[show abstract][hide abstract] ABSTRACT: Dengue is the one of the most prevalent arthropod-borne viral diseases. Dengue virus circulates between humans and mosquitoes, and causes a wide range of disease in humans. To elucidate the link between the cell tropism of dengue virus and its pathogenesis, peripheral blood cells of infected patients were analyzed by flow cytometry. The dengue virus antigen was detected in peripheral CD19+ cells (B cells) in one dengue hemorrhagic fever patient. Two dengue type-2 virus isolates were recovered from this patient using mosquito cell line C6/36 and human hematopoietic cell line K562, and designated VNHCM18-C/02 and VNHCM18-K/02, respectively. VNHCM18-K/02 exhibited strong binding ability and high infectivity to a B-lymphocyte cell line (RPMI8226) but showed poor growth in C6/36 cells, while VNHCM18-C/02 more efficiently and dominantly grew in C6/36 cells but did not efficiently bind to nor infect the B-cell line. Three amino acid differences were detected; one in an envelope protein (E-62) and two in nonstructural proteins. The distinct cell-binding to RPMI8226 was attributed to the difference between the two isolates in envelope protein E-62. Thus, we isolated two dengue type-2 virus variants with different cell-tropisms from the same patient, suggesting possible co-circulation in the patient.
Japanese journal of infectious diseases 09/2009; 62(5):343-50. · 1.51 Impact Factor
[show abstract][hide abstract] ABSTRACT: The complete nonstructural NS5 gene of Japanese encephalitis virus (JEV) was amplified and cloned into an expression vector. The NS5 protein was expressed in Escherichia coli and purified by His-tag based affinity chromatography. This recombinant NS5 protein exhibited RNA-dependent RNA polymerase (RdRp) activity in vitro in the absence of other viral or cellular factors. The RNA polymerase activity was dependent on divalent cations, and Mn(2+) was found to be 20 times more effective than Mg(2+) in coordinating the catalytic reaction of RdRp, while Ca(2+) inhibited enzyme activity. The optimal reaction conditions for the in vitro RdRp reaction were established. Characterization of the RdRp reaction products demonstrated that the JEV NS5 protein can initiate RNA synthesis through a de novo initiation mechanism in our in vitro reaction system. Comparing the efficiency of different RNA templates, we found that JEV NS5 protein was more efficient in using negative-strand RNA templates, indicating that the JEV NS5 protein is involved in regulating the ratio of positive- to negative-strand RNA. Four amino acid sequence motifs crucial for RdRp activity were also identified using site-directed mutagenesis analysis. All substitutions of the conserved residues within these motifs led to a complete inactivation or severe loss of enzyme activity.
Archives of Virology 02/2007; 152(10):1859-69. · 2.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: The Japanese encephalitis (JE) zoonotic vaccine strain ML-17 was sequenced and compared to related JE virus strains to identify genomic attenuation markers. Relative to its parental strain, JaOH0566, 25 nucleotide alterations and 10 amino acid changes to, prM/M(2), NS2A(1), NS4B(3) and NS5(4) proteins were recorded. Both structural-gene changes were in the prM/M region (127Met-->Ile and 274Asn-->Thr). To study the effects of these prM/M changes, mutants bearing the changes were prepared using an infectious clone of JaOArS982 previously established at this lab. Compared with JaOArS982, mutant 127(Met-->Ile) showed marked reduction in murine neuroinvasiveness. Mutant 274(Asn-->Thr), showed slight reduction. Neither mutant recorded ML-17-equivalent attenuation, implying that prM/M changes need to combine with other recorded genomic differences to cause attenuation. Importantly, ML-17 with its unchanged E region, presents a possible backbone candidate for preparation of "E-replacement" type live attenuated flavivirus chimeric vaccines.
[show abstract][hide abstract] ABSTRACT: The first flavivirus chimera encoding dengue 4 virus (D4) PrM and E structural proteins in a Japanese encephalitis virus (JEV) backbone was successfully generated using the long-PCR based cDNA-fragment stitching (LPCRcFS) technique, demonstrating the technique's applicability for rapid preparation of flavivirus chimeras. The JEV/D4 chimera multiplied at levels equal to JEV and D4 in the mosquito cell line C6/36, while in a mouse neuronal cell line (N2a) JEV replicated efficiently, but JEV/D4 and D4 did not. In mouse challenge experiments, JEV/D4 showed a lack of neuroinvasiveness similar to D4 when inoculated intraperitoneally, but demonstrated attenuated neurovirulence (LD50=3.17 x 10(4) f.f.u.) when inoculated intracranially. It was also noted that mice receiving intraperitoneal challenge with JEV/D4 possessed D4-specific neutralization antibody and in addition clearly showed resistance to JEV intraperitoneal challenge (at 100 x LD50). This suggests that immunity to anti-JEV non-structural protein(s) offers protection against JEV infection in vivo. Dengue secondary infection was also simulated by challenging mice pre-immunized with dengue 2 virus, with D4 or JEV/D4. Mice showed higher secondary antibody response to challenge with JEV/D4 than to D4, at 210,000 and 37,000 averaged ELISA units, respectively. Taken together, aside from demonstrating the LPCRcFS technique, it could be concluded that the PrM and E proteins are the major determinant of neuroinvasiveness for JEV. It is also expected that the JEV/D4 chimera with its pathogenicity in mice and atypical immune profile, could have applications in dengue prophylactic research, in vivo efficacy assessment of dengue vaccines and development of animal research on models of dengue secondary infection.
