Passage-attenuated strain of african swine fever virus and vaccine on basis of strain

Abstract

Provided is an African swine fever virus strain with the microbial deposit number CCTCC NO: V2023100. Also provided is a vaccine composition for preventing African swine fever, which comprises the African swine fever virus strain and a freeze-drying protective agent. Further provided is a method for preparing the vaccine composition. The virus strain has a good safety and good immune effectiveness. A pig inoculated with the virus strain can generate a high-level antibody against the African swine fever virus, and can resist challenges of strong African swine fever viruses of different genotypes. The African swine fever virus strain and the vaccine composition have clinical application prospects.

Description

A passaged attenuated strain of African swine fever virus and a vaccine based on the strain Technical Field

[0001] The present invention belongs to the field of veterinary biological products and relates to a passaged attenuated strain of African swine fever virus and a vaccine based on the strain. Background Art

[0002] African swine fever (ASF) is an acute, hemorrhagic, highly contagious disease of domestic pigs or wild boars caused by infection with the African swine fever virus (ASFV). The World Organization for Animal Health (WOAH) lists ASF as a notifiable animal disease. China classifies ASF as a Category I animal disease. The clinical manifestations of ASF include the most acute, acute, subacute, and chronic stages. Acute symptoms include: fever, depression, anorexia, petechiae on the ears, limbs, and abdominal skin, flushing and cyanosis of the visible mucous membranes, mucopurulent discharge from the eyes and nose, vomiting, constipation, feces covered with blood and mucus, diarrhea, blood in the feces, ataxia or gait stiffness, paralysis, convulsions, difficulty breathing, and abortion in pregnant sows. The mortality rate can reach 100%.

[0003] ASFV is the only member of the genus Asfivirus in the family Asfarviridae. The ASFV genome is a double-stranded linear DNA sequence ranging from 170 to 194 kb in size, containing 150 to 167 open reading frames (ORFs), encoding 54 structural proteins and over 100 non-structural proteins. ASFV has been classified into 24 genotypes based on sequence differences within the terminal 500 nucleotides of the B646L gene (encoding the p72 protein).

[0004] According to statistics from WOAH, ASF has broken out and spread in nearly 50 countries in Africa, Europe, Asia and the Caribbean, causing huge economic losses and seriously endangering the stable development of the global pig industry and related industries.

[0005] The ASF virus strain first isolated and identified in China is ASFV HLJ / 18 (GenBank: MK333180.1, full name Pig / HLJ / 2018). ASFV HLJ / 18 is a highly virulent strain, causing 100% mortality in pigs inoculated with it. Whole-genome sequencing and phylogenetic analysis confirmed that ASFV HLJ / 18 is a genotype II, highly virulent strain. As ASFV continues to spread and evolve in China, the moderately virulent genotype II strain HLJ / HRB1 / 20 (GenBank: MW656282.1, full name Pig / Heilongjiang / HRB1 / 2020) with no hematropin activity was isolated and identified in the field in China in 2020. The low-virulence genotype I strain SD / DY-I / 21 (GenBank: MZ945537.1, full name Pig / SD / DY-I / 2021) was isolated and identified in China in 2021. In 2023, the naturally recombinant, highly virulent strain JS / LG / 21 (GenBank: OQ504956.1, full name Pig / Jiangsu / LG / 2021), a combination of genotypes I and II, was first reported. This poses a greater challenge to ASF prevention and control in China. It is worth noting that the attenuated vaccine strain HLJ / 18-7GD, constructed with a genotype II virus as its parent (see Chinese patent application number CN202310363645.0), cannot provide immune protection against such genotype I and II recombinant virulent strains (for example, JS / LG / 21 strain). Such genotype I and II recombinant virulent strains (for example, JS / LG / 21 strain) were first detected in China in 2021.

[0006] Vaccination has always been the most economical and effective means of infectious disease prevention and control. The control or eradication of animal diseases such as rinderpest, bovine pleuropneumonia, classical swine fever, and equine infectious anemia have all been achieved through safe and effective vaccination. Pigs that have survived virulent ASFV infection are resistant to reinfection with the same virulent strain, suggesting the theoretical feasibility of vaccination to prevent ASF. Scientists have conducted extensive vaccine research.

[0007] The reported inactivated African swine fever virus vaccines, subunit vaccines, and recombinant vector vaccines have not achieved ideal clinical protection effects.

[0008] Vaccination with naturally mutated weak strains (e.g., genotype I NH / P68 and OUR T88 / 3 strains, genotype II Lv17 / WB / Rie1 strain) can effectively protect against attacks from homologous strong toxins, but the vaccination causes chronic symptoms such as skin ulcers and arthritis, and still has a certain amount of residual virulence.

