Study of Andes virus entry and neutralization using a pseudovirion system

Abstract

Andes virus (ANDV), a member of the Hantavirus genus in the family Bunyaviridae, causes an acute disease characteristic of New-World hantaviruses called hantavirus pulmonary syndrome (HPS). HPS is a highly pathogenic disease with a case-fatality rate of 40%. ANDV is the only hantavirus reported to spread directly from human-to-human. The aim of the present study was to develop a quantitative and high-throughput pseudovirion assay to study ANDV infection and neutralization in biosafety level 2 facilities (BSL-2).

This pseudovirion assay is based on incorporation of ANDV glycoproteins onto replication-defective vesicular stomatitis virus (VSV) cores in which the gene for the surface G protein has been replaced by that encoding Renilla luciferase. Infection by the pseudovirions can be quantified by luciferase activity of infected cell lysates. ANDV pseudovirions were neutralized by ANDV-specific antisera, and there was good concordance between specificity and neutralization titers of ANDV hamster sera as determined by our pseudovirion assay and a commonly used plaque reduction neutralization titer (PRNT) assay. In addition, the pseudovirions were used to evaluate the requirements for ANDV entry, like pH dependency and the role of β3 integrin, the reported receptor for other pathogenic hantaviruses, on entry.

Introduction

Andes virus (ANDV) is a member of the Hantavirus genus of the Bunyaviridae family of negative-sense RNA viruses (Schmaljohn et al., 1983), and a causative agent of hantavirus pulmonary syndrome (HPS) in South America (Galeno et al., 2002). The hantavirus genome is comprised of three segments L, M and S which encode the viral polymerase, two integral membrane surface proteins (G_C and G_N) and the nucleocapsid protein, respectively. The hantavirus glycoproteins (G_C and G_N) are co-translationally cleaved from a polyprotein precursor to define the highly ordered surface structure of the virion envelope (Schmaljohn et al., 1987).

Unlike the majority of other bunyaviruses, which are arthropod-borne, hantaviruses are rodent-borne viruses. Each hantavirus appears to have co-evolved with a specific primary rodent host species that it infects persistently and asymptomatically (Plyusnin and Morzunov, 2001). Transmission to non-human mammals from these natural hosts results in a non-productive infection, but transmission to humans leads to hantavirus-associated diseases. Typically the virus is spread via contact with infected rodent feces and bodily fluids as well as bites in more aggressive encounters (Glass et al., 2000, Klein et al., 2001). However, ANDV is an exception in that there is evidence it can also be directly transmitted from one human to another contact (Padula et al., 1998).

Hantaviruses are associated with two severe and sometimes fatal diseases in humans, namely hemorrhagic fever with renal syndrome (HFRS) and HPS (Hjelle et al., 1996, Khaiboullina et al., 2005, Levis et al., 1997, Lopez et al., 1996). HFRS is characterized by an incubation period of 2–3 weeks and symptoms include a combination of fever, hemorrhagic manifestations and renal impairment (Lee and van der Groen, 1989). HPS begins with a prodrome consisting of fever, myalgia and dyspnea and progresses rapidly to pulmonary microvascular leakage, respiratory distress and shock, resulting in a 40% mortality rate (Hjelle et al., 1996, Khaiboullina et al., 2005, Levis et al., 1997, Lopez et al., 1996). Old-World hantaviruses such as Hantaan virus (HTNV) and Puumala virus (PUUV) typically cause HFRS. On the other hand, New-World hantaviruses such as ANDV and Sin Nombre virus (SNV) cause HPS, and have triggered more recent interest in these viruses due to the 1993 outbreak of human HPS cases caused by SNV in the southwestern United States (Khaiboullina et al., 2005). In contrast to hantavirus strains which can cause HPS, Prospect Hill virus (PHV) is a North American strain that is non-pathogenic and does not cause any disease in humans (Yanagihara et al., 1987).

Hantaviruses have been shown to utilize integrins in order to gain entry into cells. Pathogenic hantaviruses like Sin-nombre (SNV), New York (NY-1), Puumala (PUUV), Hantaan (HTNV) and Seoul (SEOV) viruses appear to use β3 integrin whereas non-pathogenic hantaviruses (PHV) use β1 integrin (Gavrilovskaya et al., 2002, Gavrilovskaya et al., 1998). The receptor ANDV utilizes for entry has not been identified. However, entry of ANDV occurs via both apical and basolateral membranes in a polarized primary airway epithelial cell system (Rowe and Pekosz, 2006).

Since ANDV is the only hantavirus reported to spread directly from human-to-human, studying the mechanisms of entry of this virus has significant biological relevance in understanding its novel transmissibility. In addition, a deeper knowledge of ANDV entry may facilitate the development of antiviral drugs. However, studies of ANDV entry have been hampered by the need to work in biosafety level (BSL) 3 or 4 facilities (ANDV is a BSL-4 pathogen if animals are involved). To address this problem, we have developed a quantitative and high-throughput BSL-2 pseudovirion assay, which will facilitate the study of ANDV entry mechanisms and determination of neutralization titers of sera raised to this virus. Pseudovirions are replication-deficient viral particles consisting of envelope glycoprotein(s) of one virus, in this case ANDV G_N and G_C, incorporated onto the viral core of another virus, in this case VSV, in which the native envelope glycoprotein gene is either deleted or rendered inactive by stop codons.

In the present study, the development is described of a pseudovirion assay based on the incorporation of the glycoproteins (G_N and G_C) encoded by the M segment of ANDV onto replication-defective vesicular stomatitis virus (VSV) cores. In these VSV cores, the gene for the surface G protein has been replaced by that encoding Renilla luciferase, a highly sensitive and quantitative reporter that allows measurement of pseudovirus infection by luciferase activity of infected cell lysates. The ANDV pseudovirions generated were specifically neutralized by ANDV antisera but not pre-bleed antisera control and there was good concordance between neutralization titers of ANDV-specific hamster sera, as determined by plaque reduction neutralization titer (PRNT) assay. The ANDV pseudovirions demonstrated pH-dependent entry, as shown for other bunyaviruses. Pseudovirions were also used to evaluate the requirements for ANDV entry, specifically the role of the β3 integrin.