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.

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.

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.

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.

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.

Viricidal activity of several disinfectants against African swine fever virus

Prevention of African swine fever,a disease caused by African swine fever virus(ASFV),requires maintenance of high biosecurity standards,which principally relies on disinfection.Finding the perfect disinfectant against ASFV is difficult because of the lack of relevant data.Therefore,we aimed to find the most effective disinfectant and to optimise its concentration as well as contact time to confirm the viricidal effect against ASFV in vitro.We evaluated the viricidal activity of three concentrations each of six common disinfectants against ASFV using immersion disinfection assay(IDA)and spray disinfection assay(SDA);the concentrations of these disinfectants at which complete viral inactivation occurred were almost same as the manufacturer-recommended concentrations,but the exposure times for viral inactivation are different.The following disinfectants(assay:concentration,exposure time)showed complete inactivation:iodine and acid mixed solution(IDA/SDA:0.5%,10 min);compound potassium peroxymonosulfate(IDA:0.25%,30 min;SDA:0.25%,60 min);citric acid(IDA:0.25%,60 min;SDA:0.5%,60 min);sodium dichloroisocyanurate(IDA:0.125%,60 min;SDA:0.25%,60 min);and glutaral ang deciquam(IDA/SDA:0.2%,60 min);and deciquam(IDA/SDA:0.5%,60 min).However,in the presence of organic material contamination,disinfectants did not show a marked inactivation effect.Therefore,disinfection procedures should be performed in two steps:thorough mechanical cleaning followed by application of disinfectant.In conclusion,all the tested disinfectants can inactivate ASFV;these can be used as alternative disinfectants to enhance biosecurity.

A Universal Real-Time Fluorescence qPCR Method for Identifying Epidemic Strains of African Swine Fever Virus

Objective Establishing a highly sensitive real-time fluorescence quantitative PCR (qPCR) method for universal testing of epidemic African swine fever virus (ASFV) strains. Methods The ASFV p72 gene was targeted to design primer probes covering 24 p72 genotypes. The optimal amount of dimethylsulphoxide (DMSO) for qPCR amplification was determined, Various sensitivity and limit of detection (LOD) tests were performed, and clinical samples from China and imported goods were tested. Results The optimal primer-probe combination could specifically detect ASFV, 1.5% DMSO was optimal for qPCR, and LOD reached 3.2 copies/μL with good reproducibility (n = 20, p = 0.369). The method was employed to test 142 clinically suspected samples, of which 30 pig blood and 37 pig tissue samples were ASFV-positive. Moreover, the positive testing rate for ASFV was higher than for the standard qPCR method recommended by the Office International Des Epizooties (OIE), and for the commercially available kit. Thus, our method is superior for testing weakly positive samples with low virus titre, and epidemic strains present in imported goods. Conclusion Our method could be employed for universal testing of epidemic ASFV strains worldwide, ensuring wider coverage of hosts and ASFV strains/endemic strains, reducing false negatives, and benefitting early diagnosis.

The Prediction of B Cell Epitopes for VP73 Protein of African Fever Virus

[Objective] The B cell epitopes for VP73 protein of African swine fever virus was predicted. [ Method] Based on the analysis of the amino acid sequence and the flexible regions of VP73 protein, the B cell epitopes for VP73 protein of African swine fever virus were predicted by method of Kyte-Doolittie, Emini and Jameson-Wolf. [Result] The B cell epitopes were located at or adjacent to the N-terminal No. 11 - 18,26 -48,73 -82,136 - 150,159 - 174,181 - 189,191 - 210,247 - 276,279 - 295,313 - 323 and 382 - 392. [Conclusion] The multi-parameters analytic method was adopted to predict the B cell epitopes for VP73 protein of African swine fever virus, which laid solid foundation for further characterizing the protein of VP73 and researching epitope vaccine.

