Establishment of an indirect immunofluorescence assay for the detection of African swine fever virus antibodies

African swine fever(ASF)continues to cause enormous economic loss to the global pig industry.Since there is no safe and effective vaccine,accurate and timely diagnosis of ASF is essential to implement control measures.Indirect immunofluorescence assay(IFA)is a gold standard serological method recommended by the World Organization for Animal Health(WOAH).In this study,we used primary fetal kidney cells to establish a wild boar cell line(BK2258)that supported the efficient replication of ASF virus(ASFV)SD/DY-I/21 and showed visible cytopathic effect(CPE).Moreover,using BK2258,we established a sensitive and specific IFA for ASFV antibody detection.To standardize and evaluate the performance of this assay,we used serum samples from pigs infected with the low virulent genotype I SD/DY-I/21 and genotype II HLJ/HRB1/20,and immunized with the vaccine candidate HLJ/18-7GD,field samples,and negative serum samples.The IFA reacted with the ASFV-positive sera and displayed bright fluorescence foci.There was no non-specific green fluorescence due to cellular senescence or other cell damage-causing factors.Compared to a commercial indirect enzyme-linked immunosorbent assay(iELISA),ASFV antibodies were detected 1–4 days earlier using our IFA.The detection limits of the IFA and iELISA for the same ASFV-antibody positive serum samples were 1:25,600 and 1:6,400,respectively,indicating that the IFA is more sensitive than iELISA.The newly established IFA was highly specific and did not cross-react with sera positive for six other important porcine pathogens(i.e.,Classical swine fever virus(CSFV),Porcine reproductive and respiratory syndrome virus(PRRSV),Porcme circovirus type 2(PCV2),Pseudorabies virus(PRV),Foot-and-Mouth disease virus type O(FMDV/O),and Porcine epidemic diarrhea virus(PEDV)).This study thus provides a sensitive,specific,and reliable detection method that is suitable for the serological diagnosis of ASF.

A multiplex real-time PCR assay for simultaneous detection of classical swine fever virus,African swine fever virus,and atypical porcine pestivirus

With the implementation of the C-strain vaccine,classical swine fever(CSF) has been under control in China,which is currently in a chronic atypical epidemic situation.African swine fever(ASF) emerged in China in 2018 and spread quickly across the country.It is presently occurring sporadically due to the lack of commercial vaccines and farmers’ increased awareness of biosafety.Atypical porcine pestivirus(APPV) was first detected in Guangdong Province,China,in 2016,which mainly harms piglets and has a local epidemic situation in southern China.These three diseases have similar clinical symptoms in pig herds,which cause considerable losses to the pig industry.They are difficult to be distinguished only by clinical diagnosis.Therefore,developing an early and accurate simultaneous detection and differential diagnosis of the diseases induced by these viruses is essential.In this study,three pairs of specific primers and Taq-man probes were designed from highly conserved genomic regions of CSFV(5’ UTR),African swine fever virus(ASFV)(B646L),and APPV(5’ UTR),followed by the optimization of reaction conditions to establish a multiplex real-time PCR detection assay.The results showed that the method did not cross-react with other swine pathogens(porcine circovirus type 2(PCV2),porcine reproductive and respiratory syndrome virus(PRRSV),foot-and-mouth disease virus(FMDV),pseudorabies virus(PRV),porcine parvovirus(PPV),and bovine viral diarrhea virus BVDV).The sensitivity results showed that CSFV,ASFV,and APPV could be detected as low as 1 copy μL–1;the repeatability results showed that the intra-assay and interassay coefficient of variation of ASFV,CSFV,and APPV was less than 1%.Twenty-two virus samples were detected by the multiplex real-time PCR,compared with national standard diagnostic and patented method assay for CSF(GB/T 27540–2011),ASF(GB/T 18648–2020),and APPV(CN108611442A),respectively.The sensitivity of this triple real-time PCR for CSFV,ASFV,and APPV was almost the same,and the compliance results were the same(100%).A total of 451 clinical samples were detected,and the results showed that the positive rates of CSFV,ASFV,and APPV were 0.22% (1/451),1.3%(6/451),and 0%(0/451),respectively.This assay provides a valuale tool for rapid detection and accurate diagnosis of CSFV,ASFV,and APPV.

