Utility of Japanese encephalitis virus subgenomic replicon-based single-round infectious particles as antigens in neutralization tests for Zika virus and three other flaviviruses

Highlights

• Single-round infectious particles (SRIPs) of four flaviviruses were generated.

• These flaviviruses were Zika, Sepik, Wesselsbron and Usutu viruses.

• Flavivirus-crossreactive antibodies dose-dependently neutralized these four SRIPs.

• Neutralization curves for authentic Zika virus and its SRIPs were equivalent.

• SRIPs will be useful for neutralization tests for foreign flaviviral infections.

Abstract

The introduction of a foreign virus into an area may cause an outbreak, as with the Zika virus (ZIKV) outbreak in the Americas. Preparedness for handling a viral outbreak involves the development of tests for the serodiagnosis of foreign virus infections. We previously established a gene-based technology to generate some flaviviral antigens useful for functional antibody assays. The technology utilizes a Japanese encephalitis virus subgenomic replicon to generate single-round infectious particles (SRIPs) that possess designed surface antigens. In the present study, we successfully expanded the capacity of SRIPs to four human-pathogenic mosquito-borne flaviviruses that could potentially be introduced from endemic to non-endemic countries: ZIKV, Sepik virus, Wesselsbron virus, and Usutu virus. Flavivirus-crossreactive monoclonal antibodies dose-dependently neutralized these SRIPs. ZIKV-SRIPs also produced antibody-dose-dependent neutralization curves equivalent to those shown by authentic ZIKV particles using sera from a Zika fever patient. The faithful expression of designed surface antigens on SRIPs will allow their use in neutralization tests to diagnose foreign flaviviral infections.

Introduction

The genus Flavivirus contains more than 70 members (classified as 53 separate species), approximately 40 of which are pathogenic to humans (Pierson and Diamond, 2013, King et al., 2012). Dengue virus types 1–4 (DENV-1 to DENV-4), Japanese encephalitis virus (JEV), West Nile virus (WNV), Yellow fever virus (YFV) and members of Tick-born encephalitis virus (TBEV) are globally important because of their wide distribution and/or the severity of diseases they cause. However, other human pathogenic flaviviruses, such as Zika virus (ZIKV), should not be ignored. In addition to the outbreaks widely and continuously occurring in endemic areas (Ribeiro et al., 2016; Wikan and Smith, 2016), recently imported cases of ZIKV have been increasingly reported in non-endemic countries (Tappe et al., 2014, Kutsuna et al., 2014, Kwong et al., 2013, Foy et al., 2011, Zammarchi et al., 2015). The accurate identification of patients in the early phase of an outbreak can minimize disease transmission and incidence. Therefore, the diagnosis of a disease and specification of the pathogen are important for controlling potential viral outbreaks.

Because several flaviviral diseases share similar clinical manifestations, their definitive diagnoses rely on laboratory testing (Fernanda Estofolete et al., 2016). Virus isolation and/or viral RNA detection provide firm diagnoses. However, the virus is often undetectable because the period of viremia/RNAemia in the patient is limited. Therefore, serological tests are also important to accommodate this limitation. Among the several types of serological tests for flavivirus infections available, the neutralization test provides the highest specificity, and a ≥ 4-fold difference in the antibody titer between the acute and convalescent phases can be used as an alternative firm diagnosis (Calisher et al., 1989, World Health Organization, 2009). The neutralization test is a functional assay using live virus as the assay antigen. Therefore, when used to confirm imported cases of infection, it requires a viral antigen that is circulating in a foreign country, which given the rarity of the virus in non-endemic countries, is often unavailable.

Currently, the transport of live viral materials across national borders is strictly regulated under governmental security export control policies (Ministry of Economy, Trade and Industry of Japan, 2009) and the access and benefit-sharing restrictions of the Convention on Biological Diversity (United Nations, 2011). However, viral or subviral assay antigens equivalent to the authentic virus can be generated with a gene engineering technology if the corresponding nucleotide sequence information is available. We have established a system to generate flaviviral single-round infectious particles (SRIPs) (Suzuki et al., 2014). Because the pre-membrane (prM) and envelope (E) proteins constitute the virion surface proteins of flaviviruses, SRIPs can be produced by the cotransfection of two different plasmids into 293T cells: (1) a JEV subgenomic replicon plasmid lacking coding regions for the prM and E proteins; and (2) a plasmid expressing the flaviviral prM and E proteins. The most striking advantage of this system is its capacity to generate chimeric SRIPs in which the genomic RNA is derived from JEV but the surface protein is derived from other flaviviruses. Because the surface proteins of flaviviruses are the targets of neutralizing antibodies, the preparation of a small plasmid containing the prM and E genes is expected to induce the generation of SRIPs that can be used in a neutralization test. To date, we have generated chimeric SRIPs that have the surface proteins of DENV-1–4, WNV, YFV, or TBEV, SRIPs that have surface proteins of the original JEV (JEV-SRIPs) (Suzuki et al., 2014). Furthermore, JEV-SRIPs and chimeric SRIPs derived from DENV-1–3 have been shown to be useful as antigens in neutralization tests (Yamanaka et al., 2014).

The aim of the present study was to generate SRIPs of four flaviviruses and evaluate their use in neutralization tests. The four flaviviruses used in this study were ZIKV, Sepik virus (SEPV) (Kuno and Chang, 2006), Wesselsbron virus (WSLV) (Jupp and Kemp, 1998), and Usutu virus (USUV) (Nikolay et al., 2011, Vazquez et al., 2011). All these viruses cause febrile illnesses in humans (Weissenböck et al., 2010, Centers for Disease Control and Prevention, 2016). USUV has been isolated in Africa and Europe and WSLV in Africa and Asia, whereas SEPV has only been isolated in New Guinea (Weissenböck et al., 2010, Centers for Disease Control and Prevention, 2016). ZIKV was originally distributed only in Africa and Asia until 2005, but recently its distribution has been expanded to Oceania and Central and South America (Musso et al., 2015). These viruses may be imported from endemic to non-endemic countries. Therefore, the viral antigens of these viruses must be generated for laboratory diagnoses in non-endemic countries to prepare for the potential introduction of these foreign flaviviruses.