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.

An emerging hemorrhagic fever in China caused by a novel bunyavirus SFTSV

“…In Shandong Province, 83%-95% of goats were positive for SFTSV antibodies [15][16][17]; in Jiangsu Province, 57% of goats, 32% of cattle, 6% of dogs, 5% of pigs, and 1% of chickens were seropositive for SFTSV [17]; and in Hubei Province, 55.0% (6/11) of dogs, 36.7% (2/3) of goats, and 80.0% (4/5) of cattle were seropositive for SFTSV [18]. However, only a small proportion of the animals studied (1.7%-5.3%) were found to carry low levels of viral RNA in their sera [19].…”

The Pathogenesis of Severe Fever with Thrombocytopenia Syndrome Virus Infection in Alpha/Beta Interferon Knockout Mice: Insights into the Pathologic Mechanisms of a New Viral Hemorrhagic Fever

“…SFTSV is believed to be transmitted by ticks, because the virus has been detected in Haemaphysalis longicornis ticks collected from domestic animals (1). Domestic animals such as goats, dogs, and cattle within the area of endemicity have a high seroprevalence of SFTSV antibodies (7,8). However, the animal host(s) of SFTSV remains elusive.…”

Bunyamwera virus possesses a distinct nucleocapsid protein to facilitate genome encapsidation

“…Similar to other -ssRNA viruses, all members of the Bunyaviridae family contain a ribonucleoprotein (RNP) complex composed of genomic RNA enwrapped by the NP (4). After entry into the cytoplasm through membrane fusion mediated by glycoproteins, the RNP is released from the virion and serves as the template with which the copackaged RdRp transcribes mRNAs from the viral genome in the RNP.…”

“…The L segment encodes an RNA-dependent RNA polymerase (RdRp), the M segment encodes a precursor of glycoproteins (Gn and Gc), and the S segment encodes a nucleocapsid protein (NP). Moreover, a few Bunyaviridae members possess a nonstructural protein (NSs and/or NSm) using an ambisense coding strategy by the S and M segments (4).…”