[0009] Pigs vaccinated with gene-deleted attenuated strains (such as the ASFV-G-ΔI177L strain lacking the I177L gene, the BA71ΔCD2v strain lacking the CD2v gene, the ASFV SY18-ΔCD2v / UK strain lacking both the CD2v and UK genes, and the HLJ / 18-7GD strain lacking both the MGF360 / 505 and CD2v genes) showed no obvious clinical symptoms and were effectively protected against attack by homologous virulent strains. Clinical trials involving the HLJ / 18-7GD strain have been conducted, and the attenuated ASFV-G-ΔI177L vaccine has been approved for use in Vietnam. Studies have shown that the combined deletion of multiple virulence-related genes may affect the immunogenicity of ASFV and result in an inability to resist attack by homologous virulent strains. Pigs inoculated with the double-gene deletion HLJ / 18-9GL&UK-del strain, derived by combining the deletion of the 9GL and UK genes, and the JS / LG / 21-7GD deletion strain, derived by combining the deletion of the MGF360 / 505 genes and CD2v (a total of seven genes) based on the recombinant virulent strains of genotypes I and II (JS / LG / 21), showed no clinical symptoms. However, vaccination with these two gene-deleted strains was not effective against challenge with the homologous virulent strains.

[0010] Passaging and attenuating pathogens is a key technical approach in vaccine research. Vaccines for diseases such as rinderpest, bovine pleuropneumonia, classical swine fever, and equine infectious anemia all utilize animal or cell-based passage. Passaging and attenuating vaccines have played a crucial role in the prevention and control of these diseases, achieving the eradication and elimination of rinderpest, bovine pleuropneumonia, and equine infectious anemia in China, as well as the effective prevention and control of classical swine fever. After the introduction of ASFV into Portugal in 1957, studies on ASFV cell adaptation were conducted. Representative ASFV-adapted strains include the L'60BM89, BA71v, and E75CV1 strains. In 1963, Ribeiro et al. serially passaged the 1455 strain isolated in Portugal on bone marrow cells and found that its virulence gradually decreased. Inoculation of pigs with the attenuated virus after 70 passages showed significantly reduced clinical symptoms and resistance to challenge with virulent strains. In 1965, Hess et al. obtained multiple attenuated strains by passage-based passages in PK-2a cells. In 1967, Greig et al. propagated the Spencer strain to passage 35. Pigs inoculated with F35 showed no clinical symptoms and were resistant to challenge with the parental strain. Greig et al. propagated the Portuguese challenge strain to passage 34. Pigs inoculated with F34 showed slight temperature fluctuations and were resistant to challenge with the parental strain. Greig et al. propagated the Gasson strain to passage 23. F23 weakened the virulence of inoculated pigs, but this strain did not protect against challenge with the parental strain. In 1976, researchers adapted the BA71 strain to Vero cells for 100 generations, ultimately obtaining a non-virulent Vero cell-adapted strain (BA71v). In 1979, Thomson et al. used a CV cell-based attenuated strain to inoculate two pigs. The inoculated pigs were unable to effectively protect against challenge with the virulent strain. The Russian Federal Institute of Veterinary Virology and Microbiology has obtained multiple cell-based attenuated ASFV strains. The strain isolated in Congo in 1949 was propagated for 50 generations in pig kidney cells and then for 262 generations in pig bone marrow cells. The isolate obtained in France in 1964 was propagated for 135 generations in pig bone marrow cells. These strains were not tested for immunization or challenge protection. In 2015, Krug et al. inoculated pigs with the 110th generation of the Vero cell-adapted Georgian strain (ASFV-G). The inoculated pigs showed no clinical reactions, but were unable to effectively resist attack by the virulent strain. In 2015, Balysheva et al. propagated the virulent Stavropol 01 / 08 strain for 24 generations in the hybrid cell line SPEV TK with pig lymphocytes (A4C2) and 20 generations in CV cells. All pigs inoculated with the propagated virus survived, but the vaccination did not provide effective protection.The I177L gene-deficient strain (ASFV-G-ΔI177L strain) was passaged in the Plum Island porcine epithelial cell line (PIPEC) to obtain a strain with a left variable region deletion (ASFV-G-ΔI177L / ΔLVR strain). This strain showed no clinical symptoms in pigs inoculated with the strain and provided effective protection against homologous virulent strains. In 2021, Wang et al. continuously passaged the Chinese isolate (ASFV-HLJ / 18 strain) in HEK293T cells for 121 generations to obtain a highly efficient replicating strain. However, the efficiency of regressive PAM replication was extremely low, and its pathogenicity and immunogenicity require further study.

[0011] ASFV was first discovered in 1921. Currently, ASFV is endemic in over 50 countries, causing significant economic losses to the global swine industry and related sectors. Scientists have conducted extensive vaccine research and tested various vaccine development strategies. Reported protective effects of inactivated, subunit, and recombinant vector vaccines have been suboptimal. Vaccination with naturally attenuated strains can effectively protect against challenge with the same virulent strain, but can cause chronic symptoms such as skin ulcers and arthritis in vaccinated pigs, and some residual virulence remains. Vaccination with attenuated strains with certain gene deletions can effectively protect against challenge with the same virulent strain, but lacks cross-protection against heterologous strains. Some gene deletions impair ASFV immunogenicity, resulting in a lack of protection against challenge with the same virulent strain. Some attenuated strains can protect against challenge with the parental virulent strain, but retain residual virulence. Some attenuated strains completely lose their immunogenicity and therefore offer no protection. To date, no safe and effective ASFV vaccine is available for clinical use.

