTGTTCAGCAAGGTGACCCG

[1831] GGACGGCGAGACGCTGCGCCCCAACACGCTCCTGCTAAAGGAACTGGAAC

[1832] GCACGTTTAGCGGGAAGCGCCTCCTGGAGGTGATGGACAGTCTCGACGCC

[1833] AAGCAGTGGTCCGTGGCGCAGGCGCTCCCGTGCCTGGAGCCCACCCACCC

[1834] CCTCCGGCGATTCAAGACCGCGTTTGACTACGACCAGAAGTTGCTGATCG

[1835] ACCTGTGTGCGGACCGCGCCCCCTACGTCGACCATAGCCAATCCATGACC

[1836] CTGTATGTCACGGAGAAGGCGGACGGGACCCTCCCAGCCTCCACCCTGGT <00-03786>CCGCCTTCTGGTCCACGCATATAAGCGCGGACTAAAAACAGGGATGTACT

[1838] ACTGCAAGGTTCGCAAGGCGACCAACAGCGGGGTCTTTGGCGGCGACGAC

[1839] AACATTGTCTGCACGAGCTGCGCGCTGTGA

[1840] SEQ ID NO. 48: Amino acid sequence of rat cytochrome P450 2B1 in strain rRp450.

[1841] MEPSILLLLALLVGFLLLLVRGHPKSRGNFPPGPRPLPLLGNLLQL

[1842] DRGGLLNSFMQLREKYGDVFTVHLGPRPVVMLCGTDTIKEALVGQAEDFS

[1843] GRGTIAVIEPIFKEYGVIFANGERWKALRRFSLATMRDFGMGKRSVEERI

[1844] QEEAQCLVEELRKSQGAPLDPTFLFQCITANIICSIVFGERFDYTDRQFL

[1845] RLLELFYRTFSLLSSFSSQVFEFFSGFLKYFPGAHRQISKNLQEILDYIG

[1846] HIVEKHRATLDPSAPRDFIDTYLLRMEKEKSNHHTEFHHENLMISLLSLF

[1847] FAGTETSSTTLRYGFLLMLKYPHVAEKVQKEIDQVIGSHRLPTLDDRSKM

[1848] PYTDAVIHEIQRFSDLVPIGVPHRVTKDTMFRGYLLPKNTEVYPILSSAL

[1849] HDPQYFDHPDSFNPEHFLDANGALKKSEAFMPFSTGKRICLGEGIARNEL

[1850] FLFFTTILQNFSVSSHLAPKDIDLTPKESGIGKIPPTYQICFSAR*

[1851] *The sequence after *stop codon - prediction* is not expressed

[1852] SEQ ID NO. 49: DNA sequence of ICP6 in wild-type strain 17.

[1853] ATGGCCAGCCGCCCAGCCGCATCCTCTCCCGTCGAAGCGCGGGCCCCGGT

[1854] TGGGGGACAGGAGGCCGGCGGCCCCAGCGCAGCCACCCAGGGGGAGGCCG

[1855] CCGGGGCCCCTCTCGCCCACGGCCACCACGTGTACTGCCAGCGAGTCAAT

[1856] GGCGTGATGGTGCTTTCCGACAAGACGCCCGGGTCCGCGTCCTACCGCAT

[1857] CAGCGATAGCAACTTTGTCCAATGTGGTTCCAACTGCACCATGATCATCG

[1858] ACGGAGACGTGGTGCGCGGGCGCCCCCAGGACCCGGGGGCCGCGGCATCC

[1859] CCCGCTCCCTTCGTTGCGGTGACAAACATCGGAGCCGGCAGCGACGGCGG

[1860] GACCGCCGTCGTGGCATTCGGGGGAACCCCACGTCGCTCGGCGGGGACGT

[1861] CTACCGGTACCCAGACGGCCGACGTCCCCACCGAGGCCCTTGGGGGCCCC

[1862] CCTCCTCCTCCCCGCTTCACCCTGGGTGGCGGCTGTTGTTCCTGTCGCGA

[1863] CACACGGCGCCGCTCTGCGGTATTCGGGGGGGAGGGGGATCCAGTCGGCC

[1864] CCGCGGAGTTCGTCTCGGACGACCGGTCGTCCGATTCCGACTCGGATGAC

[1865] TCGGAGGACACGGACTCGGAGACGCTGTCACACGCCTCCTCGGACGTGTC

[1866] CGGCGGGGCCACGTACGACGACGCCCTTGACTCCGATTCGTCATCGGATG

[1867] ACTCCCTGCAGATAGATGGCCCCGTGTGTCGCCCGTGGAGCAATGACACC

[1868] GCGCCCCTGGATGTTTGCCCCGGGACCCCCGGCCCGGGCGCCGACGCCGG

[1869] TGGTCCCTCAGCGGTAGACCCACACGCGCCGACGCCAGAGGCCGGCGCTG

[1870] GTCTTGCGGCCGATCCCGCCGTGGCCCGGGACGACGCGGAGGGGCTTTCG

[1871] GACCCCCGGCCACGTCTGGGAACGGGCACGGCCTACCCCGTCCCCCTGGA

[1872] ACTCACGCCCGAGAACGCGGAGGCCGTGGCGCGCTTTCTGGGAGATGCCG

[1873] TGAACCGCGAACCCGCGCTCATGCTGGAGTACTTTTGCCGGTGCGCCCGC

[1874] GAGGAAACCAAGCGTGTCCCCCCCAGGACATTCGGCAGCCCCCCTCGCCT

[1875] CACGGAGGACGACTTTGGGCTTCTCAACTACGCGCTCGTGGAGATGCAGC

[1876] GCCTGTGTCTGGACGTTCCTCCGGTCCCGCCGAACGCATACATGCCCTAT

[1877] TATCTCAGGGAGTATGTGACGCGGCTGGTCAACGGGTTCAAGCCGCTGGT

[1878] GAGCCGGTCCGCTCGCCTTTACCGCATCCTGGGGGTTCTGGTGCACCTGC

[1879] GGATCCGGACCCGGGAGGCCTCCTTTGAGGAGTGGCTGCGATCCAAGGAA

[1880] GTGGCCCTGGATTTTGGCCTGACGGAAAGGCTTCGCGAGCACGAAGCCCA

[1881] GCTGGTGATCCTGGCCCAGGCTCTGGACCATTACGACTGTCTGATCCACA

[1882] GCACACCGCACACGCTGGTCGAGCGGGGGCTGCAATCGGCCCTGAAGTAT

[1883] GAGGAGTTTTACCTAAAGCGTTTTGGCGGGCACTACATGGAGTCCGTCTT

[1884] CCAGATGTACACCCGCATCGCCGGCTTTTTGGCCTGCCGGGCCACGCGCG

[1885] GCATGCGCCACATCGCCCTGGGGCGAGAGGGGTCGTGGTGGGAAATGTTC

[1886] AAGTTCTTTTTCCACCGCCTCTACGACCACCAGATCGTACCGTCGACCCC

[1887] CGCCATGCTGAACCTGGGGACCCGCAACTACTACACCTCCAGCTGCTACC

[1888] TGGTAAACCCCCAGGCCACCACAAACAAGGCGACCCTGCGGGCCATCACC

[1889] AGCAACGTCAGTGCCATCCTCGCCCGCAACGGGGGCATCGGGCTATGCGT

[1890] GCAGGCGTTTAACGACTCCGGCCCCGGGACCGCCAGCGTCATGCCCGCCC

[1891] TCAAGGTCCTTGACTCGCTGGTGGCGGCGCACAACAAAGAGAGCGCGCGT

[1892] CCGACCGGCGCGTGCGTGTACCTGGAGCCGTGGCACACCGACGTGCGGGC

[1893] CGTGCTCCGGATGAAGGGGGTCCTCGCCGGCGAAGAGGCCCAGCGCTGCG

[1894] ACAATATCTTCAGCGCCCTCTGGATGCCAGACCTGTTTTTCAAGCGCCTG

[1895] ATTCGCCACCTGGACGGCGAGAAGAACGTCACATGGACCCTGTTCGACCG

[1896] GGACACCAGCATGTCGCTCGCCGACTTTCACGGGGAGGAGTTCGAGAAGC

[1897] TCTACCAGCACCTCGAGGTCATGGGGTTCGGCGAGCAGATACCCATCCAG

[1898] GAGCTGGCCTATGGCATTGTGCGCAGTGCGGCCACGACCGGGAGCCCCTT

[1899] CGTCATGTTCAAAGACGCGGTGAACCGCCACTACATCTACGACACCCAGG

[1900] GGGCGGCCATCGCCGGCTCCAACCTCTGCACCGAGATCGTCCATCCGGCC

[1901] TCCAAGCGATCCAGTGGGGTCTGCAACCTGGGAAGCGTGAATCTGGCCCG

[1902] ATGCGTCTCCAGGCAGACGTTTGACTTTGGGCGGCTCCGCGACGCCGTGC

[1903] AGGCGTGCGTGCTGATGGTGAACATCATGATCGACAGCACGCTACAACCC

[1904] ACGCCCCAGTGCACCCGCGGCAACGACAACCTGCGGTCCATGGGAATCGG

[1905] CATGCAGGGCCTGCACACGGCCTGCCTGAAGCTGGGGCTGGATCTGGAGT

[1906] CTGTCGAATTTCAGGACCTGAACAAACACATCGCCGAGGTGATGCTGCTG

[1907] TCGGCGATGAAGACCAGCAACGCGCTGTGCGTTCGCGGGGCCCGTCCCTT

[1908] CAACCACTTTAAGCGCAGCATGTATCGCGCCGGCCGCTTTCACTGGGAGC

[1909] GCTTTCCGGACGCCCGGCCGCGGTACGAGGGCGAGTGGGAGATGCTACGC

[1910] CAGAGCATGATGAAACACGGCCTGCGCAACAGCCAGTTTGTCGCGCTGAT

[1911] GCCCACCGCCGCCTCGGCGCAGATCTCGGACGTCAGCGAGGGCTTTGCCC

[1912] CCCTGTTCACCAACCTGTTCAGCAAGGTGACCCGGGACGGCGAGACGCTG

[1913] CGCCCCAACACGCTCCTGCTAAAGGAACTGGAACGCACGTTTAGCGGGAA

[1914] GCGCCTCCTGGAGGTGATGGACAGTCTCGACGCCAAGCAGTGGTCCGTGG

[1915] CGCAGGCGCTCCCGTGCCTGGAGCCCACCCACCCCCTCCGGCGATTCAAG

[1916] ACCGCGTTTGACTACGACCAGAAGTTGCTGATCGACCTGTGTGCGGACCG

[1917] CGCCCCCTACGTCGACCATAGCCAATCCATGACCCTGTATGTCACGGAGA

[1918] AGGCGGACGGGACCCTCCCAGCCTCCACCCTGGTCCGCCTTCTGGTCCAC

[1919] GCATATAAGCGCGGACTAAAAACAGGGATGTACTACTGCAAGGTTCGCAA

[1920] GGCGACCAACAGCGGGGTCTTTGGCGGCGACGACAACATTGTCTGCACGA

[1921] GCTGCGCGCTGTGA

[1922] SEQ ID NO. 50: Amino acid sequence of ICP6 in wild-type strain 17.

