Porcine atypical pestivirus isolates adapted to cell culture and uses thereof

Abstract

The application discloses an atypical swine pestivirus isolated strain suitable for cell culture and application thereof. The application isolates an APPV isolated strain suitable for cell culture from a dead piglet determined as APPV infection, and the microbial preservation number of the APPV isolated strain is CGMCC No. 45417. The APPV isolated strain can be continuously and stably passaged and proliferated in PK-15 cells, and the virus titer gradually increases during the passage. Virus neutralization test (VNT) shows that VNT based on the APPV isolated strain can effectively distinguish APPV antibody positive serum and negative serum. The APPV isolated strain provided by the application provides key materials for biological and pathogenicity research of APPV, development of vaccines and diagnostic reagents, and has important application prospects for diagnosis of diseases caused by APPV, improvement of infection monitoring system and control of diseases caused by APPV, and can also be used for preparing diagnostic or preventive and therapeutic detection reagents or vaccines for APPV infection.

Description

Technical Field

[0001] This invention relates to porcine atypical fever virus isolates, and more particularly to porcine atypical fever virus isolates adapted for passaged cell culture and their uses, belonging to the field of porcine atypical fever virus isolates and their uses. Background Technology

[0002] Pestiviruses are enveloped, single-stranded, positive-sense RNA viruses with highly variable genomes. They belong to the genus *Pestivirus* in the family Flaviviridae and can infect pigs, ruminants, and wild animals. Classical swine fever virus (CSFV), bovine viral diarrhea virus 1 (BVDV-1), bovine viral diarrhea virus 2 (BVDV-2), and border disease virus (BDV) are currently the main members of the pestivirus family. In recent years, several novel pestiviruses have been discovered in livestock and wild animals. In 2017, the International Committee on Taxonomy of Viruses (ICCV) published a new classification guideline for phenothiazines, dividing members of the genus Pestivirus into 11 species, A through K (Smith, DB, Meyers, G., Bukh, J., Gould, EA, Monath, T., Muerhoff AS, Pletnev, A., Rico-Hesse, R., Stapleton, JT, Simmonds, P., & Becher, P. (2017). Proposed revision to the taxonomy of the genus Pestivirus, family Flaviviridae. Journal of General Virology, 98(8), 2106–2112. https: / / doi.org / 10.1099 / jgv.0.000873).

[0003] In 2015, American researchers discovered a novel porcine pestivirus (APPV) in a serum sample from a pig that tested positive for porcine reproductive and respiratory syndrome virus (PRRSV) through metagenomic sequencing. APPV is named atypical porcine pestivirus (Hause, BM, Collin, EA, Peddireddi, L., Yuan, F., Chen, Z., Hesse, RA, Gauger, PC, Clement, T., Fang, Y., & Anderson, G. (2015). Discovery of a novel putative atypicalporcine pestivirus in pigs in the USA. Journal of General Virology, 96, 2994–2998. https: / / doi.org / 10.1099 / jgv.0.000251. Subsequently, researchers inoculated serum from APPV-infected animals into pregnant sows and found that the piglets born from these sows developed paroxysmal tremors in their head and limb muscles, which affected their normal suckling and increased pre-weaning mortality (Arruda, BL, Arruda, PH, Magstadt, DR, Schwartz, KJ, Dohlman, T., Schleining, JA, Patterson, AR, Visek, CA, & Victoria, JG (2016). Identification of a divergent lineage porcine pestivirus in nursing piglets with congenital tremors and reproduction of disease following experimental inoculation. PLoS One, 11, e0150104. https: / / doi.org / 10.1371 / journal.pone.0150104). APPV belongs to the genus Pestivirus in the family Flaviviridae, and is classified as Pestivirus K. Its genome is an 11-12 kb single-stranded positive-sense RNA consisting of a 5′ untranslated region (UTR), a large open reading frame (ORF), and a 3′ UTR. The ORF encodes a polyprotein of 3,635 amino acids, which is processed and hydrolyzed by its own and host proteases to produce four structural proteins (C, E, ...). rns(E1 and E2) and 8 non-structural proteins (N pro 、p7、NS2、NS3、NS4A、NS4B、NS5A and NS5B)(Hause et al.,2015). The E2 glycoprotein of the plague virus can induce high levels of neutralizing antibodies and is a major target for diagnostic reagents and vaccine development. It is worth noting that, compared with CSFV and BVDV, the APPV E2 glycoprotein lacks two relatively complete N-terminal domains, which makes its (241aa) significantly smaller than the E2 proteins of other plague viruses (373-378aa) (Riedel,C.,Aitkenhead,H.,El Omari,K.,&Rümenapf,T.(2021).Atypical porcine pestiviruses:relationships and conserved structural features.Viruses,13(5),760.https: / / doi.org / 10.3390 / v13050760). Since the E2 protein also mediates the cell invasion of the swine fever virus, the deletion of this protein may lead to a difference in the ability of APPV to enter host cells compared to other swine fever viruses (Cagatay, GN, Antos, A., Suckstorff, O., Isken, O., Tautz, N., Becher, P., & Postel, A. (2021). Porcine complement regulatory protein CD46 is a major receptor for atypical porcine pestivirus but not for classic swine fever virus. Journal of Virology, 95(9), e02186-20. https: / / doi.org / 10.1128 / JVI.02186-20).