稳定表达猪BRD4-BD1/2蛋白的猪肺泡巨噬细胞传代细胞系的构建及其用于ASFV增殖的效果观察

前期研究发现宿主表观遗传调控蛋白——含溴结构域蛋白质4(bromodomain-containing protein 4,BRD4)有助于非洲猪瘟病毒(African swine fever virus,ASFV)的复制,为了深入研究BRD4对ASFV复制的影响,通过筛选出显著促进ASFV复制的BRD4-BD1/2结构域,利用慢病毒表达系统成功构建稳定表达BRD4-BD1/2结构域的3D4/21细胞系,分析ASFV在3D4/21-BRD4-BD1/2细胞系与WT细胞系之间的复制差异。首先,以家猪基因BRD4-BD1/2为靶标,构建了含有3x Flag标签的重组质粒pLVX-IRES-puro-3x Flag-BRD4-BD1/2,并将其与质粒pMD2.G和pSPAX2共同转染至HEK-293T细胞进行慢病毒包装,获得具有感染能力的慢病毒。使用慢病毒感染3D4/21细胞后通过嘌呤霉素药物筛选,成功获得了稳定表达BRD4-BD1/2结构域的3D4/21细胞系。利用RT-qPCR和Western blot技术分别检测了ASFV感染3D4/21-BRD4-BD1/2细胞后CP204L和B602L基因的转录水平以及相应蛋白表达水平,并通过HAD50测定评价ASFV的复制能力。研究结果表明:成功构建了稳定表达BRD4-BD1/2结构域的3D4/21细胞系。与3D4/21-WT细胞系相比,BRD4-BD1/2稳定表达细胞系能够显著促进ASFV复制。本研究提供了深入研究BRD4蛋白在ASFV复制中功能的生物材料,并为ASFV疫苗候选株的开发提供了理论基础。

Nanobodies against African swine fever virus p72 and CD2v proteins as reagents for developing two cELISAs to detect viral antibodies

African swine fever virus(ASFV)poses a significant threat to the global swine industry.Currently,there are no effective vaccines or treatments available to combat ASFV infection in pigs.The primary means of controlling the spread of the disease is through rapid detection and subsequent elimination of infected pig.Recently,a lower virulent ASFV isolate with a deleted EP402R gene(CD2v-deleted)has been reported in China,which further complicates the control of ASFV infection in pig farms.Furthermore,an EP402R-deleted ASFV variant has been developed as a potential live attenuated vaccine candidate strain.Therefore,it is crucial to develop detection methods that can distinguish wild-type and EP402R-deleted ASFV infections.In this study,two recombinant ASFV-p72 and-CD2v proteins were expressed using a prokaryotic system and used to immunize Bactrian camels.Subsequently,eight nanobodies against ASFV-p72 and ten nanobodies against ASFV-CD2v were screened.Following the production of these nanobodies with horse radish peroxidase(HRP)fusion proteins,the ASFV-p72-Nb2-HRP and ASFV-CD2v-Nb22-HRP fusions were selected for the development of two competitive ELISAs(cELISAs)to detect anti-ASFV antibodies.The two cELISAs exhibited high sensitivity,good specificity,repeatability,and stability.The coincidence rate between the two cELISAs and commercial ELISA kits was 98.6%and 97.6%,respectively.Collectively,the two cELISA for detecting antibodies against ASFV demonstrated ease of operation,a low cost,and a simple production process.The two cELISAs could determine whether pigs were infected with wild-type or CD2v-deleted ASFV,and could play an important role in monitoring ASFV infections in pig farms.

Identification of host proteins that interact with African swine fever virus pE301R

African swine fever virus(ASFV)infection poses enormous threats and challenges to the global pig industry;however,no effective vaccine is available against ASFV,attributing to the huge viral genome(approximately189 kb)and numerous encoding products(>150 genes)due to the limited understanding on the molecular mechanisms of viral pathogenesis.Elucidating the host-factor/viral-protein interaction network will reveal new targets for developing novel antiviral therapies.Using proteomic analysis,we identified 255 cellular proteins that interact with the ASFV-encoded pE301R protein when transiently expressed in HEK293T cells.Gene ontology(GO)annotation,Kyoto Encyclopedia of Genes and Genomes(KEGG)database enrichment,and protein-protein interaction(PPI)network analyses revealed that pE301R-interacting host proteins are potentially involved in various biological processes,including protein translation and folding,response to stimulation,and mitochondrial transmembrane transport.The interactions of two putative cellular proteins(apoptosis inducing factor mitochondria associated 1(AIFM1)and vimentin(VIM))with pE301R-apoptosis inducing factor have been verified by co-immunoprecipitation.Our study revealed the inhibitory role of pE301R in interferon(IFN)induction that involves VIM sequestration by pE301R,identified interactions between ASFV pE301R and cellular proteins,and predicted the potential function of pE301R and its associated biological processes,providing valuable information to enhance our understanding of viral protein function,pathogenesis,and potential candidates for the prevention and control of ASFV infection.