[1923] MASRPAASSPVEARAPVGGQEAGGPSAATQGEAAGAPLAHGHHVYCQRVN

[1924] GVMVLSDKTPGSASYRISDSNFVQCGSNCTMIIDGDVVRGRPQDPGAAAS

[1925] PAPFVAVTNIGAGSDGGTAVVAFGGTPRRSAGTSTGTQTADVPTEALGGP

[1926] PPPPRFTLGGGCCSCRDTRRRSAVFGGEGDPVGPAEFVSDDRSSDSDSDD

[1927] SEDTDSETLSHASSDVSGGATYDDALDSDSSSDDSLQIDGPVCRPWSNDT

[1928] APLDVCPGTPGPGADAGGPSAVDPHAPTPEAGAGLAADPAVARDDAEGLS

[1929] DPRPRLGTGTAYPVPLELTPENAEAVARFLGDAVNREPALMLEYFCRCAR

[1930] EETKRVPPRTFGSPPRLTEDDFGLLNYALVEMQRLCLDVPPVPPNAYMPY

[1931] YLREYVTRLVNGFKPLVSRSARLYRILGVLVHLRIRTREASFEEWLRSKE

[1932] VALDFGLTERLREHEAQLVILAQALDHYDCLIHSTPHTLVERGLQSALKY

[1933] EEFYLKRFGGHYMESVFQMYTRIAGFLACRATRGMRHIALGREGSWWEMF

[1934] KFFFHRLYDHQIVPSTPAMLNLGTRNYYTSSCYLVNPQATTNKATLRAIT

[1935] SNVSAILARNGGIGLCVQAFNDSGPGTASVMPALKVLDSLVAAHNKESAR

[1936] PTGACVYLEPWHTDVRAVLRMKGVLAGEEAQRCDNIFSALWMPDLFFKRL

[1937] IRHLDGEKNVTWTLFDRDTSMSLADFHGEEFEKLYQHLEVMGFGEQIPIQ

[1938] ELAYGIVRSAATTGSPFVMFKDAVNRHYIYDTQGAAIAGSNLCTEIVHPA

[1939] SKRSSGVCNLGSVNLARCVSRQTFDFGLRRDAVQACVLMVNIMIDSTLQP

[1940] TPQCTRGNDNLRSMGIGMQGLHTACLKLGLDLESVEFQDLNKHIAEVMLL

[1941] SAMKTSNALCVRGARPFNHFKRSMYRAGRFHWERFPDARPRYEGEWEMLR

[1942] QSMMKHGLRNSQFVALMPTAASAQISDVSEGFAPLFTNLFSKVTRDGETL

[1943] RPNTLLLKELERTFSGKRLLEVMDSLDAKQWSVAQALPCLEPTHPLRRFK

[1944] TAFDYDQKLLIDLCADRAPYVDHSQSMTLYVTEKADGTLPASTLVRLLVH

[1945] AYKRGLKTGMYYCKVRKATNSGVFGGDDNIVCTSCAL. Sequence Listing <110> Children's National Hospital Research Institute <120> Syncytic oncolytic herpes simplex mutants as effective cancer therapeutics <130> 21F-1902-CNP <150> 62 / 932,725 <151> 2019-11-08 <150> 62 / 818,577 <151> 2019-03-14 <160> 53 <170> PatentIn version 3.5 <210> 1 <211> 726 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: synthetic polynucleotide <220> <221> Mutation <222> (356)..(356) <223> c356a <400> 1 atggcccgcc gccgccgcca tcgcggcccc cgccgccccc ggccgcccgg gcccacgggc 60 gcggtcccaa ccgcacagtc ccaggtaacc tccacgccca actcggaacc cgtggtcagg 120 agcgcgcccg cggccgcccc gccgccgccc cccgccagtg ggcccccgcc ttcttgttcg 180 ctgctgctgc gccagtggct ccacgttccc gagtccgcgt ccgacgacga cgacgacgac 240 tggccggaca gccccccgcc cgagccggcg ccagaggccc ggcccaccgc cgccgccccc 300 cgcccccggt ccccaccgcc cggcgcgggc ccggggggcg gggctaaccc ctcccacccc 360 ccctcacgcc ccttccgcct tccgccgcgc ctcgccctcc gcctgcgcgt caccgcagag 420 cacctggcgc gcctgcgcct gcgacgcgcg ggcggggagg gggcgccgaa gccccccgcg 480 [[ID=2​​​cgggccgacc gggctcggtt ccggcgccgg gtggcggagg ccgaggcggt catcgggccg 660 tgcctggggc ccgaggcccg tgcccgggcc ctggcccgcg gagccggccc ggcgaactcg 720 gtctaa 726 <210> 2 <211> 241 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptide <220> <221> MISC_FEATURE <222> (119)..(119) <223> P119H mutation <400> 2 Met Ala Arg Arg Arg Arg His Arg Gly Pro Arg Arg Pro Arg Pro Pro 1 5 10 15 Gly Pro Thr Gly Ala Val Pro Thr Ala Gln Ser Gln Val Thr Ser Thr 20 25 30 Pro Asn Ser Glu Pro Val Val Arg Ser Ala Pro Ala Ala Ala Pro Pro 35 40 45 Pro Pro Pro Ala Ser Gly Pro Pro Pro Ser Cys Ser Leu Leu Leu Arg 50 55 60 Gln Trp Leu His Val Pro Glu Ser Ala Ser Asp Asp Asp Asp Asp Asp 65 70 75 80 Trp Pro Asp Ser Pro Pro Pro Glu Pro Ala Pro Glu Ala Arg Pro Thr 85 90 95 Ala Ala Ala Pro Arg Pro Arg Ser Pro Pro Pro Gly Ala Gly Pro Gly 100 105 110 Gly Gly Ala Asn Pro Ser His Pro Pro Ser Arg Pro Phe Arg Leu Pro 115 120 125 Pro Arg Leu Ala Leu Arg Leu Arg Val Thr Ala Glu His Leu Ala Arg 130 135 140 Leu Arg Leu Arg Arg Ala Gly Gly Glu Gly Ala Pro Lys Pro Pro Ala 145 150 155 160 Thr Pro Ala Thr Pro Ala Thr Pro Thr Arg Val Arg Phe Ser Pro His 165 170 175 Val Arg Val Arg His Leu Val Val Trp Ala Ser Ala Ala Arg Leu Ala 180 185 190 Arg Arg Gly Ser Trp Ala Arg Glu Arg Ala Asp Arg Ala Arg Phe Arg 195 200 205 Arg Arg Val Ala Glu Ala Glu Ala Val Ile Gly Pro Cys Leu Gly Pro 210 215 220 Glu Ala Arg Ala Arg Ala Leu Ala Arg Gly Ala Gly Pro Ala Asn Ser 225 230 235 240 Val <210> 3 <211> 846 <212> DNA <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic polynucleotide <400> 3 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagaag 720 cttagccatg gcttcccgcc ggaggtggag gagcaggatg atggcacgct gcccatgtct 780 tgtgcccagg agagcgggat ggaccgtcac cctgcagcct gtgcttctgc taggatcaat 840 gtgtag 846 <210> 4 <211> 281 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic polypeptide <400> 4 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys 65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 145 150 155 160 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu 195 200 205 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210 215 220 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Lys 225 230 235 240 Leu Ser His Gly Phe Pro Pro Glu Val Glu Glu Gln Asp Asp Gly Thr 245 250 255 Leu Pro Met Ser Cys Ala Gln Glu Ser Gly Met Asp Arg His Pro Ala 260 265 270 Ala Cys Ala Ser Ala Arg Ile Asn Val 275 280 <210> 5 <211> 767 <212> DNA <213> Artificial Sequence <220> <223> Description of artificial sequence: synthetic polynucleotide <400> 5 atggcccgcc gccgccgcca tcgcggcccc cgccgccccc ggccgcccgg gcccacgggc 60 gccgtcccaa ccgcacagtc ccaggtaacc tagactagtc tagcgtaacc tccacgccca 120 actcggaacc cgcggtcagg agcgcgcccg cggccgcccc gccgccgccc cccgccggtg 180 ggcccccgcc ttcttgttcg ctgctgctgc gccagtggct ccacgttccc gagtccgcgt 240 ccgacaacga cgatgacgac gactggccgg acagcccccc gcccgagccg gcgccagagg 300 cccggcccac cgccgccgcc ccccggcccc ggcccccacc gcccggcgtg ggcccggggg 360 gcggggctga cccctcccac cccccctcgc gccccttccg ccttccgccg cgcctcgccc 420 tccgcctgcg cgtcaccgcg gagcacctgg cgcgcctgcg cctgcgacgc gcgggcgggg 480 agggggcgcc ggagcccccc gcgacccccg cgacccccgc gacccccgcg acccccgcga 540 cccccgcgcg ggtgcgcttc tcgccccacg tccgggtgcg ccacctggtg gtctgggcct 600 cggccgcccg cctggcgcgc cgcggctcgt gggcccgcga gcgggccgac cgggctcggt 660 tccggcgccg ggtggcggag gccgaggcgg tcatcgggcc gtgcctgggg cccgaggccc 720 gtgcccgggc cctggcccgc ggagccggcc cggcgaactc ggtctaa 767 <210> 6 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic polypeptide <400> 6 Met Ala Arg Arg Arg Arg His Arg Gly Pro Arg Arg Pro Arg Pro Pro 1 5 10 15 Gly Pro Thr Gly Ala Val Pro Thr Ala Gln Ser Gln Val Thr 20 25 30 <210> 7 <211> 723 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic polynucleotide <400> 7 atggcccgcc gccgccatcg cggcccccgc cgcccccggc cgcccgggcc cacgggcgcg 60 gtcccaaccg cacagtccca ggtaacctcc acgcccaact cggaacccgt ggtcaggagc 120 gcgcccgcgg ccgccccgcc gccgcccccc gccagtgggc ccccgccttc ttgttcgctg 180 ctgctgcgcc agtggctcca cgttcccgag tccgcgtccg acgacgacga cgacgactgg 240 ccggacagcc ccccgcccga gccggcgcca