[0004] To date, congenital tremors in piglets caused by APPV have been reported in Europe, America, and Asia. Based on different genetic analyses, APPV can be divided into three main genotypes. Genotypes 2 and 3 have been found only in Asia, while genotype 1 is prevalent worldwide and shows high variability (Yuan, F., Feng, Y., Bai, J., Liu, X., Arruda, B., Anbalagan, S., & Peddireddi, L. (2022). Genetic diversity and prevalence of atypical porcine pestivirus in the Midwest of US swine herds during 2016–2018. Transboundary and Emerging Diseases, 69(2), 753–763. https: / / doi.org / 10.1111 / tbed.14046).APPV isolation in vitro is quite difficult. Hause and Arruda et al. attempted to isolate the virus from primary porcine kidney cells and passaged porcine kidney cell lines (PK-15, IBRS-2, and SK6), but without success (Hause, BM, Collin, EA, Peddireddi, L., Yuan, F., Chen, Z., Hesse, RA, Gauger, PC, Clement, T., Fang, Y., & Anderson, G. (2015). Discovery of a novel putative atypical porcinepestivirus in pigs in the USA. Journal of General Virology, 96, 2994–2998. https: / / doi.org / 10.1099 / jgv.0.000251; Arruda, BL, Arruda, PH, Magstadt, DR, Schwartz, KJ, Dohlman, T., Schleining, JA, Patterson, AR, Visek, CA, & Victoria, JG (2016). Identification of a divergent lineage porcine pestivirus in nursing piglets with congenital tremors and reproduction of disease following experimental inoculation. PLoS One, 11, e0150104. https: / / doi.org / 10.1371 / journal.pone.0150104). To date, studies on the pathogenicity of APPV in pigs have been conducted by inoculating pregnant sows with aseptically processed homogenates of infected animal tissues or serum, while the pathogenicity of cell isolates in pigs has not yet been reported.

[0005] Current research indicates that APPV strains exhibit high genetic diversity, with significant genetic differences even within a single country (Wu Weixin, Huang Jin, Zhou Lei, Yang Hanchun. Genetic variation analysis of SARS virus in some regions of my country from 2015 to 2017 [J]. Journal of Animal Husbandry and Veterinary Medicine, 2020, 51(08): 1939-1948.). To date, the origin of APPV and how it spreads to pig herds remain unclear. However, given its widespread distribution across many countries on three continents and its potential for pandemic spread, future epidemiological investigations and virus isolation and identification in more regions are necessary to monitor the genetic evolution of APPV and assess the impact of APPV infection alone or in combination with other porcine pathogens on pig herds.

[0006] Because APPV replicates very limitedly in cell lines suitable for the proliferation of classical swine fever virus and other swine fever viruses, it is very difficult to isolate it in vitro. This greatly limits the research on the pathogenic mechanism of the pathogen, vaccine development and diagnostic reagent development, thus hindering the development of APPV vaccines and diagnostic reagents as well as the research on viral infection and pathogenicity. Summary of the Invention

[0007] One of the objectives of this invention is to provide an APPV isolate that is stably passaged and adapted to passaged cell culture.

[0008] A second objective of this invention is to apply the stable, passage-adapted APPV isolates to the preparation of diagnostic reagents for APPV infection or to the preparation of vaccines for the prevention and treatment of APPV.