Optimal design of culling compensation policy under the African swine fever——Based on simulations of typical pig farms in China

This paper aims to optimize the culling compensation policy from a micro perspective through scenario simulation.Based on an investigation of 273 pig farms in eight regions,four typical pig farms were constructed according to farm size and breeding mode,representing the swine producers in China.Besides,a decision objective function of pig farms facing suspected African swine fever(ASF)outbreaks was constructed.This study used a mathematical programming model to design and simulate scenarios based on compensation standards and local implementation levels,aiming to incentivize pig farms to report epidemics.The results show that the optimal decisions on epidemic reports differed among typical farms and by herd daily age.The results suggest the following adjustments for optimizing culling compensation policies:(1)to set culling compensation standards based on the market value and(2)to maintain a high level of epidemic surveillance capability in the animal husbandry and veterinary sector.

The Lapinized Chinese Strain Vaccine Against Classical Swine Fever Virus:A Retrospective Review Spanning Half A Century

Classical swine fever （CSF）, a list A disease of Office International des Epizooties, is caused by classical swine fever virus （CSFV） belonging to the Flaviviridae family. The well-known lapinized Chinese strain of CSFV, also known as C-strain, was developed in China in the mid-1950s. In the past half a century, the vaccine has been proved to be safe and immunogenic in pigs of essentially any age. It is of high efficacy, providing immunized animals with broad-spectrum, sometimes lifelong, protection, which is contributed by both cell-mediated immunity and humoral immunity, against essentially all genotypes or subgenotypes of the virus. The maternal antibodies derived from immunized sows can confer solid protection of their offspring from disease; however, they have been proved to inhibit the successful active immunization of C-strain vaccine. The complete genomes of C-strain and dozens of established or field strains have been sequenced and annotated. Recently, the reverse genetics system of C-strain has been developed, resulting in several C- strain-derived candidate marker vaccines. Many countries manage to control or even eradicate CSF with the aid of mass vaccination with C-strain. in spite of these efforts, the eradication of the disease worldwide remains a big challenge and needs to go a long way, and provably still resorts to genetically modified C-strain vaccine. The authors present an overview of the characteristics of the vaccine, which has stood the test of half a century.

A Novel RT-LAMP Assay for Rapid and Simple Detection of Classical Swine Fever Virus

A simple and rapid assay for the detection of Classical swine fever virus(CSFV)was established using reverse transcription loop-mediated isothermal amplification(RT-LAMP).This study describes the amplification of the genomic RNA of CSFV under isothermal conditions(63℃)within one hour,using a set of six primers(two outer primers,two inner primers and two loop primers).This RT-LAMP assay showed 100-fold higher sensitivity than the standard RT-PCR method and identified eighteen additional positive cases that were negative when tested by RT-PCR.This RT-LAMP was able to detect all the 13 strains of CSFV but not the BVDV.PRRSV.SIV. PRV-PCV,thus showed a good specificity.Products amplified by RT-LAMP can be visualized by agarose gel electrophoresis and in addition,either as a white precipitate at the bottom of the tube after a pulse spin or as a color change when dyed with SYBR Green I which are visible to the naked eye.Because RT-LAMP is low-cost and produces rapid results,it has the potential to be an excellent tool for CSFV surveillance in the field,especially in developing countries.

Inhibition of African swine fever virus in liquid and feed by medium-chain fatty acids and glycerol monolaurate

