Method for rescuing type A H1N1 recombinant influenza virus carrying green fluorescent protein and preparing mouse adaptive strain

Abstract

The invention discloses a method for rescuing a type A H1N1 recombinant influenza virus carrying a green fluorescent protein and a method for preparing a mouse adaptive strain. The method comprises the following steps: rescuing through a reverse genetic manipulation technology to obtain WT-CA04-Venus carrying a Venus reporter gene; the method comprises the following steps: carrying out passage transfection on WT-CA04-Venus, and purifying and screening a bacterial strain MA-CA04-Venus adapted to a mouse; compared with WT-CA04-Venus, three amino acids, namely PB2K340T, PA M21I and PA L175L, are replaced in the genome of the MA-CA04-Venus, and the MA-CA04-Venus prepared by the invention is stable in toxicity, strong in replication ability, wide in application range, not easy to lose fluorescent protein, and suitable for popularization and application in the scenes of research, clinic, test and the like.

Description

Technical Field

[0001] The present invention relates to a method for rescuing and preparing a mouse-adapted strain, and more specifically, to a method for rescuing a recombinant influenza virus A (H1N1) carrying a green fluorescent protein and preparing a mouse-adapted strain, belonging to the technical field of biological genetic engineering. Background Art

[0002] Influenza A virus (IAV) belongs to the family Orthomyxoviridae and can cause severe respiratory diseases, especially in the elderly and children. Influenza A virus is classified based on the differences in hemagglutinin and neuraminidase, with 18 and 11 subtypes, respectively. The pdmH1N1 / 2009 influenza A virus was first discovered in Mexico in 2009 and quickly spread around the world, posing a threat to public health. In addition, H7N9, H10N8 and other new influenza viruses (H3N8, H7N4) also pose a threat to public health.

[0003] At present, pdmH1N1 or pdmH1N1-like influenza virus has become the main circulating strain of seasonal influenza. All seasonal influenza vaccines contain this component to induce specific immune responses. Therefore, understanding the pathogenicity and viral replication mechanism of pdmH1N1 influenza virus is an urgent need to prevent and control influenza virus infection. Although influenza virus has been widely studied, there is limited understanding of the dynamic process of infection and viral target cells in vivo. With the development of new technologies such as in vivo imaging system (IVIS) and micro-computed tomography (Micro-CT), real-time detection of influenza virus infection dynamics in vivo has been achieved. At present, some researchers have generated a variety of replication-competent reporter viruses that carry a variety of fluorescent or bioluminescent reporter genes, including eGFP, mCherry and Gluc, which are located in different fragments of the viral genome, mainly PB2, PA, NA and NS fragments. These reporter influenza viruses are powerful tools for identifying antiviral compounds, effective or broadly reactive monoclonal antibodies, or host factors involved in the viral life cycle in vitro. Similarly, these reporter-expressing and replication-competent IAVs can be used to monitor viral infection dynamics in tissue culture cells and animal models, and can also be used to test the effects of antiviral drugs and vaccines in vivo without invasiveness. However, these reporter viruses also have some limitations, as follows:

[0004] 1) First, most reported IAVs with replication competence are laboratory modified strain backbones, including PR8 and WSN viruses. However, the characteristics of these model IAV strains are not always consistent with current clinical isolates.

[0005] 2) Second, many reporter IAVs have poor replication capacity compared to the corresponding parental IAVs; this will affect the scope of their use and make them change compared to the real in vivo infection dynamics.

[0006] 3) Then, the foreign genes inserted into the IAV genome are partially deleted during replication and are therefore very unstable, especially in vivo.

[0007] In summary, there is an urgent need to rescue a stable, virulence reporter-expressing, replication-competent influenza A virus (IAV) based on clinical isolates. Summary of the invention

[0008] The object of the present invention is to provide a method for rescuing a recombinant influenza virus carrying green fluorescent protein and preparing a mouse-adapted strain thereof, which has the technical characteristics of strong replication ability of the prepared MA-CA04-Venus, high stability of fluorescent protein, stable virulence (toxicity), etc.

[0009] In order to achieve the above object, the present invention is implemented by the following technical solutions:

[0010] The present invention discloses a method for rescuing a recombinant influenza virus carrying green fluorescent protein (EFP) and preparing a mouse-adapted strain, the method comprising the following steps:

[0011] Step 1: Rescue the CA04 recombinant influenza virus strain carrying the Venus reporter gene by reverse genetic manipulation technology, wherein the CA04 recombinant influenza virus strain is WT-CA04-Venus;

[0012] Step 2: Subculture and transfect WT-CA04-Venus, purify and screen the mouse-adapted strain MA-CA04-Venus; compared with WT-CA04-Venus, three amino acids in the genome of MA-CA04-Venus were replaced, namely PB2 K340T, PA M21I and PA L175L.

