CN117701509A - African swine fever virus passage attenuated strain and vaccine based on strain - Google Patents
African swine fever virus passage attenuated strain and vaccine based on strain Download PDFInfo
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Abstract
The invention provides an African swine fever virus strain with a microorganism preservation number of CCTCC NO: V2023100. The invention also provides a vaccine composition containing the African swine fever virus strain and a freeze-drying protective agent for preventing African swine fever. The invention also provides a preparation method of the vaccine composition. The virus strain has good safety and immune effectiveness. Pigs inoculated with the strain can produce high-level antibodies against African swine fever virus and resist against the virulent virus attack of African swine fever of different genotypes. The African swine fever virus and vaccine composition provided by the invention has clinical application prospect.
Description
Technical Field
The invention belongs to the field of veterinary biological products, and relates to an African swine fever virus passage attenuated strain and a vaccine based on the strain.
Background
African swine fever (African Swine Fever, ASF) is an acute, hemorrhagic, virulent infectious disease of pigs or wild boars caused by infection with African swine fever virus (African Swine Fever Virus, ASFV). The world animal health organization (World Organization for Animal Health, WOAH) lists ASFs as legal report animal epidemic diseases. China lists ASF as a group of animal diseases. The clinical manifestations of ASF include most acute, subacute and chronic. The acute symptoms are mainly: the body temperature is raised, depression, anorexia, bleeding spots on the ear, limb and abdomen skin, visible mucous membrane flushing, cyanosis, mucopurulent secretion on eyes and nose, vomiting, constipation, blood and mucus coverage on the surface of feces, diarrhea, feces with blood, ataxia or gait stiffness, paralysis, convulsion, dyspnea, abortion of pregnant sows, and mortality rate of 100 percent.
ASFV is the only member of the African swine fever virus family (Asfarviridae) of the African swine fever virus genus (Asfivirus). The ASFV genome is double-stranded linear DNA of 170-194kb in size, containing 150-167 open reading frames, encoding 54 structural proteins and 100 more non-structural proteins. ASFV has been classified into 24 genotypes according to a sequence difference of about 500bp nucleotide at the end of B646L gene (encoding p72 protein).
According to WOAH statistics, ASF has been outbreaked and popular in nearly 50 countries in Africa, europe, asia and Caribbean, resulting in great economic loss and serious harm to the stable development of the global pig industry and related industries.
ASF strain identified by first isolation in China is ASFV HLJ/18 strain (GenBank: MK333180.1, herein called Pic/HLJ/2018). ASFV HLJ/18 strain is virulent strain, and mortality rate of vaccinated pigs is 100%. The whole genome sequence determination and evolution analysis prove that the ASFV HLJ/18 strain is a gene II type virulent strain. With the continuous popularity and evolution of ASFV in China, a middle virulence gene II strain HLJ/HRB1/20 strain without blood cell adsorption activity is identified in China in the field isolation of 2020 (GenBank: MW656282.1, which is called as Pig/Heilongjiang/HRB 1/2020), a low virulence gene I strain SD/DY-I/21 strain is identified in China in the field isolation of 2021 (GenBank: MZ945537.1, which is called as Pig/SD/DY-I/2021) and a naturally recombinant virulent strain JS/LG/21 strain of gene I and gene II (GenBank: OQ504956.1, which is called as Pig/Jiangsu/LG/2021) are reported for the first time in 2023, which makes Chinese ASF control more severely challenge. Of concern, attenuated vaccine strains HLJ/18-7GD (see China patent application with the application number CN 202310363645.0) constructed by taking the gene II virus as a parent cannot generate good immune protection on the recombinant virulent strains of the gene I and the gene II (such as JS/LG/21 strain). Such recombinant virulent strains of genotype I and type II (e.g., JS/LG/21 strain) were first detected in China since 2021.
Vaccine immunization has been the most economical and effective means of controlling infectious diseases. The control or elimination of animal diseases such as plague, classical swine fever and equine infectious anemia all benefit from safe and effective vaccine immunization. The virulent infection of ASFV can resist re-infection of homologous virulent by passing pigs, which suggests that vaccine immunization has theoretical feasibility for ASF prevention. Scientists have conducted a great deal of vaccine research.
No reported African swine fever virus inactivated vaccine, subunit vaccine, recombinant vector vaccine and the like have ideal clinical protection effect.
Naturally variant attenuated strains (for example, NH/P68 and OUR T88/3 strains of genotype I and Lv17/WB/Rie1 strains of genotype II) can effectively protect against attack of homologous virulence after inoculation, but the inoculation leads to chronic diseases such as skin ulcer, arthritis and the like, and still has certain residual virulence.