gaggcccggc ccaccgccgc cgccccccgc 300 ccccggtccc caccgcccgg cgcgggcccg gggggcgggg ctaacccctc cccccccccc 360 tcacgcccct tccgccttcc gccgcgcctc gccctccgcc tgcgcgtcac cgcagagcac 420 ctggcgcgcc tgcgcctgcg acgcgcgggc ggggaggggg cgccgaagcc ccccgcgacc 480 cccgcgaccc ccgcgacccc cacgcgggtg cgcttctcgc cccacgtccg ggtgcgccac 540 ctggtggtct gggcctcggc cgcccgcctg gcgcgccgcg gctcgtgggc ccgcgagcgg 600 gccgaccggg ctcggttccg gcgccgggtg gcggaggccg aggcggtcat cgggccgtgc 660 ctggggcccg aggcccgtgc ccgggccctg gcccgcggag ccggcccggc gaactcggtc 720 taa 723 <210> 8 <211> 240 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptide <400> 8 Met Ala Arg Arg Arg His Arg Gly Pro Arg Arg Pro Arg Pro Pro Gly 1 5 10 15 Pro Thr Gly Ala Val Pro Thr Ala Gln Ser Gln Val Thr Ser Thr Pro 20 25 30 Asn Ser Glu Pro Val Val Arg Ser Ala Pro Ala Ala Ala Pro Pro Pro 35 40 45 Pro Pro Ala Ser Gly Pro Pro Pro Ser Cys Ser Leu Leu Leu Arg Gln 50 55 60 Trp Leu His Val Pro Glu Ser Ala Ser Asp Asp Asp Asp Asp Asp Trp 65 70 75 80 Pro Asp Ser Pro Pro Pro Glu Pro Ala Pro Glu Ala Arg Pro Thr Ala 85 90 95 Ala Ala Pro Arg Pro Arg Ser Pro Pro Pro Gly Ala Gly Pro Gly Gly 100 105 110 Gly Ala Asn Pro Ser Pro Pro Pro Ser Arg Pro Phe Arg Leu Pro Pro 115 120 125 Arg Leu Wing Leu Arg Leu Arg Val Thr Wing Glu His Leu Wing Arg Leu 130 135 140 Arg Leu Arg Arg Ala Gly Gly Glu Gly Ala Pro Lys Pro Pro Ala Thr 145 150 155 160 Pro Ala Thr Pro Ala Thr Pro Thr Arg Val Arg Phe Ser Pro His Val 165 170 175 Arg Val Arg His Leu Val Val Trp Ala Ser Ala Ala Arg Leu Ala Arg 180 185 190 Arg Gly Ser Trp Ala Arg Glu Arg Ala Asp Arg Ala Arg Phe Arg Arg 195 200 205 Arg Val Ala Glu Ala Glu Ala Val Ile Gly Pro Cys Leu Gly Pro Glu 210 215 220 Ala Arg Ala Arg Ala Leu Ala Arg Gly Ala Gly Pro Ala Asn Ser Val 225 230 235 240 <210> 9 <211> 726 <212> DNA <213> 1-year-old <400> 9 atggcccgcc gccgccgcca tcgcggcccc cgccgccccc ggcgcccgg gcccgggc 60 gcggtcccaa ccgcacagtc ccaggtaacc tccacgccca actcggaacc cgtggtcagg 120 agcgcgcccg cggccgcccc gccgccgccc cccgccagtg ggcccccgcc ttcttgttcg 180 ctgctgctgc gccagtggct ccacgttccc gagtccgcgt ccgacgacga cgacgacgac 240 tggccggaca gccccccgcc cgagccggcg ccagaggccc ggcccaccgc cgccgccccc 300 cgcccccggt ccccaccgcc cggcgcgggc ccggggggcg gggctaaccc ctcccacccc 360 ccctcacgcc ccttccgcct tccgccgcgc ctcgccctcc gcctgcgcgt caccgcagag 420 cacctggcgc gcctgcgcct gcgacgcgcg ggcggggagg gggcgccgaa gccccccgcg 480 acccccgcga cccccgcgac ccccacgcgg gtgcgcttct cgccccacgt ccgggtgcgc 540 cacctggtgg tctgggcctc ggccgcccgc ctggcgcgcc gcggctcgtg ggcccgcgag 600 cgggccgacc gggctcggtt ccggcgccgg gtggcggagg ccgaggcggt catcgggccg 660 tgcctggggc ccgaggcccg tgcccgggcc ctggcccgcg gagccggccc ggcgaactcg 720 gtctaa 726 <210> 10 <211> 241 <212> PRT <213> Herpes simplex virus 1 <400> 10 Met Ala Arg Arg Arg Arg His Arg Gly Pro Arg Arg Pro Arg Pro Pro 1 5 10 15 Gly Pro Thr Gly Ala Val Pro Thr Ala Gln Ser Gln Val Thr Ser Thr 20 25 30 Pro Asn Ser Glu Pro Val Val Arg Ser Ala Pro Ala Ala Ala Pro Pro 35 40 45 Pro Pro Pro Ala Ser Gly Pro Pro Pro Ser Cys Ser Leu Leu Leu Arg 50 55 60 Gln Trp Leu His Val Pro Glu Ser Ala Ser Asp Asp Asp Asp Asp Asp 65 70 75 80 Trp Pro Asp Ser Pro Pro Pro Glu Pro Ala Pro Glu Ala Arg Pro Thr 85 90 95 Ala Ala Ala Pro Arg Pro Arg Ser Pro Pro Pro Gly Ala Gly Pro Gly 100 105 110 Gly Gly Ala Asn Pro Ser His Pro Pro Ser Arg Pro Phe Arg Leu Pro 115 120 125 Pro Arg Leu Ala Leu Arg Leu Arg Val Thr Ala Glu His Leu Ala Arg 130 135 140 Leu Arg Leu Arg Arg Ala Gly Gly Glu Gly Ala Pro Lys Pro Pro Ala 145 150 155 160 Thr Pro Ala Thr Pro Ala Thr Pro Thr Arg Val Arg Phe Ser Pro His 165 170 175 Val Arg Val Arg His Leu Val Val Trp Ala Ser Ala Ala Arg Leu Ala 180 185 190 Arg Arg Gly Ser Trp Ala Arg Glu Arg Ala Asp Arg Ala Arg Phe Arg 195 200 205 Arg Arg Val Ala Glu Ala Glu Ala Val Ile Gly Pro Cys Leu Gly Pro 210 215 220 Glu Ala Arg Ala Arg Ala Leu Ala Arg Gly Ala Gly Pro Ala Asn Ser 225 230 235 240 Val <210> 11 <211> 1971 <212> DNA <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic polynucleotide <400> 11 aacacattaa ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc 60 aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat 120 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact 180 catcaatgta tcttatcatg tctgctcgaa gcggccggcc gccccgactc tagactacac 240 attgatccta gcagaagcac aggctgcagg gtgacggtcc atcccgctct cctgggcaca 300 agacatgggc agcgtgccat catcctgctc ctccacctcc ggcgggaagc catggctaag 360 cttcttgtac agctcgtcca tgccgagagt gatcccggcg gcggtcacga actccagcag 420 gaccatgtga tcgcgcttct cgttggggtc tttgctcagg gcggactggg tgctcaggta 480 gtggttgtcg ggcagcagca cggggccgtc gccgatgggg gtgttctgct ggtagtggtc 540 ggcgagctgc acgctgccgt cctcgatgtt gtggcggatc ttgaagttca ccttgatgcc 600 gttcttctgc ttgtcggcca tgatatagac gttgtggctg ttgtagttgt actccagctt 660 gtgccccagg atgttgccgt cctccttgaa gtcgatgccc ttcagctcga tgcggttcac 720 cagggtgtcg ccctcgaact tcacctcggc gcgggtcttg tagttgccgt cgtccttgaa 780 gaagatggtg cgctcctgga cgtagccttc gggcatggcg gacttgaaga agtcgtgctg 840 cttcatgtgg tcggggtagc ggctgaagca ctgcacgccg taggtcaggg tggtcacgag 900 ggtgggccag ggcacgggca gcttgccggt ggtgcagatg aacttcaggg tcagcttgcc 960 gtaggtggca tcgccctcgc cctcgccgga cacgctgaac ttgtggccgt ttacgtcgcc 1020 gtccagctcg accaggatgg gcaccacccc ggtgaacagc tcctcgccct tgctcaccat 1080 ccgggaattg cggccgcggg tacaattccg cagcttttag agcagaagta acacttccgt 1140 acaggcctag aagtaaaggc aacatccact gaggagcagt tctttgattt gcaccaccac 1200 cggatccggg acctgaaata aaagacaaaa agactaaact taccagttaa ctttctggtt 1260 tttcagttcc tcgagtaccg gatcctctag agtccggagg ctggatcggt cccggtgtct 1320 tctatggagg tcaaaacagc gtggatggcg tctccaggcg atctgacggt tcactaaacg 1380 agctctgctt atatagacct cccaccgtac acgcctaccg cccatttgcg tcaatggggc 1440 ggagttgtta cgacattttg gaaagtcccg ttgattttgg tgccaaaaca aactcccatt 1500 gacgtcaatg gggtggagac ttggaaatcc ccgtgagtca aaccgctatc cacgcccatt 1560 gatgtactgc caaaaccgca tcaccatggt aatagcgatg actaatacgt agatgtactg 1620 ccaagtagga aagtcccata aggtcatgta ctgggcataa tgccaggcgg gccatttacc 1680 gtcattgacg tcaatagggg gcgtacttgg catatgatac acttgatgta ctgccaagtg 1740 ggcagtttac cgtaaatact ccacccattg acgtcaatgg aaagtcccta ttggcgttac 1800 tatgggaaca tacgtcatta ttgacgtcaa tgggcggggg tcgttgggcg gtcagccagg 1860 cgggccattt accgtaagtt atgtaacgac ctgcaggcat gcaagctcga attcgaacac 1920 gcagatgcag tcggggcggc agatcttaat taatggctgg ttgtttgttg t 19--71 <210> 12 <211> 1653 <212> DNA <213> Artificial Sequence <220> <223> Description of artificial sequence: synthetic polynucleotide <220> <221> Mutation <222> (451)..