title: Preparation and evaluation of recombinant severe fever with thrombocytopenia syndrome virus nucleocapsid protein for detection of total antibodies in human and animal sera by double-antigen sandwich enzyme-linked immunosorbent assay.
abstract: The recent emergence of the human infection confirmed to be caused by severe fever with thrombocytopenia syndrome virus (SFTSV) in China is of global concern. Safe diagnostic immunoreagents for determination of human and animal seroprevalence in epidemiological investigations are urgently needed. This paper describes the cloning and expression of the nucleocapsid (N) protein of SFTSV. An N-protein-based double-antigen sandwich enzyme-linked immunosorbent assay (ELISA) system was set up to detect the total antibodies in human and animal sera. We reasoned that as the double-antigen sandwich ELISA detected total antibodies with a higher sensitivity than traditional indirect ELISA, it could be used to detect SFTSV-specific antibodies from different animal species. The serum neutralization test was used to validate the performance of this ELISA system. All human and animal sera that tested positive in the neutralization test were also positive in the sandwich ELISA, and there was a high correlation between serum neutralizing titers and ELISA readings. Cross-reactivity was evaluated, and the system was found to be highly specific to SFTSV; all hantavirus- and dengue virus-confirmed patient samples were negative. SFTSV-confirmed human and animal sera from both Anhui and Hubei Provinces in China reacted with N protein in this ELISA, suggesting no major antigenic variation between geographically disparate virus isolates and the suitability of this assay in nationwide application. ELISA results showed that 3.6% of the human serum samples and 47.7% of the animal field serum samples were positive for SFTSV antibodies, indicating that SFTSV has circulated widely in China. This assay, which is simple to operate, poses no biohazard risk, does not require sophisticated equipment, and can be used in disease surveillance programs, particularly in the screening of large numbers of samples from various animal species.
authors.affiliation: Institute of Pathogenic Microbiology, Jiangsu Provincial Center for Disease Prevention and Control, Nanjing, China.
authors.forename: Yongjun
authors.initials: Y
authors.lastname: Jiao
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Zeng
authors.forename: Xiling
authors.initials: X
authors.lastname: Guo
authors.forename: Xian
authors.initials: X
authors.lastname: Qi
authors.forename: Xiao
authors.initials: X
authors.lastname: Zhang
authors.forename: Zhiyang
authors.initials: Z
authors.lastname: Shi
authors.forename: Minghao
authors.initials: M
authors.lastname: Zhou
authors.forename: Changjun
authors.initials: C
authors.lastname: Bao
authors.forename: Wenshuai
authors.initials: W
authors.lastname: Zhang
authors.forename: Yan
authors.initials: Y
authors.lastname: Xu
authors.forename: Hua
authors.initials: H
authors.lastname: Wang
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000097:blood*; D000956:Antigens, Viral / Q000235:genetics; D002044:Bunyaviridae Infections / Q000175:diagnosis* / Q000662:veterinary*; D002681:China; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D009500:Neutralization Tests; D019590:Nucleocapsid Proteins / Q000235:genetics; D016856:Phlebovirus / Q000276:immunology / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000235:genetics; D012680:Sensitivity and Specificity
chemical_list: D000914:Antibodies, Viral; D000956:Antigens, Viral; D019590:Nucleocapsid Proteins; D011994:Recombinant Proteins