The gene deletion attenuated strain (such as ASFV-G-delta I177L strain deleted I177L gene, BA71 delta CD2v strain deleted CD2v gene, ASFV SY 18-delta CD2v/UK strain deleted CD2v gene and UK gene, HLJ/18-7GD strain deleted MGF360/505 gene and CD2v gene) has no obvious clinical symptoms after being inoculated to pigs, and the inoculation can effectively protect the attack of homologous virulence. However, none of these gene-deleted attenuated strains provided heterologous cross-protection against the newly emerging recombinant virulent strains of genotype I and II (JS/LG/21 strains) after inoculation of pigs. Clinical trials involving the HLJ/18-7GD strain have been conducted and ASFV-G- ΔI177L attenuated vaccines have been approved for use in Vietnam. It has been found that the combined deletion of multiple virulence related genes may affect the immunogenicity of ASFV and result in failure to resist attack by homologous virulent strains. The double gene deletion HLJ/18-9GL and UK-del strains deleted by combining 9GL genes and UK genes have no clinical symptoms after pigs are inoculated by the JS/LG/21-7GD deletion strain which is used for combining deletion of MGF360/505 genes and CD2v (7 genes). However, the inoculation of the two gene deletion strains cannot effectively resist the attack of homologous virulent strains.
Pathogen passaging is an important technological path for vaccine research. Vaccine research and development of epidemic diseases such as plague, classical swine fever, equine infectious anemia and the like all adopt animal or cell passage technical paths. The passage attenuated vaccine plays an important role in prevention and control of epidemic diseases, and realizes eradication and purification of Chinese rinderpest, rinderpest and equine infectious anemia and effective prevention and control of classical swine fever. ASFV cell adaptation studies were performed after the afferent portugal of ASFV in 1957. Representative ASFV-adapted strains are L'60BM89 strain, BA71v strain, E75CV1 strain, etc. In 1963 Ribeiro et al serially passaged the 1455 strain isolated on the portugal teeth on bone marrow cells and found that its virulence was progressively impaired. The virus attenuated in passage 70 is inoculated to pigs, and the clinical symptoms of the pigs inoculated with the virus attenuated in passage 70 are greatly reduced and can resist the attack of virulent strains. The strain was obtained by passaging PK-2a cells in 1965 by Hess et al. After the Spencer strain was transferred to 35 passages in 1967 by Greig et al, the F35 vaccinated pigs were asymptomatic and were resistant to parental strain challenge. After Greig et al transferred Portuguese challenge strain to 34 passages, F34 vaccinated pigs showed small fluctuations in body temperature and were resistant to parental strain challenge. Greig et al transferred the Gasson strain to 23 passages, and vaccinated pigs had reduced virulence but were not resistant to the parental strain challenge. In 1976 researchers adapted the BA71 strain to passage on Vero cells for 100 passages, and finally obtained a non-pathogenic Vero cell adapted strain (BA 71v strain). In 1979 Thomson et al used CV cell passaged attenuated strains to vaccinate 2 pigs, which were not able to effectively combat virulent strain challenge. A plurality of cell lines were obtained for passaging ASFV attenuated strains by Russian Federal veterinary virology and microbiology studies. The isolate obtained in congo in 1949 was transferred to porcine kidney cells for 50 passages and then to porcine bone marrow cells for 262 passages. The isolate obtained in france in 1964 was uploaded on porcine bone marrow cells for 135 passages. The strains were not subjected to vaccination and challenge protection tests. In 2015, krug et al vaccinated pigs at 110 th generation of the strain Vero cell adapted strain of the Gerugia strain (ASFV-G strain), and the vaccinated pigs did not have any clinical response, but were not effective against virulent strain challenge. Balyshava et al transmitted the Stavropol 01/08 virulent strain 24 passages on the porcine kidney embryonic cell line and porcine lymphocyte hybrid cell line (the hybrid cell line SPEV TK with pig lymphocytes, A4C 2), respectively, and the vaccinated pigs survived after 20 passages on CV cells, but failed to provide effective protection. The ASFV-G-delta I177L/delta LVR strain is obtained by continuously deleting the left variable region on the basis of the I177L gene deletion strain (ASFV-G-delta I177L strain), and the strain can stably and efficiently proliferate in a plum island pig epithelial cell line (Plum Island porcine epithelial cells, PIPEC), and the vaccinated pig has no clinical symptoms and provides effective protection for homologous virulent strains. 2021, wang et al serial passage of Chinese isolates (ASFV-HLJ/18 strain) in HEK293T cells to 121 passages resulted in highly efficient replication strains, but regressing PAM cells was extremely inefficient in replication and pathogenicity and immunogenicity was still in need of further investigation.
1921 found ASFV for the first time, it has been popular in more than 50 countries at present, causing huge economic losses for pig raising and related industries worldwide. Scientists have conducted a great deal of vaccine research, trying various vaccine development strategies. The reported protection effects of the inactivated vaccine, subunit vaccine and recombinant vector vaccine are not ideal; the inoculation of the natural variation attenuated strain can effectively protect the attack of homologous virulent strain, but the inoculated pigs have chronic diseases such as skin ulcer, arthritis and the like, and still have certain residual virulence; inoculation of some gene deletion attenuated strains can effectively protect against attack by homologous virulence, but cannot provide cross protection against heterologous strains; some gene deletions affect the immunogenicity of ASFV, resulting in an inability to combat virulent homologous strains; some passaged attenuated strains are resistant to attack by the parental virulent strain but have residual virulence. Some passaged attenuated strains completely lose immunogenicity and thus fail to provide protection. So far, no safe and effective African swine fever vaccine is available for popularization and clinical application. Many attenuated african swine fever virus vaccinations do not resist the challenge of the parental african swine fever virus well. There are few reports that the African swine fever virus-inoculated pigs are effective against various African swine fever virulent attacks.