(451) <223> g451a <400> 12 atggatcgcg gggcggtggt ggggtttctt ctcggtgttt gtgttgtatc gtgcttggcg 60 ggaacgccca aaacgtcctg gagacgggtg agtgtcggcg aggacgtttc gttgcttcca --120 gctccggggc ctacggggcg cggcccgacc cagaaactac tatgggccgt ggaacccctg 180 gatgggtgcg gccccttaca cccgtcgtgg gtctcgctga tgccccccaa gcaggtgccc 240 It should be noted that there seems to be an unclear "19--71" in the original text at line ID=8, which is translated as "19--71" here. You may want to check and clarify the original content. Also, at line ID=34, there is an unclear " --120" which is translated as " --120" for now.gagacggtcg tggatgcggc gtgcatgcgc gctccggtcc cgctggcgat ggcgtacgcc 300 cccccggccc catctgcgac cgggggtcta cgaacggact tcgtgtggca ggagcgcgcg 360 gccgtggtta accggagtct ggttattcac ggggtccgag agacggacag cggcctgtat 420 accctgtccg tgggcgacat aaaggacccg actcgccaag tggcctcggt ggtcctggtg 480 gtgcaaccgg ccccagttcc gaccccaccc ccgaccccag ccgattacga cgaggatgac 540 aatgacgagg gcgaggacga aagtctcgcc ggcactcccg ccagcgggac cccccggctc 600 ccgcctcccc ccgccccccc gaggtcttgg cccagcgccc ccgaagtctc acatgtgcgt 660 ggggtgaccg tgcgtatgga gactccggaa gctatcctgt tttcccccgg ggagacgttc 720 agcacgaacg tctccatcca tgccatcgcc cacgacgacc agacctactc catggacgtc 780 gtctggttga ggttcgacgt gccgacctcg tgtgccgaga tgcgaatata cgaatcgtgt 840 ctgtatcacc cgcagctccc agaatgtctg tccccggccg acgcgccgtg cgccgcgagt 900 acgtggacgt ctcgcctggc cgtccgcagc tacgcggggt gttccagaac aaacccccca 960 ccgcgctgtt cggccgaggc tcacatggag cccgtcccgg ggctggcgtg gcaggcggcc 1020 tccgtcaatc tggagttccg ggacgcgtcc ccacaacact ccggcctgta tctgtgtgtg 1080 gtgtacgtca acgaccatat tcacgcctgg ggccacatta ccatcagcac cgcggcgcag 1140 taccggaacg cggtggtgga acagcccctc ccacagcgcg gcgcggattt ggccgagccc 1200 acccacccgc acgtcggggc ccctccccac gcgcccccaa cccacggcgc cctgcggtta 1260 ggggcggtga tgggggccgc cctgctgctg tctgcgctgg ggttgtcggt gtgggcgtgt 1320 atgacctgtt ggcgcaggcg tgcctggcgg gcggttaaaa gcagggcctc gggtaagggg 1380 cccacgtaca ttcgcgtggc cgacagcgag ctgtacgcgg actggagctc ggacagcgag 1440 ggagaacgcg accaggtccc gtggctggcc cccccggaga gacccgactc tccctccacc 1500 aatggatccg gctttgagat cttatcacca acggctccgt ctgtataccc ccgtagcgac 1560 gggcatcaat ctcgccgcca gctcacaacc tttggatccg gaaggcccga tcgccgttac 1620 tcccaggcct ccgattcgtc cgtcttctgg taa 1653 <210> 13 <211> 550 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptide <220> <221> MISC_FEATURE <222> (151)..(151) <223> A151T <400> 13 Met Asp Arg Gly Ala Val Val Gly Phe Leu Leu Gly Val Cys Val Val 1 5 10 15 Ser Cys Leu Ala Gly Thr Pro Lys Thr Ser Trp Arg Arg Val Ser Val 20 25 30 Gly Glu Asp Val Ser Leu Leu Pro Ala Pro Gly Pro Thr Gly Arg Gly 35 40 45 Pro Thr Gln Lys Leu Leu Trp Ala Val Glu Pro Leu Asp Gly Cys Gly 50 55 60 Pro Leu His Pro Ser Trp Val Ser Leu Met Pro Pro Lys Gln Val Pro 65 70 75 80 Glu Thr Val Val Asp Ala Ala Cys Met Arg Ala Pro Val Pro Leu Ala 85 90 95 Met Ala Tyr Ala Pro Pro Ala Pro Ser Ala Thr Gly Gly Leu Arg Thr 100 105 110 Asp Phe Val Trp Gln Glu Arg Ala Ala Val Val Asn Arg Ser Leu Val 115 120 125 Ile His Gly Val Arg Glu Thr Asp Ser Gly Leu Tyr Thr Leu Ser Val 130 135 140 Gly Asp Ile Lys Asp Pro Thr Arg Gln Val Ala Ser Val Val Leu Val 145 150 155 160 Val Gln Pro Ala Pro Val Pro Thr Pro Pro Pro Thr Pro Ala Asp Tyr 165 170 175 Asp Glu Asp Asp Asn Asp Glu Gly Glu Asp Glu Ser Leu Ala Gly Thr 180 185 190 Pro Ala Ser Gly Thr Pro Arg Leu Pro Pro Pro Pro Ala Pro Pro Arg 195 200 205 Ser Trp Pro Ser Ala Pro Glu Val Ser His Val Arg Gly Val Thr Val 210 215 220 Arg Met Glu Thr Pro Glu Ala Ile Leu Phe Ser Pro Gly Glu Thr Phe 225 230 235 240 Ser Thr Asn Val Ser Ile His Ala Ile Ala His Asp Asp Gln Thr Tyr 245 250 255 Ser Met Asp Val Val Trp Leu Arg Phe Asp Val Pro Thr Ser Cys Ala 260 265 270 Glu Met Arg Ile Tyr Glu Ser Cys Leu Tyr His Pro Gln Leu Pro Glu 275 280 285 Cys Leu Ser Pro Ala Asp Ala Pro Cys Ala Ala Ser Thr Trp Thr Ser 290 295 300 Arg Leu Ala Val Arg Ser Tyr Ala Gly Cys Ser Arg Thr Asn Pro Pro 305 310 315 320 Pro Arg Cys Ser Ala Glu Ala His Met Glu Pro Val Pro Gly Leu Ala 325 330 335 Trp Gln Ala Ala Ser Val Asn Leu Glu Phe Arg Asp Ala Ser Pro Gln 340 345 350 His Ser Gly Leu Tyr Leu Cys Val Val Tyr Val Asn Asp His Ile His 355 360 365 Ala Trp Gly His Ile Thr Ile Ser Thr Ala Ala Gln Tyr Arg Asn Ala 370 375 380 Val Val Glu Gln Pro Leu Pro Gln Arg Gly Ala Asp Leu Ala Glu Pro 385 390 395 400 Thr His Pro His Val Gly Ala Pro Pro His Ala Pro Pro Thr His Gly 405 410 415 Ala Leu Arg Leu Gly Ala Val Met Gly Ala Ala Leu Leu Leu Ser Ala 420 425 430 Leu Gly Leu Ser Val Trp Ala Cys Met Thr Cys Trp Arg Arg Arg Ala 435 440 445 Trp Arg Ala Val Lys Ser Arg Ala Ser Gly Lys Gly Pro Thr Tyr Ile 450 455 460 Arg Val Ala Asp Ser Glu Leu Tyr Ala Asp Trp Ser Ser Asp Ser Glu 465 470 475 480 Gly Glu Arg Asp Gln Val Pro Trp Leu Ala Pro Pro Glu Arg Pro Asp 485 490 495 Ser Pro Ser Thr Asn Gly Ser Gly Phe Glu Ile Leu Ser Pro Thr Ala 500 505 510 Pro Ser Val Tyr Pro Arg Ser Asp Gly His Gln Ser Arg Arg Gln Leu 515 520 525 Thr Thr Phe Gly Ser Gly Arg Pro Asp Arg Arg Tyr Ser Gln Ala Ser 530 535 540 Asp Ser Ser Val Phe Trp 545 550 <210> 14 <211> 1653 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: synthetic polynucleotide <400> 14 atggatcgcg gggcggtggt ggggtttctt ctcggtgttt gtgttgtatc gtgcttggcg 60 ggaacgccca aaacgtcctg gagacgggtg agtgtcggcg aggacgtttc gttgcttcca 120 gctccggggc ctacggggcg cggcccgacc cagaaactac tatgggccgt ggaacccctg 180 gatgggtgcg gccccttaca cccgtcgtgg gtctcgctga tgccccccaa gcaggtgccc 240 gagacggtcg tggatgcggc gtgcatgcgc gctccggtcc cgctggcgat ggcgtacgcc 300 cccccggccc catctgcgac cgggggtcta cgaacggact tcgtgtggca ggagcgcgcg 360 gccgtggtta accggagtct ggttattcac ggggtccgag agacggacag cggcctgtat 420 accctgtccg tgggcgacat aaaggacccg gctcgccaag tggcctcggt ggtcctggtg 480 gtgcaaccgg ccccagttcc gaccccaccc ccgaccccag ccgattacga cgaggatgac 540 aatgacgagg gcgaggacga aagtctcgcc ggcactcccg ccagcgggac cccccggctc 600 ccgcctcccc ccgccccccc gaggtcttgg cccagcgccc ccgaagtctc acatgtgcgt 660 ggggtgaccg tgcgtatgga gactccggaa gctatcctgt tttcccccgg ggagacgttc 720 agcacgaacg tctccatcca tgccatcgcc cacgacgacc agacctactc catggacgtc 780 gtctggttga ggttcgacgt gccgacctcg tgtgccgaga tgcgaatata cgaatcgtgt 840 ctgtatcacc cgcagctccc agaatgtctg tccccggccg acgcgccgtg cgccgcgagt 900 acgtggacgt ctcgcctggc cgtccgcagc tacgcggggt gttccagaac aaacccccca 960 ccgcgctgtt cggccgaggc tcacatggag cccgtcccgg ggctggcgtg gcaggcggcc 1020 tccgtcaatc tggagttccg ggacgcgtcc ccacaacact