| Japanese encephalitis virus (JEV) is a positive-sense single-stranded RNA virus, which belongs to the genus Flavivirus of Flaviviridae family. The JEV genome is approximately 11Kb in length. It presents a single ORF encoding a polyprotein that is processed into more than 10 structural proteins and non-structural proteins. JEV is an important cause of anthropozoonosis. Japanese encephalitis could results in permanent neuropsychiatric sequelae with high fatality rate. There are billions of people living in the JE endemic area. Hence, with worldwide concerns, JE poses a public heath importance. The study aims to analyze recombination and molecular evolution of JEV genomes, which benefits the understanding of the pathogenic mechanism of JEV, viral molecular epidemiological characters, vaccine developments and molecular diagnosis of Japanese encephalitis.1. Sequencing of JEV genomesIn this study, the full genome sequence of 121 JEV isolates in China from 1950s to 2007 were completed. The JEV isolates isolated from more than 20 mosquito species, bloods and cerebrospinal fluids of Japanese encephalitis patients, midges and bats samples. And the isolation places cover major Japanese encephalitis endemic regions of China: Heilongjiang Province represents the northern extremity of JE endemic area in China, Fujian Province represents the southern extremity of JE endemic area in China, Gansu Province represents the western extremity of JE endemic area in China, and Shanghai city represents the eastern extremity of JE endemic area in China. The JEV isolates belongs to GI and GUI JEV. The statistics of sequencing quality shows that the average coverage rate is 7.41423927 and the average quality value is 40.34710744 namely 99.99% accuracy and 0.01% error, which is closed to the Human Genome Project sequencing quality criteria and is equivalent to the quality of relevant large scale sequencing of viral genomes. 2. Establishment of virus databaseBased on the MySQL language and the phpMyAdmin software with the graphical management interface, the database (159,179 sequences in total) of Family Flaviviridae virus information was established to facilitate future study, including all the available genes and genomes sequences of the entire Family Flaviviridae viruses in GenBank and the 121 JEV genomes sequenced in this study.3. Recombination of JEV genomesWith 12 softwares of recombination analysis, the genome wide screen was performed among 178 genomes of JEV strains (57 genome of JEV download from GenBank, while 121 genome of JEV were sequenced in this study.). The result showed a large amount of inter-genotype and intra-genotype recombination signals in 139 JEV strains, taking 78% of the total strains (139/178). There is no recombination signals in GII and GIV JEV. By further analyzing recombination signals with bioinformatics and statistic methods, 3 strains with inter-Genotype I / III homologous recombination were identified. Among the 3 strains, the SC04-25 isolated from a mosquito sample in China contains 9 recombinant breakpoints that distribute every region within genome except NS2b and 3'non-coding region. The rest two strains (HLJ02-144 and K94P05) isolated from the mosquitoes in Heilongjiang Province, China and South Korea respectively. By calculating the positions of recombinant regions, the most similar paternal strains are isolated from Japanese encephalitis patients and mosquito samples. The similarities and genetic distances between recombinant strains and reference strains were compared. Based on the finding above, it indicates the recombinant JEV is existed in nature. The recombinant events could happen in single gene level or genomic level. The study is the first report of naturally existed inter-Genotype I/III recombination of JEV.4. Molecular evolution of JEVWith the molecular phylogeny and bioinformatics technology, the nucleotide substitution model, molecular clock model, mean substitution rate and mean growth rate of JEV genomes were calculated. Based on genetic distances and evolutionary time, phylogenetic trees for JEV genes and genomes were built separately. Result showed that the best substitution models for structural proteins coding region and non-structural proteins are HKY model and GTR model separately, while the best substitution model for JEV genomes is GTR model. The mean substitution rate of JEV is around an order of magnitude of 10-4 and the mean growth rate is verge on zero, which suggesting mean growth rate of JEV is slow comparing to other RNA virus, such as influenza virus. Phylogenetic analysis on viral genomes showed that evolution of Genotype III JEV is related to the source of different host, and Genotype I JEV has the with molecular characteristic among the virus isolated from different region. These results provide the molecular evidence for research on JEV genotyping, sub-genotype classification and the molecular difference of JEV among varied hosts and isolation places. According to the biothermodynamics, the biological entropy (0-1.08694) for every nucleotide site in JEV genomes were calculated, which offers data to analyze conserved region and highly variable sites of JEV. Besides, the results virus evolutionary show that the origin of JEV strains is from 801 BC and the common ancestor for Genotype III and Genotype I JEV are approximately appear on 1742 and 1798 respectively. In short, it is the first study on the molecular evolution of JEV based on the whole genome... |
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