[0009] The above-mentioned objective of the present invention is achieved through the following technical solution:

[0010] This invention first provides a stable, passage-adapted APPV isolate, classified as porcine atypical fever virus; its microbial accession number is CGMCC No. 45417; its accession date is February 8, 2023; its depositary institution is the China General Microbiological Culture Collection Center; and its address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0011] This invention used RT-PCR and sequencing to detect and sequence diseased and dead piglets from a pig farm in Heilongjiang Province, confirming APPV infection. APPV-positive samples were further inoculated into PK-15 cells and blindly passaged multiple times for virus isolation. RT-PCR results showed that the cell cultures were APPV positive at each passage. Subsequent indirect immunofluorescence assay (IFA) confirmed that the isolate could be stably passaged in PK-15 cells, with the viral titer increasing with each passage. It has now been stably passaged to passage 30. Further transmission electron microscopy revealed enveloped spherical virus particles with a diameter of approximately 40-50 nm, consistent with the morphology and size of hepatitis B virus. These results indicate that this invention successfully isolated a stably passageable APPV strain, named isolate China / HLJ491 / 2017.

[0012] The present invention further analyzed the in vitro proliferation characteristics of the isolate China / HLJ491 / 2017. The results showed that the viral titer of this strain could reach 10 after 72 hours of infection with PK-15 cells. 5.5 TCID 50 / mL.

[0013] To further identify the isolate China / HLJ491 / 2017, this invention determined the whole genome sequence of the China / HLJ491 / 2017 strain. A phylogenetic tree was constructed based on the APPV isolate's whole genome sequence, and homology analysis was performed with the nucleotide and amino acid sequences of the APPV reference strain published by NCBI. The results showed that the China / HLJ491 / 2017 strain belongs to APPV genotype 2, with 93.3%–97.6% homology with strains of the same genotype and 80.9%–83.5% nucleotide homology with strains of different genotypes. Furthermore, the isolate China / HLJ491 / 2017 (genotype 2) isolated in this invention has a different genotype than the German isolate (Ger-NRW_L277, genotype 1). Genotype 2 strains have currently only been found in Asia (mainly China). Additionally, the two isolates have significant differences in nucleotide sequences, with only 83.4% homology. These differences may lead to differences in biological characteristics such as replication ability and pathogenicity.

[0014] This invention further utilizes the isolated APPV China / HLJ491 / 2017 strain to perform a virus neutralization test (VNT), demonstrating that the VNT based on this isolate can effectively distinguish between APPV antibody-positive and negative sera. Given that VNT is the gold standard method for APPV, the APPV isolate provided by this invention can offer a gold standard reference method for the development of APPV diagnostic reagents.

[0015] In summary, this invention successfully isolated an APPV isolate that can be continuously passaged in PK-15 cells. This isolate can be stably passaged and proliferated in PK-15 cells, and the viral titer gradually increases during passage. This APPV isolate will provide key materials for the study of the virus's biology and pathogenicity, as well as the development of vaccines and diagnostic reagents. It has important application value for the diagnosis of diseases caused by APPV, the improvement of infection monitoring systems, and the control of the epidemic caused by APPV. It can also be used to prepare rapid diagnostic reagents for APPV infection and for vaccine research. Attached Figure Description

[0016] Figure 1 The infection status of different passages of the APPV isolate China / HLJ491 / 2017 in PK-15 cells.

[0017] Figure 2 To observe the morphology of APPV isolate China / HLJ491 / 2017 virus particles under an electron microscope; (A) Viral particles in the supernatant of cell culture infected by APPV China / HLJ491 / 2017 strain; (B) Viral particles in cells infected by APPV China / HLJ491 / 2017 strain.

[0018] Figure 3 The multi-step growth curve of APPV isolate China / HLJ491 / 2017 on PK-15 cells.

[0019] Figure 4 The amplified whole genome fragment of APPV isolate China / HLJ491 / 2017; M: DL2000 DNA Marker; 1: APPV-1F / 1R; 2: APPV-2F / 2R; 3: APPV-3F / 3R; 4: APPV-4F / 4R; 5: APPV-5F / 5R; 6: APPV-6F / 6R; 7: APPV-7F / 7R; 8: APPV-8F / 8R; 9: APPV-9F / 9R; 10: APPV-10F / 10R.

