title: Inclusion of an Arg-Gly-Asp receptor-recognition motif into the capsid protein of rabbit hemorrhagic disease virus enables culture of the virusin vitro.
abstract: The fact that rabbit hemorrhagic disease virus (RHDV), an important member of the Caliciviridae family, cannot be propagatedin vitrohas greatly impeded the progress of investigations into the mechanisms of pathogenesis, translation, and replication of this and related viruses. In this study, we have successfully bypassed this obstacle by constructing a mutant RHDV (mRHDV) by using a reverse genetics technique. By changing two amino acids (S305R,N307D), we produced a specific receptor-recognition motif (Arg-Gly-Asp; called RGD) on the surface of the RHDV capsid protein. mRHDV was recognized by the intrinsic membrane receptor (integrin) of the RK-13 cells, which then gained entry and proliferated as well as imparted apparent cytopathic effects. After 20 passages, the titers of RHDV reached 1 × 104.350% tissue culture infectious dose (TCID50)/ml at 72 h. Furthermore, mRHDV-infected rabbits showed typical rabbit plague symptoms and died within 48-72 h. After immunization with inactivated mRHDV, the rabbits survived wild-type RHDV infection, indicating that mRHDV could be a candidate virus strain for producing a vaccine against RHDV infection. In summary, this study offers a novel strategy for overcoming the challenges of proliferating RHDVin vitroBecause virus uptake via specific membrane receptors, several of which specifically bind to the RGD peptide motif, is a common feature of host cells, we believe that this the strategy could also be applied to other RNA viruses that currently lack suitable cell lines for propagation such as hepatitis E virus and norovirus.
authors.affiliation: From the Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Zhu
authors.affiliation: From the Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China.
authors.forename: Qiuhong
authors.initials: Q
authors.lastname: Miao
authors.affiliation: From the Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China.
authors.forename: Yonggui
authors.initials: Y
authors.lastname: Tan
authors.affiliation: From the Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China.
authors.forename: Huimin
authors.initials: H
authors.lastname: Guo
authors.affiliation: From the Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China.
authors.forename: Teng
authors.initials: T
authors.lastname: Liu
authors.affiliation: From the Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China.
authors.forename: Binbin
authors.initials: B
authors.lastname: Wang
authors.affiliation: From the Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China.
authors.forename: Zongyan
authors.initials: Z
authors.lastname: Chen
authors.affiliation: From the Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China.
authors.forename: Chuanfeng
authors.initials: C
authors.lastname: Li
authors.affiliation: From the Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China liugq@shvri.ac.cn.
authors.forename: Guangqing
authors.initials: G
authors.lastname: Liu
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D017250:Caliciviridae Infections / Q000276:immunology / Q000517:prevention & control / Q000662:veterinary; D036022:Capsid Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D002522:Chlorocebus aethiops; D006224:Cricetinae; D004285:Dogs; D017929:Hemorrhagic Disease Virus, Rabbit / Q000235:genetics / Q000254:growth & development / Q000276:immunology; D006801:Humans; D061985:Madin Darby Canine Kidney Cells; D011817:Rabbits; D011971:Receptors, Immunologic / Q000235:genetics / Q000276:immunology; D018000:Receptors, Peptide / Q000235:genetics / Q000276:immunology; D014709:Vero Cells; D014765:Viral Vaccines / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D014776:Virus Cultivation / Q000379:methods*
chemical_list: D036022:Capsid Proteins; D011971:Receptors, Immunologic; D018000:Receptors, Peptide; D014765:Viral Vaccines; C051382:arginyl-glycyl-aspartic acid directed cell adhesion receptor
keywords: RK-13 cells; integrin; rabbit hemorrhagic disease virus; reverse genetics technique; viral DNA; viral protein; viral replication; virus entry