Indirect Immunofluorescence Assay for the Simultaneous Detection of Antibodies against Clinically Important Old and New World Hantaviruses

Sabine Lederer,Raluca Panculescu-Gatej,Paul Heyman,Erik Lattwein,Winfried Stoecker,Antti Vaheri,Paula Padula,Heinz Feldmann,Karen Sonnenberg,Merle Hanke,Tatjana Avšič-Županc,Åke Lundkvist,Olli Vapalahti,Daryl Dick,Matthias Niedrig,Jonas Schmidt‐Chanasit ,Pablo A Víal ,Paul K.s Chan ,Cornelia Svetlana Ceianu

doi:10.1371/journal.pntd.0002157

Abstract

In order to detect serum antibodies against clinically important Old and New World hantaviruses simultaneously, multiparametric indirect immunofluorescence assays (IFAs) based on biochip mosaics were developed. Each of the mosaic substrates consisted of cells infected with one of the virus types Hantaan (HTNV), Puumala (PUUV), Seoul (SEOV), Saaremaa (SAAV), Dobrava (DOBV), Sin Nombre (SNV) or Andes (ANDV). For assay evaluation, serum IgG and IgM antibodies were analyzed using 184 laboratory-confirmed hantavirus-positive sera collected at six diagnostic centers from patients actively or previously infected with the following hantavirus serotypes: PUUV (Finland, n=97); SEOV (China, n=5); DOBV (Romania, n=7); SNV (Canada, n=23); ANDV (Argentina and Chile, n=52). The control panel comprised 89 sera from healthy blood donors. According to the reference tests, all 184 patient samples were seropositive for hantavirus-specific IgG (n=177; 96%) and/or IgM (n=131; 72%), while all control samples were tested negative. In the multiparametric IFA applied in this study, 183 (99%) of the patient sera were IgG and 131 (71%) IgM positive (accordance with the reference tests: IgG, 96%; IgM, 93%). Overall IFA sensitivity for combined IgG and IgM analysis amounted to 100% for all serotypes, except for SNV (96%). Of the 89 control sera, 2 (2%) showed IgG reactivity against the HTNV substrate, but not against any other hantavirus. Due to the high cross-reactivity of hantaviral nucleocapsid proteins, endpoint titrations were conducted, allowing serotype determination in >90% of PUUV- and ANDV-infected patients. Thus, multiparametric IFA enables highly sensitive and specific serological diagnosis of hantavirus infections and can be used to differentiate PUUV and ANDV infection from infections with Murinae-borne hantaviruses (e.g. DOBV and SEOV).

Highlights

Hantaviruses are enveloped and negative-sense single-stranded RNA viruses of the Bunyaviridae family
Hantaviruses are the causative agents of hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS) — serious emerging diseases, with case-fatality rates of up to 15% and about 35%, respectively
Over 21 human pathogenic serotypes have been described, which are classified into New World and Old World (Asia and Europe) hantaviruses

Summary

Introduction

Hantaviruses are enveloped and negative-sense single-stranded RNA viruses of the Bunyaviridae family. The hantavirus genome consists of three segments (L, M, and S), coding for the viral RNA polymerase (L protein), glycoproteins (Gn and Gc) and the nucleocapsid (N) protein, respectively [1,2,3,4,5]. The majority of hantaviruses are etiologic agents of either hemorrhagic fever with renal syndrome (HFRS) or hantavirus cardiopulmonary syndrome (HCPS). The number of hantavirus infections is increasing, as reflected by a very recent outbreak at Yosemite National Park (USA; June–August 2012), which put an estimated 10,000 persons at risk of infection and caused several fatal cases [6]. Transmission to humans occurs through the respiratory tract by inhalation of dust and aerosols containing virus-contaminated particles shed by persistently infected viral reservoir species (primarily mice, voles and rats)

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