ASFV、PRV和PCV2多重荧光定量PCR检测方法的建立和应用

非洲猪瘟病毒(ASFV)、伪狂犬病毒(PRV)、猪圆环病毒2型(PCV2)是危害养猪业的3种重要病毒,但目前国内外尚无可同时检测这3种病原的方法。为建立一种可同时检测上述3种病原的方法,本研究以ASFV的B646L基因、PRV的gE基因及PCV2的rep基因为靶基因,采用Prime 3 web软件分别设计特异性引物和探针。采用相应引物分别构建上述3种靶基因的重组质粒,经测序鉴定后分别作为质粒标准品,并均稀释到1×105拷贝/μL后按照体积比1∶1∶1混匀后作为模板,采用方阵法对引物、探针、酶和各反应条件等的优化,初步建立ASFV、PRV和PCV2多重荧光定量PCR检测方法。结果显示,该方法标准曲线Ct值与相应质粒标准品浓度均存在良好的线性关系,相关系数(R2)均大于0.99。利用该方法同时检测猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)、口蹄疫病毒(FMDV)、猪细小病毒(PPV)、牛病毒性腹泻病毒(BVDV)、传染性胃肠炎病毒(TGEV)等病原,结果显示,本实验建立的该方法仅特异性扩增ASFV、PRV和PCV2,与其他病原均无交叉反应,特异性强;分别利用该方法与已公布的中华人民共和国农业农村部公告第172号中ASFV荧光定量PCR检测方法、PCV2荧光定量PCR方法(GB/T 35901-2018)、PRV荧光定量PCR检测方法(GB/T 35911-2018)对10倍倍比稀释(1.0×10^(4)拷贝/μL~1.0×10_(0)拷贝/μL)后的pUC57-ASFV-B646L、pUC57-PRV-gE、pGEMT-PCV2-rep质粒标准品1∶1∶1的混合物检测,结果显示该方法对3种质粒标准品的最低检测限均为10拷贝/μL,相同或略高于上述各病原的单一qPCR检测结果,表明本实验建立的该方法敏感性较高;选取3个稀释度的质粒标准品分别按1∶1∶1比例混合后进行组内和组间重复性试验,结果显示组内组间变异系数均小于3%,重复性好。利用本实验建立的方法和上述3种病原标准检测方法对114份临床样品检测,结果显示,本实验建立的方法对ASFV、PRV和PCV2检测的的阳性样品数分别为10份、10份和42份,未发现混合感染现象,与3种病原的标准方法相比符合率均为100%。以上结果表明,本研究建立的ASFV、PRV和PCV2多重荧光定量PCR方法特异性强、敏感性高、重复性好,可用于这3种常见猪病的鉴别诊断和流行病学调查。

靶向非洲猪瘟病毒EP152R基因sgRNA细胞系的构建及其对ASFV复制影响的研究

有报道证实,缺失重要功能基因EP152R的非洲猪瘟病毒(ASFV)不能有效复制。为了筛选靶向ASFV EP152R基因的小向导RNA(sgRNA),分析EP152R基因对ASFV复制的影响,本研究利用簇状规则间隔的短回文重复序列及其相关蛋白9(CRISPR-Cas9)核酸酶系统(CCTop)并利用猪的基因组作为脱靶对象设计并筛选靶向ASFV EP152R基因(72387nt~72845nt)的sg RNA,并采用非随机整合报告系统(TIDE)计算各对sg RNA的编辑效率确定最佳sg RNA。结果显示,筛选了5对靶向敲除ASFV EP152R基因的sg RNA,其中3对sg RNA对EP152R基因的编辑效率达30%~42%,另外两对sg RNA的编辑效率低于10%。将筛选的3对sg RNA退火后分别克隆至p-LentiCRISPRv2载体中,构建3个重组慢病毒质粒p-LentiCRISPRv2-gREP152R-1/2/3,并采用菌液PCR及测序鉴定正确后分别与2个辅助质粒共转染HEK293T细胞,72 h后获得各慢病毒并分别感染野猪肺细胞(WSL细胞),通过嘌呤霉素筛选表达各sg RNA的细胞WSL-g REP152R,并采用western blot鉴定。结果显示,构建的各细胞在40 ku处均出现特异性条带,而WSL细胞无该条带,表明正确构建表达3种EP152R sg RNA的细胞系WSL-g REP152R-1/2/3。将携带m Cherry报告基因的重组ASFV(mCherry-ASFV)以MOI 1感染各WSL-g REP152R细胞系,分别于不同时间观察荧光;收集各细胞上清液,采用荧光定量PCR(qPCR)检测ASFV P72基因的Ct值,并测定上清液中的病毒效价(感染后72 h)。观察结果显示,随感染时间的延长WSL-g REP152R-2/3中的红色荧光均明显减少;WSL-g REP152R-1及空白对照WSL细胞中的红色荧光则逐渐增多,但前者的红色荧光均少于后者。q PCR和TCID50测定结果显示,与空白对照WSL细胞相比,3种WSL-g REP152R细胞上清液中病毒扩增的Ct值均显著或极显著升高(P<0.05、P<0.01),而病毒效价均显著或极显著降低(P<0.05、P<0.01),且以WSL-g REP152R-2中的病毒效价最低。将m Cherry-ASFV感染各WSLg REP152R细胞并连续传3代,采用q PCR检测F2、F3代细胞上清液中的病毒扩增的Ct值。结果显示,与WSL细胞相比,各WSL-g REP152R细胞F2代上清液中ASFV扩增的Ct值均极显著升高(P<0.01),且WSL-g REP152R-2 F2代及WSL-g REP152R-1/2 F3代细胞上清液中均检测不到病毒扩增的Ct值。上述结果表明,本研究设计的sg RNA能够有效靶向ASFV EP152R基因并敲除该基因进而抑制ASFV及其子代病毒的复制,且g REP152R-2抑制ASFV复制的效果最好。本研究为ASFV疫苗的开发及抗ASF育种猪提供重要参考数据。