ccggcctgta tctgtgtgtg 1080 gtgtacgtca acgaccatat tcacgcctgg ggccacatta ccatcagcac cgcggcgcag 1140 taccggaacg cggtggtgga acagcccctc ccacagcgcg gcgcggattt ggccgagccc 1200 acccacccgc acgtcggggc ccctccccac gcgcccccaa cccacggcgc cctgcggtta 1260 ggggcggtga tgggggccgc cctgctgctg tctgcactgg ggttgtcggt gtgggcgtgt 1320 atgacctgtt ggcgcaggcg tgcctggcgg gcggttaaaa gcagggcctc gggtaagggg 1380 cccacgtaca ttcgcgtggc cgacagcgag ctgtacgcgg actggagctc ggacagcgag 1440 ggagaacgcg accaggtccc gtggctggcc cccccggaga gacccgactc tccctccacc 1500 aatggatccg gctttgagat cttatcacca acggctccgt ctgtataccc ccgtagcgat 1560 gggcatcaat ctcgccgcca gctcacaacc tttggatccg gaaggcccga tcgccgttac 1620 tcccaggcct ccgattcgtc cgtcttctgg taa 1653 <210> 15 <211> 550 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptide <400> 15 Met Asp Arg Gly Ala Val Val Gly Phe Leu Leu Gly Val Cys Val Val 1 5 10 15 Ser Cys Leu Ala Gly Thr Pro Lys Thr Ser Trp Arg Arg Val Ser Val 20 25 30 Gly Glu Asp Val Ser Leu Leu Pro Ala Pro Gly Pro Thr Gly Arg Gly 35 40 45 Pro Thr Gln Lys Leu Leu Trp Ala Val Glu Pro Leu Asp Gly Cys Gly 50 55 60 Pro Leu His Pro Ser Trp Val Ser Leu Met Pro Pro Lys Gln Val Pro 65 70 75 80 Glu Thr Val Val Asp Ala Ala Cys Met Arg Ala Pro Val Pro Leu Ala 85 90 95 Met Ala Tyr Ala Pro Pro Ala Pro Ser Ala Thr Gly Gly Leu Arg Thr 100 105 110 Asp Phe Val Trp Gln Glu Arg Ala Ala Val Val Asn Arg Ser Leu Val 115 120 125 Ile His Gly Val Arg Glu Thr Asp Ser Gly Leu Tyr Thr Leu Ser Val 130 135 140 Gly Asp Ile Lys Asp Pro Ala Arg Gln Val Ala Ser Val Val Leu Val 145 150 155 160 Val Gln Pro Ala Pro Val Pro Thr Pro Pro Pro Thr Pro Ala Asp Tyr 165 170 175 Asp Glu Asp Asp Asn Asp Glu Gly Glu Asp Glu Ser Leu Ala Gly Thr 180 185 190 Pro Ala Ser Gly Thr Pro Arg Leu Pro Pro Pro Pro Ala Pro Pro Arg 195 200 205 Ser Trp Pro Ser Ala Pro Glu Val Ser His Val Arg Gly Val Thr Val 210 215 220 Arg Met Glu Thr Pro Glu Ala Ile Leu Phe Ser Pro Gly Glu Thr Phe 225 230 235 240 Ser Thr Asn Val Ser Ile His Ala Ile Ala His Asp Asp Gln Thr Tyr 245 250 255 Ser Met Asp Val Val Trp Leu Arg Phe Asp Val Pro Thr Ser Cys Ala 260 265 270 Glu Met Arg Ile Tyr Glu Ser Cys Leu Tyr His Pro Gln Leu Pro Glu 275 280 285 Cys Leu Ser Pro Ala Asp Ala Pro Cys Ala Ala Ser Thr Trp Thr Ser 290 295 300 Arg Leu Ala Val Arg Ser Tyr Ala Gly Cys Ser Arg Thr Asn Pro Pro 305 310 315 320 Pro Arg Cys Ser Ala Glu Ala His Met Glu Pro Val Pro Gly Leu Ala 325 330 335 Trp Gln Ala Ala Ser Val Asn Leu Glu Phe Arg Asp Ala Ser Pro Gln 340 345 350 His Ser Gly Leu Tyr Leu Cys Val Val Tyr Val Asn Asp His Ile His 355 360 365 Ala Trp Gly His Ile Thr Ile Ser Thr Ala Ala Gln Tyr Arg Asn Ala 370 375 380 Val Val Glu Gln Pro Leu Pro Gln Arg Gly Ala Asp Leu Ala Glu Pro 385 390 395 400 Thr His Pro His Val Gly Ala Pro Pro His Ala Pro Pro Thr His Gly 405 410 415 Ala Leu Arg Leu Gly Ala Val Met Gly Ala Ala Leu Leu Leu Ser Ala 420 425 430 Leu Gly Leu Ser Val Trp Ala Cys Met Thr Cys Trp Arg Arg Arg Ala 435 440 445 Trp Arg Ala Val Lys Ser Arg Ala Ser Gly Lys Gly Pro Thr Tyr Ile 450 455 460 Arg Val Ala Asp Ser Glu Leu Tyr Ala Asp Trp Ser Ser Asp Ser Glu 465 470 475 480 Gly Glu Arg Asp Gln Val Pro Trp Leu Ala Pro Pro Glu Arg Pro Asp 485 490 495 Ser Pro Ser Thr Asn Gly Ser Gly Phe Glu Ile Leu Ser Pro Thr Ala 500 505 510 Pro Ser Val Tyr Pro Arg Ser Asp Gly His Gln Ser Arg Arg Gln Leu 515 520 525 Thr Thr Phe Gly Ser Gly Arg Pro Asp Arg Arg Tyr Ser Gln Ala Ser 530 535 540 Asp Ser Ser Val Phe Trp 545 550 <210> 16 <211> 1653 <212> DNA <213> artificial sequence <220> <223> Artificial Sequence Description: Synthetic polynucleotide <400> 16 atggatcgcg gggcggtggt ggggtttctt ctcggtgttt gtgttgtatc gtgcttggcg 60 ggaacgccca aaacgtcctg gagacgggtg agtgtcggcg aggacgtttc gttgcttcca 120 gctccggggc ctacggggcg cggcccgacc cagaaactac tatgggccgt ggaacccctg 180 gatgggtgcg gccccttaca cccgtcgtgg gtctcgctga tgccccccaa gcaggtgccc 240 gagacggtcg tggatgcggc gtgcatgcgc gctccggtcc cgctggcgat ggcgtacgcc 300 cccccggccc catctgcgac cgggggtcta cgaacggact tcgtgtggca ggagcgcgcg 360 gccgtggtta accggagtct ggttattcac ggggtccgag agacggacag cggcctgtat 420 accctgtccg tgggcgacat aaaggacccg gctcgccaag tggcctcggt ggtcctggtg 480 gtgcaaccgg ccccagttcc gaccccaccc ccgaccccag ccgattacga cgaggatgac 540 aatgacgagg gcgaggacga aagtctcgcc ggcactcccg ccagcgggac cccccggctc 600 ccgcctcccc ccgccccccc gaggtcttgg cccagcgccc ccgaagtctc acatgtgcgt 660 ggggtgaccg tgcgtatgga gactccggaa gctatcctgt tttcccccgg ggagacgttc 720 agcacgaacg tctccatcca tgccatcgcc cacgacgacc agacctactc catggacgtc 780 gtctggttga ggttcgacgt gccgacctcg tgtgccgaga tgcgaatata cgaatcgtgt 840 ctgtatcacc cgcagctccc agaatgtctg tccccggccg acgcgccgtg cgccgcgagt 900 acgtggacgt ctcgcctggc cgtccgcagc tacgcggggt gttccagaac aaacccccca 960 ccgcgctgtt cggccgaggc tcacatggag cccgtcccgg ggctggcgtg gcaggcggcc 1020 tccgtcaatc tggagttccg ggacgcgtcc ccacaacact ccggcctgta tctgtgtgtg 1080 gtgtacgtca acgaccatat tcacgcctgg ggccacatta ccatcagcac cgcggcgcag 1140 taccggaacg cggtggtgga acagcccctc ccacagcgcg gcgcggattt ggccgagccc 1200 acccacccgc acgtcggggc ccctccccac gcgcccccaa cccacggcgc cctgcggtta 1260 ggggcggtga tgggggccgc cctgctgctg tctgcgctgg ggttgtcggt gtgggcgtgt 1320 atgacctgtt ggcgcaggcg tgcctggcgg gcggttaaaa gcagggcctc gggtaagggg 1380 cccacgtaca ttcgcgtggc cgacagcgag ctgtacgcgg actggagctc ggacagcgag 1440 ggagaacgcg accaggtccc gtggctggcc cccccggaga gacccgactc tccctccacc 1500 aatggatccg gctttgagat cttatcacca acggctccgt ctgtataccc ccgtagcgac 1560 gggcatcaat ctcgccgcca gctcacaacc tttggatccg gaaggcccga tcgccgttac 1620 tcccaggcct ccgattcgtc cgtcttctgg taa 1653 <210> 17 <211> 550 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic polypeptide <400> 17 Met Asp Arg Gly Ala Val Val Gly Phe Leu Leu Gly Val Cys Val Val 1 5 10 15 Ser Cys Leu Ala Gly Thr Pro Lys Thr Ser Trp Arg Arg Val Ser Val 20 25 30 Gly Glu Asp Val Ser Leu Leu Pro Ala Pro Gly Pro Thr Gly Arg Gly 35 40 45 Pro Thr Gln Lys Leu Leu Trp Ala Val Glu Pro Leu Asp Gly Cys Gly 50 55 60 Pro Leu His Pro Ser Trp Val Ser Leu Met Pro Pro Lys Gln Val Pro 65 70 75 80 Glu Thr Val Val Asp Ala Ala Cys Met Arg Ala Pro Val Pro Leu Ala 85 90 95 Met Ala Tyr Ala Pro Pro Ala Pro Ser Ala Thr Gly Gly Leu Arg Thr 100 105 110 Asp Phe Val Trp Gln Glu Arg Ala Ala Val Val Asn Arg Ser Leu Val 115 120 125 Ile His Gly Val Arg Glu Thr Asp Ser Gly Leu Tyr Thr Leu Ser Val 130 135 140 Gly Asp Ile Lys Asp Pro Ala Arg Gln Val Ala Ser Val Val Leu Val 145 150 155 160 Val Gln Pro Ala Pro Val Pro Thr Pro Pro Pro Thr Pro Ala