Background:The ongoing African swine fever virus(ASFv)epidemic has had a major impact on pig production globally and biosecurity efforts to curb ASFv infectivity and transmission are a high priority.It has been recently identified that feed and feed ingredients,along with drinking water,can serve as transmission vehicles and might facilitate transboundary spread of ASFv.Thus,it is important to test the antiviral activity of regulatory compatible,antiviral feed additives that might inhibit ASFv infectivity in feed.One promising group of feed additive candidates includes medium-chain fatty acids(MCFA)and monoglyceride derivatives,which are known to disrupt the lipid membrane surrounding certain enveloped viruses and bacteria.Results:The antiviral activities of selected MCFA,namely caprylic,capric,and lauric acids,and a related monoglyceride,glycerol monolaurate(GML),to inhibit ASFv in liquid and feed conditions were investigated and suitable compounds and inclusion rates were identified that might be useful for mitigating ASFv in feed environments.Antiviral assays showed that all tested MCFA and GML inhibit ASFv.GML was more potent than MCFA because it worked at a lower concentration and inhibited ASFv due to direct virucidal activity along with one or more other antiviral mechanisms.Dose-dependent feed experiments further showed that sufficiently high GML doses can significantly reduce ASFv infectivity in feed in a linear manner in periods as short as 30 min,as determined by infectious viral titer measurements.Enzyme-linked immunosorbent assay(ELISA)experiments revealed that GML treatment also hinders antibody recognition of the membrane-associated ASFv p72 structural protein,which likely relates to protein conformational changes arising from viral membrane disruption.Conclusion:Together,the findings in this study indicate that MCFA and GML inhibit ASFv in liquid conditions and that GML is also able to reduce ASFv infectivity in feed,which may help to curb disease transmission.

An Indirect ELISA of Classical Swine Fever Virus Based on Quadruple Antigenic Epitope Peptide Expressed in E.coli

In this study,a synthesized quadruple antigenic epitope gene region of the classical swine fever virus (CSFV)E2 glycoprotein was expressed in E.coli to a obtain target protein.This target protein was used as a coating antigen to establish an indirect ELISA for specifically detecting anti-CSFV antibodies in serum samples from pigs.The P/N cut-off value of this assay was 1.92 by receiver operating characteristic curve(ROC)analysis based on 30 negative sera and 80 positive samples.The test gave 97.5%sensitivity and 96.7%specificity compared with the indirect hemagglutination(IHA)test.The inter-assay and intra-assay coefficients of variation (CVs)for 16 sera were both≤6.8%.No cross-reactivity between the coating antigen and anti-bovine viral diarrhoea virus(BVDV)antibodies was observed.

African swine fever and meat prices fluctuation:An empirical study in China based on TVP-VAR model

African swine fever(ASF),a fatal disease outbroken in China in August 2018,has widely attracted social concern especially in the information era.The occurrence of ASF led to an imbalance between supply and demand in pork and other meat markets.As a result,meat prices fluctuated greatly during the past year in 2019.To measure ASF quantitatively,the internet public concern index about ASF was created using web crawler methods.The relationships between ASF and meat prices were analyzed based on time-varying parameter vector auto-regressive(TVP-VAR)model.The results showed that there were some differences in the impact size,direction and duration of ASF on the prices of pork,chicken,beef and mutton,and the characteristics of time variability and heterogeneity were obvious.At the same time,the impact of ASF on meat prices is not consistent with the trend and degree of ASF.The impulse intensity is strongly correlated with the strength and duration of ASF,and it is generally weak in the early stage and much stronger in the middle and late periods.The results indicate that macro regulations,monitoring and early-warning system,standardizing production and circulation,and the public opinion monitoring and guidance about ASF should be given more attention in future to stabilize the market expectations and to promote a smooth functioning of the livestock markets.

Genomic Sequence Determination of Classical Swine Fever Virus Persistent Infection Strain

Full genomic sequence of a newly isolated persistent infection strain of classical swine fever virus was firstly determined. It was demonstrated by sequence analyses that nucleotides homologies of this strain compared with virulent Shimen and vaccine HCLV were 89.7%and 87.7%, and homologies of amino acids were 94.8%and 93.3%, respectively. The sequencing results primarily suggest a tighter relationship between this persistent infection strain and virulent Shimen strain than vaccine HCLV strain.

Discrepancy of Classical Swine Fever Virus in Different Tissues by One-Step RT-PCR and Nested RT-PCR

Reverse transcription polymerase chain reaction (RT-PCR) was used for the detection of classical swine fever virus (CSFV) in blood and tissue samples of field cases and experimentally inoculated pigs. The distribution of CSFV in different organ samples showed some discrepancies in infected pigs. Four weaner pigs were inoculated with C-strain vaccine virus, then samples of spleen, tonsil, lung, mesenteric lymph node, kidney and brain were collected after slaughter and tested for E2 and NS5B genes using one-step RT-PCR and nested RT-PCR. Using the same method, 12 field cases were simultaneously studied. A discrepancy of CSFV in different samples was found upon detecting the target gene. The most reliable diagnostic organs were spleen and tonsil, and the nested RT-PCR assay provided a highly sensitive and specific method with comparable performance to the one-step RT-PCR assay.