[0012] Summary of the Invention

[0013] The present invention uses wild boar kidney cells (Boar kidney cells) BK2258 to continuously passage genotype I and II recombinant African swine fever virus strains (JS / LG / 21 strain) for 93 generations, and then returns F93 to primary porcine alveolar macrophages (Porcine alveolar macrophages, PAM) for 10 generations to obtain a cell-passaged adapted strain (JS4821 strain) of the African swine fever virus JS / LG / 21 strain. Pigs inoculated with the cell-passaged adapted strain JS4821 strain do not experience fever, depression, decreased food intake, skin ulcers, or joint swelling, and the inoculated pigs can simultaneously resist attacks from the virulent genotype I and II recombinant African swine fever virus strains (JS / LG / 21 strain) and the virulent genotype II African swine fever virus epidemic strain (HLJ / 18 strain), showing good cross-protection effects. The invention has great application value in vaccine development.

[0014] In order to solve the problems existing in the prior art, the first aspect of the present invention provides an African swine fever virus strain, which is an African swine fever virus strain with a microbial preservation number of CCTCC NO: V2023100.

[0015] The second aspect of the present invention provides a vaccine composition for preventing, mitigating or controlling African swine fever, wherein the vaccine composition uses the African swine fever virus strain described in the first aspect of the present invention as an immunogen.

[0016] In some embodiments, the vaccine composition is a live vaccine composition.

[0017] In some embodiments, the raw materials of the vaccine composition include the African swine fever virus strain and excipients.

[0018] In some embodiments, the excipient is a lyoprotectant.

[0019] In some embodiments, in the raw materials of the vaccine composition, the lyoprotectant is an aqueous solution containing 4-12 w / w% gelatin and 30-50 w / w% sucrose.

[0020] In some embodiments, in the raw materials of the vaccine composition, the dry weight ratio of the African swine fever virus strain to the lyophilized protective agent is 1-20×10 6.0 TCID 50 : 1g (for example, selected from 1×10 6.0 TCID 50 , 2×10 6.0 TCID 50 , 3×10 6.0 TCID 50 , 4×10 6.0 TCID 50 , 5×10 6.0 TCID 50 , 6×10 6.0 TCID 50 , 7×10 6.0 TCID 50 , 8×10 6.0 TCID 50 , 9×10 6.0 TCID 50 , 10×10 6.0 TCID 50 , 11×10 6.0 TCID 50 , 12×10 6.0 TCID 50 , 13×10 6.0 TCID 50 , 14×106.0 TCID 50 , 15×10 6.0 TCID 50 , 16×10 6.0 TCID 50 , 17×10 6.0 TCID 50 , 18×10 6.0 TCID 50 , 19×10 6.0 TCID 50 With 20×10 6.0 TCID 50 Any value or range between any two values ​​in: 1g).

[0021] In some embodiments, the TCID of the African swine fever virus strain is 50 It is calculated based on the Reed-Muench method using immunofluorescence analysis of primary porcine alveolar macrophage culture.

[0022] The third aspect of the present invention provides a method for preparing the vaccine composition described in the second aspect of the present invention, wherein the preparation method comprises: using the African swine fever virus strain as an immunogen to prepare the vaccine composition.

[0023] In some embodiments, the preparation method is: mixing the African swine fever virus strain with the lyoprotectant to obtain a mixture, and freeze-drying the mixture to obtain the vaccine composition.

[0024] In some embodiments, the temperature change program of the freeze-drying is: -4 to -6°C, 0.8-1.2 hours; -35 to -45°C, 1.5-2.5 hours; -14 to -18°C, 14-18 hours; -5 to -7°C, 2-4 hours; 4-6°C, 1.5-2.5 hours; 26-30°C, 5-7 hours.

[0025] Advantages of the present invention:

[0026] 1. The ASFV JS4821 strain, which can stably proliferate on BK2258 cells, was obtained.

[0027] 2. Pigs inoculated with the cell-passaged ASFV JS4821 strain did not develop fever and had no clinical symptoms.

[0028] 3. The cell-passaged strain ASFV JS4821 can simultaneously resist attacks from the homologous virulent strain JS / LG / 21 (genotype I according to the B646L genotyping) and the heterologous virulent strain HLJ / 18 (genotype II), and has cross-immune protection effects against different ASFV genotypes. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 shows CPE images of F93 generation virus obtained by culturing ASFV JS / LG / 21 strain in BK2258 cells and a control image. Scale bar: 100 μm.

[0030] Figure 2 shows CPE photos of ASFV JS4821 strain cultured with PAM and a control photo. The scale bar is 100 μm.

[0031] Figure 3 shows fluorescence and bright field images of IFA tests of ASFV JS4821 and JS / LG / 21 strains cultured with PAM. Scale bar: 100 μm.

[0032] Figure 4 shows the statistical data on the pathogenicity and transmissibility of ASFV JS / LG / 21 strain to pigs.

[0033] Figure 5 shows that the ASFV JS4821 strain was 10 6 TCID 50 / pig dose after immunization of the pig's body temperature.

[0034] Figure 6 shows electrophoresis patterns characterizing the deletion of genomic segments in ASFV JS4821 strain relative to JS / LG / 21 strain.

[0035] FIG7 shows the situation of antibody induction by ASFV JS4821 strain.

[0036] Figure 8 shows the body temperature of pigs after being challenged with the virulent genotype I strain JS / LG / 21.

[0037] Figure 9 shows the survival rate of pigs after infection with the virulent genotype I strain JS / LG / 21.

[0038] Figure 10 shows the body temperature of pigs after being challenged with the virulent genotype II strain HLJ / 18.