[0013] Preferably, step 1 comprises rearranging the NS segment of CA04, expressing NS1-Venus and NEP as a polyprotein, connecting them through the 2A autoprotein cleavage site of porcine terser virus-1 (PTV-1), releasing NEP from the upstream NS1-Venus protein during translation, and introducing two silent mutations at the splicing acceptor site to avoid splicing of CA04's NS mRNA.

[0014] Preferably, the method comprises mixing 8 pHH21 recombinant plasmids, 4 pCAGGS recombinant plasmids and Lipofectamine LTXplus reagent with Opti-MEM, and culturing at room temperature for 20 minutes; adding the mixture of plasmids and transfection reagent to a 6-well plate containing 293T cells using Opti-MEM, and adding TPCK trypsin with a final concentration of 0.5 μg / mL 24 hours after transfection.

[0015] Preferably, the eight pHH21 recombinant plasmids are: pHH21-CA04-PB2, pHH21-CA04-PB1, pHH21-CA04-PA, pHHH21-CA04-HA, pHHH21-CA04-NP, pHHH21-CA04-NA, pHHH21-CA04-M, pHH21-CA04--NS-Venus;

[0016] The four pCAGGS recombinant plasmids are pCAGGS-CA04-PB2, pCAGGS-CA04-PB1, pCAGGS-CA04-PA and pCAGGS-CA04-NP.

[0017] Preferably, in step 2, WT-CA04-Venus is transfected into mice for 9 generations, and starting from the fifth generation of CA04-Venus, MDCK cells are inoculated every other generation, and finally, a mouse-adapted CA04-Venus strain is obtained.

[0018] The present invention provides an application of WT-CA04-Venus, and the applications of WT-CA04-Venus include monitoring the dynamics of viral infection in tissue culture cells and animal models; testing the non-invasive effects of antiviral drugs and vaccines in vivo; and identifying antiviral compounds, effective or broadly responsive monoclonal antibodies, and host factors that can participate in and affect the viral life cycle in an in vitro environment.

[0019] Beneficial effects: The prepared MA-CA04-Venus has stable toxicity, strong replication ability, wide range of applications, and is not easy to lose fluorescent protein. It is suitable for promotion and use in research, clinical, experimental and other scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of CA04-NS carrying the Venus reporter gene of the present invention.

[0021] Figure 2 This is one of the images of detecting the expression of fluorescent protein after 293T cells were transfected with the plasmid of the present invention.

[0022] Figure 3This is the second figure of detecting the expression of fluorescent protein after 293T cells were transfected with the plasmid of the present invention.

[0023] Figure 4 The blood coagulation diagram is obtained after the cells and cell culture supernatant of the present invention are inoculated into chicken embryos 72 hpi.

[0024] Figure 5 This is one of the passage diagrams of WT-CA04-Venus in chicken embryos and mammalian cells of the present invention.

[0025] Figure 6 This is the second diagram of the passage of WT-CA04-Venus of the present invention in chicken embryos and mammalian cells.

[0026] Figure 7 This is a diagram of the WT-CA04-Venus of the present invention being serially passaged from lung to lung of mice.

[0027] Figure 8 It is a schematic diagram of the fluorescent protein of WT-CA04-Venus of the present invention during the mouse passage process.

[0028] Fig. 9 This is a picture of fluorescent protein expressed in lung homogenate of the fifth generation WT-CA04-Venus of the present invention.

[0029] Fig.10 It is a growth kinetic diagram of WT-CA04-Venus and MA-CA04-Venus of the present invention in MDCK cells at 33°C.

[0030] Fig.11 It is a growth kinetic diagram of WT-CA04-Venus and MA-CA04-Venus of the present invention in MDCK cells at 37°C.

[0031] Fig.12 The present invention is a graph of analyzing the firefly and Renilla luciferase activities of cell lysates after incubating the transfected cells at 33°C for 24 hours.

[0032] Fig.13 The present invention is a graph of analyzing the firefly and Renilla luciferase activities of cell lysates after incubating the transfected cells at 37° C. for 24 hours.

[0033] In the figure: Figure 1 The spliceosome acceptor site (SA) in NS was mutated to prevent mRNA splicing. NS1 was fused to Venus via a linker (GSGG) followed by the PTV-1 2A autoproteolytic cleavage site.

[0034] Figure 5The WT-CA04-Venus virus was serially diluted 10-fold and inoculated into the allantoic cavity of SPF chicken embryos in a volume of 200 μL from 10-3 to 10-5, with 3 eggs per dilution, and incubated at 35°C for 72 hours, and then the above process was repeated. The E1-E4 generation viruses were inoculated into MDCK cells with a final concentration of 1 μg / mL TPCK-trypsin.