OGG1 inhibition suppresses African swine fever virus replication

African swine fever virus(ASFV)is an important pathogen that causes a highly contagious and lethal disease in swine,for which neither a vaccine nor treatment is available.The DNA repair enzyme 8-oxoguanine DNA glycosylase 1(OGG1),which excises the oxidative base lesion 8-oxo-7,8-dihydroguanine(8-oxoG),has been linked to the pathogenesis of different diseases associated with viral infections.However,the role of OGG1-base excision repair(BER)in ASFV infection has been poorly investigated.Our study aimed to characterize the alteration of host reactive oxygen species(ROS)and OGG1 and to analyse the role of OGG1 in ASFV infection.We found that ASFV infection induced high levels and dynamic changes in ROS and 8-oxoG and consistently increased the expression of OGG1.Viral yield,transcription level,and protein synthesis were reduced in ASFV-infected primary alveolar macrophages(PAMs)treated by TH5487 or SU0268 inhibiting OGG1.The expression of BER pathway associated proteins of ASFV was also suppressed in OGG1-inhibited PAMs.Furthermore,OGG1 was found to negatively regulate interferonβ(IFN-β)production during ASFV infection and IFN-βcould be activated by OGG1 inhibition with TH5487 and SU0268,which blocked OGG1 binding to 8-oxoG.Additionally,the interaction of OGG1 with viral MGF360-14-L protein could disturb IFN-βproduction to further affect ASFV replication.These results suggest that OGG1 plays the crucial role in successful viral infection and OGG1 inhibitors SU0268 or TH5487 could be used as antiviral agents for ASFV infection.

African swine fever virus S273R protein antagonizes type I interferon production by interfering with TBK1 and IRF3 interaction

African swine fever(ASF)is originally reported in East Africa as an acute hemorrhagic fever.African swine fever virus(ASFV)is a giant and complex DNA virus with icosahedral structure and encodes a variety of virulence factors to resist host innate immune response.S273R protein(pS273R),as a SUMO-1 specific cysteine protease,can affect viral packaging by cutting polymeric proteins.In this study,we found that pS273R was an important antagonistic viral factor that suppressed cGAS-STING-mediated type I interferon(IFN-I)production.A detailed analysis showed that pS273R inhibited IFN-I production by interacting with interferon regulatory factor 3(IRF3).Subsequently,we showed that pS273R disrupted the association between TBK1 and IRF3,leading to the repressed IRF3 phosphorylation and dimerization.Deletion and point mutation analysis verified that pS273R impaired IFN-I production independent of its cysteine protease activity.These findings will help us further understand ASFV pathogenesis.

Identification of African swine fever virus MGF505-2R as a potent inhibitor of innate immunity in vitro

African swine fever(ASF)is etiologically an acute,highly contagious and hemorrhagic disease caused by African swine fever virus(ASFV).Due to its genetic variation and phenotypic diversity,until now,no efficient commercial vaccines or therapeutic options are available.The ASFV genome contains a conserved middle region and two flexible ends that code for five multigene families(MGFs),while the biological functions of the MGFs are not fully characterized.Here,ASFV MGF505-2R-deficient mutant ASFV-Δ2R was constructed based on a highly virulent genotype II field isolate ASFV CN/GS/2018 currently circulating in China.Transcriptomic profiling demonstrated that ASFV-Δ2R was capable of inducing a larger number of differentially expressed genes(DEGs)compared with ASFV CN/GS/2018.Hierarchical clustering of up-regulated DEGs revealed that ASFV-Δ2R induced the most dramatic expression of interferon-related genes and inflammatory and innate immune genes,as further validated by RT-qPCR.The GO and KEGG pathway analysis identified significantly enriched pathways involved in pathogen recognition and innate antiviral immunity.Conversely,pharmacological activation of those antiviral immune responses by exogenous cytokines,including type I/II IFNs,TNF-αand IL-1β,exerted combinatory effects and synergized in antiviral capacity against ASFV replication.Collectively,MGF505-2R is a newly identified inhibitor of innate immunity potentially implicated in immune evasion.

