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.

Developing a duplex ARMS-qPCR method to differentiate genotype Ⅰ and Ⅱ African swine fever viruses based on their B646L genes

Africanswinefever(ASF),causedbythe African swine fever virus (ASFV), is an acute, hemorrhagic, and contagious disease of domestic pigs and wild boars.The disease is notifiable and listed by the World Organization for Animal Health (WOAH)(Wang N et al. 2019).

非洲猪瘟病毒pA104R蛋白单克隆抗体的制备与应用

为了给非洲猪瘟的临床诊断技术和实验室研究提供物质基础,本研究以我国首次分离的非洲猪瘟病毒(ASFV)Pig/HLJ/2018株基因组为模板,扩增pA104R基因,并将其克隆至pET-30a载体,构建重组原核表达质粒pET-30a-A104R,表达并纯化重组pA104R蛋白(rpA104R),以其为抗原免疫BALB/c小鼠制备单克隆抗体(MAb),并采用ELISA、western blot、间接免疫荧光法(IFA)和免疫组化(IHC)对所获得的MAb进行鉴定。结果显示,正确构建p ET-30a-A104R重组质粒,并获得大小约为12 ku的可溶性蛋白。通过间接ELISA方法筛选到2株阳性杂交瘤细胞株为4G2和4E10,并且2株MAb的重链均为IgG1,轻链为Kappa链。Western blot和IFA结果显示,MAb 4G2株能够特异性地识别ASFV pA104R蛋白和病毒感染的猪肺泡巨噬细胞中表达的pA104R蛋白。IHC结果表明,MAb 4G2可以与病毒感染的阳性猪组织发生特异性反应。本研究也为ASFV基因缺失疫苗的开发提供了技术储备。

Novel P22-monoclonal antibody based blocking ELISA for the detection of African swine fever virus antibodies in serum

African swine fever(ASF)is a highly infectious,transboundary viral disease of domestic and wild pigs,and is currently the most serious threat to world swine production,resulting in significant economic loss.In the absence of vaccines and treatments,the control of the disease entirely depends on accurate and early diagnosis accompanied by the culling of infected pigs.Thus,a highly specific and sensitive diagnostic assay is required during an outbreak and surveillance of the disease.In this study,a highly sensitive,specific,rapid and repeatable P22-monoclonal antibody-based blocking enzyme-linked immunosorbent assay(bELISA)assay was developed for the detection of antibodies against genotype I and II African swine fever viruses(ASFVs).A total of 806 pig serum samples were tested to evaluate the performance of the diagnostic assay.To determine the PI(percent Inhibition)cut-off value,receiver-operating characteristic(ROC)analysis was applied.According to the ROC analysis of the data,98.10%specificity and 100%sensitivity were recorded when the threshold cut-off value of PI was established at 47%.In addition,the assay was able to detect ASFV antibodies as early as 9 days post-infection when serum samples from experimentally infected pigs were used.Taking all together,the results of the present study indicated that the P22-mAb based bELISA assay can be used for rapid and accurate detection of antibodies against ASFV,which could play a valuable role in the containment and prevention of ASFV as an alternative to other serological diagnostic methods.Also,this study will assist researchers to further investigate the immunogenic importance of P22 protein in ASFV infection.

Host protein ABCE1 interacts with the viral phosphoprotein and promotes rabies virus replication

Rabies virus(RABV)phosphoprotein(P)plays an important role in disrupting host interferon(IFN)-mediated antiviral immune response.ABCE1 is known as an RNase L inhibitor that negatively regulates the 2′,5′-oligoadenylate(2-5A)/RNase L antiviral system related to the IFN signaling pathway.In this study,we screened the host factors associated with RABV P protein,while focusing on ABCE1 because of its role in the life cycle of several viruses.Our results showed that knockout of ABCE1 in HEK293T cells inhibited RABV replication.In contrast,the overexpression of ABCE1 in HEK293T cells enhanced RABV replication.Notably,the co-immunoprecipitation assay showed that ABCE1 interacted with RABV P protein.Since ABCE1 and RABV P proteins are related to the IFN signaling pathway,we propose that the interaction of viral P protein with ABCE1 leads to the disruption of host antiviral immune response.In summary,our research showed that ABCE1 is an important host protein that interacts with viral P protein and regulates RABV replication.Moreover,the interaction between ABCE1 and RABV P protein may facilitate the viral P protein-mediated disruption of host antiviral immune response.