[0039] Figure 11 shows the survival rate of pigs after infection with the virulent genotype II strain HLJ / 18. DETAILED DESCRIPTION

[0040] To make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0041] Strains and cells

[0042] The JS / LG / 21 strain of African swine fever virus (ASFV JS / LG / 21, JS / LG / 21) is a naturally isolated and virulent strain of African swine fever virus. Based on the B646L genotyping, JS / LG / 21 is a genotype 1 ASFV. Genomic sequence analysis indicates that JS / LG / 21 is a natural recombinant of genotype 1 and genotype 2 ASFV. JS / LG / 21 was isolated, identified, and preserved by the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The literature (Highly lethal genotype I and II recombinant African swine fever viruses detected in pigs. Dongming Zhao et al, Nature Communications. Volume 14, Article number: 3096(2023). https: / / doi.org / 10.1038 / s41467-023-38868-w) records the isolation and characterization of the Pig / Jiangsu / LG / 2021 strain (JS / LG / 21 strain). GenBank No. OQ504956.1 discloses the genome sequence of the JS / LG / 21 strain, the full name of which is Pig / Jiangsu / LG / 2021.

[0043] The GenBank accession number of ASFV HLJ / 18 strain is MK333180.1, where its full name is Pig / HLJ / 2018.

[0044] BK2258 cells are derived from wild boar kidneys and are produced and maintained by the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The preparation method for BK2258 cells is described in the Chinese patent application number CN202211137583.3.

[0045] Primary porcine alveolar macrophages (PAMs) were prepared and used by Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0046] Example 1: Subculture adaptation of ASFV JS / LG / 21 strain

[0047] Method for culturing virus in BK2258 cells: ASFV JS / LG / 21 strain was diluted to 10,000 TCID using DMEM (purchased from Thermo Fisher Scientific Biochemicals (Beijing) Co., Ltd., catalog number: C11995500BT) maintenance medium (containing 3 v / v% fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin). 50 / ml. Inoculate the diluted virus strain into a well-grown monolayer of BK2258 cells. Culture the flask in a 37°C, 5% CO2 incubator and observe the cell monolayer daily for cytopathic effect (CPE). If no obvious CPE is observed after 8 days of culture, harvest the virus after 8 days. If obvious CPE is observed after 3 days of culture, harvest the virus when CPE reaches 80%.

[0048] The ASFV JS / LG / 21 strain was cultured in BK2258 cells using the previously described BK2258 cell culture method. No CPE was observed after 8 days of culture in the first passage. The F1 passage culture was freeze-thawed twice at -70°C / 37°C, and the virus culture was harvested. The ASFV JS / LG / 21 strain was serially passaged using the same method using BK2258 cells. Obvious CPE began to appear at F6 and gradually increased with each passage. Figure 1, Panel A, shows a photograph of BK2258 cells inoculated with the F93 strain 96 hours after inoculation. The characteristics of F93 culture are cell aggregation, cell shrinkage, cell lysis, and finally the appearance of circular plaques. Figure 1, Panel B, shows a control of uninoculated BK2258 cells cultured under the same conditions. This demonstrates that the ASFV JS / LG / 21 strain F93 is fully adapted to BK2258 cells.

[0049] Method for primary PAM virus culture: ASFV JS4821 strain was diluted to 10000 TCID using RPMI Medium 1640 (purchased from Thermo Fisher Scientific Biochemical Products (Beijing) Co., Ltd., catalog number: C11875500BT) complete culture medium (containing 10 v / v% fetal bovine serum, 100 U / ml penicillin, 100 μg / ml streptomycin). 50 / ml. Inoculate the primary PAM with the diluted virus strain. Place the culture flask in a 37°C, 5% CO2 incubator and observe the cells daily for cytopathic effect (CPE). If significant CPE is observed after two days of culture, harvest the virus when CPE reaches 80%.

[0050] The F93 strain was inoculated into primary PAM using the aforementioned method for culturing the virus using primary PAM and then passaged for 10 consecutive generations. Significant CPE was observed in each passage. Figure 2, Panel A, shows a photograph of the F10 strain cultured in primary PAM 72 hours after inoculation with the primary PAM. The F10 strain cultured in primary PAM is characterized by cell aggregation and cell lysis. Figure 2, Panel B, shows a control cultured in primary PAM under the same conditions without virus inoculation. The F10 strain, derived from the F93 strain cultured on BK2258 cells and passaged in primary PAM, was designated ASFV JS4821.

[0051] The present invention deposits the African swine fever virus JS4821 strain, obtained through continuous in vitro passage, with a patent application-approved depository institution. The depository is the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China. The microbial accession number is CCTCC NO: V2023100. The strain is classified as African Swine Fever Virus and the culture is named African swine fever virus ASFV JS4821. The date of deposit is November 6, 2023, and the identified survival time is November 7, 2023.

[0052] Example 2: Titer determination of ASFV JS4821 strain and ASFV JS / LG / 21 strain

[0053] ASFV JS4821 strain and ASFV JS / LG / 21 strain were diluted 10-fold to 10 in RPMI Medium 1640 culture medium. -8 Dilution. -4 , 10 -5 , 10 -6 , 10 -7 and 10 -8 Each of the five virus dilutions was inoculated into 96-well plates containing primary PAM. Eight wells were plated for each dilution, with 100 μl added per well. As a normal cell control, RPMI Medium 1640 culture medium was added to the primary PAM plates in eight replicates, with 100 μl added per well. The 96-well plates were incubated at 37°C in a 5% CO2 incubator for 7 days.

