Megumi Okumura

Flavivirus Encephalitis Pathological Aspects of Mouse and Other Animal Models

Encephalitic flaviviruses are important arthropod-borne pathogens of humans and other animals. In particular, the recent emergence of the West Nile virus (WNV) and Japanese encephalitis virus (JEV) in new geographic areas has caused a considerable public health alert and international concern. Among the experimental in vivo models of WNV and JEV infection, mice and other laboratory rodents are the most thoroughly studied and well-characterized systems, having provided data that are important for understanding the infectious process in humans. Macaca monkeys have also been used as a model for WNV and JEV infection, mainly for the evaluation of vaccine efficacy, although a limited number of published studies have addressed pathomorphology. These animal models demonstrate the development of encephalitis with many similarities to the human disease; however, the histological events that occur during infection, especially in peripheral tissues, have not been fully characterized.

Development of Serological Assays for Thottapalayam Virus, an Insectivore-Borne Hantavirus

ABSTRACT Thottapalayam virus (TPMV), a member of the genus Hantavirus in the family Bunyaviridae, was isolated from an insectivore, Suncus murinus (musk shrew), captured in southern India in 1964. While the isolation of TPMV predates the discovery of the prototype Hantaan virus, little is known about its genetics and biology. To date, preliminary evidence suggests that TPMV differs significantly, both antigenically and genetically, from all known rodent-borne hantaviruses. However, since detailed epizootiological studies have not been conducted, it is unclear if TPMV is naturally harbored by an insectivore host or if TPMV represents a “spillover” from its natural rodent reservoir host. Moreover, to what extent TPMV causes infection and/or disease in humans is not known. To address these issues, we first studied the antigenic profile of TPMV using monoclonal antibodies against Hantaan and Seoul viruses and polyclonal immune sera against Puumala virus and TPMV. Armed with this newfound information, we developed an enzyme-linked immunosorbent assay system for the diagnosis of TPMV infections in shrews and humans, using a recombinant TPMV N antigen manipulated to have an E5/G6 epitope to be captured by monoclonal antibody clone E5/G6. Using this assay, we found anti-TPMV antibodies in sera from a patient with high fever of unknown etiology in Thailand and from two shrews captured in Indonesia. Seropositivity was verified by the indirect immunofluorescence antibody test, Western blotting analysis, and focus reduction neutralization test. Collectively, our data indicate that TPMV is harbored by Suncus murinus as its host in nature and is capable of infecting humans.

Cell fusion activities of Hantaan virus envelope glycoproteins.

ABSTRACT Hantaan virus (HTNV)-infected Vero E6 cells undergo cell fusion with both infected and uninfected cells under low-pH conditions. Flow cytometry and fluorescence microscopy of HTNV-infected Vero E6 cells showed that envelope glycoproteins (GPs) were located both on the cell surface and in the cytoplasm. Neutralizing monoclonal antibodies (MAbs) against the G1 and G2 envelope GPs inhibited cell fusion, whereas nonneutralizing MAbs against G1 or G2 and MAbs against the nucleocapsid protein (NP) did not. Transfected Vero E6 cells that expressed GPs but not those that expressed NP fused and formed syncytia. These results indicate that HTNV GPs act as fusogens at the cell surface. No fusion activity was observed either in infected Vero cells that were passaged more than 150 times or in BHK-21 cells, although GPs appeared to localize to the cell surface. This variability in fusion induction suggests the involvement of host cell factors in the process of cell membrane fusion.

Development of a serotyping ELISA system for Thailand virus infection.

To distinguish Thailand virus infection from infections with other hantaviruses, we established an ELISA serotyping system using a truncated nucleocapsid protein of Thailand virus lacking 49 amino acids at the N-terminus. In evaluations using patient and rodent sera, Thailand virus infection was readily distinguished from Hantaan and Seoul virus infections. Therefore, this ELISA system is an effective alternative to neutralization tests.

Hantavirus species in India: a retrospective study.

Hantaviruses cause hemorrhagic fever with renal syndrome in Europe and Asia. There are about 20 documented hantavirus species and newer species are being described worldwide, especially in non-rodent reservoirs, i.e shrews. Focus reduction neutralization test is the classical serotyping technique for hantavirus. However, this study employs a previously established serotyping ELISA, to retrospectively analyze known hantavirus IgG reactive samples for infecting serotypes. The result suggests presence of Thailand virus- like and Hantaan virus -like strains in India.

Epitope analysis of monoclonal antibody E5/G6, which binds to a linear epitope in the nucleocapsid protein of hantaviruses

Summary.Monoclonal antibody E5/G6 recognized a linear epitope common to hantavirus nucleocapsid proteins. Using synthetic peptides, we identified epitope E5/G6 as the 9 mer YEDVNGIRK (NP 165–173), in which D167, G170, I171, and R172 are indispensable. Furthermore, all the peptides synthesized using various hantavirus sequences bound MAb E5/G6 consistently, despite the existence of several amino acid variations in this region. These results indicate that MAb E5/G6 is a useful tool for detecting hantavirus antigen in rodent or patient tissues using Western blotting or other immunohistochemical assays.

