Tick-borne encephalitis virus(TBEV)is a re-emerging pathogen in Kazakhstan,where the increasing risk of its spread underscores the need for improved healthcare preparedness,including the development of local vaccines....Tick-borne encephalitis virus(TBEV)is a re-emerging pathogen in Kazakhstan,where the increasing risk of its spread underscores the need for improved healthcare preparedness,including the development of local vaccines.However,the absence of reference TBEV strains in the country presented a major challenge.To address this,we generated a prototype strain(Vasilchenko)of the Siberian TBEV genotype,predominant in Kazakhstan,using synthetic genome and molecular infectious clone technology.A DNA-launched TBEV molecular clone was assembled from DNA fragments,enabling virus rescue upon plasmid transfection.During the propagation of the post-transfection virus in cell culture,a single amino acid substitution(E51K)in the envelope protein emerged,resulting in a 100-fold increase in the titer of the mutant variant.In vivo,this mutation significantly attenuated virulence:while wild-type TBEV caused 100%mortality in BALB/c mice,the E51K variant was non-lethal and exhibited reduced viremia,suggesting impaired neuroinvasiveness.To further exploit this attenuated,high-titer virus,we developed a GFP-expressing reporter TBEV variant.Using this reporter system,we demonstrated that favipiravir possesses antiviral activity against TBEV,with inhibitory concentrations within a pharmacologically relevant range.In conclusion,synthetic genomics enabled the generation of a reference TBEV strain to replenish Kazakhstan's collections.The E51K mutation enhances viral replication in vitro while attenuating pathogenicity in vivo,and the derived reporter virus is suitable for antiviral compound screening.展开更多
Tick-borne encephalitis virus(TBEV) is a pathogenic virus known to cause central nervous system(CNS) diseases in humans, and has become an increasing public health threat nowadays. The rates of TBEV infection in the e...Tick-borne encephalitis virus(TBEV) is a pathogenic virus known to cause central nervous system(CNS) diseases in humans, and has become an increasing public health threat nowadays. The rates of TBEV infection in the endemic countries are increasing. However, there is no effective antiviral against the disease. This underscores the urgent need for tools to study the emergence and pathogenesis of TBEV and to accelerate the development of vaccines and antivirals. In this study, we reported an infectious c DNA clone of TBEV that was isolated in China(the WH2012 strain). A beta-globin intron was inserted in the coding region of nonstructural protein 1(NS1) gene to improve the stability of viral genome in bacteria. In mammalian cells, the inserted intron was excised and spliced precisely, which did not lead to the generation of inserted mutants. High titers of infectious progeny viruses were generated after the transfection of the infectious clone. The cDNA-derived TBEV replicated efficiently, and caused typical cytopathic effect(CPE) and plaques in BHK-21 cells. In addition, the CPE and growth curve of cDNA-derived virus were similar to that of its parental isolate in cells. Together, we have constructed the first infectious TBEV cDNA clone in China, and the clone can be used to investigate the genetic determinants of TBEV virulence and disease pathogenesis, and to develop countermeasures against the virus.展开更多
Tick-borne encephalitis virus(TBEV)is an important tick-borne pathogen that poses as a serious public health concern.The coverage and immunogenicity of the currently available vaccines against TBEV are relatively low;...Tick-borne encephalitis virus(TBEV)is an important tick-borne pathogen that poses as a serious public health concern.The coverage and immunogenicity of the currently available vaccines against TBEV are relatively low;therefore,it is crucial to develop novel and effective vaccines against TBEV.The present study describes a novel strategy for the assembly of virus-like particles(VLPs)by co-expressing the structural(core/prM/E)and non-structural(NS2B/NS3Pro)proteins of TBEV.The efficacy of the VLPs was subsequently evaluated in C57BL/6 mice,and the resultant IgG serum could neutralize both Far-Eastern and European subtypes of TBEV.These findings indicated that the VLP-based vaccine elicited the production of cross-subtype reactive antibodies.The VLPs provided protection to mice lacking the type I interferon receptor(IFNAR^(-/-))against lethal TBEV challenge,with undetectable viral load in brain and intestinal tissues.Furthermore,the group that received the VLP vaccine did not exhibit significant pathological changes and the inflammatory factors were significantly suppressed compared to the control group.Immunization with the VLP vaccine induced the production of multiple-cytokine-producing antiviral CD4+T cells in vivo,including TNF-α^(+),IL-2^(+),and IFN-γ^(+)T cells.Altogether,the findings suggest that noninfectious VLPs can serve as a potentially safe and effective vaccine candidate against diverse subtypes of TBEV.展开更多
基金supported by funds from the scientific and technical program BR218004/0223“Improving measures to ensure biological safety in Kazakhstan:counteracting dangerous and especially dangerous infections”.Part of this work carried out in Russian Federation was supported by internal grant funding from the Scientific Centre for Family Health and Human Reproduction Problems:grant number 121022500179-0.Title:“Molecular,Organismal,and Population Patterns of the Epidemic Process of Anthropozoonotic and Transmissible Infections in the Territory of Northern Asia and Adjacent Areas”.