[0035] Figure 10-11 MDCK cells were infected with the indicated recombinant influenza viruses at an MOI of 0.01, and cell supernatants were collected every 12 h until 72 h post-infection. Virus titers were determined by plaque assay. Data shown were triplicates, and values ​​are standard deviation ± mean.

[0036] Figure 12-13 In the table, the values ​​shown are the mean ± SD of the data from three independent experiments and normalized to the WT-CA04-Venus activity (100%). *, p < 0.05 compared with WT-CA04-Venus virus. **, p < 0.01 compared with WT-CA04-Venus virus. ns, no significant difference. DETAILED DESCRIPTION

[0037] The present invention will be further described below in conjunction with the accompanying drawings, but the present invention is not limited to the following embodiments.

[0038] The present application rescued a recombinant A / California / 04 / 2009 (H1N1, CA04) virus expressing fluorescent protein and having strong replication ability through reverse genetic manipulation technology, and its NS segment contained Venus gene, which was called WT-CA04-Venus. By rearranging the NS segment of CA04, NS1-Venus and NEP were expressed as a polyprotein, and they were connected through the porcine terser virus-1 (PTV-1) 2A self-protein hydrolysis cleavage site, so that NEP was released from the upstream NS1-Venus protein during translation. Two silent mutations were introduced at the splicing acceptance site to avoid splicing of CA04's NS mRNA. After the WT-CA04-Venus reporter virus was transmitted from lung to lung for 9 times, the infected mice showed severe symptoms on the 3rd day after inoculation. A mouse-adapted CA04-Venus (MA-CA04-Venus) was purified from the lungs of mice infected by the ninth passage.

[0039] like Figure 1-13 The figure shows a specific embodiment of a method for rescuing a recombinant influenza virus A (H1N1) carrying green fluorescent protein and preparing a mouse-adapted strain. The embodiment is a method for rescuing a recombinant influenza virus A (H1N1) carrying green fluorescent protein and preparing a mouse-adapted strain. The preparation method comprises the following steps:

[0040] Step 1: Rescue the CA04 recombinant influenza virus strain carrying the Venus reporter gene by reverse genetic manipulation technology, wherein the CA04 recombinant influenza virus strain is WT-CA04-Venus;

[0041] Step 2: Subculture WT-CA04-Venus and purify and select the mouse-adapted strain MA-CA04-Venus; subculture WT-CA04-Venus to mice for 9 times, and inoculate MDCK cells every other generation starting from the fifth generation CA04-Venus, and finally obtain a mouse-adapted CA04-Venus strain. Compared with WT-CA04-Venus, three amino acids in the genome of the MA-CA04-Venus have been replaced, namely PB2 K340T, PA M21I and PA L175L.

[0042] The present application rescues the recombinant pdm H1N1 CA04 reporter influenza virus WT-CA04-Venus expressing the Venus fluorescent protein. The recombinant influenza virus can maintain the stability of the Venus gene inserted into the genome in limited in vitro passages. Compared with WT-CA04-Venus, MA-CA04-Venus has brighter fluorescence, higher growth ability and stronger virulence. And through specific examples, it is shown that PA M21I is a potential virulence determinant, and the PB2 K340T and PAL175F mutations are the reasons for the enhanced stability of the Venus gene inserted into the MA-CA04-Venus genome. These results broaden the understanding of influenza virus replication, and provide a powerful tool for exploring the interaction between viruses and hosts, evaluating the efficacy of antiviral drugs and broad-spectrum anti-reactive monoclonal antibodies, and combining new imaging technologies to detect the dynamics of viral infection caused by 2009H1N1 influenza viruses.

[0043] In a preferred embodiment, step 1 includes rearranging the NS segment of CA04, expressing NS1-Venus and NEP as a polyprotein, connecting them through the 2A autoprotein cleavage site of porcine terseter virus-1 (PTV-1), releasing NEP from the upstream NS1-Venus protein during translation, and introducing two silent mutations at the splicing acceptance site to avoid splicing of NS mRNA of CA04. It includes mixing 8 pHH21 recombinant plasmids, 4 pCAGGS recombinant plasmids and Lipofectamine LTX plus reagent with Opti-MEM, culturing at room temperature for 20 minutes; adding the mixture of plasmids and transfection reagent to a 6-well plate containing 293T cells with Opti-MEM, and adding TPCK trypsin with a final concentration of 0.5μg / mL 24 hours after transfection. The 8 pHH21 recombinant plasmids are: pHH21-CA04-PB2, pHH21-CA04-PB1, pHH21-CA04-PA, pHHH21-CA04-HA, pHHH21-CA04-NP, pHHH21-CA04-NA, pHHH21-CA04-M, pHH21-CA04-HA, pHH21-CA04-NP, pHH21-CA04-NA, pHH21-CA04-M; the 4 pCAGGS recombinant plasmids are pCAGGS-CA04-PB2, pCAGGS-CA04-PB1, pCAGGS-CA04-PA and pCAGGS-CA04-NP.