[0020] Figure 5 Phylogenetic analysis of the whole genome of APPV isolate China / HLJ491 / 2017.

[0021] Figure 6 Neutralization of APPV China / HLJ491 / 2017 strain in serum from clinically infected swine farms; (A) Neutralization of APPV China / HLJ491 / 2017 strain at 200 TCID50. 50(A) PK-15 cells were infected with a dose of 100 μL, and uninfected PK-15 cells were used as a negative control; (B) Clinical APPV-infected porcine serum was continuously diluted 2-fold starting from 1:4 for VNT, and the highest titer of APPV neutralizing antibody in the serum was 128. Detailed Implementation

[0022] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions to the details and form of the present invention can be made without departing from the spirit and scope of the invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0023] Example 1: Isolation and identification of porcine atypical fever virus strains adapted to passaged cell culture, and APPV virus neutralization test.

[0024] 1. Experimental Materials and Methods

[0025] 1.1 Clinical Samples, Antibodies, and Cells

[0026] In July and August 2017, 60 weaned piglets at a pig farm in Heilongjiang Province fell ill, with a mortality rate of 10%. All pigs had been vaccinated with routine vaccines, including swine fever vaccine, porcine reproductive and respiratory syndrome (PRRS) vaccine, porcine circovirus type 2 (PCV2) vaccine, and pseudorabies vaccine. The sick piglets exhibited progressive emaciation, slow growth, respiratory symptoms such as coughing and wheezing, abdominal breathing, and eyelid swelling or dermatitis. Tissue samples from the dead pigs were sent by the local veterinarian to the Laboratory of the Innovative Team for Highly Infectious Swine Diseases at the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences (hereinafter referred to as "the Inventor's Laboratory") for diagnosis. The Inventor's Laboratory tested for CSFV, PRRSV, pseudorabies virus (PRV), PCV2, porcine parvovirus (PPV), swine influenza virus (SIV), and APPV pathogens using PCR / RT-PCR or quantitative real-time PCR (qPCR) / RT-qPCR. APPV and PCV2 were positive, while other pathogens were negative.

[0027] PK-15 cells were preserved in the inventor's laboratory and cultured in DMEM medium containing 10% fetal bovine serum (FBS), 100 μg / mL streptomycin and 100 IU / mL penicillin at 37°C and 5% CO2. The monoclonal antibody (MAb) 6E2 against APPV E2 protein was prepared and preserved in the inventor's laboratory. PCV2 antiserum was provided by Harbin Guosheng Biotechnology Co., Ltd.

[0028] 1.2 Pathogen processing, RNA extraction and cDNA synthesis

[0029] The tissue sample was chopped and placed in a mortar. An appropriate amount of sterile quartz sand was added and the sample was ground thoroughly. Then, 1 mL of DMEM medium was added to make a suspension and transferred to a 1.5 mL centrifuge tube. The suspension was frozen and thawed three times. The sample was centrifuged at 4°C and 2000×g for 10 min. 200 μL of the supernatant was collected and viral RNA was extracted according to the instructions of the Simply P Total RNA Extraction Kit (BioFlux, China). Finally, 40 μL of RNase-free DEPC-treated water was added for elution. Reverse transcription was performed. The system consisted of 6 μL RNA template, 0.5 μL AMV, 0.5 μL RRI, 4 μL dNTP, 4 μL AMV Buffer, 1 μL random primer 9N, and 5 μL DEPC water. After mixing, the mixture was incubated in a 42°C water bath for 1 h before use.

[0030] 1.3 RT-PCR and Sequencing

[0031] RT-PCR was performed using primers published by Postel et al. (Postel, A., Meyer, D., Petrov, A., & Becher, P. (2017). Recent emergence of a novel porcine pestivirus: interference with classical swine fever diagnosis? Emerging Microbes & Infections, 6, 1-2. https: / / doi.org / 10.1038 / emi.2017.5). The forward primer APPV_5030-fw (5′-CCCCAGGCAATACCTCACAAC-3′) and the reverse primer APPV_5469-rev (5′-CCCCCTTTTTGGTTCCTCCC-3′) were used to amplify a portion of the NS3 gene (440 bp) of APPV using Ex Taq polymerase (TaKaRa, China). The PCR reaction program was: 95℃ pre-denaturation for 5 min, 35 amplification cycles (56℃ for 30 s and 72℃ for 45 s), and extension at 72℃ for 10 min. The PCR products were purified using an Omega gel extraction kit (Omega, USA) and then sequenced.

