Encephalomyocarditis virus(EMCV),a potential zoonotic pathogen,poses significant socioeconomic and public health challenges across various host species.Although EMCV rarely triggers severe clinical symptoms in humans,...Encephalomyocarditis virus(EMCV),a potential zoonotic pathogen,poses significant socioeconomic and public health challenges across various host species.Although EMCV rarely triggers severe clinical symptoms in humans,its widespread prevalence and unique biological characteristics underscore the need for continuous surveillance and the development of effective therapeutics and prophylactics.In this study,we evaluated the neutralizing effects of a monoclonal antibody derived from the spleens of mice immunized with EMCV virus-like particles(VLPs),both in vitro and in vivo.Using recombinant DNA technology,we engineered a baculovirus system to express EMCVs P12A and 3C,facilitating the production of VLPs in Sf9 cells.These VLPs serve as antigens to immunize mice,leading to the isolation of the monoclonal antibody 45G3.This antibody exhibited high specificity for EMCV confor-mational epitopes,excluding linear epitopes,and demonstrated potent in vitro neutralizing activity,with an IC50 of 0.01873μg/mL.Immunoelectron microscopy(IEM)revealed a strong direct interaction between the 45G3 antibody and EMCV particles.Virus adsorption inhibition assays demonstrated that 45G3 effectively blocked viral attachment,thereby preventing further infection of host cells.These findings further support the notion of a robust interaction between the virus and the antibody.Moreover,in vivo assessments revealed that 45G3 significantly reduced viral loads in treated mice and improved survival outcomes following EMCV exposure.Additionally,posttreatment analysis revealed reduced tissue damage and a markedly decreased inflammatory response in the brain,indicating that the 45G3 antibody effectively blocked viral infection,thereby mitigating tissue damage and enhancing survival.These findings position 45G3 as a promising candidate for EMCV management and provide a strong foundation for the future development of antiviral drugs targeting this widespread virus.展开更多
The objective of this study was to construct the infectious clone of encephalomyocarditis virus (EMCV) BJC3 strain.The genomic cDNA of the virus was amplified by three overlapping segments using RT-PCR,and cloned into...The objective of this study was to construct the infectious clone of encephalomyocarditis virus (EMCV) BJC3 strain.The genomic cDNA of the virus was amplified by three overlapping segments using RT-PCR,and cloned into low-copy plasmid pWSK29 to construct the full-length cDNA clone pWSKBJC3/ w.The pWSKBJC3/w was in vitro transcribed and transfected into BHK-21 cells to rescue the virus.The results showed that the full-length cDNA clone was infectious and the virus could be rescued in BHK-21 cells.The rescued virus designated RvBJC3W was identified by RT-PCR and indirect immunofluorescence assay(IFA).The rescued virus had similar growth characteristics to its parental virus BJC3 and retained pathogenicity for mice.Our results indicate that the first infectious cDNA clone of EMCV in China has been successfully established and provides an essential tool for investigating the molecular basis of pathogenicity of EMCV.展开更多
Encephalomyocarditis virus(EMCV)is a positive single-stranded small RNA virus without envelope,which can infect a variety of mammals.Swines are the most susceptible animals,which can cause acute myocarditis and respir...Encephalomyocarditis virus(EMCV)is a positive single-stranded small RNA virus without envelope,which can infect a variety of mammals.Swines are the most susceptible animals,which can cause acute myocarditis and respiratory failure in piglets and reproductive failure in pregnant sows.Diseases caused by EMCV have a wide range of effects on the global swine industry.In this study,a strain of EMCV was isolated from a swine aborted fetus in northeast China.It was identified by reverse transcriptase polymerase chain reaction(RT-PCR),electron microscopic observation and indirect immunofluorescence assay.The subsequent results showed that the virus titer of HLJ strain grew to 8.3 lgTCID50 on baby hamster kidney 21(BHK-21)cells.And HLJ strain caused the specific cytopathic effect(CPE)on BHK-21 cells and severe pathological changes in mice.Complete genome sequencing and multiple sequence alignment showed that the homology between HLJ strain and other isolates worldwide was 71.5%-99.7%.Phylogenetic analysis showed that EMCV isolates fell into five clusters:lineageⅠ,Ⅱ,Ⅲ,ⅣandⅤ,based on the nucleotide sequences of the entire open reading frame(ORF)and VP1 gene.HLJ isolate was grouped into lineage I.The analyses of amino acid mutation sites of VP1 protein showed that the amino acids at positions 20 and 54 in VP1 junction were unique to HLJ strain.The isolation of HLJ strain enriched the epidemiological database of EMCV.展开更多
Encephalomyocarditis virus (EMCV) infection leads to many diseases including encephalitis,myocarditis and diabetes in its natural host,the mouse.In this study,we generated four cDNA clones with a point mutation at pos...Encephalomyocarditis virus (EMCV) infection leads to many diseases including encephalitis,myocarditis and diabetes in its natural host,the mouse.In this study,we generated four cDNA clones with a point mutation at position 100 of VP1.The amino acids isoleucine,alanine,serine and proline were substituted with threonine in the four different clones of EMCV strain BJC3 by site-specific mutagenesis,and viable viruses were rescued.Although all mutants and wild-type viruses display different plaque morphologies,they replicate comparably in BHK-21 cells.The pathogenicity of the mutated viruses was systematically analyzed to investigate the importance of this amino acid in the viral pathogenicity and disease phenotype of EMCV infection in mice.The results showed that the isoleucine(T1100I) and proline-mutated viruses (T1100P) exhibited a reduced mortality,lower cerebral virus loads and alleviated brain damage while the viruses with serine (T1100S) and alanine (T1100A) substitutions displayed similar properties as the wild-type virus.These findings indicate that the amino acid at position 100 of VP1 is important for EMCV in vivo infection,and its mutation alters the pathogenicity of viral infection in mice.展开更多