[0044] Example 1: The MA-CA04-Venus prepared in the present application was identified in vitro and in vivo, and the molecular determinants of the virulence and stability of the replication-competent reporter virus MA-CA04-Venus were studied. The details are as follows:

[0045] 1. Materials and Methods

[0046] 1.1 Cells and viruses

[0047] Human embryonic kidney epithelial cells (293T, Cat. No. CRL-3216) were obtained from the American Type Culture Collection (ATCC) and cultured in DMEM (Gibco) containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin solution (Gibco). Madin-Darby canine kidney cells (MDCK, Cat. No: CCL-34) obtained from ATCC were cultured in DMEM (Gibco) containing 5% FBS and 1% penicillin / streptomycin in an incubator at 37°C with 5% CO2.

[0048] A / California / 04 / 09 (CA04, H1N1) and CA04 recombinant influenza virus strain carrying the Venus reporter gene (WT-CA04-Venus) were rescued by reverse genetic manipulation technology.

[0049] 1.2 Plasmid

[0050] Method for constructing virus rescue plasmid: 8 gene fragments of CA04 were amplified by reverse transcription-PCR, and then 7 of them were inserted into the pHH21 vector by BsmBI restriction enzyme digestion, namely pHH21-CA04-PB2, pHH21-CA04-PB1, pHH21-CA04-PA, pHHH21-CA04-HA, pHHH21-CA04-NP, pHHH21-CA04-NA, pHHH21-CA04-M, and pHH21-CA04--NS-Venus; the Venus gene was inserted into the NS fragment by overlapping extension PCR, and then integrated into the pHH21 vector by homologous recombination, named pHH21-CA04 NS-Venus. Next, the ORFs of the polymerase and NP genes (PB2 / PB1 / PA and NP) were inserted into the pCAGGS vectors, named pCAGGS-CA04-PB2, pCAGGS-CA04-PB1, pCAGGS-CA04-PA, and pCAGGS-CA04-NP. Mutant genes were generated using PCR-directed mutagenesis with point mutation primers. All plasmids were sequenced to ensure that there were no unwanted mutations.

[0051] 1.3 Virus rescue

[0052] One day before transfection of mice, 293T cells were seeded into six-well plates. Eight pHH21 recombinant plasmids, four pCAGGS recombinant plasmids, and Lipofectamine LTX plus reagent (ThermoFisher Scientific, USA) were mixed with Opti-MEM (Gibco, New York, USA) and incubated at room temperature for 20 minutes. The mixture of plasmids and transfection reagents was added to the 6-well plate with Opti-MEM (Gibco, New York, USA). TPCK trypsin was added at a final concentration of 0.5 μg / mL 24 hours after transfection. The supernatant and cells were then collected 48 hours after transfection, and the mixture was inoculated into 10-day-old specific pathogen-free (SPF) chicken embryo eggs (Boehringer Ingelheim, Beijing, China) for virus propagation at 35°C. The virus was detected by hemagglutinin test, and the whole genome was sequenced by Sangle method to avoid unnecessary mutations.

[0053] WT-Venus-CA04 was cultured for 4 generations in 10-day-old SPF chicken embryo eggs. Meanwhile, WT-CA04-Venus was subcultured in MEM (Gibco, New York, USA) at a concentration of MOI = 0.01 for MDCK cells, MEM supplemented with 0.3% bovine serum albumin (BSA, Sigma, Commonwealth of Pennsylvania, United Kingdom) and 1 μg / mL TPCK-trypsin, and subcultured 6 times at 33°C, and the supernatant and cells were collected when the cell lesion reached more than 80%.

[0054] 1.4 Transplantation of WT-CA04-Venus into mouse lungs

[0055] To obtain the mouse-adapted CA04-virus, 6-week-old SPF female BALB / c mice (SLAC, Shanghai, China) were anesthetized with dry ice, and the WT-CA04-virus and WT-CA04-Venus were inoculated intranasally and passaged in the mouse lungs for nine times, and the mouse-adapted CA04-Venus strain (MA-CA04-Venus) was selected by plaque purification.