Rapid Recovery of Classical Swine Fever Virus Directly from Cloned cDNA

The reverse genetics for classical swine fever virus （CSFV） is currently based on the transfection of in vitro transcribed RNA from a viral genomic cDNA clone, which is inefficient and time-consuming. This study was aimed to develop an improved method for rapid recovery of CSFV directly from cloned cDNA. Full-length genomic cDNA from the CSFV Shimen strain, which was flanked by a T7 promoter, the hepatitis delta virus ribozyme and T7 terminator sequences, was cloned into the low- copy vector pOK12, producing pOKShimen-RzTФ. Direct transfection of pOKShimen-RzTqb into PK/T7 cells, a PK-15- derived cell line stably expressing bacteriophage T7 RNA polymerase, allowed CSFV to be rescued rapidly and efficiently, i.e., at least 12 h faster and 31.6-fold greater viral titer when compared with the in vitro transcription-based rescue system. Furthermore, the progeny virus rescued from PK/T7 cells was indistinguishable, both in vitro and in vivo, from its parent virus and the virus rescued from classical reverse genetics. The reverse genetics based on intracellular transcription is efficient, convenient and cost-effective. The PK/T7 cell line can be used to rescue CSFV directly from cloned cDNA and it can also be used as an intracellular transcription and expression system for studying the structure and function of viral genes.

Insights into African swine fever virus immunoevasion strategies

African swine fever(ASF) is an acute and highly contagious disease that causes severe economic losses to the swine industry. ASF is caused by infection of African swine fever virus(ASFV) in domestic pigs, leading to almost 100% mortality. However, no effective vaccines and pharmacologic treatment against ASF are available. ASF poses a severe threat to the swine industry and the economy. Here we summarize potential virus-host cell interaction mechanisms involving the suppression of innate and adaptive immune responses to ASFV entry and infection. These mechanisms include modulation of apoptosis, inhibition of inflammatory responses, reduction of IFN production, inhibition of autophagy, and suppression of MHC-I expression. Insights into immunoevasion strategies by ASFV may shed light on the development of vaccines, as well as preventive and therapeutic drugs.

Development of a recombinant pB602L-based indirect ELISA assay for detecting antibodies against African swine fever virus in pigs

African swine fever(ASF),caused by the African swine fever virus(ASFV),is a devastating disease of domestic and wild pigs.There is no effective vaccine,and the control of the disease relies mainly on surveillance and early detection of infected pigs.Previously,serological assays,such as ELISA,have been developed mainly based on recombinant structural viral proteins of ASFV,including p72,p54,and p30.However,the antibodies against these proteins do not provide efficient protection against ASFV infection in pigs.Therefore,new serological assays that can be applied for clinical diagnosis and evaluating serological immune response in vaccinated pigs are still required.In this study,we expressed and purified a recombinant p B602 L protein.The purified p B602 L protein was then used as an antigen to develop an indirect ELISA assay.This assay has no cross-reaction with the anti-sera against the 15 most common pig pathogens in China,such as classical swine fever virus,pseudorabies virus,and porcine parvovirus.This assay and a commercial ELISA kit were then used to detect 60 field pig serum samples,including an unknown number of antiASFV sera.The coincidence of the two assays was 95%.Furthermore,the p B602 L-based ELISA was employed to test the antibody responses to the seven-gene-deleted ASFV strain HLJ/18-7 GD in pigs.The results showed that the antibody levels in all vaccinated pigs,starting from the 10 th day post-inoculation,have increased continuously during the observation period of 45 days.Our results indicate that this p B602 L-based indirect ELISA assay can be employed potentially in the field of ASFV diagnosis.