Journal of General Virology 10/2004; 85(Pt 9):2503-13. · 3.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: In this study, the complete genomic sequence of chikungunya virus (CHIK; S27 African prototype) was determined and the presence of an internal polyadenylation [I-poly(A)] site was confirmed within the 3' non-translated region (NTR) of this strain. The complete genome was 11805 nucleotides in length, excluding the 5' cap nucleotide, an I-poly(A) tract and the 3' poly(A) tail. It comprised two long open reading frames that encoded the non-structural (2474 amino acids) and structural polyproteins (1244 amino acids). The genetic location of the non-structural and structural proteins was predicted by comparing the deduced amino acid sequences with the known cleavage sites of other alphaviruses, located at the C-terminal region of their virus-encoded proteins. In addition, predicted secondary structures were identified within the 5' NTR and repeated sequence elements (RSEs) within the 3' NTR. Amino acid sequence homologies, phylogenetic analysis of non-structural and structural proteins and characteristic RSEs revealed that although CHIK is closely related to o'nyong-nyong virus, it is in fact a distinct virus. The existence of I-poly(A) fragments with different lengths (e.g. 19, 36, 43, 91, 94 and 106 adenine nucleotides) at identical initiation positions for each clone strongly suggests that the polymerase of the alphaviruses has a capacity to create poly(A) by a template-dependant mechanism such as 'polymerase slippage', as has been reported for vesicular stomatitis virus.
Journal of General Virology 01/2003; 83(Pt 12):3075-84. · 3.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: A reverse transcription-polymerase chain reaction (RT-PCR) was developed for the detection of Chikungunya virus infection. Based on the nonstructural protein 1 (nsP1) and glycoprotein E1 (E1) genes of Chikungunya, two primer sets were designed. Total RNA were extracted from the cell culture fluid of Aedes albopictus C6/36 cells inoculated with the S27 prototype virus, isolated in Tanzania in 1953, and the Malaysian strains (MALh0198, MALh0298, and MALh0398), isolated in Malaysia in 1998. For both sets of RNA samples, the expected 354- and 294-base pair (bp) cDNA fragments were amplified effectively from the nsP1 and E1 genes, respectively. Phylogenetic analysis was conducted for the Malaysian strain and other virus strains isolated from different regions in the world endemic for Chikungunya, using partial E1 gene sequence data. The Malaysian strains isolated during the epidemics of 1998 fell into a cluster with other members of the Asian genotype.
Journal of Medical Virology 08/2002; 67(3):370-4. · 2.37 Impact Factor
[show abstract][hide abstract] ABSTRACT: Apoptosis is a highly regulated process of cellular self-destruction with diverse functions in multicellular organisms. It is known to be one of the mechanisms of viral pathogenesis. St. Louis encephalitis virus (SLEV), an arthropod-borne flavivirus, causes encephalitis disease of varying severity mostly in North America and in some regions of South America. This virus induces cytopathic effects in vertebrate cell lines, however, the mechanism by which this occurs is yet to be elucidated. SLEV induced cytopathic effects in K562 cells, a human mononuclear cell line, and in Neuro 2a cells, a mouse neuroblastoma cell line. SLEV-infected K562 and Neuro 2a cells underwent apoptotic cell death, whereas neither the cells inoculated with UV-inactivated virus nor the mock-infected cells developed cytopathic effects. The gene expression of regulators of apoptosis was investigated in K562 cells. A rise in the expression of the pro-apoptotic bax gene was detected specifically in the SLEV-infected K562 cells. These findings suggest that up-regulation of bax mRNA is correlated with cytopathic effects in SLEV-infected K562 cells.
Archives of Virology 07/2002; 147(6):1105-19. · 2.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: We prepared recombinant Japanese encephalitis (JE) virus populations possessing random mutations at the envelope (E) protein region by a long PCR-based method. Neutralization-resistant mutants were selected from these populations by application of JE-specific virus neutralizing monoclonal antibody (mAb) 503, which possessed a 51,200-fold neutralization titer. We classified the mutants into three groups, each bearing two amino acid alterations at the E protein region: 52, Gln-Arg, and 136, Lys-Glu; 136, Lys-Glu, and 275, Ser-Pro; and 126, Ile-Thr, and 136, Lys-Glu, respectively. Three different genetically engineered variants, each bearing a single mutation, 126, Ile-Thr; 136, Lys-Glu; and 275, Ser-Pro, respectively, showed partial but not complete recovery of reactivity to mAb 503. Our results indicate that the amino acid substitutions at amino acid positions 52, 126, 136, and 275 altered the structure of the neutralization epitope for mAb 503 on the E protein. All these mutations were clustered at the junction of domains I and II of the E protein and it is likely that the epitope for mAb 503 is composed of at least E(0)-e, D(0)-a, and k strands of the E protein. We also demonstrated the efficacy of the long PCR-based recombinant virus technique as a useful tool for the creation of a variety of mutants bearing random mutations at targeted areas of the virus genome.