Disclosure of Invention
The invention uses wild boar kidney cells (Boar kidney cells) BK2258 to continuously passage 93 generations of gene I type and II type recombinant African swine fever virus strain (JS/LG/21 strain), and then F93 is returned to primary pig alveolar macrophages (Primary porcine alveolar macrophages, PAM) to pass 10 generations, thus obtaining the cell passage adapted strain (namely JS4821 strain) of the African swine fever virus JS/LG/21 strain. Pigs inoculated by the strain JS4821 strain with the cell passage adaptive strain have no clinical symptoms such as increased body temperature, mental depression, food intake decline, skin ulceration or arthrocele, and the inoculated pigs can resist attack of the virulent strain (JS/LG/21 strain) of the gene I type and the recombinant African swine fever virus type II type and the epidemic virulent strain (HLJ/18 strain) of the gene II type. Artificially weakened African swine fever virus strains with cross protection effect are rarely reported. The JS4821 strain has great application value in developing vaccines.
In order to solve the problems in the prior art, the first aspect of the invention provides an African swine fever virus strain, wherein the African swine fever virus strain is an African swine fever virus strain with a microorganism preservation number of CCTCC NO: V2023100.
In a second aspect, the invention provides a vaccine composition for preventing, slowing or controlling African swine fever, which comprises the African swine fever virus strain according to the first aspect of the invention as an immunogen.
In some embodiments, the vaccine composition is a live vaccine composition.
In some embodiments, the vaccine composition comprises the african swine fever virus strain and an adjuvant as raw materials.
In some embodiments, the adjuvant is a lyoprotectant.
In some embodiments, the lyoprotectant is an aqueous solution containing 4-12w/w% gelatin and 30-50w/w% sucrose in the starting material of the vaccine composition.
In some embodiments, the dry weight ratio of the African swine fever virus strain to the lyoprotectant in the starting material of the vaccine composition is 1-20 x 10 6.0 TCID 50 ：1g。
In some embodiments, the TCID of the african swine fever virus strain 50 Is calculated according to the Reed-Muench method by adopting an immunofluorescence analysis method based on primary pig alveolar macrophage culture.
In a third aspect, the present invention provides a method of preparing a vaccine composition according to the second aspect of the invention, the method comprising: the vaccine composition is prepared by taking the African swine fever virus strain as an immunogen.
In some embodiments, the method of making 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.
In some embodiments, the freeze-drying temperature variation procedure is: -4 to-6 ℃,0.8 to 1.2 hours; -35 to-45 ℃ for 1.5-2.5 hours; -14 to-18 ℃,14-18 hours; -5 to-7 ℃,2-4 hours; 4-6 ℃ for 1.5-2.5 hours; 26-30 ℃ for 5-7 hours.
The invention has the advantages that:
1. a passaging strain ASFV JS4821 strain capable of stably proliferating on BK2258 cells was obtained.
2. The ASFV JS4821 strain does not generate fever after being inoculated into pigs, and has no clinical symptoms.
3. The cell passage strain ASFV JS4821 strain can resist the attack of homologous virulent strain JS/LG/21 strain (gene I type) and heterologous virulent strain HLJ/18 strain (gene II type) simultaneously, and has the effect of cross protection of different genotypes of ASFV.
Drawings
FIG. 1 shows CPE photographs of F93-generation viruses obtained by culturing ASFV JS/LG/21 strain with BK2258 cells, in a scale of 100. Mu.m.
FIG. 2 shows photographs of CPE of ASFV JS4821 strain cultured with PAM, with a scale of 100. Mu.m, compared to a control photograph.
FIG. 3 shows a fluorescence photograph and a bright field photograph based on an IFA test of culturing ASFV JS4821 strain and JS/LG/21 strain with PAM, with a scale of 100. Mu.m.
FIG. 4 shows statistical data of pathogenicity and transmissibility of ASFV JS/LG/21 strain on pigs.
FIG. 5 shows ASFV JS4821 strain at 10 6 TCID 50 Body temperature of pigs after immunization of the first pig dose.
FIG. 6 shows an electropherogram of ASFV JS4821 relative to the deletion of genomic fragments of JS/LG/21 strain.
Fig. 7 shows the case of ASFV JS4821 strain-induced antibody.
FIG. 8 shows the body temperature of the virulent strain JS/LG/21 strain of genotype I after challenge.
FIG. 9 shows the survival rate of the virulent strain JS/LG/21 strain of genotype I after challenge.
FIG. 10 shows body temperature after challenge of the virulent strain HLJ/18 strain of genotype II.
FIG. 11 shows the survival rate of the virulent strain HLJ/18 strain of genotype II after challenge.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the embodiments of the present invention will be described in further detail with reference to the accompanying drawings.
