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Objective In order to investigate etiology and molecular-epidemiological characteristics of enterovirus associated encephalitis (EAE) in Zhejiang,2008-2012.Method Cerebrospinal fluid and stool specimens were collected from suspected EAE patients,who were admitted to our hospitals.RD and Hep-2 cell lines were used to isolate enterovirus (EV).Serotypes of these EV isolates were identified through neutralization test by using serotype specific anti-sera.VP 1 genes of these isolates were sequenced,compared and used for the construction of phylogenetic tree.Results 127 (20.6％) human enterovirus (HEV) strains were isolated from 616 samples,which were collected from 610 patients.Serotypes of these EV isolates,including 60 coxsackievirus (CV),and 67 Echovirus (E) appeared to be CVA9,CVB1,CVB3-5,E3,E4,E6,E9,El4,E25 and E30,respectively.Predominant EV serotypes on EAE from 2008 to 2012 were seen as CVB3,CVB5,E6,E30 and E30,respectively.The full length of VP1 genes from different EV isolates was between 834 and 918 nucleotides.The VP1 gene similarities between these isolates and the reference strains were from 76.7％ to 85.0％ (nucleotides level) and 91.1％ to 97.9％ (amino acids level).The VP1 genes from E6 serotype isolates appeared most diverged,reaching 20.4％ (nucleotides level) attd 4.8％ (amino acids level).Based on the generated phylogenetic tree,all the EV isolates were fallen on the same branch of HEV-B,and the isolates in the same serotype formed one sub-branch,suggesting there existed geographical and temporal effects.E6 isolates diverged into two branchlets.Conclusion EVs from HEV-B were the etiologic agents for EAE in Zhejiang province from 2008 to 2012.All these EV isolates showed 12 serotypes,with predominant isolates varied every year.E30 was determined as the most dominant serotype while serotype E6 diverged into two sub-genetypes.
RESUMO
<p><b>OBJECTIVE</b>To investigate the molecular characteristics and genotype of Japanese encephalitis virus (JEV) found in vector mosquitoes in Zhejiang province from 1982 to 1983.</p><p><b>METHODS</b>A total of 3188 mosquitoes were collected in Dinghai district and Yiwu city in Zhejiang province, during year 1982 and 1983. The virus was isolated by C6/36 cell, and then identified by hemagglutination inhibition test. The isolated strains were activated in year 2011, and plaque forming unit (PFU) were applied to test the virus titer. The suckling rats were tested under intracranial inoculation, where PrM and E genes were amplified and sequenced. Their nucleotide and amino acid sequences were analyzed and compared with the JEV vaccine strain SA14-14-2 and the JEV isolated in Zhejiang province during 2007 and 2010; and phylogenetic tree were constructed by bioinformatic software.</p><p><b>RESULTS</b>From the 3188 mosquitoes captured, eleven virus strains were isolated and found to be able to cause cytopathogenic effect (CPE) in C6/36 cells within 72 hours. Virus titer ranged from 2.5 to 6.47 lg PFU/ml. The suckling rats would die within 72 hours since the inoculation. The phylogenetic analysis with the PrM and E genes showed that the JEV isolated in Zhejiang during 1982 and 1983 belonged to genotype III; while the JEV isolated in Zhejiang during 2007 and 2010 belonged to genotype I. The analysis of E genes from 5 isolated strains found that the homology of nucleotide sequence was over 98.9%, and the homology of amino acid sequence was over 99.8%. The compare between the 5 virus strains and the vaccine strain SA14-14-2 found 10 common amino acid variation sites, and showed that the homology of nucleotide sequence was over 97.7%, and the homology of amino acid sequence was 99.2%. The compare between the 5 virus strains and the JEV isolated in Zhejiang during 2007 and 2010 found a 87.7% - 87.9% nucleotide homology and an over 98.8% amino acid homology.</p><p><b>CONCLUSION</b>The JEV isolated from the mosquitoes in Dinghai district and Yiwu city between year 1982 and 1983, were genotype III.</p>