基金funded by the National Key Research and Development Program of China(grant number:2023YFC2306501)the Hubei Provincial Fund for Supporting High-Quality Development of the Seed Industry"Conservation and Utilization of Agricultural Germplasm Resources"Project(grant number:HBZY2023A001-16)。
文摘Encephalomyocarditis virus(EMCV),a potential zoonotic pathogen,poses significant socioeconomic and public health challenges across various host species.Although EMCV rarely triggers severe clinical symptoms in humans,its widespread prevalence and unique biological characteristics underscore the need for continuous surveillance and the development of effective therapeutics and prophylactics.In this study,we evaluated the neutralizing effects of a monoclonal antibody derived from the spleens of mice immunized with EMCV virus-like particles(VLPs),both in vitro and in vivo.Using recombinant DNA technology,we engineered a baculovirus system to express EMCVs P12A and 3C,facilitating the production of VLPs in Sf9 cells.These VLPs serve as antigens to immunize mice,leading to the isolation of the monoclonal antibody 45G3.This antibody exhibited high specificity for EMCV confor-mational epitopes,excluding linear epitopes,and demonstrated potent in vitro neutralizing activity,with an IC50 of 0.01873μg/mL.Immunoelectron microscopy(IEM)revealed a strong direct interaction between the 45G3 antibody and EMCV particles.Virus adsorption inhibition assays demonstrated that 45G3 effectively blocked viral attachment,thereby preventing further infection of host cells.These findings further support the notion of a robust interaction between the virus and the antibody.Moreover,in vivo assessments revealed that 45G3 significantly reduced viral loads in treated mice and improved survival outcomes following EMCV exposure.Additionally,posttreatment analysis revealed reduced tissue damage and a markedly decreased inflammatory response in the brain,indicating that the 45G3 antibody effectively blocked viral infection,thereby mitigating tissue damage and enhancing survival.These findings position 45G3 as a promising candidate for EMCV management and provide a strong foundation for the future development of antiviral drugs targeting this widespread virus.
基金supported by the Key Project of National Natural Science Foundation of China(30530550)
文摘The objective of this study was to construct the infectious clone of encephalomyocarditis virus (EMCV) BJC3 strain.The genomic cDNA of the virus was amplified by three overlapping segments using RT-PCR,and cloned into low-copy plasmid pWSK29 to construct the full-length cDNA clone pWSKBJC3/ w.The pWSKBJC3/w was in vitro transcribed and transfected into BHK-21 cells to rescue the virus.The results showed that the full-length cDNA clone was infectious and the virus could be rescued in BHK-21 cells.The rescued virus designated RvBJC3W was identified by RT-PCR and indirect immunofluorescence assay(IFA).The rescued virus had similar growth characteristics to its parental virus BJC3 and retained pathogenicity for mice.Our results indicate that the first infectious cDNA clone of EMCV in China has been successfully established and provides an essential tool for investigating the molecular basis of pathogenicity of EMCV.
基金Supported by the National Natural Science Foundation of China(31372438)。
文摘Encephalomyocarditis virus(EMCV)is a positive single-stranded small RNA virus without envelope,which can infect a variety of mammals.Swines are the most susceptible animals,which can cause acute myocarditis and respiratory failure in piglets and reproductive failure in pregnant sows.Diseases caused by EMCV have a wide range of effects on the global swine industry.In this study,a strain of EMCV was isolated from a swine aborted fetus in northeast China.It was identified by reverse transcriptase polymerase chain reaction(RT-PCR),electron microscopic observation and indirect immunofluorescence assay.The subsequent results showed that the virus titer of HLJ strain grew to 8.3 lgTCID50 on baby hamster kidney 21(BHK-21)cells.And HLJ strain caused the specific cytopathic effect(CPE)on BHK-21 cells and severe pathological changes in mice.Complete genome sequencing and multiple sequence alignment showed that the homology between HLJ strain and other isolates worldwide was 71.5%-99.7%.Phylogenetic analysis showed that EMCV isolates fell into five clusters:lineageⅠ,Ⅱ,Ⅲ,ⅣandⅤ,based on the nucleotide sequences of the entire open reading frame(ORF)and VP1 gene.HLJ isolate was grouped into lineage I.The analyses of amino acid mutation sites of VP1 protein showed that the amino acids at positions 20 and 54 in VP1 junction were unique to HLJ strain.The isolation of HLJ strain enriched the epidemiological database of EMCV.
基金supported by the National Natural Science Foundation of China (Grant No. 30530550)the Program for Innovative Research Team in University of China (Grant No. IRT0866)
文摘Encephalomyocarditis virus (EMCV) infection leads to many diseases including encephalitis,myocarditis and diabetes in its natural host,the mouse.In this study,we generated four cDNA clones with a point mutation at position 100 of VP1.The amino acids isoleucine,alanine,serine and proline were substituted with threonine in the four different clones of EMCV strain BJC3 by site-specific mutagenesis,and viable viruses were rescued.Although all mutants and wild-type viruses display different plaque morphologies,they replicate comparably in BHK-21 cells.The pathogenicity of the mutated viruses was systematically analyzed to investigate the importance of this amino acid in the viral pathogenicity and disease phenotype of EMCV infection in mice.The results showed that the isoleucine(T1100I) and proline-mutated viruses (T1100P) exhibited a reduced mortality,lower cerebral virus loads and alleviated brain damage while the viruses with serine (T1100S) and alanine (T1100A) substitutions displayed similar properties as the wild-type virus.These findings indicate that the amino acid at position 100 of VP1 is important for EMCV in vivo infection,and its mutation alters the pathogenicity of viral infection in mice.
