Adaptation of BHK-21 cells to growth in shaker culture and subsequent challenge by Japanese encephalitis virus.
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Baby hamster kidney (BHK-21) cells were adapted to grow in shaker culture using Waymouth medium 752/1 containing 20 mM N-2-hydroxyethyl-piperazine-N'-2'-ethanesulfonic acid buffer and supplemented with 2.5% (vol/vol) calf serum, 0.002% (wt/vol) sodium oleate, and 0.2% fatty acid-free bovine serum albumin (WO2.5). Infectivity of Japanese encephalitis virus grown in the cells adapted to WO2.5 approached 2 x 10(8) plaque-forming units per ml. The culture volume of infected cells was reduced fivefold 12 h after infection. This step resulted in a 10-fold increase in infectivity over that obtained from infected cultures not subjected to volume reduction. (+info)
Characterization and E protein expression of mutant strains during persistent infection of KN73 cells with Japanese encephalitis virus.
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OBJECTIVE: To study the character of mutants originating from Japanese encephalitis viruses and the relationship between the characterization of mutant strains and E protein expression. METHODS: Persistent infection was established with standard strains of Japanese encephalitis viruse, known as parental viruse, in a human hepatoma cell line, KN73. Cells were subcultured weekly using trypsinization techniques. Cell-associated viruses of persistently infected cells were collected by a freeze and thaw method. Virus titers were examined by plaque method using baby hamster kidney (BHK) cells. Indirect immunofluorescence assays were used to examine E and NS3 protein antigens. Western blot analysis was used to test expression of E and NS3 proteins. RESULTS: In the early phase (24 - 36 h) post-infection, virus titer in culture fluid from KN73 cells infected with parental viruses were 10(6) PFU/ml. They were 10(3 - 4) PFU/ml in the late phase (3 years) post-infection. The titer of cell-associated viruse was 10(2 - 3) PFU/ml. A virus super-infection assay found that virus titers in culture fluid from persistently infected KN73 cells acutely super- infected with parental viruses were much lower than that of culture fluids in acutely infected normal KN73 at the same phase. Indirect immunoflurescence assay revealed that the quantity of viral antigens in persistently infected KN73 cells was lower than that in acutely infected KN73 cells with parental viruses. Western blot analyses indicated that the molecular weights of E and NS3 proteins were 53 kD and 73 kD, respectively. Expression of NS3 protein in persistently infected KN73 cells was stable but expression of E protein was markedly suppressed. CONCLUSIONS: The virulence and reproduction of viruses obtained from persistently infected KN73 cells, which have some features of DI viruses and were involved in persistent infection, was lower than that of parental viruses. These mutants may have be related to the decrease in E protein expression. (+info)
Detection and stability of Japanese encephalitis virus RNA and virus viability in dead infected mosquitoes under different storage conditions.
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A semi-nested polymerase chain reaction (PCR) was evaluated for detection of Japanese encephalitis (JE) virus in infected mosquitoes stored under simulated northern Australian summer conditions. The effect of silica gel, thymol, and a combination of the two on RNA stability and virus viability in dead mosquitoes were also examined. While JE virus RNA was relatively stable in mosquitoes held for up to 14 days after death, viable virus was not detected after day 1. Thymol vapor inhibited fungal contamination. Detection of single mosquitoes infected with JE virus in large pools of mosquitoes was also investigated. Single laboratory-infected mosquitoes were detected in pools of < or = 200 mosquitoes and in pools diluted to 0.2/100 and 0.1/100 mosquitoes, using the semi-nested PCR. However, the ability to detect live virus decreased as pool size increased. The semi-nested PCR proved more expensive than virus isolation for pools of 100 mosquitoes. However, the semi-nested PCR was faster and more economical using larger pools. Results indicate that surveillance of JE virus in mosquitoes using the semi-nested PCR is an alternative to monitoring seroconversions in sentinel pigs. (+info)
A functional epitope determinant on domain III of the Japanese encephalitis virus envelope protein interacted with neutralizing-antibody combining sites.
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The envelope (E) protein of Japanese encephalitis virus (JEV) is associated with viral binding to cellular receptors, membrane fusion, and the induction of protective neutralizing-antibody responses in hosts. Most previous studies have not provided detailed molecular information about the spatial configuration of the functional epitopes on domain III of the E protein. Here site-directed mutagenesis was performed to demonstrate that the functional epitope determinants at Ser331 and Asp332 on domain III of the JEV E protein interacted with neutralizing monoclonal antibody (MAb) E3.3. Bacterial expression of the recombinant Fab E3.3 confirmed the molecular interactions of Arg94 in complementary determining region H3 with Ser331 and Asp332 on domain III. This study elucidates the detailed molecular structures of the neutralizing epitope determinants on JEV domain III, which can provide useful information for designing new vaccines. (+info)
The extent of homologous recombination in members of the genus Flavivirus.