Strains and cells
African swine fever virus JS/LG/21 strain (ASFV JS/LG/21 strain, JS/LG/21 strain) is a naturally isolated virulent strain of African swine fever virus. According to B646L genotyping, JS/LG/21 strain is gene I type African swine fever virus. Based on genome sequence feature analysis, JS/LG/21 strain is a natural recombinant virus of gene I type African swine fever virus and gene II type African swine fever virus. The JS/LG/21 strain was isolated, identified and stored by the Harbin veterinary institute of China academy of agricultural sciences. The isolation and characterization of Pig/Jiangsu/LG/2021 (JS/LG/21 strain) is described in the literature (Highly lethal genotype I and II recombinant African swine fever viruses detected in Pig. Donging Zhao et al, nature communications. Volume 14,Article number:3096 (2023): https:// doi. Org/10.1038/s 41467-023-38868-w). The genome sequence of JS/LG/21 strain is shown in GenBank No. OQ504956.1, wherein the strain is called Pic/Jiangsu/LG/2021.
GenBank of ASFV HLJ/18 strain is MK333180.1, herein designated as Pic/HLJ/2018.
BK2258 cell is a wild boar kidney-derived cell (Boar kidney cells) prepared and stored by the Harbin veterinary institute of the national academy of agricultural sciences. The preparation of BK2258 cells is described in the application document of the Chinese patent application No. CN 202211137583.3.
Primary PAM cells (Primary porcine alveolar macrophages, PAMs) were prepared and used by the Harbin veterinary institute of the national academy of agricultural sciences.
Example 1: passage adaptation of ASFV JS/LG/21 strain
Method of BK2258 cell culture virus: with DMEM (purchased from Simer Feishier Biochemical Co., ltd., beijing), cat# C11995500 BT)Maintaining the culture solution (containing 3v/v% fetal bovine serum, 100U/ml penicillin, 100 μg/ml streptomycin) to dilute ASFV JS/LG/21 strain to 10000TCID 50 /ml. The diluted virus strain was inoculated with BK2258 cells grown as a good monolayer. The flask was placed at 37℃with 5% CO 2 Is cultured in an incubator of (2), and the presence or absence of cytopathic effect (CPE) on a cell monolayer is observed every day. If no apparent CPE was found for 8 days of culture, the virus was harvested after 8 days of culture. If significant CPE was seen 3 days of culture, the virus was harvested when CPE reached 80%.
The ASFV JS/LG/21 strain was cultured with BK2258 cells by the method of virus culture with BK2258 cells as described above. CPE was not present at day 8 of the generation 1 culture. After freeze thawing the F1-generation cultures 2 times at-70℃and 37℃the virus culture broth was harvested and serial passaging of ASFV JS/LG/21 strain was continued using BK2258 cells in the same way. Apparent CPE appears from F6, and increases with increasing number of generations. In fig. 1, a shows a photograph of an F93 strain 96h after inoculation of BK2258 cells. F93 strain culture is characterized by cell aggregation, shrinkage, lysis, and finally formation of circular plaques. In fig. 1, B shows a BK2258 cell control cultured under the same conditions without virus inoculation. As can be seen, ASFV JS/LG/21 strain F93 was fully adapted to BK2258 cells.
Method for primary PAM cell culture of virus: ASFV JS4821 strain was diluted to 10000TCID with RPMI Medium 1640 (purchased from Semer Feisher Biochemical Co., ltd., product number: C11875500 BT) complete broth (containing 10v/v% fetal bovine serum, 100U/ml penicillin, 100. Mu.g/ml streptomycin) 50 /ml. The diluted virus strain was inoculated with primary PAM cells. The flask was placed at 37℃with 5% CO 2 In the incubator of (a), cells were observed daily for cytopathic effect (CPE). If significant CPE was seen on day 2 of culture, the virus was harvested when CPE reached 80%.
The F93 strain is inoculated with primary PAM cells by adopting the method for culturing viruses by the primary PAM cells and continuously transmitted for 10 generations, and obvious CPE is visible in each generation. In fig. 2, a shows a photograph of the F10 strain after 72h inoculation with primary PAM cells. F10 strain culture is characterized by cell aggregation and lysis. In fig. 2, B shows a primary PAM cell control cultured under the same conditions without virus inoculation. The F10 strain obtained by passaging the primary PAM cells of ASFV JS/LG/21 is named ASFV JS4821 strain.
The African swine fever virus JS4821 strain obtained through serial in vitro passage is submitted to a patent program approval preservation institution for preservation. The preservation unit is China Center for Type Culture Collection (CCTCC); addresses are China, wuhan, university of Wuhan; the microorganism has a preservation number of CCTCC NO: V2023100; the classification is named: african Swine Fever Virus the culture is named African swine fever virus ASFV JS4821 African swine fever virus ASFV JS4821; the preservation time is 2023, 11 and 6; survival time was identified as 2023, 11, 7.
Example 2: titer determination of ASFV JS4821 strain and ASFV JS/LG/21 strain
Respectively diluting ASFV JS4821 strain and ASFV JS/LG/21 strain with RPMI Medium 1640 culture solution 10 times to 10 times -8 Dilution. Will 10 -4 、10 -5 、10 -6 、10 -7 And 10 -8 These 5 dilutions of virus were inoculated with primary PAM cells in 96 wells, respectively, and 8-well replicates were made for each dilution, 100 μl/well. With normal cell controls, primary PAM cells in 96 wells were added using RPMI Medium 1640 Medium and 8-well replicates were performed at 100 μl/well. Placing 96-well plate at 37deg.C, containing 5% CO 2 Culturing in an incubator for 7 days.