[0056] In order to study the pathogenicity of MA-Venus-CA04, this application uses 10 5 PFU / 50 μL MA-Venus-CA04 or WT-Venus-CA04 were used to anesthetize mice. Five 6-week-old SPF female BALB / c mice (SLAC, Shanghai, China) were used in each group, and the body weight and survival rate of the mice were observed every day for 14 consecutive days.

[0057] 1.5 Growth kinetics assay

[0058] MDCK cells were infected with WT-Venus-CA04 or MA-Venus-CA04 at an MOI of 0.01 in triplicate and cultured at 33°C or 37°C with MEM containing 0.3% BSA (Sigma, Commonwealth of Pennsylvania, UK) and 1 μg / ml TPCK-trypsin. Supernatants were collected at 12, 24, 36, 48, 60 and 72 hours after infection. Subsequently, the virus titer in the supernatant at each time point was determined by plaque assay.

[0059] 1.6 Microgenome testing

[0060] pCAGGS-CA04 PB2, pCAGGS-CA04 PB1, pCAGGS-CA04 PA and pCAGGS-CA04 NP (0.25 μg each) of WT-CA04-Venus or MA-CA04-Venus were co-transfected with a plasmid expressing firefly luciferase protein (pPolⅠ-NP(0)Luc(0), 0.25 μg) and a plasmid expressing Renilla luciferase gene (pRL-null, 0.25 μg) into 293T cells using Lipofectamine LTX and Plus Reagent (ThermoFisher Scientific, USA). 48 hours after transfection, the supernatant was removed and cell lysate was prepared. The activities of firefly luciferase and Renilla luciferase were detected using the Dual-Glo Luciferase Assay System and GloMax The activity was measured using a 96-well microplate luminometer (Promega) (30).

[0061] 1.77 Statistical analysis

[0062] Statistical analysis was performed using GraphPad Prism 6.0 (GraphPad Software Inc., San Diego, CA, USA) and Student's T test was used.

[0063] 2. Research results of this embodiment

[0064] 2.1 Insertion of Venus reporter gene into the NS segment of pdmH1N1 virus

[0065] The Venus fluorescent reporter gene is a GFP variant with eight mutations that effectively improve the formation of the chromophore and the fluorescence intensity. In this study, the Venus reporter gene was inserted into the C-terminus of the pdmH1N1CA04 influenza virus NS1 and fused to it via a GSGG linker. Two mutations were introduced at the splice acceptor (SA) site to prevent splicing by mRNA processing (e.g. Figure 1 The transfection process was as described in the Materials and Methods section. 48 hours after transfection, cells were first examined using a fluorescence microscope, and the supernatant and cells (eg Figure 2-3 ). Subsequently, the mixture was inoculated into 10-day-old SPF chicken embryos and incubated at 35°C. 72 hours after inoculation, the allantoic fluid was harvested, and the allantoic fluid had hemagglutination activity (such as Figure 4 ). Sanger sequencing of the whole genome of WT-CA04-Venus confirmed the absence of unwanted mutations.

[0066] 2.2 In vitro characteristics of WT-CA04-Venus virus

[0067] At present, many influenza viruses with different fluorescent reporter genes and locations have been constructed. However, these influenza viruses carrying fluorescent reporter genes are very easy to lose foreign genes. Initially, WT-CA04-Venus embryonic eggs were passaged four times in succession. These four generations of viruses infected MDCK cells with the same dose, and fluorescent protein expression was observed 24 hours after infection. However, less fluorescent expression was observed in the fourth generation (such as Figure 5 ). WT-CA04-Venus can be stably present in the first few generations in chicken embryos, but the stability of its fluorescent protein decreases with the increase of the number of passages. MDCK cells were infected with 0.01 MOI and the fluorescence expression was observed for six consecutive generations. It was found that the fluorescence intensity of the sixth generation was lower than that of the first five generations. It was concluded that the fluorescent protein of the sixth generation WT-CA04-Venus may be partially lost (such as Figure 6 ). Therefore, WT-CA04-Venus can only maintain the stability of the fluorescent gene inserted into the genome in a limited number of in vitro passages.

[0068] 2.3 The mouse-adapted strain MA-CA04-Venus was obtained by mouse lung-to-lung passage and plaque purification.

[0069] pdm H1N1 CA04 influenza virus is less pathogenic to mice. To generate a mouse-adapted CA04-Venus strain, Figure 7 As shown, the present application performed continuous passage of WT-CA04-Venus from lung to lung. From the fifth passage, the body weight began to decrease, and WT-CA04-Venus was continuously passaged to the ninth passage. The mice infected with the ninth passage lung homogenate had a significant decrease in body weight on the third day after infection.