Generation and Immunogenicity of a Recombinant Adenovirus Co-Expressing the E2 Protein of Classical Swine Fever Virus and the GP5 Protein of Porcine Reproduction and Respiratory Syndrome Virus

Classical swine fever （CSF） and porcine reproduction and respiratory syndrome （PRRS） are both economically important, highly contagious diseases of swine worldwide. To develop an effective vaccine to control these two diseases, we constructed a recombinant adenovirus rAdV-GP52AE2, using a replication-defective human adenovirus serotype 5 as a delivery vector, to co-express the GP5 protein of highly pathogenic porcine reproduction and respiratory syndrome virus （PRRSV） and the E2 protein of classical swine fever virus （CSFV）. Foot-and-mouth disease virus （FMDV） 2A peptide was used as a linker between the GP5 and E2 proteins to allow automatic self-cleavage of the polyprotein. The GP5 and E2 genes were expressed as demonstrated by immunofluorescence assay and Western blotting. Immunization of mice resulted in a CSFV-neutralizing antibody titer of 1：128 and a PRRSV-neutralizing antibody titer of 1：16. The lymphoproliferative responses were detected by Cell Counting Kit-8 assay and the stimulation index of CFSV-specific and PRRSV-specific lymphocytes in the rAdV-GP52AE2 group was significantly higher than that in the negative control group. The results show that rAdV-GP52AE2 can induce both effective humoral and cell-mediated immune responses in mice. The protective efficacy of the recombinant virus against CSF was evaluated in immunized rabbits, which were protected from fever induced by challenge with C-strain. Our study provides supporting evidence for the use of FMDV 2A to develop a bivalent genetically-engineered vaccine.

Development of Multiple ELISAs for the Detection of Antibodies against Classical Swine Fever Virus in Pig Sera

The major immunogenic proteins （Ems, E2 and NS3） of classical swine fever virus （CSFV） （Shimen strain） were expressed in E. coli and purified by affinity chromatography. The recombinant antigens were applied to develop multiple enzyme-linked immunosorbent assays （ELISAs） for the detection of specific antibodies in pig sera. Optimum cut-off values were determined by receiver operating characteristic （ROC） analysis after testing 201 sera of vaccinated pigs and 64 negative sera of unvaccinated piglets. The multiple ELISAs were validated with 265 pig sera yielding high sensitivity and specificity in comparison with the virus neutralization results. The results demonstrated that multiple ELISAs can be a valuable tool for the detection of CSFV infection and serological surveys in CSFV-free countries or for the evaluation of the antibody responses in pigs induced by a live attenuated C-strain vaccination

Development of a Recombinase-aided Amplification Combined With Lateral Flow Dipstick Assay for the Rapid Detection of the African Swine Fever Virus

Objective To establish a sensitive,simple and rapid detection method for African swine fever virus(ASFV)B646L gene.Methods A recombinase-aided amplification-lateral flow dipstick(RAA-LFD)assay was developed in this study.Recombinase-aided amplification(RAA)is used to amplify template DNA,and lateral flow dipstick(LFD)is used to interpret the results after the amplification is completed.The lower limits of detection and specificity of the RAA assay were verified using recombinant plasmid and pathogenic nucleic acid.In addition,30 clinical samples were tested to evaluate the performance of the RAA assay.Results The RAA-LFD assay was completed within 15 min at 37°C,including 10 min for nucleic acid amplification and 5 minutes for LFD reading results.The detection limit of this assay was found to be 200 copies per reaction.And there was no cross-reactivity with other swine viruses.Conclusion A highly sensitive,specific,and simple RAA-LFD method was developed for the rapid detection of the ASFV.

Heterogeneity and Secondary Structure Analysis of 3' Untranslated Region in Classical Swine Fever Viruses

