Based on the infectious full-length cDNA clone of rabbit hemorrhagic disease virus (RHDV), the in vitro transcripts are introduced into RK13 cells. 12 h later, CPE could be observed clearly, and virual antigen could a...Based on the infectious full-length cDNA clone of rabbit hemorrhagic disease virus (RHDV), the in vitro transcripts are introduced into RK13 cells. 12 h later, CPE could be observed clearly, and virual antigen could also be detected by IFA. The titre of the recovered virus is 104.6/mL. Immune electron micro-scopic observation of the virus particles revealed that the particles were rotund with a diameter of about 30 nm. Besides, virus titre quantification obtained by qRT-PCR showed a correlation between time from infection and virus titre. All these results showed that we have recovered RHDV from RK13 cells by re-verse genetics technology successfully, and this would be very useful in studies of the antigenicity, virulence, pathogenesis, maturation and new type vaccines of RHDV.展开更多
Rabbit hemorrhagic disease was described in China in 1984 and can cause hemorrhagic necrosis of the liver within two or three days after infection.The etiological agent,rabbit hemorrhagic disease virus(RHDV),belongs t...Rabbit hemorrhagic disease was described in China in 1984 and can cause hemorrhagic necrosis of the liver within two or three days after infection.The etiological agent,rabbit hemorrhagic disease virus(RHDV),belongs to the Lagovirus genus in the Caliciviridae family.Compared to other calicivirus,such as rNV and SMSV,the structure of Lagovirus members is not well characterized.In this report,structures of two types of wild RHDV particles,the intact virion and the core-like particle(CLP),were reconstructed by cryo-electron microscopy at 11Åand 17Å,respectively.This is the first time the 3D structure of wild caliciviruses CLP has been provided,and the 3D structure of intact RHDV virion is the highest resolution structure in Lagovirus.Comparison of the intact virion and CLP structures clearly indicated that CLP was produced from the intact virion with the protrusion dissociated.In contrast with the crystal structures of recombinant Norovirus and San Miguel sea lion virus,the capsomers of RHDV virion exhibited unique structural features and assembly modes.Both P1 and P2 subdomains have interactions inside the AB capsomer,while only P2 subdomains have interaction inside CC capsomer.The pseudo atomic models of RHDV capsomers were constructed by homology modeling and density map fitting,and the rotation of RHDV VP60 P domain with respect to its S domain,compared with SMSV,was observed.Collectively,our cryo-electron microscopic studies of RHDV provide close insight into the structure of Lagovirus,which is important for functional analysis and better vaccine development in the future.展开更多
Rabbit hemorrhagic disease virus(RHDV)is a member of the Caliciviridae family and cannot be propagated in vitro,which has impeded the progress of investigating its replication mechanism.Construction of an RHDV replico...Rabbit hemorrhagic disease virus(RHDV)is a member of the Caliciviridae family and cannot be propagated in vitro,which has impeded the progress of investigating its replication mechanism.Construction of an RHDV replicon system has recently provided a platform for exploring RHDV replication in host cells.Here,aided by this replicon system and using two-step affinity purification,we purified the RHDV replicase and identified its associated host factors.We identified rabbit nucleolin(NCL)as a physical link,which mediating the interaction between other RNA-dependent RNA polymerase(Rd Rp)-related host proteins and the viral replicase Rd Rp.We found that the overexpression or knockdown of NCL significantly increased or severely impaired RHDV replication in RK-13 cells,respectively.NCL was identified to directly interact with RHDV Rd Rp,p16,and p23.Furthermore,NCL knockdown severely impaired the binding of Rd Rp to Rd Rp-related host factors.Collectively,these results indicate that the host protein NCL is essential for RHDV replication and acts as a physical link between viral replicase and host proteins.展开更多
用RHDV人工感染易感兔,每隔4小时剖杀一组,整个病程大多为24小时,且病变典型.血凝试验查得,主要脏器病毒出现时间顺序是肝(攻毒后12小时)、脾、心、肾(16小时)、肺(20小时);病程后期,病毒含量从高到低依次为肝、脾、肺、肾、心。免疫荧...用RHDV人工感染易感兔,每隔4小时剖杀一组,整个病程大多为24小时,且病变典型.血凝试验查得,主要脏器病毒出现时间顺序是肝(攻毒后12小时)、脾、心、肾(16小时)、肺(20小时);病程后期,病毒含量从高到低依次为肝、脾、肺、肾、心。免疫荧光间接法检查感染兔各脏器,发现病毒经肌注兔体后4小时,即可定位于肝细胞,并在肝细胞核内复制增殖;兔感染病毒后16小时,在肝、脾、肾小球中可检出有免疫复合物沉着或抗体附着。电镜检查,肝细胞核内发现有病毒粒子,直径为25～30nm,细胞结构破坏严重。作者认为,RHDV 是一种 DNA 病毒;免疫复合物引起的免疫损伤及肝细胞结构、功能被破坏是兔发生病毒性出血症的重要原因。展开更多
We investigated the ability of the rabbit hemorrhagic disease virus(RHDV) capsid protein(VP60) to interact specifically with the minor structural protein VP10,using an in vivo cell-based CheckMate Mammalian Two-Hybrid...We investigated the ability of the rabbit hemorrhagic disease virus(RHDV) capsid protein(VP60) to interact specifically with the minor structural protein VP10,using an in vivo cell-based CheckMate Mammalian Two-Hybrid System.RHDV VP60 protein interacted specifically with VP10.Immunofluorescence analysis and co-immunoprecipitation with specific antibodies revealed the existence of biologically important VP60/VP10 complexes.However,when VP60 was divided into two fragments,the interaction between VP60 and VP10 was impaired dramatically.These results will be helpful for further investigating the mechanism of RHDV particle assembly.展开更多
基金This work was supported by the Natural Science Foundation of Zhejiang Province(Grant No.Y305047).