[0070] As mentioned above, WT-CA04-Venus is very likely to lose fluorescent protein during mouse passage. Therefore, starting from the fifth generation of CA04-Venus, MDCK cells were inoculated every other generation (e.g. Figure 8 ). The lung homogenate of the ninth passage expressed fluorescent protein, and the weight of the mice also decreased significantly. Therefore, the present application used the lung homogenate of the ninth passage to purify the plaques with high intensity of Venus fluorescent reporter protein with a fluorescence positive rate of 100% and no loss of fluorescent protein in five rounds. Finally, the present application obtained a mouse-adapted CA04-Venus strain, called MA-CA04-Venus (such as Fig. 9 ).

[0071] In order to confirm the toxicity of MA-CA04-Venus to mice, this application used 10 5PFU MA-CA04-Venus or WT-CA04-Venus virus was inoculated intranasally to infect mice, and body weight and survival rate were measured every day for 14 days. It was finally found that mice infected with MA-CA04-Venus virus had a significant decrease in weight, and 2 of 5 infected mice died, while mice infected with WT-CA04-Venus virus had only a slight decrease in weight. These results indicate that MA-CA04-Venus is significantly more toxic than WT-CA04-Venus.

[0072] 2.4The replication ability of MA-CA04-Venus in mammalian cells is significantly enhanced

[0073] In order to study the replication ability of MA-CA04-Venus in mammalian cells, the present application infected MDCK cells with WT-CA04-Venus or MA-CA04-Venus at an MOI of 0.01, and cultured the infected cells at 33°C or 37°C, respectively. The supernatant was collected at the specified time points, and the virus titer was determined by plaque assay. It was finally found that the virus titer of MA-CA04-Venus was significantly higher than that of WT-CA04-Venus at both 33°C and 37°C. The virus titers of the two viruses at 24hpi at 33°C were similar, but at 48, 60 and 72hpi at 33°C, the virus titer of MA-CA04-Venus was significantly higher than that of WT-CA04-Venus ( Fig.10 ). At 37°C, 24, 36, 48, 60, and 72 hpi, the viral titers of MA-CA04-Venus were higher than those of WT-CA04-Venus (e.g. Fig.11 ). These results are consistent with the high pathogenicity of MA-CA04-Venus in mice.

[0074] 2.5 Amino acid mutations in PB2 and PA polymerase proteins lead to the adaptation of mice to MA-CA04-Venus

[0075] In order to reveal the mechanism by which MA-CA04-Venus has high replication ability in mammalian cells and high pathogenicity in mice, the present application compared the whole genome sequences of WT-CA04-Venus and MA-CA04-Venus, and found that 3 amino acids had mutated (PB2 K340T, PA M21I, PAL175L) (as shown in Table 1 below). These mutations are mainly located in the PB2 and PA subunits of the influenza virus polymerase. The present application compares the polymerase activity of WT-CA04-Venus and MA-CA04-Venus viruses, and uses the mini-genome detection method to detect the effects of single amino acid mutations (PB2 K340T, PA M21I, PAL175F) and double amino acid mutations (PB2 K340T+PA M21I, PB2 K340T+PAL175F) on polymerase activity.

[0076] It was found that in 293T cells, the polymerase activity of MA-CA04-Venus was significantly lower than that of WT-CA04-Venus at both 33°C and 37°C (e.g. Fig.13 ). PA M21I can significantly increase the polymerase activity of WT-CA04-Venus, increasing by 3.6 times at 33°C and 4.9 times at 37°C. PA L175F does not affect the polymerase activity of WT-CA04-Venus. PB2 K340T does not change the polymerase activity at 33°C, but significantly enhances the polymerase activity of WT-CA04-Venus at 37°C. The PB2K340T+PAM21I double mutation significantly increased the polymerase activity at 33°C, while the difference was not obvious at 37°C. However, the PB2K340T+PAL175F double mutation significantly reduced the polymerase activity of WT-CA04-Venus at both 33°C and 37°C, which is similar to the polymerase activity of MA-CA04-Venus.

[0077] Therefore, this indicates that PA M21I leads to a significant increase in influenza virus polymerase activity, while the PB2K340T+PA L175F double amino acid substitution leads to a decrease in viral polymerase activity. These results suggest that PA M21I is associated with the high virulence of MA-CA04-Venus, while PB2 K340T+PA L175F is associated with maintaining the stability of Venus in the influenza virus genome.

[0078] 3. Conclusion of this Example (Application)

[0079] Through the technical solution and specific examples of this application, it is concluded that influenza viruses carrying reporter genes including fluorescent proteins or luciferase proteins play an important role in high-throughput screening of antiviral drugs, dynamic visualization of invading influenza virus replication in vivo, and virus-host interaction. However, inserting foreign genes into the influenza virus genome often leads to reduced pathogenicity and reduced stability of the reporter gene, especially for seasonal influenza viruses.