The attenuated vaccine strains of CSFV have a 12-nucleotides （nt） insertion in the 3＇-UTR of genome as compared to that of CSFV virulent strains. In this study, we found a distinct heterogeneity in the 3＇-UTR of attenuated Thiverval and HCLV strains. The longest 3＇-UTR of Thiverval strain was 259 base pairs （bp） with a 32-nt insertion, the shortest 3＇-UTR had only 233 bp with a 6-nt insertion. The longest 3＇-UTR of HCLV strain was 244 bp with a 17-nt insertion and the shortest 3＇ UTR was 235 bp with a 8-nt insertion. Compared with the published sequences of 3＇-UTR of vaccine and virulent strains, the 3＇-UTR of CSFV vaccine strains have two variable regions where insertion among the different vaccine strains were frequently found. The first is located between the second conservative TALk codon and the start of T-rich region where we found the variable length insertion in the same vaccine strain Thiveral or HCLV and the second is located between the end of T-rich region and the front of GAA eodon, however, a 4-nt deletion was found in this region in the virulent Shimen strain. These two regions may represent the ＂hot spot＂ for mutation. Modeling the secondary structures of the 3＇-UTR suggests that the T-rich insertion could result in the change of structure and free energy, thus affecting the stability of the 3＇-UTR structure. These findings will help to understand the mechanism of attenuated vaccines and improve vaccine safety, stability, and efficacy.

Blocking Effects of Spleen Vaccine on Vertical Transmission of Classical Swine Fever Virus in Sows

[ Objective] To investigate the blocking effects of spleen vaccine on vertical transmission of classical swine fever virus （CSFV） in sows. [ Method] Sows infected by CSFV were selected from three large-scale pig farms and they were randomly divided into group Ⅰ, group Ⅱ and control group. The sows in the group Ⅰ were vaccinated with CSF spleen vaccine at a 1.5 times normal dose per pig; those in the group Ⅱ were vaccinated with CSF spleen vaccine at a 2.0 times normal dose per pig; and those in the control group were vaccinated with cell vaccine at a 4.0 times normal- dose per pig. The CSF antigens of piglets were detected by enzyme-linked immunosorbent assay （ELISA）. [ Result] The antigen positive rate of piglets in the experimental group （18.5%） was significantly lower than that in the control group （48.1% ）. No significant difference was found be- tween the group Ⅰ and the group Ⅱ. [ Condmion] CSF spleen vaccine has good blocking effects on vertical transmission of CSFV in sows.

Diagnosis of Mixed Infection of Classical Swine Fever Virus and Porcine Reproductive and Respiratory Syndrome Virus and Pathogenic Characteristics

[ Objective] To diagnose swine diseases caused by CSFV （ Classical swine fever virus）, PRRSV （ Porcine reproductive and respiratory syndrome virus） and PRV （Pseudo-rabies virus） and analyze the pathogenic characteristics. [ Methodl The tissues and viscera of the diseased swine were collected from a hoggery in Fujian Province. DNA and RNA were extracted for PCR amplification and sequencing. ELISA method was used to determine CSFV, PRRSV and PRV infection. [ Result] The sequencing analysis and ELISA results showed that the mixed infection was caused by CSFV, PRRSV and PRV. [ Conclusion] The swine epidemic situation was mainly caused by CSFV and PRRSV.

African swine fever virus MGF505-3R inhibits cGAS-STING-mediated IFN-βpathway activation by degrading TBK1

African swine fever virus(ASFV)is an important pathogen causing acute infectious disease in domestic pigs and wild boars that seriously endangers the global swine industry.As ASFV is structurally complex and encodes a large number of functional proteins,no effective vaccine has been developed to date.Thus,dissecting the mechanisms of immune escape induced by ASFV proteins is crucial.A previous study showed that the ASFV-encoded protein is an important factor in host immunity.In this study,we identified a negative regulator,MGF505-3R,that significantly downregulated cGAS/STING-and poly(dG:dC)-mediated IFN-βand interferon stimulation response element(ISRE)reporter activity and suppressed IFNB1 and IFIT2 mRNA levels.In addition,TBK1,IRF3 and IκBαphosphorylation levels were also inhibited.Mechanistically,MGF505-3R interacted with cGAS/TBK1/IRF3 and targeted TBK1 for degradation,thereby disrupting the cGAS-STING-mediated IFN-βsignaling pathway,which appears to be highly correlated with autophagy.Knockdown MGF505-3R expression enhanced IFN-βand IL-1βproduction.Taken together,our study revealed a negative regulatory mechanism involving the MGF505-3R-cGAS-STING axis and provided insights into an evasion strategy employed by ASFV that involves autophagy and innate signaling pathways.