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.

Immunogenicity of a recombinant VSV-Vectored SARS-CoV vaccine induced robust immunity in rhesus monkeys after single-dose immunization

Severe acute respiratory syndrome(SARS)is a highly contagious zoonotic disease caused by SARS coronavirus(SARS-Co V).Since its outbreak in Guangdong Province of China in 2002,SARS has caused 8096 infections and774 deaths by December 31st,2003.Although there have been no more SARS cases reported in human populations since 2004,the recent emergence of a novel coronavirus disease(COVID-19)indicates the potential of the recurrence of SARS and other coronavirus disease among humans.Thus,developing a rapid response SARS vaccine to provide protection for human populations is still needed.Spike(S)protein of SARS-Co V can induce neutralizing antibodies,which is a pivotal immunogenic antigen for vaccine development.Here we constructed a recombinant chimeric vesicular stomatitis virus(VSV)VSVΔG-SARS,in which the glycoprotein(G)gene is replaced with the SARS-Co V S gene.VSVΔG-SARS maintains the bullet-like shape of the native VSV,with the heterogeneous S protein incorporated into its surface instead of G protein.The results of safety trials revealed that VSVΔG-SARS is safe and effective in mice at a dose of 1×10^(6)TCID_(50).More importantly,only a single-dose immunization of 2×10^(7)TCID_(50)can provide high-level neutralizing antibodies and robust T cell responses to non-human primate animal models.Thus,our data indicate that VSVΔG-SARS can be used as a rapid response vaccine candidate.Our study on the recombinant VSV-vectored SARS-Co V vaccines can accumulate experience and provide a foundation for the new coronavirus disease in the future.

A seven-gene-deleted African swine fever virus is safe and effective as a live attenuated vaccine in pigs

African swine fever(ASF)is a devastating infectious disease in swine that is severely threatening the global pig industry.An efficacious vaccine is urgently required.Here,we used the Chinese ASFV HLJ/18 as a backbone and generated a series of genedeleted viruses.The virulence,immunogenicity,safety,and protective efficacy evaluation in specific-pathogen-free pigs,commercial pigs,and pregnant sows indicated that one virus,namely HLJ/18-7GD,which has seven genes deleted,is fully attenuated in pigs,cannot convert to the virulent strain,and provides complete protection of pigs against lethal ASFV challenge.Our study shows that HLJ/-18-7GD is a safe and effective vaccine against ASFV,and as such is expected to play an important role in controlling the spread of ASFV.

Emergence and prevalence of naturally occurring lower virulent African swine fever viruses in domestic pigs in China in 2020

African swine fever virus(ASFV)has been circulating in China for more than two years,and it is not clear whether the biological properties of the virus have changed.Here,we report on our surveillance of ASFVs in seven provinces of China,from June to December,2020.A total of 22 viruses were isolated and characterized as genotype II ASFVs,with mutations,deletions,insertions,or short-fragment replacement occurring in all isolates compared with Pig/HLJ/2018(HLJ/18),the earliest isolate in China.Eleven isolates had four different types of natural mutations or deletion in the EP402R gene and displayed a nonhemadsorbing(non-HAD)phenotype.Four isolates were tested for virulence in pigs;two were found to be as highly lethal as HLJ/18.However,two non-HAD isolates showed lower virulence but were highly transmissible;infection with 106 TCID50 dose was partially lethal and caused acute or sub-acute disease,whereas 103 TCID50 dose caused non-lethal,sub-acute or chronic disease,and persistent infection.The emergence of lower virulent natural mutants brings greater difficulty to the early diagnosis of ASF and creates new challenges for ASFV control.

TRIM35 mediates protection against influenza infection by activating TRAF3 and degrading viral PB2

Tripartite motif(TRIM)family proteins are important effectors of innate immunity against viral infections.Here we identified TRIM35 as a regulator of TRAF3 activation.Deficiency in or inhibition of TRIM35 suppressed the production of type I interferon(IFN)in response to viral infection.777m35-deficient mice were more susceptible to influenza A virus(IAV)infection than were wild-type mice.TRIM35 promoted the RIG-Imediated signaling by catalyzing Lys63-linked polyubiquitination of TRAF3 and the subsequent formation of a signaling complex with VISA and TBK1.IAV PB2 polymerase countered the innate antiviral immune response by impeding the Lys63-linked polyubiquitination and activation of TRAF3.TRIM35 mediated Lys48-linked polyubiquitination and proteasomal degradation of IAV PB2,thereby antagonizing its suppression of TRAF3 activation.Our in vitro and in vivo findings thus reveal novel roles of TRIM35,through catalyzing Lys63-or Lys48-linked polyubiquitination,in RIG-I antiviral immunity and mechanism of defense against IAV infection.

Generation and application of replication-competent Venus-expressing H5N1,H7N9,and H9N2 influenza A viruses

The generation and application of replication-competent influenza A virus(IAV) expressing a reporter gene represent a valuable tool to elucidate the mechanism of viral pathogenesis and establish new countermeasures to combat the threat of influenza. Here, replication-competent IAVs with a neuraminidase(NA) segment harboring a fluorescent reporter protein, Venus, were generated in the background of H5N1, H7N9, and H9N2 influenza viruses, the three subtypes of viruses with imminent pandemic potential. All three reporter viruses maintained virion morphology, replicated with similar or slightly reduced titers relative to their parental viruses, and stably expressed the fluorescent signal for at least two passages in embryonated chicken eggs. As a proof of concept, we demonstrated that these reporter viruses,used in combination with a high-content imaging system, can serve as a convenient and rapid tool for the screening of antivirals and host factors involved in the virus life cycle. Moreover, the reporter viruses demonstrated similar growth properties and tissue tropism as their parental viruses in mice, among which the H7N9 NA-Venus virus could potentially be used in ex vivo studies to better understand H7N9 pathogenesis or to develop novel therapeutics.

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.

A novel linear and broadly neutralizing peptide in the SARS-CoV-2 S2 protein for universal vaccine development

As humans continue to develop COVID-19 widely,numerous novel variants of SARS-CoV-2 have emerged[1,2].These variants,which may possess enhanced transmissibility and often result in breakthrough infections in the vaccinated population,pose great challenges to the current vaccine strategies targeting the immunodominance of the receptor-binding domain(RBD)of the spike(S)protein[2].

Single cell RNA and immune repertoire prowling of COVID-19 patients reveal novel neutralizing antibody

There is no doubt that COVID-19 outbreak is currently the biggest public health threat,which has caused catastrophic con sequences in many countries and regions.As host immunity is key to fighting against virus infection,it is important to characterize the immunologic changes in the COVID-19 patients,and to explore potential therapeutic candidates.The most efficient ways to end this pandemic are to vaccinate the susceptible population,and to use specific drugs,such as monoclonal antibodies against the viral spike protein(S protein),to treat the affected individuals.Several promising neutralizing antibodies have recently been reported(Cao et al.,2020;Lv et al.,2020).

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.