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The family Flaviviridae includes important human pathogens, such as dengue (DEN) virus, yellow fever (YF) virus and hepatitis C virus, many of which have emerged or re-emerged in recent years. Until recently, flavivirus evolution was thought to proceed in a clonal manner, with diversity generated mainly through the accumulation of mutational changes. However, this assumption has now been shown to be invalid, with homologous recombination demonstrated in all three genera of the FLAVIVIRIDAE: Since recombination has important implications for the study of virus evolution, a survey of recombination in the viruses of the genus Flavivirus was carried out. Using envelope gene sequence data and a combination of graphical and phylogenetic analyses, hitherto unreported recombination in Japanese encephalitis virus and St Louis encephalitis virus was detected, as well as further recombinants in DEN virus. However, no evidence for recombination was found in West Nile or YF viruses, or in the tick-borne flavivirus group. It is proposed that the difference between the mosquito- and tick-borne viruses can be accounted for by their differing modes of transmission, whilst the variation among the mosquito-borne flaviviruses reflects both the ecology of the particular host and vector species and also bias in the sampling process. (+info)
Longitudinal studies in South Indian villages on Japanese encephalitis virus infection in mosquitoes and seroconversion in goats.
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Japanese encephalitis (JE) is endemic in Cuddalore district, Tamil Nadu, where Culex tritaeniorhynchus Giles was the major vector. We screened 45 100 adult female Cx. tritaeniorhynchus (902 pools) by enzyme-linked immunosorbent assay and isolated and confirmed JE virus (JEV) by using an insect bioassay system. We had 69 isolates of which 62 (90%) were identified as JEV. The average vector abundance per man hour for Cx. tritaeniorhynchus was 324.5 per month for the period June 1998-May 2000. The average minimum infection rate (MIR) per month in Cx. tritaeniorhynchus was 1.4 (range 0.0-5.6). Every year, a new batch of goats, 20 in the first year and 31 in the second year, born during the non-JE transmission period (January-June), aged <6 months and negative for haemagglutination inhibition (HI) antibodies were procured and placed in the villages as sentinels. Fortnightly, blood specimens were collected from these goats and tested for JE antibodies by HI test. Seroconversions (SCs) were recorded in 14 goats (70%) in the first year and 23 goats (74%) in the second year. JE HI antibody titres in goats were low (1:10-1:80) and these levels declined to undetectable levels in about 4 weeks following SCs. The time sequence of events indicated that four of five peaks of MIR in mosquitoes were followed 1-3 months later by peaks in the proportion of seroconverted goats. We suggest the screening of goats and cattle as a more feasible tool to stratify areas according to JE infection risk to the human population through the regular health system rather than screening mosquitoes using monoclonal antibodies, which is possible only in specialized laboratories. (+info)
Origin and evolution of Japanese encephalitis virus in southeast Asia.
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Since it emerged in Japan in the 1870s, Japanese encephalitis has spread across Asia and has become the most important cause of epidemic encephalitis worldwide. Four genotypes of Japanese encephalitis virus (JEV) are presently recognized (representatives of genotypes I to III have been fully sequenced), but its origin is not known. We have determined the complete nucleotide and amino acid sequence of a genotype IV Indonesian isolate (JKT6468) which represents the oldest lineage, compared it with other fully sequenced genomes, and examined the geographical distribution of all known isolates. JKT6468 was the least similar, with nucleotide divergence ranging from 17.4 to 19.6% and amino acid divergence ranging from 4.7 to 6.5%. It included an unusual series of amino acids at the carboxy terminus of the core protein unlike that seen in other JEV strains. Three signature amino acids in the envelope protein (including E327 Leu-->Thr/Ser on the exposed lateral surface of the putative receptor binding domain) distinguished genotype IV strains from more recent genotypes. Analysis of all 290 JEV isolates for which sequence data are available showed that the Indonesia-Malaysia region has all genotypes of JEV circulating, whereas only more recent genotypes circulate in other areas (P < 0.0001). These results suggest that JEV originated from its ancestral virus in the Indonesia-Malaysia region and evolved there into the different genotypes which then spread across Asia. Our data, together with recent evidence on the origins of other emerging viruses, including dengue virus and Nipah virus, imply that tropical southeast Asia may be an important zone for emerging pathogens. (+info)
A Japanese encephalitis virus peptide present on Johnson grass mosaic virus-like particles induces virus-neutralizing antibodies and protects mice against lethal challenge.
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Protection against Japanese encephalitis virus (JEV) is antibody dependent, and neutralizing antibodies alone are sufficient to impart protection. Thus, we are aiming to develop a peptide-based vaccine against JEV by identifying JEV peptide sequences that could induce virus-neutralizing antibodies. Previously, we have synthesized large amounts of Johnson grass mosaic virus (JGMV) coat protein (CP) in Escherichia coli and have shown that it autoassembled to form virus-like particles (VLPs). The envelope (E) protein of JEV contains the virus-neutralization epitopes. Four peptides from different locations within JEV E protein were chosen, and these were fused to JGMV CP by recombinant DNA methods. The fusion protein autoassembled to form VLPs that could be purified by sucrose gradient centrifugation. Immunization of mice with the recombinant VLPs containing JEV peptide sequences induced anti-peptide and anti-JEV antibodies. A 27-amino-acid peptide containing amino acids 373 to 399 from JEV E protein, present on JGMV VLPs, induced virus-neutralizing antibodies. Importantly, these antibodies were obtained without the use of an adjuvant. The immunized mice showed significant protection against a lethal JEV challenge. (+info)