[0032] 1.4 Virus isolation

[0033] Porcine tissue identified as positive for APPV nucleic acid by RT-PCR was homogenized and freeze-thawed. The homogenate was then centrifuged at 2000×g for 10 min at 4°C. The supernatant was sterilized by filtration through a 0.45 μm membrane (Millipore, USA), and then inoculated into PK-15 cells at a density of approximately 80%. The cells were cultured at 37°C and 5% CO2 for 72 h. The culture was then repeatedly freeze-thawed three times, and the supernatant was harvested by centrifugation at 2000×g for 10 min at 4°C. The supernatant was then re-inoculated into PK-15 cells and passaged blindly for 5 generations. The culture supernatant harvested from each generation was used to detect APPV and PCV2 using RT-qPCR and qPCR, respectively (Liu, H., Shi, K., Zhao, J., Yin, Y., Chen, Y., Si, H., Qu, S., Long, F., & Lu, W. (2022). Development of a one-step multiplexqRT-PCR assay for the detection of African swine fever virus, classical swinefever virus and atypical porcine pestivirus. BMC Veterinary Research, 18(1), 43. https: / / doi.org / 10.1186 / s12917-022-03144-4; Olvera, A., Sibila, M., Calsamiglia, M., Segalés, J., & Domingo, M. (2004). Comparison of porcine circovirus type 2 load in serum quantified by a real-time PCR in postweaningmultisystemic wasting syndrome and porcine dermatitis and nephropathy syndrome naturally affected pigs. Journal of Virological Methods, 117(1), 75–80. https: / / doi.org / 10.1016 / j.jviromet.2003.12.007).

[0034] To ensure the purity of the isolated APPV isolates, the 11th generation virus culture was mixed with an equal volume of anti-PCV2 serum (diluted 1:10 with DMEM) and incubated at 37°C for 2 hours to neutralize any PCV2 present in the culture. The neutralized virus culture was then inoculated into PK-15 cells and cultured at 37°C for 72 hours. After three freeze-thaw cycles, the supernatant was harvested and passaged in PK-15 cells. APPV and PCV2 levels in the 12th to 20th generation virus cultures were detected by RT-qPCR and qPCR, respectively.

[0035] 1.5 Observation using indirect immunofluorescence assay (IFA) and transmission electron microscopy (TEM)

[0036] PK-15 cells were seeded in 96-well plates and inoculated with APPV-positive viral solution by RT-PCR after 12 hours. Normal cells without viral inoculation were also included as a control. The cells were cultured at 37°C and 5% CO2 for 72 hours. The culture medium was discarded from the 96-well plates, and 100 μL of pre-chilled anhydrous ethanol was added to each well for fixation at 4°C for 30 minutes. Then, MAb 6E2, an anti-APPV E2 protein diluted 1:200 with PBS containing 5% bovine serum albumin, was added and incubated at 37°C for 2 hours. After washing five times with PBST, FITC-labeled goat anti-mouse IgG (Invitrogen, USA) diluted 1:300 was added and incubated at 37°C for 1 hour. After washing five times with PBST, 50 μL of glycerol-PBS buffer (1:1) was added, and fluorescence was observed using an inverted fluorescence microscope (Nikon TE200, Japan). Viral titers were calculated using the Reed and Muench method (Reed, LJ, Muench, H. (1983). A simple method of estimating fifty percent endpoints. American Journal of Hygiene, 27, 493–4973).

[0037] After three freeze-thaw cycles, the virus culture was centrifuged at 8000×g for 30 min at 4°C to remove cell debris. Then, it was centrifuged at 25,000×g for 3 h, the supernatant was discarded, and the precipitate was resuspended in 100 μL PBS. The culture was then negatively stained with 2% phosphotungstic acid, and the virus morphology was observed using a transmission electron microscope (Hitachi, Tokyo, Japan). APPV-infected PK-15 cells were fixed in 2.5% glutaraldehyde and then prepared into ultrathin sections according to the published literature (Zhao, C., Chen, C., Li, Y., Dong, S., Tan, K., Tian, ​​Y., Zhang, L., Huang, J., & Zhang, L. (2019). Genomic characterization of a novel recombinant porcine astrovirus isolated in northeastern China. Archives of Virology, 164(5), 1469–1473. https: / / doi.org / 10.1007 / s00705-019-04162-8) and observed using a transmission electron microscope.