[0054] The fluorescence in each well was detected by IFA (immunofluorescence assay), which includes the following steps: (1) fixing the cells in each well of the 96-well plate with formaldehyde, (2) washing each well with PBS, and then adding rabbit anti-ASFV p72 protein polyclonal antibody to each well, the antibody concentration was 1:1000, the addition amount was 100 μl / well, and the 96-well plate was incubated at 37°C for 30 minutes. (3) washing each well with PBS, and then adding FITC-labeled rabbit anti-swine IgG (Sigma-Aldrich, F0382-2ML) to each well, the antibody concentration was 1:200, the addition amount was 100 μl / well, and the 96-well plate was incubated at 37°C for 30 minutes. (4) washing each well with PBS, and then performing fluorescence detection and bright field detection on each well. See Figure 3 for some photos. In Figure 3, A shows the ASFV JS4821 strain was infected with 10 -5 IFA fluorescence photos of the primary PAM7d after inoculation with 10 dilutions; A# shows the ASFV JS4821 strain inoculated with 10 -5 B shows the bright field photo of the primary PAM7d after inoculation with 10 dilutions; -8 IFA fluorescence photos of the primary PAM7d after inoculation with 10 dilutions; B# shows the ASFV JS4821 strain inoculated with 10 -8 Bright field photographs after inoculation of primary PAM7d at 10 dilutions; C shows the ASFV JS / LG / 21 strain at 10 -5 IFA fluorescence photos of the primary PAM7d after inoculation with 10 dilutions; C# shows the ASFV JS / LG / 21 strain inoculated with 10 -5 D shows the bright field photo of IFA detection after the primary PAM7d was inoculated with 10 dilutions; -8 IFA fluorescence photos of the primary PAM7d after inoculation with 10 dilutions; D# shows the ASFV JS / LG / 21 strain inoculated with 10 -8 Bright field photographs of primary PAM7d after inoculation with different dilutions.

[0055] The number of positive fluorescence wells for each treatment was recorded. -4 , 10 -5 and 10 -6 All 8 wells corresponding to the dilution showed IFA positive; 10 -7 and 10 -8 The 8 wells of IFA corresponding to the dilution were all negative, and the titer of ASFV JS4821 strain was calculated according to the Reed-Muench method to be 10 7.50 TCID 50 / ml. For ASFV JS / LG / 21 strain, 10 -4 , 10 -5 and 10 -6 All 8 wells corresponding to the dilution showed IFA positive; 10 -7 The dilutions corresponding to the two wells showed IFA positive, and the six wells showed IFA negative; 10 -8 All 8 wells corresponding to the dilution showed IFA negative, and the titer of ASFV JS / LG / 21 strain was calculated according to the Reed-Muench method to be 10 7.67 TCID 50 / ml.

[0056] It can be seen that there is no obvious difference in the titer between ASFV JS4821 strain and ASFV JS / LG / 21 strain on target cells (primary PAM), indicating that cell passage of ASFV JS / LG / 21 strain has no effect on its in vitro reproduction ability.

[0057] Example 3: Pathogenicity analysis of ASFV JS / LG / 21 strain

[0058] To evaluate the virulence and transmissibility of ASFV JS / LG / 21 strain to pigs, 7-week-old SPF pigs were selected from the Experimental Animal Center of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, and injected intramuscularly for 10 3 HAD 50 with 10 6 HAD 50 The ASFV JS / LG / 21 strain was diluted with RPMI Medium 1640 culture medium (PAM culture of ASFV JS / LG / 21 strain), and 6 pigs were inoculated with each dose.

[0059] Starting from the first day of infection, two SPF pigs were selected from each group to be housed with infected pigs to assess the transmission ability of the ASFV JS / LG / 21 strain. The survival and clinical signs of all pigs were monitored daily. At the specified time after inoculation (pi) or post-contact (p.ct), the African swine fever virus p72 gene in the oral swab, rectal swab and EDTA anticoagulated blood of each pig was detected by qPCR to reflect the viral load. The dead or euthanized pigs were autopsied, and samples of the brain, heart, liver, spleen, lung, kidney, tonsil, inguinal lymph node, submandibular lymph node and mediastinal lymph node were collected. The p72 gene in each sample was detected by qPCR to reflect the viral load.

[0060] The corresponding test results are shown in Figure 4. In Figure 4, af shows 10 6 HAD 50 The corresponding indicators of the dose-vaccinated pigs and the companion pigs; gI shows 10 3 HAD 50Corresponding indicators of dose-vaccinated pigs and companion pigs; a and g show the rectal temperature of the pigs after vaccination; b and h show the survival rate of the pigs after vaccination; c and i show the viral DNA content of the pigs' oral swabs; d and j show the viral DNA content of the pigs' rectal swabs; e and k show the viral DNA content of the pigs' blood; f and l show the viral DNA content of 10 tissues of the pigs. The black dotted line represents the normal rectal temperature of the pigs (40°C). LN1 represents the inguinal lymph node; LN2 represents the submandibular lymph node; and LN3 represents the mediastinal lymph node.

