key: cord-0994011-cv6vsmzz
authors: Guo, Li; Liu, Shasha; Song, Jingdong; Han, Lianlian; Zhang, Hu; Wu, Chao; Wang, Conghui; Zhou, Hongli; Wang, Jianwei
title: Seroprevalence of Wenzhou virus in China
date: 2020-07-15
journal: Biosaf Health
DOI: 10.1016/j.bsheal.2020.07.004
sha: 6b182c2854786d165bb581cbfd77866012ff64cb
doc_id: 994011
cord_uid: cv6vsmzz

Wenzhou virus (WENV) was first identified in rodents and Asian house shrews in Wenzhou, Zhejiang province, China. However, little is known about the prevalence of WENV infections in humans in China. To determine the threat that WENV may pose to humans, we determine the seroprevalence of WENV in healthy individuals in China in this study. Cross-reactivities of nucleoprotein (NP) were detected between Lymphocytic choriomeningitis virus (LCMV) and WENV using Western blot and ELISA assy. The prevalence of specific IgG antibodies against WENV NP was investigated in different age groups of 830 healthy individuals aged 0–70 years old in China using a competition ELISA assay. The results indicate that WENV and LCMV share cross-reactive epitopes between NPs. The total seroprevalence of WENV in healthy adults was 4.6%, with 3.6% (8/221) for individuals 15–44 years of age, 5.4% (17/317) for individuals 45–59 years of age, and 4.1% (4/98) for older adults over 60. The total seroprevalence of WENV in children under age 15 was 1.5%, with 2.9% (1/34) in children aged 2–5 years, and 2.2% in 5–14 years (2/91). The finding suggests that WENV or WENV-like virus may sporadically infect humans of China.

J o u r n a l P r e -p r o o f

Wenzhou virus (WENV), a novel arenavirus, was first identified in rodents and Asian house shrews in Wenzhou, Zhejiang Province, China in 2014 [1] .

Arenaviruses are enveloped viruses with a bi-segmented, negative-sense, single-stranded RNA genome. The genome organization is well conserved across the arenaviruses family, which contains a large (L) and a small (S) segment [2] . The L segment encodes the viral RNA-dependent RNA polymerase (RdRp) and a small RING finger protein that functions as a matrix protein, whereas the S segment encodes the viral glycoprotein precursor (GPC) and the nucleoprotein (NP) [3] . NP, the main structural element of the viral ribonucleoprotein, is the most abundant viral polypeptide in virions and infected cells [2] .

Arenaviruses cause asymptomatically chronic infections in rodents, but some of them can infect humans when host rodents invade areas of human habitation. To date, several arenaviruses have been associated with human disease with different clinical symptoms. Lassa [4] , Junin [5] , Machupo [6] , J o u r n a l P r e -p r o o f common than what is realized [12] . After its initial characterization, WENV was 

J o u r n a l P r e -p r o o f system and mouse antisera against WENV and LCMV NPs which were produced by E.coli expression system using Western blot and ELISA assays.

Western blot analysis showed that the antisera against WENV or LCMV NPs reacted with LCMV NP and WENV NP ( Figure 1A) . Similar cross-reactivities were also detected by ELISA assays (Figure 1B and 1C) . Mouse sera against WENV and LCMV NPs reacted strongly with the homologous NP. Moreover, antisera reacted with the heterologous NP when the antibody dilutions of the mice antiserum were low (<1:5000). These results indicate that WENV and LCMV share cross-reactive epitopes between NPs. Therefore, a WENV IgG cELISA assay was developed by using LCMV NP as a competing antigen to minimize the cross-reactivity for WENV seroprevalence determination.

To determine the seroprevalence of WENV in humans, we developed a cELISA protocol for detecting IgG antibodies against WENV using NP as the coating antigen. The parameters for the ELISA assay including the amount of NP coating (12.5 ng/well) and serum dilutions (1:400) were optimized using chessboard titration tests. We determined the cELISA cut-off value of 0.27 by determining the inflection point of absorbance values (Abs) for WENV NP as previously described [15, 16] . A tested sample was considered positive if its A450 was above the cut-off value.

To provide positive and negative controls for cELISA, we identified J o u r n a l P r e -p r o o f WENV-positive and WENV-negative serum samples by Western blot analysis using purified WENV NP. A single serum sample that was positive for WENV NP protein was obtained from a healthy child (Figure 2A) . To determine the concentration of LCMV NP required for exhaustive antibody competition of WENV, the serum samples were competed with concentrations of LCMV NP ranging from 0-16 mg/mL. Using this positive control serum, we determined that 200 ng/well of competing LCMV NP was sufficient for the cELISA competition assays ( Figure 2B ).

We then used the cELISA protocol to screen for anti-NP IgG in sera from 830 healthy individuals. The total seroprevalence of WENV in 636 healthy adults >14 years of age was 4.6% (Table 1 ). The seroprevalence of WENV was 3.6% for individuals 15-44 years of age, 5.4% for individuals 45-59 years of age, and 4.1% for older adults over 60 ( Figure 3 ). The total seroprevalence of WENV in 194 healthy children under age 15 was 1.5% (Table 1) 

In this study, we determined the seroprevalence of WENV, which was first identified in rodents and Asian house shrews, to be 1.5% in children and 4.6%

in adults in China. The finding suggests that WENV or WENV-like virus may sporadically infect humans.

Serology can be used as an indicator of the circulation of given viruses for specific area or population. To avoid false positives caused by cross-reactivity, we used an NP-based cELISA to assess the seroprevalence of WENV in healthy individuals in China. This method is an optimal approach for evaluating the seroprevalence of a virus and for carrying out serodiagnosis of the multiple viruses where cross-reactivities occur, such as human bocaviruses [15, 17] and polyomaviruses [16] . We observed seroprevalences of anti-WENV IgG ranging from 2.2% in older children (5-14 years of age) to 5.4% in middle-aged adults (45-59 years of age). These results suggest that a low seroprevalence of WENV in China. These findings differ from those of a recent study of individuals in Southeastern Asia, where the seroprevalence of WENV was reported to be 13.2% in healthy individuals and 17.4% in individuals with Dengue-like/influenza-like illness [10] . The discrepancies between these two studies may be attributed to the method of detection; in this study, WENV IgG antibodies were detected using a competitive ELISA, whereas Blasdell et al. 

J o u r n a l P r e -p r o o f 

Isolation and characterization of a novel arenavirus harbored by Rodents and Shrews in Zhejiang province

ICTV Virus Taxonomy Profile: Arenavirida

Pathogenesis of Lassa fever

Human infection with arenaviruses in the Americas

Reemergence of Bolivian Hemorrhagic Fever

Genetic detection and characterization of Lujo virus, a new hemorrhagic fever-associated arenavirus from southern Africa

Evidence of human infection by a new mammarenavirus endemic to Southeastern Asia

Zoonotic aspects of arenavirus infections

Lymphocytic choriomeningitis virus: an emerging obstetric pathogen?

Study on cross-reactivity between nucleocapsid proteins of two arenaviruses

Secretory expression of all 16 subtypes of the hemagglutinin 1 protein of influenza A virus in insect cells

Differential seroprevalence of human bocavirus species 1-4 in Beijing

Seroepidemiology of human polyomaviruses

Seroepidemiology of human bocaviruses 1-4

The authors declare that there are no conflicts of interest.Journal Pre-proof J o u r n a l P r e -p r o o f