[0038] 1.6 Multi-step growth curve

[0039] PK-15 cells were seeded in 24-well plates. Once the cell confluence reached 80%, virus cultured to passage 18 was seeded into the cells at a multiplicity of infection (MOI) of 0.1. The cells were incubated at 37°C and 5% CO2 for 2 hours to allow for virus adsorption. The supernatant was then discarded, and the cells were washed three times with serum-free DMEM. 500 μL of DMEM containing 10% FBS was added, and the cells were cultured further at 37°C and 5% CO2. Cell cultures were harvested at different time points (12, 24, 36, 48, 60, 72, and 84 hours) and stored at -80°C. After three freeze-thaw cycles, the supernatant was collected by centrifugation. The TCID value at different time points was determined using IFA. 50 ), and plot multi-step growth curves.

[0040] 1.7 Whole genome amplification

[0041] Based on the complete genome sequence of APPV_VIRES_NM01_C1 strain (GenBank accession number MK378658), 10 pairs of specific primers with overlapping amplification regions were designed using SnapGene software 4.1.9 to amplify the complete genome of APPV. Sequence information is detailed in Table 1.

[0042] The PCR reaction program was as follows: 95℃ pre-denaturation for 5 min, amplification for 35 cycles (56℃ for 30 s and 72℃ for 1 min 45 s), and extension at 72℃ for 10 min. The PCR amplification products were purified using an Omega gel extraction kit (Omega, USA) and cloned into the pMD18-T vector (TaKaRa, China). The ligation product was transformed into *E. coli* (DH5α) competent cells. Positive colonies were picked and sequenced. The complete full-length genome sequence of APPV was generated using the SeqMan program in DNASTAR 7.1 software.

[0043] Table 1 Primer sequences for the entire APPV genome

[0044]

[0045]

[0046] 1.8 Sequence Analysis

[0047] The obtained APPV whole genome sequence was compared with the sequences of 24 APPV strains published in the GenBank database to analyze their homology and construct a phylogenetic tree. Multiple sequence alignment and sequence similarity calculations were performed using DNASTAR 7.1 software, and phylogenetic analysis was conducted using the Neighbor-Joining algorithm in MEGA7 software, with 1000 bootstrap replicates.

[0048] 1.9 Virus Neutralization Test (VNT)

[0049] VNT is considered the gold standard method for detecting porcine pestivirus. In this experiment, the isolated APPV China / HLJ491 / 2017 strain was used for VNT. Four swine serum samples from a local pig farm (where APPV infection had occurred before and the infected pigs were confirmed to be APPV positive by RT-PCR and sequencing as described in section 2.2) were tested for APPV antibodies. At the same time, two SPF swine serum samples were used as controls. The specific steps were performed according to the method published by Cagatay et al. (Cagatay, GN, Meyer, D., Wendt, M., Becher, P., & Postel, A. (2019). Characterization of the humoral immune response induced after infection with atypical porcine pestivirus (APPV). Viruses, 11(10), 880. https: / / doi.org / 10.3390 / v11100880).

[0050] 2. Experimental Results

[0051] 2.1 In vitro isolation and identification of APPV isolates adapted to passaged cell culture

[0052] Porcine tissue homogenates identified as positive for APPV nucleic acid (and also positive for PCV2) were inoculated into PK-15 cells and passaged blindly for 5 generations. RT-qPCR and qPCR detection revealed that cell cultures from generations 1 to 5 were positive for both APPV and PCV2. To obtain a pure APPV isolate, the 11th generation virus culture was treated with anti-PCV2 serum and then passaged again in PK-15 cells. RT-qPCR and qPCR results showed that the Ct value of PCV2 gradually increased with each generation, and PCV2 was undetectable from generation 17 onwards, with only APPV detected, indicating that PCV2 was completely eliminated (Table 2). Notably, the Ct value of PCV2 in the untreated group (untreated group) also gradually increased in subsequent generations, and PCV2 was also undetectable from generation 17 onwards, indicating that even without anti-PCV2 serum treatment, PCV2 gradually disappears with increasing passage number.