[0080] The present application firstly used reverse genetics to generate WT-CA04-Venus recombinant influenza virus carrying Venus fluorescent reporter gene in NS segment, and then obtained a mouse-adapted CA04-Venus by lung-to-lung and plaque purification. MA-CA04-Venus was continuously passaged in SPF chicken embryos and mammalian cells in vitro, and maintained stability in limited passages (e.g. Figure 5 and 6 Compared with WT-CA04-Venus, MA-CA04-Venus grew better (e.g. Figure 10-11 ). Further studies showed that compared with WT-CA04-Venus, three amino acids were replaced in the genome of MA-CA04-Venus, including PB2 K340T, PA M21I, and PA L175L (Table 1).

[0081] Table 1 Amino acid differences between WT-CA04-Venus and MA-CA04-Venus in PB2 and PA.

[0082]

[0083] The present application found that PAM21I significantly enhanced the polymerase activity of influenza virus, while the PB2K340T+PAL175F double mutation reduced the polymerase activity of the virus. Therefore, in this study, the present application generated a mouse-adapted CA04-Venus that stably carries the Venus fluorescent gene, has good in vitro replication ability and relatively high mouse virulence, and further explored the molecular mechanism of maintaining high virulence and stable reporter gene.

[0084] Rescued recombinant influenza viruses carrying fluorescent proteins or luciferases usually have low replication ability in vitro and weak pathogenicity in vivo. Therefore, technicians have now developed the technical idea of ​​PR8-Gluc, a recombinant PR8 influenza virus carrying Gluc in the NA segment. It was found that the replication rate of PR8-Gluc in SPF embryonic eggs and MDCK cells was lower than that of WT-PR8 virus. In comparison, the virulence of PR8-Gluc to mice was reduced by 1000 times. At present, technicians have generated a variety of colored influenza viruses carrying different fluorescent proteins (such as eGFP, eCFP, mCherry and Venus), and found that the toxicity of recombinant fluorescent influenza viruses (such as WT-PR8-Venus, WT-H5N1-Venus) in mice was significantly reduced. These attenuated viruses are suitable for in vitro antiviral screening, but they cannot reflect the true immune response of mice or other animals after infection. Subsequently, these fluorescent influenza viruses acquired higher pathogenicity after continuous passage through the lungs of mice. However, the current fluorescent seasonal influenza virus has not been produced in existing studies.

[0085] In the current study, the applicant generated a recombinant pandemic seasonal H1N1 influenza virus expressing the Venus protein and obtained the more pathogenic MA-CA04-Venus reporter virus (e.g. Figure 1-4 ). This suggests that the attenuation caused by foreign gene insertion may be independent of the type of fluorescent protein and the influenza virus backbone. Serial passage in mouse lungs is an effective and widely applicable strategy to address this issue.

[0086] The choice of fluorescent protein or luciferase depends on the application of the replication-composing influenza virus. Recombinant IAVs expressing fluorescent proteins are suitable for studying cellular localization and viral interactions with host cells in vivo and in vitro due to their penetrating properties. However, reporter viruses expressing bioluminescence require systemic injection of substrates before each observation, which may interfere with the immune response, and this application does not allow the use of recombinant influenza viruses expressing luciferase to track target cells infected with influenza viruses. EGFP is the most widely used fluorescent protein in biology and imaging. However, Venus is 30 times brighter than YFP when the spectrum is the same. On the other hand, the spectrum of Venus is narrower than that of EGFP and is not easily overlapped with other fluorescent proteins in multicolor experiments. PR8 influenza virus expressing Venus, combined with dual-light microscopy, can clearly track the movement of neutrophils labeled with Cy3 and Ly6G in the lungs of infected mice.

[0087] Therefore, the present application selected Venus as a fluorescent protein to generate seasonal reporter influenza viruses with replication ability in the study. Revealing the mechanism of pathogenicity and stability of recombinant influenza viruses expressing foreign genes will help broaden the present application's understanding of the virus life cycle and the development of new influenza virus vector vaccines. HAT380A and PB2 E712D enhanced the viral replication and pathogenicity of PR8 Venus-expressing reporter influenza viruses in mice, and PB2 E712D played a key role in enhancing viral replication and pathogenicity.

[0088] At present, technicians have found that PB2 E712D promotes the stability of Venus insertion by improving the efficiency of modifying the NS segment. Technicians have also found that the combination of PB2 V25A and PAR443K can significantly enhance the pathogenicity of WT-Venus-H5N1 virus carrying PR8 modified NS segment to mice. From the above research, it can be seen that the polymerase complex of influenza virus must mutate to enhance pathogenicity to mice and stabilize the insertion of foreign fluorescent genes into its genome.