综合洗消体系对阻断非洲猪瘟病毒传入猪场的作用

切断非洲猪瘟病毒(ASFV)传播途径是防控非洲猪瘟的有效手段。本试验旨在通过在某种猪场建立综合洗消体系以阻断ASFV传入。对拟进入猪场的车辆在出发前和距猪场1 km处清洗和消毒,在猪场门口进行第3次消毒;人员进入猪场前洗澡和隔离,并将其衣物消毒;输入物资进场前消毒。通过实时荧光定量PCR检测经洗消的车辆、人员、物资和猪场内猪只携带ASFV核酸情况,酶联免疫吸附测定(ELISA)检测猪只的ASFV抗体水平。结果显示,2019、2020、2021和2022年,车辆ASFV检出率分别为2.6%、1.3%、0和0,人员ASFV检出率分别为3.0%、1.1%、0和0.9%,物资ASFV检出率分别为12.5%、0、5.9%和0;4年间,猪场内猪只的ASFV核酸和抗体检测均为阴性。结果表明,通过建立完善的洗消体系并开展ASFV检测,能有效阻断ASFV传入猪场。

非洲猪瘟病毒p30蛋白氨基酸序列分段表达及反应原性分析

为比较非洲猪瘟病毒(African swine fever virus,ASFV)p30蛋白氨基酸序列中不同片段的反应原性,探究p30蛋白氨基酸序列中可能的抗原优势片段。本研究通过PCR、克隆技术将p30蛋白氨基酸序列中不同片段的编码基因分别插入表达质粒pET32a,经IPTG诱导、Ni柱纯化、透析后,获得p30蛋白氨基酸序列中8~101 aa片段(P-1#)、58~101 aa片段(P-2#)、101~158 aa片段(P-3#)、8~194 aa片段(P-4#),用酶联免疫吸附试验(ELISA)分析p30蛋白氨基酸序列中不同片段与p30蛋白免疫兔血清和ASFV阳性猪血清的反应原性。结果显示,上述片段均获得诱导表达及纯化,表达形式有可溶性表达(P-1#、P-3#)和包涵体表达(P-2#、P-4#)。各片段以1.0 mg/L包被时,p30免疫兔血清与p30蛋白氨基酸序列中4种片段均能发生免疫反应,ASFV阳性猪血清与P-4#、P-1#反应较佳,与P-3#反应中等,与P-2#反应最弱。综上,p30蛋白氨基酸序列中不同片段反应原性的差异为认识p30抗原优势区域提供了数据,这将有助于非洲猪瘟血清学诊断抗原的科学筛选。

半固体培养法制备非洲猪瘟病毒pA104R蛋白的单克隆抗体

为了快速、高效制备非洲猪瘟病毒(ASFV)单克隆抗体,本研究通过大肠杆菌系统表达并纯化了ASFV重组蛋白pA104R。以ASFV重组蛋白pA104R为抗原,分别比较了CpG ODN联合氢氧化铝佐剂和常规弗氏佐剂两种免疫策略,并重点比较半固体培养法和常规有限稀释法来制备ASFV pA104R单克隆抗体的效率。结果显示,本研究获得了相对分子质量为3.5×104的ASFV重组可溶性蛋白pA104R,通过其与CpG ODN联合氢氧化铝佐剂免疫小鼠,在第21 d即可达到融合要求,本试验方法(重组蛋白pA104R与CpG ODN联合氢氧化铝佐剂免疫)较重组蛋白pA104R与常规弗氏佐剂免疫节省14 d时间。通过半固体培养法筛选单克隆的试验周期比有限稀释法缩短28 d,并减少了亚克隆的工作量。半固体培养法获得5株阳性杂交瘤细胞,挑选效价较高的3株(9A4、9H6、11F5)进行鉴定,重链均为IgG,轻链均为KAPPA。纯化后的3株单克隆抗体针对pA104蛋白和全病毒蛋白质的效价分别达1∶160000~1∶320000和1∶200~1∶400。本研究优选了CpG ODN联合氢氧化铝佐剂结合半固体培养法筛选pA104R的单克隆抗体,为单克隆抗体制备提供了快速高效的新策略。