[0053] To further identify the APPV isolate, virus cultures from passages 10, 20, and 30 were inoculated into PK-15 cells. IFA detection was then performed using a specific monoclonal antibody against the APPV E2 protein prepared in the inventors' laboratory. The results showed that specific fluorescent foci were visible in virus cultures from different passages, and the number of fluorescent foci gradually increased with each passage, indicating that the strain could be continuously and stably passaged in PK-15 cells. Furthermore, the viral titer gradually increased during passage. These results demonstrate that an APPV isolate adapted to passaged cell culture was successfully isolated in this experiment, and it was named China / HLJ491 / 2017 strain. Figure 1 Its microbial preservation number is CGMCC No.45417.

[0054] To confirm the morphology of the isolated APPV China / HLJ491 / 2017 strain, electron microscopy was performed on the virus particles in the cell culture supernatant and virus-infected cells. The results showed that the isolated APPV isolate consisted of enveloped spherical particles with a diameter of approximately 50 nm, similar in morphology and size to phenotypic viruses. Figure 2 ).

[0055] Table 2. Nucleic acid detection results of APPV and PCV2 in different generations of viral cultures.

[0056]

[0057] 2.2 In vitro replication kinetics of APPV isolates

[0058] The 18th generation APPV China / HLJ491 / 2017 strain was inoculated into PK-15 cells at an MOI of 0.1. Viral supernatant was harvested at different time points (12, 24, 36, 48, 60, 72, and 84 h), and viral titer was determined by IFA. The results showed that the APPV China / HLJ491 / 2017 isolate could effectively proliferate in PK-15 cells, and the viral titer reached its peak of 10 at 72 h post-infection. 5.5 TCID 50 / mL( Figure 3 ).

[0059] 2.3 Genome-wide characteristics and sequence analysis of APPV isolates

[0060] RNA was extracted from the 10th generation viral fluid of APPV China / HLJ491 / 2017 strain and amplified by RT-PCR using whole-genome sequencing primers. Ten target fragments were successfully amplified, and the band sizes were consistent with expectations. Figure 4 The sequencing results of 10 specifically amplified fragments were assembled using DNASTAR 7.1 software to obtain the complete genome sequence of the China / HLJ491 / 2017 isolate. Phylogenetic analysis based on the viral complete genome sequence showed that APPV isolates are currently mainly divided into 3 genotypes, while the China / HLJ491 / 2017 isolate belongs to genotype 2. Figure 5 The nucleotide and amino acid sequence homology between China / HLJ491 / 2107 and the reference strain on NCBI was analyzed using the MegAlign Clustal W algorithm. The results showed that the homology between the China / HLJ491 / 2107 isolate and strains of the same genotype ranged from 93.3% to 97.6%, with the highest nucleotide homology (97.6%) observed with the APPV_VIRES_NM01_C1 strain. The nucleotide homology with strains of different genotypes ranged from 80.9% to 83.5% (Table 3).

[0061]

[0062]

[0063] 2.4 Detection of neutralizing antibodies in APPV isolates

[0064] The virus neutralization test (VNT) is the gold standard method for APPV. This experiment used the isolated APPV China / HLJ491 / 2017 strain for VNT to detect antibodies against APPV in porcine serum. Four swine serum samples from a pig farm that had previously experienced APPV infection (where infected pigs were confirmed to be APPV positive using the RT-PCR method and sequencing described in section 1.2), and two samples of SPF swine serum were used for APPV antibody detection.

[0065] VNT detection results showed that the highest APPV neutralizing antibody titer in the serum of APPV-infected pigs detected using APPV isolates was 128 ( Figure 6 ), while SPF porcine serum did not show detectable APPV antibodies.

Claims

1. An atypical porcine pestivirus (APPV) isolate adapted to cell culture, characterized in that, Its microbial preservation number is: CGMCC No. 45417.

2. The use of the porcine atypical fever virus isolate of claim 1 in the preparation of reagents for diagnosing porcine atypical fever virus infection.

3. Use according to claim 2, characterized in that, The reagents used to diagnose SARS virus infection are antigens or antibodies that detect SARS virus.

4. The use of the SARS virus isolate of claim 1 in the preparation of a vaccine for the prevention and treatment of SARS virus infection.

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