The fluorescence in each well was detected by IFA (immunofluorescence assay ) method comprising the steps of: (1) Fixing the cells in each well of a 96-well plate with formaldehyde, (2) after washing each well with PBS, adding a rabbit anti-ASFV p72 protein polyclonal antibody to each well at an antibody concentration of 1:1000 at 100 μl/well, and incubating at 37deg.C for 30min. (3) After washing each well with PBS, FITC-labeled rabbit anti-pig IgG (Sigma-Aldrich, F0382-2 ML) was added at an antibody concentration of 1:200 in an amount of 100. Mu.l/well and incubated at 37℃for 30min. (4) After washing each well with PBS, fluorescence and bright field detection was performed for each well. See fig. 3 for a partial photograph. In FIG. 3, A shows ASFV JS4821 strain at 10 -5 IFA detection fluorescence photograph after 7d dilution inoculation of primary PAM cells; a# shows ASFV JS4821 strain at 10 -5 After 7d of dilution inoculation of primary PAM cellsBright field photographs; b shows ASFV JS4821 strain to 10 -8 IFA detection fluorescence photograph after 7d dilution inoculation of primary PAM cells; b# shows ASFV JS4821 strain at 10 -8 Bright field photographs 7d after dilution inoculation of primary PAM cells; c shows ASFV JS/LG/21 strain at 10 -5 IFA detection fluorescence photograph after 7d dilution inoculation of primary PAM cells; c# shows ASFV JS/LG/21 strain at 10 -5 IFA detection bright field photograph 7d after dilution inoculation of primary PAM cells; d shows ASFV JS/LG/21 strain at 10 -8 IFA detection fluorescence photograph after 7d dilution inoculation of primary PAM cells; d# shows ASFV JS/LG/21 strain at 10 -8 Bright field photographs 7d after dilution inoculation of primary PAM cells.
The number of positive fluorescent wells per treatment was recorded. For ASFV JS4821 strain, 10 -4 、10 -5 And 10 -6 The 8 wells corresponding to the dilutions all showed IFA positive; 10 -7 And 10 -8 The 8-hole IFA corresponding to the dilution is negative, and the titer of ASFV JS4821 strain is calculated to be 10 according to the Reed-Muench method 7.50 TCID 50 /ml. For ASFV JS/LG/21 strain, 10 -4 、10 -5 And 10 -6 The 8 wells corresponding to the dilutions all showed IFA positive; 10 -7 The 2 holes corresponding to the dilution all show IFA positive, and the 6 holes show IFA negative; 10 -8 8 wells corresponding to dilution all showed IFA negative, and the titer of ASFV JS/LG/21 strain was calculated to be 10 according to the Reed-Muench method 7.67 TCID 50 /ml。
As can be seen, the titer of ASFV JS4821 strain on target cells (primary PAM) was not significantly different from that of ASFV JS/LG/21 strain. This indicates that passage of cells did not significantly affect their ability to propagate in vitro, and that passage did not affect the ability of the virus to propagate in vitro.
Example 3: pathogenicity analysis of ASFV JS/LG/21 strain
To evaluate virulence and transmissibility of ASFV JS/LG/21 strain to pigs, SPF-grade pigs of 7 weeks of age were selected from the national academy of agricultural sciences Harbin veterinary institute of laboratory animal center and respectively intramuscular injected 10 3 HAD 50 And 10 (V) 6 HAD 50 Is inoculated with 6 pigs per dose.
From day 1 of infection, 2 additional SPF-grade pigs were selected for each group and raised with the infected pigs to assess the transmission capacity of ASFV JS/LG/21 strain. All pigs were monitored daily for survival and clinical signs. qPCR was used to test individual pigs for african swine fever virus p72 gene in oral swab, rectal swab and EDTA anticoagulated blood at designated times post inoculation (p.i) or post exposure (p.ct) to reflect viral load. The dead or euthanized pigs were dissected and samples were taken of brain, heart, liver, spleen, lung, kidney, tonsils, inguinal, mandibular and mediastinal lymph nodes, and the p72 gene in each sample was detected using qPCR to reflect viral load.
The corresponding test results are shown in fig. 4. In FIG. 4, a-f show 10 6 HAD 50 Corresponding indexes of the dose inoculation pigs and concomitant pigs; g-I shows 10 3 HAD 50 Corresponding indexes of the dose inoculation pigs and concomitant pigs; a and g show the rectal body temperature of the pigs after inoculation; b and h show the survival rate of pigs after inoculation; c and i show the oral swab viral DNA content of pigs; d and j show the rectal swab viral DNA content of pigs; e and k show the blood viral DNA content of pigs; f and l show the viral DNA content of 10 tissues of pigs. The black line dashed line represents the normal rectal body temperature (40 ℃) of the pig. LN1 represents inguinal lymph node; LN2 represents a mandibular lymph node; LN3 represents the mediastinal lymph node.