Asp Tyr 165 170 175 Asp Glu Asp Asp Asn Asp Glu Gly Glu Asp Glu Ser Leu Ala Gly Thr 180 185 190 Pro Ala Ser Gly Thr Pro Arg Leu Pro Pro Pro Pro Ala Pro Pro Arg 195 200 205 Ser Trp Pro Ser Ala Pro Glu Val Ser His Val Arg Gly Val Thr Val 210 215 220 Arg Met Glu Thr Pro Glu Ala Ile Leu Phe Ser Pro Gly Glu Thr Phe 225 230 235 240 Ser Thr Asn Val Ser Ile His Ala Ile Ala His Asp Asp Gln Thr Tyr 245 250 255 Ser Met Asp Val Val Trp Leu Arg Phe Asp Val Pro Thr Ser Cys Ala 260 265 270 Glu Met Arg Ile Tyr Glu Ser Cys Leu Tyr His Pro Gln Leu Pro Glu 275 280 285 Cys Leu Ser Pro Ala Asp Ala Pro Cys Ala Ala Ser Thr Trp Thr Ser 290 295 300 Arg Leu Ala Val Arg Ser Tyr Ala Gly Cys Ser Arg Thr Asn Pro Pro 305 310 315 320 Pro Arg Cys Ser Ala Glu Ala His Met Glu Pro Val Pro Gly Leu Ala 325 330 335 Trp Gln Ala Ala Ser Val Asn Leu Glu Phe Arg Asp Ala Ser Pro Gln 340 345 350 His Ser Gly Leu Tyr Leu Cys Val Val Tyr Val Asn Asp His Ile His 355 360 365 Ala Trp Gly His Ile Thr Ile Ser Thr Ala Ala Gln Tyr Arg Asn Ala 370 375 380 Val Val Glu Gln Pro Leu Pro Gln Arg Gly Ala Asp Leu Ala Glu Pro 385 390 395 400 Thr His Pro His Val Gly Ala Pro Pro His Ala Pro Pro Thr His Gly 405 410 415 Ala Leu Arg Leu Gly Ala Val Met Gly Ala Ala Leu Leu Leu Ser Ala 420 425 430 Leu Gly Leu Ser Val Trp Ala Cys Met Thr Cys Trp Arg Arg Arg Ala 435 440 445 Trp Arg Ala Val Lys Ser Arg Ala Ser Gly Lys Gly Pro Thr Tyr Ile 450 455 460 Arg Val Ala Asp Ser Glu Leu Tyr Ala Asp Trp Ser Ser Asp Ser Glu 465 470 475 480 Gly Glu Arg Asp Gln Val Pro Trp Leu Ala Pro Pro Glu Arg Pro Asp 485 490 495 Ser Pro Ser Thr Asn Gly Ser Gly Phe Glu Ile Leu Ser Pro Thr Ala 500 505 510 Pro Ser Val Tyr Pro Arg Ser Asp Gly His Gln Ser Arg Arg Gln Leu 515 520 525 Thr Thr Phe Gly Ser Gly Arg Pro Asp Arg Arg Tyr Ser Gln Ala Ser 530 535 540 Asp Ser Ser Val Phe Trp 545 550 <210> 18 <211> 1653 <212> DNA <213> Herpes simplex virus 1 <400> 18 atggatcgcg gggcggtggt ggggtttctt ctcggtgttt gtgttgtatc gtgcttggcg 60 ggaacgccca aaacgtcctg gagacgggtg agtgtcggcg aggacgtttc gttgcttcca 120 gctccggggc ctacggggcg cggcccgacc cagaaactac tatgggccgt ggaacccctg 180 gatgggtgcg gccccttaca cccgtcgtgg gtctcgctga tgccccccaa gcaggtgccc 240 gagacggtcg tggatgcggc gtgcatgcgc gctccggtcc cgctggcgat ggcgtacgcc 300 cccccggccc catctgcgac cgggggtcta cgaacggact tcgtgtggca ggagcgcgcg 360 gccgtggtta accggagtct ggttattcac ggggtccgag agacggacag cggcctgtat 420 accctgtccg tgggcgacat aaaggacccg gctcgccaag tggcctcggt ggtcctggtg 480 gtgcaaccgg ccccagttcc gaccccaccc ccgaccccag ccgattacga cgaggatgac 540 aatgacgagg gcgaggacga aagtctcgcc ggcactcccg ccagcgggac cccccggctc 600 ccgcctcccc ccgccccccc gaggtcttgg cccagcgccc ccgaagtctc acatgtgcgt 660 ggggtgaccg tgcgtatgga gactccggaa gctatcctgt tttcccccgg ggagacgttc 720 agcacgaacg tctccatcca tgccatcgcc cacgacgacc agacctactc catggacgtc 780 gtctggttga ggttcgacgt gccgacctcg tgtgccgaga tgcgaatata cgaatcgtgt 840 ctgtatcacc cgcagctccc agaatgtctg tccccggccg acgcgccgtg cgccgcgagt 900 acgtggacgt ctcgcctggc cgtccgcagc tacgcggggt gttccagaac aaacccccca 960 ccgcgctgtt cggccgaggc tcacatggag cccgtcccgg ggctggcgtg gcaggcggcc 1020 tccgtcaatc tggagttccg ggacgcgtcc ccacaacact ccggcctgta tctgtgtgtg 1080 gtgtacgtca acgaccatat tcacgcctgg ggccacatta ccatcagcac cgcggcgcag 1140 taccggaacg cggtggtgga acagcccctc ccacagcgcg gcgcggattt ggccgagccc 1200 acccacccgc acgtcggggc ccctccccac gcgcccccaa cccacggcgc cctgcggtta 1260 ggggcggtga tgggggccgc cctgctgctg tctgcgctgg ggttgtcggt gtgggcgtgt 1320 atgacctgtt ggcgcaggcg tgcctggcgg gcggttaaaa gcagggcctc gggtaagggg 1380 cccacgtaca ttcgcgtggc cgacagcgag ctgtacgcgg actggagctc ggacagcgag 1440 ggagaacgcg accaggtccc gtggctggcc cccccggaga gacccgactc tccctccacc 1500 aatggatccg gctttgagat cttatcacca acggctccgt ctgtataccc ccgtagcgac 1560 gggcatcaat ctcgccgcca gctcacaacc tttggatccg gaaggcccga tcgccgttac 1620 tcccaggcct ccgattcgtc cgtcttctgg taa 1653 <210> 19 <211> 550 <212> PRT <213> Herpes simplex virus 1 <400> 19 Met Asp Arg Gly Ala Val Val Gly Phe Leu Leu Gly Val Cys Val Val 1 5 10 15 Ser Cys Leu Ala Gly Thr Pro Lys Thr Ser Trp Arg Arg Val Ser Val 20 25 30 Gly Glu Asp Val Ser Leu Leu Pro Ala Pro Gly Pro Thr Gly Arg Gly 35 40 45 [[ID=3,4]]Pro Thr Gln Lys Leu Leu Trp Ala Val Glu Pro Leu Asp Gly Cys Gly 50 55 60 Pro Leu His Pro Ser Trp Val Ser Leu Met Pro Pro Lys Gln Val Pro 65 70 75 80 Glu Thr Val Val Asp Ala Ala Cys Met Arg Ala Pro Val Pro Leu Ala 85 90 95 Met Ala Tyr Ala Pro Pro Ala Pro Ser Ala Thr Gly Gly Leu Arg Thr 100 105 110 Asp Phe Val Trp Gln Glu Arg Ala Ala Val Val Asn Arg Ser Leu Val 115 120 125 Ile His Gly Val Arg Glu Thr Asp Ser Gly Leu Tyr Thr Leu Ser Val 130 135 140 Gly Asp Ile Lys Asp Pro Ala Arg Gln Val Ala Ser Val Val Leu Val 145 150 155 160 Val Gln Pro Ala Pro Val Pro Thr Pro Pro Pro Thr Pro Ala Asp Tyr 165 170 175 Asp Glu Asp Asp Asn Asp Glu Gly Glu Asp Glu Ser Leu Ala Gly Thr 180 185 190 Pro Ala Ser Gly Thr Pro Arg Leu Pro Pro Pro Pro Ala Pro Pro Arg 195 200 205 Ser Trp Pro Ser Ala Pro Glu Val Ser His Val Arg Gly Val Thr Val 210 215 220 Arg Met Glu Thr Pro Glu Ala Ile Leu Phe Ser Pro Gly Glu Thr Phe 225 230 235 240 Ser Thr Asn Val Ser Ile His Ala Ile Ala His Asp Asp Gln Thr Tyr 245 250 255 Ser Met Asp Val Val Trp Leu Arg Phe Asp Val Pro Thr Ser Cys Ala 260 265 270 Glu Met Arg Ile Tyr Glu Ser Cys Leu Tyr His Pro Gln Leu Pro Glu 275 280 285 Cys Leu Ser Pro Ala Asp Ala Pro Cys Ala Ala Ser Thr Trp Thr Ser 290 295 300 Arg Leu Ala Val Arg Ser Tyr Ala Gly Cys Ser Arg Thr Asn Pro Pro 305 310 315 320 Pro Arg Cys Ser Ala Glu Ala His Met Glu Pro Val Pro Gly Leu Ala 325 330 335 Trp Gln Ala Ala Ser Val Asn Leu Glu Phe Arg Asp Ala Ser Pro Gln 340 345 350 His Ser Gly Leu Tyr Leu Cys Val Val Tyr Val Asn Asp His Ile His 355 360 365 Ala Trp Gly His Ile Thr Ile Ser Thr Ala Ala Gln Tyr Arg Asn Ala 370 375 380 Val Val Glu Gln Pro Leu Pro Gln Arg Gly Ala Asp Leu Ala Glu Pro 385 390 395 400 Thr His Pro His Val Gly Ala Pro Pro His Ala Pro Pro Thr His Gly 405 410 415 Ala Leu Arg Leu Gly Ala Val Met Gly Ala Ala Leu Leu Leu Ser Ala 420 425 430 Leu Gly Leu Ser Val Trp Ala Cys Met Thr Cys Trp Arg Arg Arg Ala 435 440 445 Trp Arg Ala Val Lys Ser Arg Ala Ser Gly Lys Gly Pro Thr Tyr Ile 450 455 460 Arg Val Ala Asp Ser Glu Leu Tyr Ala Asp Trp Ser Ser Asp Ser Glu 465 470 475 480 Gly Glu Arg Asp Gln Val Pro Trp Leu Ala Pro Pro Glu Arg Pro Asp 485 490 495 Ser Pro Ser Thr Asn Gly Ser Gly Phe Glu Ile Leu Ser Pro Thr Ala 500 505 510 Pro Ser Val Tyr Pro Arg Ser Asp Gly His Gln Ser Arg Arg Gln Leu 515 520 525 Thr Thr Phe Gly Ser Gly Arg Pro Asp Arg Arg Tyr Ser Gln Ala Ser 530 535 540 Asp Ser Ser Val Phe Trp 545 550 <210> 20 <211> 3267 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: synthetic polynucleotide <220> <221> introns <222> (58)..