[0061] In 10 6 HAD 50 In the group, all 6 vaccinated pigs developed fever on day 4 pi and died between days 5 and 8 pi; 2 contact pigs developed fever on day 9 after contact and died on day 12 (a, b); viral DNA was detected in oral swabs, rectal swabs and blood of all vaccinated pigs and contact pigs (c, d, e); high levels of viral DNA were detected in the organs of the dead pigs (brain, heart, liver, spleen, lungs, kidneys, tonsils, inguinal lymph nodes, submandibular lymph nodes and mediastinal lymph nodes) (f).

[0062] In 10 3 HAD 50 In the group, all pigs died between the 6th and 15th days (h); two contact pigs developed fever on the 9th and 10th days after infection and died on the 12th and 14th days after vaccination, respectively (g, h); viral DNA was detected in the oral swabs, rectal swabs, and blood of all vaccinated and contact pigs, but the level was slightly lower than 10 6 HAD 50 Groups (i, j, k); high levels of viral DNA were detected in organs of dead pigs (brain, heart, liver, spleen, lungs, kidneys, tonsils, inguinal lymph nodes, submandibular lymph nodes and mediastinal lymph nodes) (l). These results indicate that the recombinant virus isolate ASFV JS / LG / 21 is highly lethal and transmissible.

[0063] Example 4: Safety study of pigs vaccinated with ASFV JS4821 strain

[0064] To evaluate the safety of ASFV JS4821 strain to pigs, 10 6 TCID 50Seven-week-old pigs (SPF-grade pigs provided by the Experimental Animal Center of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences) were inoculated intramuscularly with ASFV JS4821 (PSA culture of ASFV JS4821 diluted in sterile PBS) at a dose of 100 / pig. The inoculations were performed independently in two groups of five pigs each. Following inoculation, rectal temperature was measured daily for each inoculated pig, and their food and water intake, mental state, and body surface symptoms were observed. Results showed that within 28 days after inoculation, all pigs maintained normal body temperatures (Figure 5), food and water intake, and mental state, with no adverse clinical symptoms observed. This demonstrates the good safety of ASFV JS4821.

[0065] Example 5: Study on the differences between ASFV JS4821 strain and ASFV JS / LG / 21 strain

[0066] According to Example 3, both groups of pigs inoculated with the ASFV JS / LG / 21 strain died within 14 days of inoculation. According to Example 4, no symptoms were observed 28 days after inoculation with the ASFV JS4821 strain. Therefore, it can be inferred that the ASFV JS4821 strain has significantly attenuated its virulence. To further examine the genetic differences between the ASFV JS4821 strain and the ASFV JS / LG / 21 strain, the genome was divided into 25 segments based on the full genome sequence of the ASFV JS / LG / 21 strain (GenBank accession number: OQ504956.1). Upstream and downstream primers were designed based on the aforementioned 25 genomic fragments. The genomic templates of the parental strain (JS / LG / 21) and the attenuated strain (JS 4821) were extracted using the AXYGEN Viral Nucleic Acid Miniprep Kit (purchased from Corning Life Sciences (Wujiang) Co., Ltd., Catalog No.: AP-MN-BF-VNA-250) for PCR amplification. The amplification results were characterized by electrophoresis. The electropherogram corresponding to the primer pair with the most significant difference in the length of the two genomic amplification products is shown in Figure 6. In Figure 6, lane 1: DL15000 DNA Marker, lane 2: amplification product of the parental strain (JS / LG / 21), lane 3: amplification product of the attenuated strain (JS 4821), and lane C: HO control amplification. As shown in Figure 6, the length of the amplification product of the parental strain (JS / LG / 21) and the amplification product of the JS4821 strain differs by approximately 2 kb.

[0067] The results in FIG6 are generated by amplification using an upstream primer sequence F (SEQ ID NO. 1) and a corresponding downstream primer sequence R (SEQ ID NO. 2).

[0068] The F sequence is: 5'-TGTTTAGTGAGCCGTTTCT-3'.

[0069] The R sequence is: 5'-TGTCACCATCAGAGAGTTC-3'.

[0070] These results indicate that the ASFV JS4821 strain has a 2kb deletion relative to the ASFV JS / LG / 21 strain genome. This deletion is believed to be the primary cause of the weakening of the ASFV JS4821 strain. The ASFV JS4821 strain has the potential to be used in the development of a live vaccine.

[0071] Example 6: ASFV JS4821 strain immunogenicity study

[0072] To evaluate the immunogenicity of the passaged strain (ASFV JS4821 strain), 10 3 TCID 50 / pig and 10 6 TCID 50 Seven-week-old pigs (SPF pigs provided by the Experimental Animal Center of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences) were inoculated intramuscularly with JS4821 strain (PSA culture of ASFV JS4821 strain diluted in sterile PBS) at a dose of 1:1 / pig. Five pigs were inoculated with each dose. A non-inoculated control group (negative control) of five pigs was also established. Blood was collected every five days after inoculation, and serum was prepared. The p22 protein blocking ELISA antibody test method was used to determine the p22 antibody blocking rate. The test procedure is as follows:

[0073] 1. Coating: Dilute the prokaryotic expressed African swine fever virus p22 protein antigen into 4 μg / ml antigen dilution solution with antigen coating solution (carbonate buffer), add the antigen dilution solution to a 96-well plate, 50 μl / well, and coat at 4°C overnight.