文摘Tick-borne encephalitis virus(TBEV)is a re-emerging pathogen in Kazakhstan,where the increasing risk of its spread underscores the need for improved healthcare preparedness,including the development of local vaccines.However,the absence of reference TBEV strains in the country presented a major challenge.To address this,we generated a prototype strain(Vasilchenko)of the Siberian TBEV genotype,predominant in Kazakhstan,using synthetic genome and molecular infectious clone technology.A DNA-launched TBEV molecular clone was assembled from DNA fragments,enabling virus rescue upon plasmid transfection.During the propagation of the post-transfection virus in cell culture,a single amino acid substitution(E51K)in the envelope protein emerged,resulting in a 100-fold increase in the titer of the mutant variant.In vivo,this mutation significantly attenuated virulence:while wild-type TBEV caused 100%mortality in BALB/c mice,the E51K variant was non-lethal and exhibited reduced viremia,suggesting impaired neuroinvasiveness.To further exploit this attenuated,high-titer virus,we developed a GFP-expressing reporter TBEV variant.Using this reporter system,we demonstrated that favipiravir possesses antiviral activity against TBEV,with inhibitory concentrations within a pharmacologically relevant range.In conclusion,synthetic genomics enabled the generation of a reference TBEV strain to replenish Kazakhstan's collections.The E51K mutation enhances viral replication in vitro while attenuating pathogenicity in vivo,and the derived reporter virus is suitable for antiviral compound screening.
基金This study was supported by grants from the National Key R&D Program of China(2018YFA0507201)。
文摘Tick-borne encephalitis virus(TBEV) is a pathogenic virus known to cause central nervous system(CNS) diseases in humans, and has become an increasing public health threat nowadays. The rates of TBEV infection in the endemic countries are increasing. However, there is no effective antiviral against the disease. This underscores the urgent need for tools to study the emergence and pathogenesis of TBEV and to accelerate the development of vaccines and antivirals. In this study, we reported an infectious c DNA clone of TBEV that was isolated in China(the WH2012 strain). A beta-globin intron was inserted in the coding region of nonstructural protein 1(NS1) gene to improve the stability of viral genome in bacteria. In mammalian cells, the inserted intron was excised and spliced precisely, which did not lead to the generation of inserted mutants. High titers of infectious progeny viruses were generated after the transfection of the infectious clone. The cDNA-derived TBEV replicated efficiently, and caused typical cytopathic effect(CPE) and plaques in BHK-21 cells. In addition, the CPE and growth curve of cDNA-derived virus were similar to that of its parental isolate in cells. Together, we have constructed the first infectious TBEV cDNA clone in China, and the clone can be used to investigate the genetic determinants of TBEV virulence and disease pathogenesis, and to develop countermeasures against the virus.
基金This work was supported by grants from the National Key Research and Development Program of China(grant number:2018YFA0507201 to X.W.C.)the National Science Foundation of China(grant number:32000111 to Q.Y.)the China Postdoctoral Science Foundation(grant number:2020T130021ZX to Q.Y.and grant number:2020M672580 to Q.Y.).
文摘Tick-borne encephalitis virus(TBEV)is an important tick-borne pathogen that poses as a serious public health concern.The coverage and immunogenicity of the currently available vaccines against TBEV are relatively low;therefore,it is crucial to develop novel and effective vaccines against TBEV.The present study describes a novel strategy for the assembly of virus-like particles(VLPs)by co-expressing the structural(core/prM/E)and non-structural(NS2B/NS3Pro)proteins of TBEV.The efficacy of the VLPs was subsequently evaluated in C57BL/6 mice,and the resultant IgG serum could neutralize both Far-Eastern and European subtypes of TBEV.These findings indicated that the VLP-based vaccine elicited the production of cross-subtype reactive antibodies.The VLPs provided protection to mice lacking the type I interferon receptor(IFNAR^(-/-))against lethal TBEV challenge,with undetectable viral load in brain and intestinal tissues.Furthermore,the group that received the VLP vaccine did not exhibit significant pathological changes and the inflammatory factors were significantly suppressed compared to the control group.Immunization with the VLP vaccine induced the production of multiple-cytokine-producing antiviral CD4+T cells in vivo,including TNF-α^(+),IL-2^(+),and IFN-γ^(+)T cells.Altogether,the findings suggest that noninfectious VLPs can serve as a potentially safe and effective vaccine candidate against diverse subtypes of TBEV.
