Identification and characterization of RNA-dependent RNA polymerase activity in recombinant Japanese encephalitis virus NS5 protein.

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.

Combined detection and genotyping of Chikungunya virus by a specific reverse transcription-polymerase chain reaction.

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.

St. Louis encephalitis virus induced pathology in cultured cells.

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.

Locus of a virus neutralization epitope on the Japanese encephalitis virus envelope protein determined by use of long PCR-based region-specific random mutagenesis.

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.

Detection of HBV-DNA and HCV-RNA viral sequences by polymerase chain reaction in selected Kenyan samples and the relationship to HBV seromarkers.

We undertook a study on selected samples from patients who had presented with viral hepatitis and conditions of the liver (liver cirrhosis, chronic hepatitis and hepatocellular carcinoma). Diagnosis, screening and confirmation for viral hepatitis was done using a battery of techniques: ultrasound, conventional serological methods (Hepatitis B surface Antigen [HBsAg] - Reverse Passive Haemagglutination [RPHA], Hepatitis B core Antibody [HBcAb] - Passive Haemagglutination [PHA], Alpha-feto Protein - RPHA), Hepatitis B e Antigen/Antibody [HBeAg/Ab] - Radioimmunoassay [RIA], Hepatitis C antibody [HCV-Ab] - Enzyme Immunosorbent Assay [EIA]. Due to the high specificity and sensitivity of the Polymerase Chain Reaction technique [PCR] in detecting the viral genomes, it was used to establish the presence of the HBV-DNA and HCV-RNA to correlate the serological diagnosis of their respective seromarkers. A total of 39 serum samples were tested comprising 11 blood donors, 8 chronic liver disease patients and 20 hepatocellular carcinoma cases. 4/19 (21%) HCV-antibody (C-l) reactive samples were found to be positive for HCV-RNA by PCR. 14 of the 19 (73.7%) including the 4 HCV-RNA positive cases tested positive for HBcAb. 6 of 11 (55%) HBsAg positive cases also tested positive for HBV-DNA by PCR, In 8 of 20 (40%) hepatocellular carcinoma cases, no aetiological role could be assigned to hepatitis B or C as only HBcAb was demonstrated in those cases.