[0089] In this study, WT-CA04-Venus PB2 K340T, PAM21I and PAL175L mutations occurred during the continuous passage of mice. The PAM21I mutation can enhance the virulence of CA04 influenza virus. The present application creatively discovered that the PAM21I mutation greatly enhanced the polymerase activity of WT-CA04-Venus (e.g. Figure 12-13 ). Therefore, the PA M21I mutation is a virulence determinant of MA-CA04-Venus. On the other hand, the polymerase complex plays a key role in the replication and genome stability of influenza viruses. The present application also found that the polymerase activity of MA-CA04-Venus was significantly lower than that of WT-CA04-Venus (e.g. Figure 12-13 ). Minigenome assays showed that the PB2 K340T and PAL175F double mutations resulted in lower polymerase activity in MA-CA04-Venus. In previous studies, the polymerase activity of MA-Venus-PR8 or MA-H5N1-Venus was also significantly reduced compared with their respective WT counterparts. Therefore, the PA M21I substitution was responsible for the high virulence, while the PB2 K340T and PAL175F double mutations contributed to the stability of the Venus gene in the MA-CA04-Venus NS fragment.

[0090] Finally, it should be noted that the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by ordinary technicians in this field should be considered as the protection scope of the present invention.

Claims

1. A method for rescuing a recombinant influenza virus carrying green fluorescent protein (H1N1) and preparing a mouse-adapted strain, characterized in that The preparation method comprises the following steps: Step 1: Rescue the CA04 recombinant influenza virus strain carrying the Venus reporter gene by reverse genetic manipulation technology, wherein the CA04 recombinant influenza virus strain is WT-CA04-Venus; Step 2: Subculture and transfect WT-CA04-Venus, purify and screen the mouse-adapted strain MA-CA04-Venus; compared with WT-CA04-Venus, three amino acids in the genome of MA-CA04-Venus were replaced, namely PB2 K340T, PA M21I and PA L175L.

2. The method for rescuing a recombinant influenza virus carrying green fluorescent protein (H1N1) and preparing a mouse-adapted strain according to claim 1, characterized in that: The step 1 includes rearranging the NS segment of CA04, expressing NS1-Venus and NEP as a polyprotein, connecting them through the 2A autoprotein cleavage site of porcine terser virus-1 (PTV-1), releasing NEP from the upstream NS1-Venus protein during translation, and introducing two silent mutations at the splicing acceptor site to avoid splicing of CA04's NS mRNA.

3. The method for rescuing a recombinant influenza virus carrying green fluorescent protein (H1N1) and preparing a mouse-adapted strain according to claim 2, characterized in that: It includes mixing 8 pHH21 recombinant plasmids, 4 pCAGGS recombinant plasmids and Lipofectamine LTX plus reagent with Opti-MEM and incubating at room temperature for 20 minutes; The mixture of plasmid and transfection reagent was added to a 6-well plate containing 293T cells using Opti-MEM, and TPCK trypsin was added at a final concentration of 0.5 μg / mL 24 hours after transfection.

4. The method for rescuing a recombinant influenza virus carrying green fluorescent protein (H1N1) and preparing a mouse-adapted strain according to claim 3, characterized in that: The eight pHH21 recombinant plasmids are: pHH21-CA04-PB2, pHH21-CA04-PB1, pHH21-CA04-PA, pHHH21-CA04-HA, pHHH21-CA04-NP, pHHH21-CA04-NA, pHHH21-CA04-M, pHH21-CA04--NS-Venus; The four pCAGGS recombinant plasmids are pCAGGS-CA04-PB2, pCAGGS-CA04-PB1, pCAGGS-CA04-PA and pCAGGS-CA04-NP.

5. The method for rescuing a recombinant influenza virus carrying green fluorescent protein (H1N1) and preparing a mouse-adapted strain according to claim 1, 2, 3 or 4, characterized in that: In step 2, WT-CA04-Venus was transfected into mice for 9 generations, and starting from the fifth generation of CA04-Venus, MDCK cells were inoculated every other generation. Finally, a mouse-adapted CA04-Venus strain was obtained.

6. A WT-CA04-Venus application as claimed in any one of claims 1 to 5, characterized in that: Applications of WT-CA04-Venus include monitoring viral infection dynamics in tissue culture cells and animal models; testing the effects of antiviral drugs and vaccines in vivo without invasiveness; and identifying antiviral compounds, potent or broadly reactive monoclonal antibodies, and host factors that can engage and influence the viral life cycle in an in vitro environment.

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