Byoung-Hee Lee

Use of Vesicular Stomatitis Virus Pseudotypes Bearing Hantaan or Seoul Virus Envelope Proteins in a Rapid and Safe Neutralization Test

ABSTRACT A vesicular stomatitis virus (VSV) pseudotype bearing hantavirus envelope glycoproteins was produced and used in a neutralization test as a substitute for native hantavirus. The recombinant VSV, in which the enveloped protein gene (G) was replaced by the green fluorescent protein gene and complemented with G protein expressed in trans (VSVΔG*G), was kindly provided by M. A. Whitt. 293T cells were transfected with plasmids for the expression of envelope glycoproteins (G1 and G2) of HTNV or SEOV and were then infected with VSVΔG*G. Pseudotype VSV with the Hantaan (VSVΔG*-HTN) or Seoul (VSVΔG*-SEO) envelope glycoproteins were harvested from the culture supernatant. The number of infectious units (IU) of the pseudotype VSVs ranged from 105 to 106/ml. The infectivity of VSVΔG*-HTN and VSVΔG*-SEO was neutralized with monoclonal antibodies, immune rabbit sera, and sera from patients with hemorrhagic fever with renal syndrome, and the neutralizing titers were similar to those obtained with native hantaviruses. These results show that VSVΔG*-HTN and -SEO can be used as a rapid, specific, and safe neutralization test for detecting hantavirus-neutralizing antibodies as an effective substitute for the use of native hantaviruses. Furthermore, the IU of VSVΔG*-HTN and -SEO did not decrease by more than 10-fold when stored at 4°C for up to 30 days. The stability of the pseudotype viruses allows distribution of the material to remote areas by using conventional cooling boxes for use as a diagnostic reagent.

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.

The Multimerization of Hantavirus Nucleocapsid Protein Depends on Type-Specific Epitopes

ABSTRACT Multimerization of the Hantaan virus nucleocapsid protein (NP) in Hantaan virus-infected Vero E6 cells was observed in a competitive enzyme-linked immunosorbent assay (ELISA). Recombinant and truncated NPs of Hantaan, Seoul, and Dobrava viruses lacking the N-terminal 49 amino acids were also detected as multimers. Although truncated NPs of Hantaan virus lacking the N-terminal 154 amino acids existed as a monomer, those of Seoul and Dobrava formed multimers. The multimerized truncated NP antigens of Seoul and Dobrava viruses could detect serotype-specific antibodies, whereas the monomeric truncated NP antigen of Hantaan virus lacking the N-terminal 154 amino acids could not, suggesting that a hantavirus serotype-specific epitope on the NP results in multimerization. The NP-NP interaction was also detected by using a yeast two-hybrid assay. Two regions, amino acids 100 to 125 (region 1) and amino acids 404 to 429 (region 2), were essential for the NP-NP interaction in yeast. The NP of Seoul virus in which the tryptophan at amino acid number 119 was replaced by alanine (W119A mutation) did not multimerize in the yeast two-hybrid assay, indicating that tryptophan 119 in region 1 is important for the NP-NP interaction in yeast. However, W119A mutants expressed in mammalian cells were detected as the multimer by using competitive ELISA. Similarly, the truncated NP of Seoul virus expressing amino acids 155 to 429 showed a homologous interaction in a competitive ELISA but not in the yeast two-hybrid assay, indicating that the C-terminal region is important for the multimerization detected by competitive ELISA. Combined, the results indicate that several steps and regions are involved in multimerization of hantavirus NP.

Hantavirus-specific CD8+-T-cell responses in newborn mice persistently infected with Hantaan virus

ABSTRACT The relationship between virus-specific CD8+-T-cell responses and viral persistence was studied in mice by using Hantaan virus (HTNV). We first established a simple method for measuring levels of virus-specific CD8+ T cells by flow cytometry. Next, to produce a mouse model of persistent HTNV infection, newborn mice were inoculated subcutaneously within 24 h of birth with 1 or 0.1 50% newborn mouse lethal dose of HTNV. All mice that escaped lethal infection were persistently infected with HTNV until at least 30 days after virus inoculation and had no virus-specific CD8+ T cells producing gamma interferon (IFN-γ). Subsequently, the virus was eliminated from some of the mice, depending on the appearance of functional virus-specific CD8+ T cells, which have the ability to produce IFN-γ and tumor necrosis factor alpha (TNF-α) and have cytotoxic activity. Neutralizing antibodies were detected in all mice, regardless of the presence or absence of virus. In the acute phase, which occurs within 30 days of infection, IFN-γ-producing HTNV-specific CD8+ T cells were detected on day 15 after virus inoculation. However, TNF-α production and the cytotoxic activity of these specific CD8+ T cells were impaired and HTNV was not removed. Almost all of these specific CD8+ T cells disappeared by day 18. These results suggest that functional HTNV-specific CD8+ T cells are important for clearance of HTNV.

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.

Detection of antibody for the serodiagnosis of hantavirus infection in different rodent species.

Summary Peroxidase-labeled staphylococcal protein A, streptococcal protein G, and antibodies directed against Mus musculus (mouse), Rattus norvegicus (rat), Mesocretus auratus (hamster), and Peromyscusleucopus were examined for their reactivity with immunoglobulin G (IgG) from various rodent species. The purpose of this study was to identify the optimal secondary antibodies or reagents for specific serodiagnosis of hantavirus infection in various rodent species. Using ELISA, a total of 65 sera from 29 rodent species of the family Muridae and one serum sample from family Octodontidae were compared for IgG reactivity with the six different reagents. The results demonstrate that the reactivities of the secondary antibodies and reagents to the sera varied, even among sera from rodents of the same genus. Hantavirus-specific antibody ELISA revealed that hantavirus-infected rodent sera obtained from M. musculus, R. norvegicus, Apodemus agrarius, A. peninsulae, and Bandicota indica bound to the six different conjugates in a similar pattern as that detected in IgG ELISA. These results indicate that the applicability of secondary antibodies and protein A and G should be carefully evaluated before use for serodiagnosis in different rodent species.

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.

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.