Paradoxical effects of chondroitin sulfate-E on Japanese encephalitis viral infection.

Glycosaminoglycans (GAGs) have diverse functions in the body and are involved in viral infection. The purpose of this study was to evaluate the possible roles of the E-disaccharide units GlcAβ1-3GalNAc(4,6-O-disulfate) of chondroitin sulfate (CS), a GAG involved in neuritogenesis and neuronal migration, in Japanese encephalitis virus (JEV) infection. Soluble CS-E (sCS-E) derived from squid cartilage inhibited JEV infection in African green monkey kidney-derived Vero cells and baby hamster kidney-derived BHK cells by interfering with viral attachment. In contrast, sCS-E enhanced viral infection in the mouse neuroblastoma cell line Neuro-2a, despite the fact that viral attachment to Neuro-2a cells was inhibited by sCS-E. This enhancement effect in Neuro-2a cells seemed to be related to increased viral RNA replication and was also observed in a rat infection model in which intracerebral coadministration of sCS-E with JEV in 17-day-old rats resulted in higher brain viral loads than in rats infected without sCS-E administration. These results show the paradoxical effects of sCS-E on JEV infection in different cell types and indicate that potential use of sCS-E as an antiviral agent against JEV infection should be approached with caution considering its effects in the neuron, the major target of JEV.

Age-dependent hantavirus-specific CD8+ T-cell responses in mice infected with Hantaan virus

Summary.To investigate age-dependent differences in hantavirus-specific CD8+ T-cell responses, mice were inoculated with 0.1 50% newborn mouse lethal dose of Hantaan virus (HTNV) at 0, 3, 7, 14, or 35 days after birth. HTNV-specific CD8+ T cells producing gamma interferon (IFN-γ) were measured on day 30 after HTNV inoculation. Although no IFN-γ-producing HTNV-specific CD8+ T cells were detected in most of the mice inoculated with HTNV on day 0 after birth, most mice inoculated at 3, 7, 14, or 35 days had HTNV-specific CD8+ T cells. The production of tumor necrosis factor alpha (TNF-α) by IFN-γ-producing CD8+ T cells and the cytotoxic activity against HTNV-infected target cells were similar in immature and adult mice. However, the number of IFN-γ-producing HTNV-specific CD8+ T cells was significantly less in mice inoculated with HTNV at 3 days than in older mice. In addition, a strong correlation between HTNV persistence and a lack of HTNV-specific CD8+ T cells was observed. These results suggest that mice over 7 days old have the ability to induce functional HTNV-specific CD8+ T-cell responses that are indistinguishable from the responses of adult mice, and that HTNV-specific CD8+ T cells are important for clearance of HTNV.

Lack of vertical transmission of Hantaan virus from persistently infected dam to progeny in laboratory mice

It is unclear how the hantaviruses are transferred from infected to uninfected rodents. We studied the status of persistently infected laboratory mice and examined the frequency of viral transmission to their offspring. Expression of Hantaan virus nucleocapsid protein was detected in the lungs of persistently infected dams. None of the progeny displayed viral antigen, although they were strongly positive for IgG antibodies against hantavirus. There was neither hantavirus RNA nor virus-specific IgM antibodies or virus-specific CD8+ T cells in the progeny. These results did not show any indication for a vertical transmission of hantaviruses, at least in the laboratory mouse model studied.

Characterization of Japanese encephalitis virus infection in an immortalized mesencephalic cell line, CSM14.1.

Neurons are the major target cell of Japanese encephalitis virus (JEV). Rats intracerebrally inoculated with JEV show an age‐dependent pattern of resistance to infection in which resistance is closely associated with neuronal maturation. However, because there is no reliable and convenient cell culture system that mimics the in vivo properties of JEV infection of immature and mature neurons, the mechanisms underlying this association remain poorly understood. The aim of the present study was to examine JEV infection in immortalized CSM14.1 rat neuronal cells, which can be induced to differentiate into neurons by culture under non‐permissive conditions. JEV infected undifferentiated CSM14.1 cells more efficiently than differentiated cells, resulting in production of more progeny virus in the former setting than in the latter. An infectious virus recovery assay detected more internalized virions in undifferentiated cells. On the other hand, JEV infection of differentiated cells induced more rapid and stronger expression of interferon‐β gene, along with smaller amounts of JEV RNA. Taken together, these results show that the initial phase of viral infection and the later IFN response play roles in the viral susceptibility of undifferentiated and differentiated CSM14.1 cells. Because CSM14.1 cells became more resistant to JEV infection as they mature, this culture system can be used as an in vitro model for studying age‐dependent resistance of neurons to JEV infection.