[0074] 2. Washing: Wash the 96-well plate 5 times with 300 μl / well of PBST (phosphate-buffered saline containing 0.05 v / v% Tween 20), each time for 1 minute.

[0075] 3. Blocking: Block a 96-well plate with 5 w / v% skim milk, 200 μl / well, and incubate the 96-well plate at 37°C for 2 hours.

[0076] 4. Washing: Same as 2.

[0077] 5. Sample addition: Dilute each serum sample in a 1:1 volume ratio with PBS. Add the dilution to a 96-well plate at 50 μl / well and incubate the plate at 37°C for 30 minutes.

[0078] 6. Washing: Same as 2.

[0079] 7. Add enzyme-labeled antibody: Use secondary antibody diluent (purchased from Huzhou Yingchuang Biotechnology Co., Ltd., product number: HRP-SD-001) to dilute the enzyme-labeled antibody (mouse anti-African swine fever virus p22 protein porcine IgG monoclonal antibody labeled with horseradish peroxidase) at a volume ratio of 1:2000, 50 μl / well, and incubate the 96-well plate at 37°C for 30 minutes.

[0080] 8. Washing: Same as 2.

[0081] 9. Color development: Add 50 μl TMB substrate to each well for color development and incubate at 37°C in the dark for 10 minutes.

[0082] 10. Stop: Add 50 μl of stop solution (2M H2SO4) to each well.

[0083] 11. Reading: Determine OD 450nm value.

[0084] The blocking rate was calculated according to the following formula: blocking rate % = [(negative control average OD 450nm - Sample OD 450nm ) / average OD of negative control 450nm The judgment criteria are: when the blocking rate is higher than 47%, the serum is ASFV antibody positive; when the blocking rate is lower than 47%, the serum is ASFV antibody negative.

[0085] The results showed that 5 days after vaccination, 10 3 TCID 50 / pig group and 10 6 TCID 50 / pig group's antibodies did not turn positive; 10 days after vaccination, 10 3 TCID 50 / 3 / 5 of the pigs in the group turned positive for antibodies, 10 6 TCID 50 5 / 5 of the pigs in the group turned positive for antibodies; 20 days after vaccination, 10 3 TCID 50 / pig group and 10 6 TCID 50 All pigs in the immunization group tested positive for antibodies, and antibody titers reached peak levels. Antibody levels in both groups remained at peak levels 25 days after vaccination, while antibody levels in the non-immunized control group remained negative. The results of immunization duration and blocking efficiency are shown in Figure 7. These results demonstrate that the attenuated strain (ASFV JS4821) possesses strong immunogenicity and is valuable for developing a vaccine against African swine fever virus.

[0086] Example 7: Study on the protection of ASFV JS4821 strain against the homologous virulent strain JS / LG / 21 strain (genotype I according to B646L genotyping)

[0087] This example is used to evaluate the effectiveness of immune protection of the attenuated strain ASFV JS4821 against the homologous virulent strain. 6 TCID 50 ASFV JS4821 strain (PSF culture of ASFV JS4821 strain diluted with sterile PBS) was injected intramuscularly into 7-week-old pigs (SPF pigs provided by the Experimental Animal Center of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences), a total of 5 pigs. 28 days after inoculation, 100LD 50 Pigs were challenged with a homologous virulent strain (ASFV JS / LG / 21 strain, genotype I) of 100 mg / pig by intramuscular injection. A non-immunized control group of 5 pigs was challenged with the virus.

[0088] After the infection, the pigs' body temperature was measured daily, and their feeding, drinking, mental state, body surface and other clinical symptoms were observed, and deaths were recorded. Results of the immunization group after the ASFV JS / LG / 21 strain attack: Except for one pig that had a body temperature higher than 40.0°C on the 4th day after the infection, the body temperature of all other pigs was not higher than 40.0°C, and the body temperature was normal (Figure 8); all 5 vaccinated pigs had normal feeding, drinking, mental state, etc.; 100% of the pigs survived (Figure 9). Results of the control group after the ASFV JS / LG / 21 strain infection: All 5 pigs had a body temperature higher than 40.5°C (Figure 8), and they showed symptoms such as reduced feeding, depression, and lying down; 2 pigs died on the 5th day after the infection, 1 pig died on the 6th day, and 2 pigs died on the 7th day, with a survival rate of 0% (Figure 9). The above results show that the immune protection rate of JS4821 strain immunity against the virulent JS / LG / 21 strain is 100%. Compared with the JS / LG / 21 strain, the ASFV JS4821 strain has lost its virulence due to reasons such as the deletion of large genomic fragments. The cell culture titer of the ASFV JS4821 strain has not changed compared with the JS / LG / 21 strain, so the attenuation does not affect the artificial reproduction of the virus. Vaccination with the ASFV JS4821 strain can produce immune protection against the JS / LG / 21 strain. These characteristics indicate that the ASFV JS4821 strain has the ability to prepare live vaccines.