文摘Based on the infectious full-length cDNA clone of rabbit hemorrhagic disease virus (RHDV), the in vitro transcripts are introduced into RK13 cells. 12 h later, CPE could be observed clearly, and virual antigen could also be detected by IFA. The titre of the recovered virus is 104.6/mL. Immune electron micro-scopic observation of the virus particles revealed that the particles were rotund with a diameter of about 30 nm. Besides, virus titre quantification obtained by qRT-PCR showed a correlation between time from infection and virus titre. All these results showed that we have recovered RHDV from RK13 cells by re-verse genetics technology successfully, and this would be very useful in studies of the antigenicity, virulence, pathogenesis, maturation and new type vaccines of RHDV.
基金This work was supported by National Natural Science Foundation of China(Grant Nos.30700029,30721003)Chinese Academy of Sciences(KGCX1-YW-13)+1 种基金the National Basic Research Program(973 Program)(Nos.2006CB806506,2006CB911001)the National Programs for High Technology Research and Development Program(863 Program)(No.2006AA02Z173).
文摘Rabbit hemorrhagic disease was described in China in 1984 and can cause hemorrhagic necrosis of the liver within two or three days after infection.The etiological agent,rabbit hemorrhagic disease virus(RHDV),belongs to the Lagovirus genus in the Caliciviridae family.Compared to other calicivirus,such as rNV and SMSV,the structure of Lagovirus members is not well characterized.In this report,structures of two types of wild RHDV particles,the intact virion and the core-like particle(CLP),were reconstructed by cryo-electron microscopy at 11Åand 17Å,respectively.This is the first time the 3D structure of wild caliciviruses CLP has been provided,and the 3D structure of intact RHDV virion is the highest resolution structure in Lagovirus.Comparison of the intact virion and CLP structures clearly indicated that CLP was produced from the intact virion with the protrusion dissociated.In contrast with the crystal structures of recombinant Norovirus and San Miguel sea lion virus,the capsomers of RHDV virion exhibited unique structural features and assembly modes.Both P1 and P2 subdomains have interactions inside the AB capsomer,while only P2 subdomains have interaction inside CC capsomer.The pseudo atomic models of RHDV capsomers were constructed by homology modeling and density map fitting,and the rotation of RHDV VP60 P domain with respect to its S domain,compared with SMSV,was observed.Collectively,our cryo-electron microscopic studies of RHDV provide close insight into the structure of Lagovirus,which is important for functional analysis and better vaccine development in the future.
基金sponsored by Shanghai Sailing Program(20YF1457700)the National Natural Science Foundation of China(32000109 and 31672572)the China Postdoctoral Science Foundation(2019M660885 and 2021T140718)
文摘Rabbit hemorrhagic disease virus(RHDV)is a member of the Caliciviridae family and cannot be propagated in vitro,which has impeded the progress of investigating its replication mechanism.Construction of an RHDV replicon system has recently provided a platform for exploring RHDV replication in host cells.Here,aided by this replicon system and using two-step affinity purification,we purified the RHDV replicase and identified its associated host factors.We identified rabbit nucleolin(NCL)as a physical link,which mediating the interaction between other RNA-dependent RNA polymerase(Rd Rp)-related host proteins and the viral replicase Rd Rp.We found that the overexpression or knockdown of NCL significantly increased or severely impaired RHDV replication in RK-13 cells,respectively.NCL was identified to directly interact with RHDV Rd Rp,p16,and p23.Furthermore,NCL knockdown severely impaired the binding of Rd Rp to Rd Rp-related host factors.Collectively,these results indicate that the host protein NCL is essential for RHDV replication and acts as a physical link between viral replicase and host proteins.
文摘用RHDV人工感染易感兔,每隔4小时剖杀一组,整个病程大多为24小时,且病变典型.血凝试验查得,主要脏器病毒出现时间顺序是肝(攻毒后12小时)、脾、心、肾(16小时)、肺(20小时);病程后期,病毒含量从高到低依次为肝、脾、肺、肾、心。免疫荧光间接法检查感染兔各脏器,发现病毒经肌注兔体后4小时,即可定位于肝细胞,并在肝细胞核内复制增殖;兔感染病毒后16小时,在肝、脾、肾小球中可检出有免疫复合物沉着或抗体附着。电镜检查,肝细胞核内发现有病毒粒子,直径为25～30nm,细胞结构破坏严重。作者认为,RHDV 是一种 DNA 病毒;免疫复合物引起的免疫损伤及肝细胞结构、功能被破坏是兔发生病毒性出血症的重要原因。
基金supported by the National Natural Science Foundation of China (30870114)the Zhejiang Natural Science Foundation (3100396)the Fundamental Research Funds for the Central Institutes Program(2012JB08)
文摘We investigated the ability of the rabbit hemorrhagic disease virus(RHDV) capsid protein(VP60) to interact specifically with the minor structural protein VP10,using an in vivo cell-based CheckMate Mammalian Two-Hybrid System.RHDV VP60 protein interacted specifically with VP10.Immunofluorescence analysis and co-immunoprecipitation with specific antibodies revealed the existence of biologically important VP60/VP10 complexes.However,when VP60 was divided into two fragments,the interaction between VP60 and VP10 was impaired dramatically.These results will be helpful for further investigating the mechanism of RHDV particle assembly.