(861) <223> introns <220> <221> mutation <222> (848)..(848) <223> a848c <220> <221> introns <222> (1529)..(1663) <223> introns <220> <221> mutation <222> (1712)..(1712) <223> g1712a <400> 20 atggagcccc gccccggagc gagtacccgc cggcctgagg gccgccccca gcgcgaggtg 60 aggggccggg cgccatgtct ggggcgccat gtctggggcg ccatgtctgg ggcgccatgt 120 ctggggcgcc atgttggggg gcgccatgtt gggggcgcc atgttggggg acccccgacc 180 cttacactgg aaccggccgc catgttgggg gacccccact catacacggg agccgggcgc 240 catgttgggg cgccatgtta gggggcgtgg aaccccgtga cactatatat acagggaccg 300 ggggcgccat gttagggggc gcggaacccc ctgaccctat atatacaggg accggggtcg 360 ccctgttagg ggtcgccatg tgaccccctg actttatata tacagacccc caacacctac 420 acatggcccc tttgactcag acgcagggcc cggggtcgcc gtgggacccc cctgactcat 480 acacagagac acgcccccac aacaaacaca cagggaccgg ggtcgccgtg ttagggggcg 540 tggtccccac tgactcatac gcagggcccc cttactcaca cgcatctagg ggggtgggga 600 ggagccgccc gccatatttg ggggacgccg tgggaccccc gactccggtg cgtctggagg 660 gcgggagaag agggaagaag aggggtcggg atccaaagga cggacccaga ccacctttgg 720 ttgcagaccc ctttctcccc cctcttccga ggccagcagg ggggcaggac tttgtgaggc 780 ggggggggag ggggaactcg tgggcgctga ttgacgcggg aaatcccccc attcttaccc 840 gccccccctt tttcccctca gcccgccccg gatgtctggg tgtttccctg cgaccgagac 900 ctgccggaca gcagcgactc ggaggcggag accgaagtgg gggggcgggg ggacgccgac 960 caccatgacg acgactccgc ctccgaggcg gacagcacgg acacggaact gttcgagacg 1020 gggctgctgg ggccgcaggg cgtggatggg ggggcggtct cgggggggag ccccccccgc 1080 gaggaagacc ccggcagttg cgggggcgcc ccccctcgag aggacggggg gagcgacgag 1140 ggcgacgtgt gcgccgtgtg cacggatgag atcgcgcccc acctgcgctg cgacaccttc 1200 ccgtgcatgc accgcttctg catcccgtgc atgaaaacct ggatgcaatt gcgcaacacc 1260 tgcccgctgt gcaacgccaa gctggtgtac ctgatagtgg gcgtgacgcc cagcgggtcg 1320 ttcagcacca tcccgatcgt gaacgacccc cagacccgca tggaggccga ggaggccgtc 1380 agggcgggca cggccgtgga ctttatctgg acgggcaatc agcggttcgc cccgcggtac 1440 ctgaccctgg gggggcacac ggtgagggcc ctgtcgccca cccacccgga acccaccacg 1500 gacgaggatg acgacgacct ggacgacggt gaggcggggg gcggcaagga ccctggggga 1560 ggaggaggag gagggggggg agggaggaat aggcgggcgg gcgaggaaag ggcgggccgg 1620 ggagggggcg taacctgatc gcgccccccg ttgtctcttg cagcagacta cgtaccgccc 1680 gccccccgcc ggacgccccg cgcccccccca cacagaggcg ccgccgcgcc ccccgtgacg 1740 ggcggggcgt ctcacgcagc cccccagccg gccgcggctc ggacagcgcc cccctcggcg 1800 cccatcgggc cacacggcag cagtaacacc aacaccacca ccaacagcag cggcggcggc 1860 ggctcccgcc agtcgcgagc cgcggcgccg cggggggcgt ctggcccctc cgggggggtt 1920 ggggttgggg ttggggttgt tgaagcggag gcggggcggc cgaggggccg gacgggcccc 1980 cttgtcaaca gacccgcccc ccttgcaaac aacagagacc ccatagtgat cagcgactcc 2040 cccccggcct ctccccacag gccccccgcg gcgcccatgc caggctccgc cccccgcccc 2100 gggccccccg cgtccgcggc cgcgtcggga cccgcgcgcc cccgcgcggc cgtggccccg 2160 tgcgtgcgag cgccgcctcc ggggcccggc ccccgcgccc cggccccccgg ggcggagccg 2220 gccgccccgcc ccgcggacgc gcgccgtgtg ccccagtcgc actcgtccct ggctcaggcc 2280 gcgaaccaag aacagagtct gtgccgggcg cgtgcgacgg tggcgcgcgg ctcgggggggg 2340 ccgggcgtgg agggtgggca cgggccctcc cgcggcgccg ccccctccgg cgccgccccg 2400 ctcccctccg ccgcctctgt cgagcaggag gcggcggtgc gtccgaggaa gaggcgcggg 2460 tcgggccagg aaaacccctc cccccagtcc acgcgtcccc ccctcgcgcc ggcaggggcc 2520 aagagggcgg cgacgcaccc cccctccgac tcagggccgg gggggcgcgg ccagggtggg 2580 cccgggaccc ccctgacgtc ctcggcggcc tccgcctctt cctcctctgc ctcttctccc 2640 tcggccccga cccccgcggg ggccgcctct tccgccgccg gggccgcgtc ctcctccgct 2700 tccgcctcct cgggcggggc cgtcggtgcc ctgggaggga gaagaagga aacctccctc 2760 ggcccccgcg ctgcttctgg gccgcgggg ccgaggaagt gtgcccggaa gacgcgccac 2820 gcggagactt ccggggccgt ccccgcgggc ggcctcacgc gctacctgcc catctcgggg 2880 gtctctagcg tggtcgccct gtcgcccttac gtgaacaaga cgatcacggg ggactgcctg 2940 cccatcctgg acatggagac ggggaacatc ggggcgtacg tggtcctggt ggaccagacg 3000 ggaaacatgg cgacccggct gcgggccgcg gtccccggct ggagccgccg caccctgctc 3060 cccgagaccg cgggtaacca cgtgatgccc cccgagtacc cgacggcccc cgcgtcggag 3120 tggaacagcc tctggatgac ccccgtgggg aacatgctgt tcgaccaggg caccctagtg 3180 ggcgccctgg acttccgcag cctgcggtct cggcacccgt ggtccgggga gcaggggggcg 3240 tcgacccggg acgagggaaa acaataa 3267 <210> twenty one <211> 775 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptide <220> <221> MISC_FEATURE <222> (258)..(258) <223> R258H <400> twenty one Met Glu Pro Arg Pro Gly Ala Ser Thr Arg Arg Pro Glu Gly Arg Pro 1 5 10 15 Gln Arg Glu Pro Ala Pro Asp Val Trp Val Phe Pro Cys Asp Arg Asp 20 25 30 Leu Pro Asp Ser Ser Asp Ser Glu Ala Glu Thr Glu Val Gly Gly Arg 35 40 45 Gly Asp Ala Asp His His Asp Asp Asp Ser Ala Ser Glu Ala Asp Ser 50 55 60 Thr Asp Thr Glu Leu Phe Glu Thr Gly Leu Leu Gly Pro Gln Gly Val 65 70 75 80 Asp Gly Gly Ala Val Ser Gly Gly Ser Pro Pro Arg Glu Glu Asp Pro 85 90 95 Gly Ser Cys Gly Gly Ala Pro Pro Arg Glu Asp Gly Gly Ser Asp Glu 100 105 110 Gly Asp Val Cys Ala Val Cys Thr Asp Glu Ile Ala Pro His Leu Arg 115 120 125 Cys Asp Thr Phe Pro Cys Met His Arg Phe Cys Ile Pro Cys Met Lys 130 135 140 Thr Trp Met Gln Leu Arg Asn Thr Cys Pro Leu Cys Asn Ala Lys Leu 145 150 155 160 Val Tyr Leu Ile Val Gly Val Thr Pro Ser Gly Ser Phe Ser Thr Ile 165 170 175 Pro Ile Val Asn Asp Pro Gln Thr Arg Met Glu Ala Glu Glu Ala Val 180 185 190 Arg Ala Gly Thr Ala Val Asp Phe Ile Trp Thr Gly Asn Gln Arg Phe 195 200 205 Ala Pro Arg Tyr Leu Thr Leu Gly Gly His Thr Val Arg Ala Leu Ser 210 215 220 Pro Thr His Pro Glu Pro Thr Thr Asp Glu Asp Asp Asp Asp Leu Asp 225 230 235 240 Asp Ala Asp Tyr Val Pro Pro Ala Pro Arg Arg Thr Pro Arg Ala Pro 245 250 255 Pro His Arg Gly Ala Ala Ala Pro Pro Val Thr Gly Gly Ala Ser His 260 265 270 Ala Ala Pro Gln Pro Ala Ala Ala Arg Thr Ala Pro Pro Ser Ala Pro 275 280 285 Ile Gly Pro His Gly Ser Ser Asn Thr Asn Thr Thr Thr Asn Ser Ser 290 295 300 Gly Gly Gly Gly Ser Arg Gln Ser Arg Ala Ala Ala Pro Arg Gly Ala 305 310 315 320 Ser Gly Pro Ser Gly Gly Val Gly Val Gly Val Gly Val Val Glu Ala 325 330 335 Glu Ala Gly Arg Pro Arg Gly Arg Thr Gly Pro Leu Val Asn Arg Pro 340 345 350 Ala Pro Leu Ala Asn Asn Arg Asp Pro Ile Val Ile Ser Asp Ser Pro 355 360 365 Pro Ala Ser Pro His Arg Pro Pro Ala Ala Pro Met Pro Gly Ser Ala 370 375 380 Pro Arg Pro Gly Pro Pro Ala Ser Ala Ala Ala Ser Gly Pro Ala Arg 385 390 395 400 Pro Arg Ala Ala Val Ala P...