At 10 6 HAD 50 In the group, all 6 pigs vaccinated started fever on day 4 of p.i. and died between day 5 and day 8 of p.i.; 2 pigs exposed to swine started to fever on day 9 after exposure and died on day 12 (a, b); viral DNA was detected in all vaccinated and contact pigs in oral swabs, rectal swabs and blood (c, d, e); high levels of viral DNA (f) were detected in organs of dead pigs (brain, heart, liver, spleen, lung, kidney, tonsils, inguinal lymph nodes, submaxillary lymph nodes and mediastinal lymph nodes).
At 10 3 HAD 50 In the group, all pigs died (h) from day 6 to day 15; the 2 contact pigs began to heat up on days 9 and 10 after infection, and died (g, h) on days 12 and 14 after inoculation, respectively; all jointsViral DNA was detected in both the oral swab, rectal swab, and blood of the breeding and contact pigs, but at levels slightly below 10 6 HAD 50 Group (i, j, k); high levels of viral DNA were detected in organs of dead pigs (brain, heart, liver, spleen, lung, kidney, tonsils, inguinal lymph nodes, submaxillary lymph nodes and mediastinal lymph nodes) (l). These results indicate that the recombinant virus isolate ASFV JS/LG/21 has high lethality and transmissibility.
Example 4: safety study of ASFV JS4821 strain inoculated pigs
To evaluate the safety of ASFV JS4821 strain against pigs, the strain was used as a vaccine at 10 6 TCID 50 Dose of first pigs (PAM cultures of ASFV JS/LG/21 strain diluted with sterile PBS), 7 week old pigs (SPF grade pigs supplied by the experimental animal center of haerbin veterinary institute of academy of agricultural sciences) were vaccinated with ASFV JS4821 strain intramuscular injection. The two groups were inoculated independently, 5 pigs per group. After inoculation, rectal body temperature is measured daily for each inoculated pig, and clinical symptoms such as feeding, drinking water, mental state, body surface and the like are observed. The results show that all pigs have normal body temperature (figure 5) after inoculation, normal feeding, drinking water, mental state and the like, and no adverse clinical symptoms are seen, which indicates that ASFV JS/LG/21 strain has good safety.
Example 5: differential research of ASFV JS4821 strain and ASFV JS/LG/21 strain
As can be seen from example 3, both groups of pigs vaccinated with ASFV JS/LG/21 strain died within 14 days after vaccination. As can be seen from example 4, no symptoms were seen in ASFV JS4821 strain at 28 days of inoculation. From this, it is assumed that ASFV JS4821 strain developed significant virulence attenuation. To further examine the genetic differences between ASFV JS4821 strain and ASFV JS/LG/21 strain, the gene component was divided into 25 fragments by reference to the whole genome sequence of ASFV JS/LG/21 strain (GenBank accession number: OQ 504956.1). The upstream and downstream primers were designed based on 25 genome fragments, and PCR amplification was performed on genome templates extracted from the parent strain JS/LG/21 strain and the passaging attenuated strain JS4821 strain by means of an AXYGEN virus nucleic acid miniprep kit (purchased from Corning Life sciences (Wu Jiang) Co., ltd., cat# AP-MN-BF-VNA-250), and the amplification results were characterized by electrophoresis. Two genomic amplification product lengthsThe electropherograms corresponding to the most distinct primer pairs are shown in FIG. 6. In fig. 6, lane 1: DL15000 DNA Marker, lane 2: amplified product of parent strain JS/LG/21, lane 3: amplified product of passaging attenuated strain JS4821 strain, lane C: h 2 O control amplification. As can be seen from FIG. 6, the parent strain JS/LG/21 strain amplification product is approximately 2kb in length from the JS4821 strain amplification product.
FIG. 6 shows the results obtained by amplification of the upstream primer sequence F (SEQ ID NO. 1) and the downstream primer sequence (SEQ ID NO. 2).
The F sequence is as follows: 5'-TGTTTAGTGAGCCGTTTCT-3'.
The R sequence is as follows: 5'-TGTCACCATCAGAGAGTTC-3'.
As a result, the ASFV JS4821 strain was deleted about 2kb relative to the genome of the ASFV JS/LG/21 strain. It is estimated that this deletion of genomic fragments is the main cause of attenuation of ASFV JS4821 strain. The ASFV JS4821 strain has the prospect of preparing live vaccines.
Example 6: immunogenicity study of ASFV JS4821 strain
To evaluate immunogenicity of the passaged strain ASFV JS4821 strain, 10 each 3 TCID 50 Pig head/pig head 10 6 TCID 50 Virus dose of JS4821 strain of first pigs (PAM culture of ASFV JS/LG/21 strain diluted with sterile PBS) 7-week-old pigs (SPF grade pigs supplied by the experimental animal center of haerbin veterinary institute of academy of agricultural sciences) were inoculated intramuscularly, each dose inoculating 5 pigs. Meanwhile, 5 non-inoculated control groups (negative control) were set. Blood was collected every 5 days after inoculation, serum was prepared, and the p22 antibody blocking rate was measured by a p22 protein blocking ELISA antibody detection method. The detection procedure was as follows:
1. coating: prokaryotic expression of African swine fever virus p22 protein antigen was diluted to 4 μg/ml antigen dilution with antigen coating solution (carbonate buffer), added to 96-well plates, 50 μl/well, and coated overnight at 4deg.C.