[0089] Example 8: Study on the protective effect of ASFV JS4821 strain against the virulent heterologous strain ASFV HLJ / 18 strain (genotype II)

[0090] This example is used to evaluate the effectiveness of immune protection of the attenuated strain ASFV JS4821 against heterologous virulent strains. 6 TCID 50ASFV JS4821 strain (PSF culture of ASFV JS4821 strain diluted with sterile PBS) was injected intramuscularly into 7-week-old pigs (SPF pigs provided by the Experimental Animal Center of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences), a total of 5 pigs. 28 days after inoculation, 100LD 50 Pigs were challenged with a heterologous virulent strain (ASFV HLJ / 18 strain, genotype II) of 100 mg / pig by intramuscular injection. A non-immunized control group of 5 pigs was challenged with the virus.

[0091] After the infection, the pigs' body temperature was measured daily, and clinical symptoms such as feeding, drinking, mental state, and body surface were observed, and deaths were recorded. Results of the immunization group after the ASFV HLJ / 18 strain attack: All 5 pigs had a body temperature below 40.0°C, which was normal (Figure 10); all 5 pigs had normal feeding, drinking, mental state, etc., and 100% of the pigs survived (Figure 11). Results of the control group after the ASFV HLJ / 18 strain infection: All 5 pigs had a body temperature above 40.5°C (Figure 10), and they showed symptoms such as reduced feeding, depression, and lying down. One pig died on the 7th day after infection, one pig died on the 9th day, one pig died on the 10th day, and two pigs died on the 11th day. The survival rate was 0% (Figure 11). This shows that the immune protection rate of JS4821 strain against the genotype II virulent strain HLJ / 18 is 100%.

[0092] Based on Examples 7 and 8, it can be seen that the vaccination with ASFV JS4821 strain has immune protection against JS / LG / 21 strain and ASFV HLJ / 18 strain, which shows that the live vaccine prepared with ASFV JS4821 strain as the immunogen can resist the attack of at least two ASFV epidemic strains and has good protective efficacy for pigs.

[0093] Example 9: Preparation of vaccine

[0094] Preparation of lyoprotectant: Prepare an aqueous solution containing 8 w / w% gelatin and 40 w / w% sucrose. Autoclave at 116°C for 20 minutes and store at 2-8°C until ready for use.

[0095] Virus culture: ASFV JS4821 strain was diluted to 10,000 TCID using RPMI Medium 1640 (purchased from Thermo Fisher Scientific Biochemical Products (Beijing) Co., Ltd., catalog number: C11875500BT) complete culture medium (containing 10 v / v% fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin). 50 / ml. Inoculate the diluted virus strain into the primary PAM. Place the culture flask in a 37°C, 5% CO2 incubator and harvest the virus when the CPE reaches 80%.

[0096] Mix: African swine fever virus JS4821 culture medium (virus content not less than 10 6.0 TCID 50 / ml) and freeze-dried protective agent were mixed evenly at a volume ratio of 8.5:1.

[0097] Lyophilization: freeze-dry the mixture at the following conditions: -5°C, 1 hour; -40°C, 2 hours; -16°C, 14-18 hours; -6°C, 3 hours; 5°C, 2 hours; and 28°C, 6 hours.

[0098] Physical property characterization: The freeze-dried vaccine obtained is loose and porous, sponge-like, and dissolves quickly after adding water.

[0099] It is understood from common technical knowledge that the present invention may be implemented by other embodiments that do not depart from its spirit or essential features. Therefore, the embodiments disclosed above are, in all respects, merely illustrative and not exclusive. All modifications within the scope of the present invention or equivalent to the scope of the present invention are intended to be encompassed by the present invention.

Claims

1. An African swine fever virus strain, wherein the African swine fever virus strain is an African swine fever virus strain with a microbial preservation number of CCTCC NO: V2023100.

2. A vaccine composition for preventing, mitigating or controlling African swine fever, wherein the vaccine composition uses the African swine fever virus strain described in claim 1 as an immunogen.

3. The vaccine composition according to claim 2, characterized in that The vaccine composition is a live vaccine composition.

4. The vaccine composition according to claim 2, characterized in that The raw materials of the vaccine composition include the African swine fever virus strain and auxiliary materials.

5. The vaccine composition according to claim 4, characterized in that The auxiliary material is a freeze-drying protective agent.

6. The vaccine composition according to claim 5, characterized in that In the raw materials of the vaccine composition, the lyophilization protectant is an aqueous solution containing 4-12 w / w% gelatin and 30-50 w / w% sucrose.

7. The vaccine composition according to claim 5, characterized in that In the raw materials of the vaccine composition, the dry weight ratio of the African swine fever virus strain to the lyophilized protective agent is 1-20×10 6.0 TCID 50 : 1g.

8. The vaccine composition according to claim 7, characterized in that TCID of the African swine fever virus strain 50 It is calculated based on primary porcine alveolar macrophage culture using immunofluorescence analysis according to the Reed-Muench method.

9. A method for preparing the vaccine composition according to claim 5, wherein the method comprises: using the African swine fever virus strain as an immunogen to prepare the vaccine composition.

10. The preparation method according to claim 9, characterized in that: The preparation method comprises: mixing the African swine fever virus strain with the lyophilization protectant to obtain a mixture, and freeze-drying the mixture to obtain the vaccine composition.

11. The preparation method according to claim 10, characterized in that: The temperature change program of the freeze-drying is: -4 to -6°C, 0.8-1.2 hours; -35 to -45°C, 1.5-2.5 hours; -14 to -18°C, 14-18 hours; -5 to -7°C, 2-4 hours; 4-6°C, 1.5-2.5 hours; 26-30°C, 5-7 hours.

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