Claims

1. A non-natural herpes simplex virus ("HSV") obtained by the following method: infecting host cells with 17TermA and rRp450, and culturing the host cells after serial passages to isolate HSV Mut-3 mutants containing a WT-like genotype, wherein the Mut-3 mutants have the following non-synonymous mutations compared to the parent virus: alanine to threonine mutation at position 151 of gE protein, arginine to histidine mutation at position 258 of ICP0 protein, alanine to threonine mutation at position 376 of DNA packaging terminase subunit 1 protein, threonine to methionine mutation at position 1155 of ICP8 protein, and proline to histidine mutation at position 119 of ICP34.5 protein, In the Mut-3 mutant, the amino acid sequence of gE protein is SEQ ID NO: 13, the amino acid sequence of ICP0 is SEQ ID NO: 21, the amino acid sequence of DNA packaging terminase subunit 1 protein is SEQ ID NO: 36, the amino acid sequence of ICP8 protein is SEQ ID NO: 28, and the amino acid sequence of ICP34.5 protein is SEQ ID NO:

2.

2. A non-natural herpes simplex virus ("HSV") obtained by: Host cells were infected with 17TermA and rRp450 and cultured after serial passages to isolate HSV Mut-3 mutants containing a WT-like genotype, the Mut-3 mutants having the following non-synonymous mutations compared to the parental virus: alanine to threonine mutation at position 151 of gE protein, arginine to histidine mutation at position 258 of ICP0 protein, alanine to threonine mutation at position 376 of DNA packaging terminase subunit 1 protein, threonine to methionine mutation at position 1155 of ICP8 protein, and proline to histidine mutation at position 119 of ICP34.5 protein, wherein in the Mut-3 mutant, the amino acid sequence of the gE protein is SEQ ID NO: 13, the amino acid sequence of ICP0 is SEQ ID NO: 21, the amino acid sequence of the DNA packaging terminase subunit 1 protein is SEQ ID NO: 36, the amino acid sequence of the ICP8 protein is SEQ ID NO: 28, and the amino acid sequence of the ICP34.5 protein is SEQ ID NO: 2; Furthermore, the ICP34.5 protein coding region is removed from the Mut-3 mutant to generate the Mut 3Δ34.5 mutant.

3. A non-natural herpes simplex virus ("HSV") obtained by: Host cells were infected with 17TermA and rRp450 and cultured after serial passages to isolate HSV Mut-3 mutants containing a WT-like genotype, the Mut-3 mutants having the following non-synonymous mutations compared to the parental virus: alanine to threonine mutation at position 151 of gE protein, arginine to histidine mutation at position 258 of ICP0 protein, alanine to threonine mutation at position 376 of DNA packaging terminase subunit 1 protein, threonine to methionine mutation at position 1155 of ICP8 protein, and proline to histidine mutation at position 119 of ICP34.5 protein, wherein in the Mut-3 mutant, the amino acid sequence of the gE protein is SEQ ID NO: 13, the amino acid sequence of ICP0 is SEQ ID NO: 21, the amino acid sequence of the DNA packaging terminase subunit 1 protein is SEQ ID NO: 36, the amino acid sequence of the ICP8 protein is SEQ ID NO: 28, and the amino acid sequence of the ICP34.5 protein is SEQ ID NO: 2; Furthermore, the ICP6 protein coding region is removed from the Mut-3 mutant to generate the Mut 3ΔICP6 mutant.

4. The non-natural HSV of any one of claims 1-3, wherein the HSV is derived from an HSV type 1 ("HSV-1") or HSV type 2 ("HSV-2") strain.

5. The non-natural HSV of any one of claims 1-3, wherein the HSV is derived from the HSV-1 KOS strain.

6. The non-natural HSV of any one of claims 1-3, further comprising a transgene.

7. A composition comprising the non-natural HSV according to any one of claims 1-6 and a carrier.

8. The composition of claim 7, wherein the carrier is a pharmaceutically acceptable carrier.

9. The composition of claim 7 or 8, further comprising a cryopreservative that facilitates freezing and thawing of the non-natural HSV without significant loss of virulence.

10. A cell infected with the non-natural HSV according to any one of claims 1 to 6.

11. The cell of claim 10, wherein the cell is a lymphocyte.

12. An in vitro method for infecting a cell, comprising contacting the cell with a non-natural HSV as described in any one of claims 1-6 or a composition as described in any one of claims 7-9.

13. The method of claim 12, wherein the cells are lymphocytes.

14. The method of claim 12 or 13, wherein the cells have been infected with Epstein-Barr virus ("EBV").

15. A method for preparing a non-natural HSV having a transgene, comprising introducing the transgene into the non-natural HSV according to any one of claims 1-5.

16. Use of the non-natural HSV according to any one of claims 1 to 6 or the composition according to any one of claims 7 to 9 in the preparation of a medicament for inhibiting the growth or metastasis of neuroblastoma.

17. Use of the non-natural HSV of any one of claims 1-6 or the composition of any one of claims 7-9 in the preparation of a medicament for treating cancer in a subject.

18. An in vitro method for inducing lysis of neuroblastoma cells, comprising contacting the cells with the non-natural HSV of any one of claims 1-6 or the composition of any one of claims 7-9.

19. A kit comprising the non-natural HSV of any one of claims 1-6 or the composition of any one of claims 7-9, and instructions for use.

Images