2. Washing: 300 μl/Kong Xi with PBST (phosphate buffered saline containing 0.05v/v% Tween 20) for 1 minute each.
3. Closing: 5w/v% skim milk was blocked, 200. Mu.l/well, and incubated at 37℃for 2 hours.
4. Washing: and 2.
5. Sample adding: the serum was diluted 1:1 by volume in PBS, added to a 96-well plate, 50. Mu.l/well, and incubated at 37℃for 30 minutes.
6. Washing: and 2.
7. Enzyme-labeled antibody: the enzyme-labeled antibody (horseradish peroxidase-labeled mouse anti-African swine fever virus p22 protein porcine IgG monoclonal antibody) was diluted 1:2000 by volume with a secondary antibody diluent (purchased from Ind Biotechnology Co., ltd., huzhou, cat# HRP-SD-001) and incubated at 37℃for 30 minutes.
8. Washing: and 2.
9. Color development: mu.l of TMB substrate was added to each well and developed, and the reaction was carried out at 37℃for 10 minutes in the absence of light.
10. And (3) terminating: mu.l of stop solution (2M H) was added to each well 2 SO 4 )。
11. Reading: determination of OD 450nm Values.
The% blocking was calculated according to the following formula = [ (average OD of negative control) 450nm Sample OD 450nm ) Average OD of negative control 450nm ]X 100. The judgment standard is as follows: the blocking rate was positive above 47% and negative below 47%.
The results show that: 5 days after inoculation, 10 3 TCID 50 Pig group/head 10 6 TCID 50 The first pig group antibody does not transfer positive; 10 days after inoculation, 10 3 TCID 50 3/5 antibody transfer to yang, 10 in the first pig group 6 TCID 50 The first pig group 5/5 antibody changes positive; 20 days after inoculation, 10 3 TCID 50 Pig group/head 10 6 TCID 50 The first pig group antibody totally changes positive, and the antibody titer reaches the peak; 25 days after inoculation, both groups continued to peak; the non-immunized control antibody was always negative. Results of immunization time and blocking rate are shown in fig. 7. The result shows that the strain ASFV JS4821 with the attenuated passage has strong immunogenicity and has the value of preparing the vaccine against the African swine fever virus.
Example 7: protective study of ASFV JS4821 strain on homologous virulent strain JS/LG/21 strain (Gene I)
This example was used to evaluate passaging attenuated strain ASFV JS4821 strainImmunoprotection against homologous virulent strains. At 10 6 TCID 50 Dose of first pigs (PAM cultures of ASFV JS/LG/21 strain diluted with sterile PBS) ASFV JS4821 strain was intramuscular inoculated with 5 first 7 week old pigs (SPF grade pigs supplied by the institute of laboratory animals, haerbin veterinary sciences, national academy of agricultural sciences). After 28d inoculation, 100LD was obtained with homologous virulent strain (ASFV JS/LG/21 strain, genotype I) 50 The dosage of pigs is intramuscular injection to counteract toxic substances. And 5 pigs in a non-immune control group are provided for detoxification.
After toxin expelling, the pig body temperature is measured every day, clinical symptoms such as feeding, drinking water, mental state, body surface and the like are observed, and death cases are recorded. Post-challenge immune panel results for ASFV JS/LG/21 strain: except that 1 pig had 1 time body temperature higher than 40.0 ℃ on day 4 after the challenge, all other pigs had body temperatures not higher than 40.0 ℃ and were normothermic (fig. 8); all 5 pigs had normal symptoms such as feeding, drinking water, mental status, etc.; 100% survival (figure 9). Results of ASFV JS/LG/21 strain post-challenge control group: all 5 pigs had a body temperature above 40.5deg.C (figure 8) and had symptoms of reduced feeding, mental depression, and decubitus; 2 pigs died on day 5 after challenge, 1 pig on day 6, 2 pigs died on day 7, and survival rate was 0% (fig. 9). The result shows that the JS4821 strain has 100% of immune protection rate to the virulent strain JS/LG/21 after immunization. The ASFV JS4821 strain loses toxicity due to the fact that the ASFV JS4821 strain has large genome segment deletion and the like relative to the JS/LG/21 strain, the cell culture titer of the ASFV JS4821 strain is unchanged relative to the JS/LG/21 strain, so that artificial propagation is not affected, and immune protection against the JS/LG/21 strain can be generated by inoculating the ASFV JS4821 strain, which indicates that the ASFV JS4821 strain has the capability of preparing live vaccines.
