title: A recombinant rabies virus expressing a phosphoprotein-eGFP fusion is rescued and applied to the rapid virus neutralization antibody assay.
abstract: Rabies remains a worldwide concern, and dogs are a major vector for rabies virus (RABV) transmission. Vaccination is used in China to control the spread of rabies in dogs, a practice which necessitates effective, efficient, and high-throughput methods to confirm vaccination. The current rapid fluorescent focus inhibition test (RFFIT) method to measure virus-neutralizing antibody titers in the serum involves multiple steps, and more efficient methods are needed to match the increasing demand for this type of monitoring. In this study, based on the parental rRC-HL strain, a recombinant RABV rRV-eGFP expressing enhanced green fluorescent protein (eGFP) fused with RABV P protein was generated by a reverse genetic technique. The rRV-eGFP grew stably and successfully expressed P-eGFP fusion in Neuro-2A (NA) host cells. Furthermore, the P protein was shown to co-localize with eGFP in rRV-eGFP-infected NA cells. Since eGFP is easily detected in infected cells under a fluorescence microscope, rRV-eGFP could be used to establish a more rapid virus-neutralizing antibody titers assay based on RFFIT, designated as the RFFIT-eGFP method. From 69 canine serum samples, the RFFIT-eGFP method was shown to be as specific and as sensitive as the RFFIT method, suggesting that it might represent a faster tool than conventional RFFIT for measuring RABV virus-neutralizing antibody titers in canine sera without sacrificing accuracy.
authors.affiliation: The Key Laboratory of Ministry of Education for Microbial and Plant Genetic Engineering, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresourses, Guangxi University, Nanning 530004, Guangxi, China; Laboratory of Animal Infectious Diseases, College of Animal Sciences and Veterinary Medicine, Guangxi University, Nanning 530004, Guangxi, China.
authors.forename: Hai-Bo
authors.initials: HB
authors.lastname: Tang
authors.affiliation: The Key Laboratory of Ministry of Education for Microbial and Plant Genetic Engineering, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresourses, Guangxi University, Nanning 530004, Guangxi, China; Laboratory of Animal Infectious Diseases, College of Animal Sciences and Veterinary Medicine, Guangxi University, Nanning 530004, Guangxi, China.
authors.forename: Zhuan-Ling
authors.initials: ZL
authors.lastname: Lu
authors.affiliation: The Key Laboratory of Ministry of Education for Microbial and Plant Genetic Engineering, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresourses, Guangxi University, Nanning 530004, Guangxi, China; Laboratory of Animal Infectious Diseases, College of Animal Sciences and Veterinary Medicine, Guangxi University, Nanning 530004, Guangxi, China.
authors.forename: Xian-Kai
authors.initials: XK
authors.lastname: Wei
authors.affiliation: Laboratory of Animal Infectious Diseases, College of Animal Sciences and Veterinary Medicine, Guangxi University, Nanning 530004, Guangxi, China.
authors.forename: Yi-Zhi
authors.initials: YZ
authors.lastname: Zhong
authors.affiliation: Laboratory of Animal Infectious Diseases, College of Animal Sciences and Veterinary Medicine, Guangxi University, Nanning 530004, Guangxi, China.
authors.forename: Tao-Zhen
authors.initials: TZ
authors.lastname: Zhong
authors.affiliation: The Key Laboratory of Ministry of Education for Microbial and Plant Genetic Engineering, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresourses, Guangxi University, Nanning 530004, Guangxi, China; Laboratory of Animal Infectious Diseases, College of Animal Sciences and Veterinary Medicine, Guangxi University, Nanning 530004, Guangxi, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Pan
authors.affiliation: Laboratory of Animal Infectious Diseases, College of Animal Sciences and Veterinary Medicine, Guangxi University, Nanning 530004, Guangxi, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Luo
authors.affiliation: The Key Laboratory of Ministry of Education for Microbial and Plant Genetic Engineering, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresourses, Guangxi University, Nanning 530004, Guangxi, China; Laboratory of Animal Infectious Diseases, College of Animal Sciences and Veterinary Medicine, Guangxi University, Nanning 530004, Guangxi, China.
authors.forename: Su-Huan
authors.initials: SH
authors.lastname: Liao
authors.affiliation: Laboratory of Animal Infectious Diseases, College of Animal Sciences and Veterinary Medicine, Guangxi University, Nanning 530004, Guangxi, China.
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Minamoto
authors.affiliation: The Key Laboratory of Ministry of Education for Microbial and Plant Genetic Engineering, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresourses, Guangxi University, Nanning 530004, Guangxi, China; Laboratory of Animal Infectious Diseases, College of Animal Sciences and Veterinary Medicine, Guangxi University, Nanning 530004, Guangxi, China. Electronic address: tingrongluo@gxu.edu.cn.
authors.forename: Ting Rong
authors.initials: TR
authors.lastname: Luo
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000276:immunology*; D000914:Antibodies, Viral / Q000276:immunology*; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics*; D051379:Mice; D018832:Molecular Chaperones; D009500:Neutralization Tests; D010750:Phosphoproteins / Q000235:genetics* / Q000276:immunology*; D012367:RNA, Viral; D011820:Rabies virus / Q000235:genetics* / Q000276:immunology* / Q000472:pathogenicity; D011993:Recombinant Fusion Proteins; D015678:Viral Structural Proteins / Q000235:genetics* / Q000276:immunology*; D014779:Virus Replication
chemical_list: D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D018832:Molecular Chaperones; C103961:P phosphoprotein, Rabies virus; D010750:Phosphoproteins; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D015678:Viral Structural Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
keywords: Rabies virus (RABV); Rapid fluorescent focus inhibition test (RFFIT)-eGFP method