Effects of the number of amino acid residues in the signal segment upstream or downstream of the NS2B-3 cleavage site on production and secretion of prM/M-E virus-like particles of West Nile virus.

Expression of genes for precursor M (prM) and envelope (E) proteins of West Nile virus (WNV) leads to the production of small, capsidless, and non-infectious virus-like particles (VLPs) possessing the E antigen which is responsible for viral entry and immune protection. It has been reported that processing of the secretion signal affects viral release. We examined the secretion efficiency of VLPs into the culture medium from RK13 or 293T cells transfected with expression vectors for prM and E proteins of WNV which were constructed to comprise different lengths of signal peptides upstream of the prM-E domain.

DNA sequence analysis of varicella-zoster virus gene 62 from subclinical infections in healthy children immunized with the Oka varicella vaccine.

A live attenuated varicella vaccine, the Oka vaccine strain (vOka), is routinely administered to children in Japan and other countries, including the United States. vOka consists of a mixture of genotypically distinct variants, but little is known about the growth potential of each variants in vivo. We isolated varicella-zoster virus (VZV) DNA sequences from the peripheral blood mononuclear cells (PBMCs) of asymptomatic healthy children immunized with the Oka varicella vaccine.

Crystal structure of the catalytic domain of Japanese encephalitis virus NS3 helicase/nucleoside triphosphatase at a resolution of 1.8 A.

The NS3 protein of Japanese encephalitis virus (JEV) is a large multifunctional protein possessing protease, helicase, and nucleoside 5'-triphosphatase (NTPase) activities, and plays important roles in the processing of a viral polyprotein and replication. To clarify the enzymatic properties of NS3 protein from a structural point of view, an enzymatically active fragment of the JEV NTPase/helicase catalytic domain was expressed in bacteria and the crystal structure was determined at 1.8 A resolution.

Japanese encephalitis subunit vaccine composed of virus-like envelope antigen particles purified from serum-free medium of a high-producer J12#26 cell clone.

A stable cell clone, J12#26, which continuously secretes large amounts of the envelope (E) antigen of Japanese encephalitis (JE) virus (J. Virol. 77 (2003) 8745) was adapted to serum-free medium.

High-level expression of clostridial sialidase using a ferredoxin gene promoter-based plasmid.

A "large" sialidase isozyme (NanI) from Clostridium perfringens is a representative microbial sialidase with broad substrate specificity, being used for the analysis of sialoglycoconjugates. It is also a possible virulence factor. However, purification of the native enzyme in a large quantity is not practical due to its low productivity.

Enhancement of immunity against VZV by giving live varicella vaccine to the elderly assessed by VZV skin test and IAHA, gpELISA antibody assay.

The enhancement of immunity against varicella-zoster vaccine (VZV) by subcutaneous injection of a live varicella vaccine was assessed by the VZV skin test for cell-mediated immunity (CMI), and immunoadherence hemagglutination assay (IAHA) and gpELISA antibody assays in the elderly people of 50-79 years of age. A total of 127 subjects were examined: 79 aged 50-59, 25 aged 60-69, and 25 aged 70-79. All were seropositive by the gpELISA assay (one was seronegative in the IAHA antibody assay).

Stable high-producer cell clone expressing virus-like particles of the Japanese encephalitis virus e protein for a second-generation subunit vaccine.

We produced and characterized a cell clone (J12#26 cells) that stably expresses Japanese encephalitis virus (JEV) cDNA, J12, which encodes the viral signal peptide, premembrane (prM), and envelope (E) proteins (amino acid positions 105 to 794). Rabbit kidney-derived RK13 cells were transfected with a J12 expression plasmid, selected by resistance to marker antibiotics, and cloned by two cycles of a limiting-dilution method in the presence of antibiotics, a procedure that prevents the successful generation of E-producing cell clones. J12#26 cells secreted virus-like particles containing the authentic E antigen (E-VLP) into the culture medium in a huge enzyme-linked immunosorbent assay-equivalent amount (2.

Measurement of HCV RdRp activity with C-terminal 21 aa truncated NS5b protein: optimization of assay conditions.

The non-structural protein 5b (NS5b) of hepatitis C virus (HCV), bearing an RNA-dependent RNA polymerase (RdRp) activity, is considered as a new target of antiviral therapy. We expressed and purified the C-terminal 21 amino acid truncated NS5b protein fused with glutathione S-transferase (GST-5bC21) using Escherichia coli. With the highly purified GST-5bC21 protein, we established an in vitro assay system for RdRp activity by using poly(C) as the template and a 12 mer oligo(rG) as the primer.