Example 8: protective study of ASFV JS 4812 strain on heterologous virulent strain ASFV HLJ/18 strain (Gene II)
This example was used to evaluate the immunoprotection effectiveness of passaged attenuated strain ASFV JS4821 strain against heterologous virulent strains. At 10 6 TCID 50 Dose of first pigs (PAM cultures of ASFV JS/LG/21 strain diluted with sterile PBS) ASFV JS4821 strain was intramuscular inoculated with 5 first 7 week old pigs (SPF grade pigs supplied by the institute of laboratory animals, haerbin veterinary sciences, national academy of agricultural sciences). After 28d inoculation, the strain was used with a heterologous virulent strain (ASFV HLJ/18 strain, geneType II) at 100LD 50 The dosage of pigs is intramuscular injection to counteract toxic substances. And 5 pigs of a non-immune control group are provided for detoxification.
After toxin is removed, the body temperature is measured every day, clinical symptoms such as feeding, drinking water, mental state, body surface and the like are observed, and death cases are recorded. post-ASFV HLJ/18 strain challenge immunization group results: all 5 pigs had a body temperature below 40.0 ℃ and were normothermic (figure 10); all 5 pigs had normal symptoms of feeding, drinking water, mental status, etc., 100% survived (fig. 11). Results of ASFV HLJ/18 strain post-challenge control group: all 5 pigs had a body temperature higher than 40.5 ℃ (figure 10), had symptoms of reduced feeding, mental depression, happy lying and the like, and died 1 pig on day 7, 1 pig on day 9, 1 pig on day 10, 2 pigs on day 11, and the survival rate was 0% after challenge. The JS4821 strain shows that the immune protection rate of the strain on the gene type II virulent strain HLJ/18 strain is 100 percent after immunization. The inoculated ASFV JS4821 strain has the immune protection against JS/LG/21 strain and ASFV HLJ/18 strain, which shows that the live vaccine prepared by taking the ASFV JS4821 strain as immunogen can resist attack of at least two epidemic strains of the ASFV, and has good protective effect on pigs.
Example 9: preparation of vaccine
Preparation of lyoprotectant: an aqueous solution containing 8w/w% gelatin and 40w/w% sucrose is autoclaved at 116 ℃ for 20 minutes and then placed at 2-8 ℃ for standby.
Virus culture: ASFV JS4821 strain was diluted to 10000TCID with RPMI Medium 1640 (purchased from Semer Feisher Biochemical Co., ltd., product number: C11875500 BT) complete broth (containing 10v/v% fetal bovine serum, 100U/ml penicillin, 100. Mu.g/ml streptomycin) 50 /ml. The diluted virus strain was inoculated with primary PAM cells. The flask was placed at 37℃with 5% CO 2 Is cultured in an incubator, and the virus is harvested when the CPE reaches 80%.
Mixing: mixing African swine fever virus JS4821 culture solution (virus content not less than 10) 6.0 TCID 50 And/ml) and the freeze-drying protective agent are uniformly mixed according to the volume ratio of 8.5:1.
And (3) freeze-drying: freeze-drying the mixed solution, wherein the procedures are as follows: -5 ℃,1 hour; -40 ℃,2 hours; -16 ℃,14-18 hours; -6 ℃,3 hours; 5 ℃ for 2 hours; 28℃for 6 hours.
Characterization of physical properties: the obtained freeze-dried vaccine is loose and porous and is spongy, and can be dissolved rapidly after water is added.
It will be appreciated by those skilled in the art that the present invention can be carried out in other embodiments without departing from the spirit or essential characteristics thereof. Accordingly, the above disclosed embodiments are illustrative in all respects, and not exclusive. All changes that come within the scope of the invention or equivalents thereto are intended to be embraced therein.
Claims (10)
1. An African swine fever virus strain, wherein the African swine fever virus strain is an African swine fever virus strain with a microorganism collection number of CCTCC NO: V2023100.
2. A vaccine composition for preventing, slowing or controlling african swine fever, the vaccine composition comprising the african swine fever virus strain of claim 1 as an immunogen.
3. The vaccine composition of claim 2, wherein the vaccine composition is a live vaccine composition.
4. The vaccine composition of claim 2, wherein the vaccine composition comprises the african swine fever virus strain and an adjuvant as raw materials.
5. The vaccine composition of claim 4, wherein the adjuvant is a lyoprotectant;
preferably, in the starting materials of the vaccine composition, the lyoprotectant is an aqueous solution containing 4-12w/w% gelatin and 30-50w/w% sucrose.
6. The vaccine composition of claim 5, wherein the dry weight ratio of African swine fever virus strain to the lyoprotectant in the starting material of the vaccine composition is 1-20 x 10 6.0 TCID 50 ：1g。
7. The vaccine composition of claim 6, wherein the TCID of the african swine fever virus strain 50 Is calculated according to the Reed-Muench method by adopting an immunofluorescence analysis method based on primary pig alveolar macrophage culture.
8. A process for the preparation of a vaccine composition according to any one of claims 2 to 7, which process comprises: the vaccine composition is prepared by taking the African swine fever virus strain as an immunogen.
9. The preparation method according to claim 8, wherein 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.
10. The method of claim 9, wherein the freeze-drying temperature variation procedure is: -4 to-6 ℃,0.8 to 1.2 hours; -35 to-45 ℃ for 1.5-2.5 hours; -14 to-18 ℃,14-18 hours; -5 to-7 ℃,2-4 hours; 4-6 ℃ for 1.5-2.5 hours; 26-30 ℃ for 5-7 hours.
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