Objective: To identify the pathogen caused an outbreak of aseptic meningitis in Tancheng county of Shandong province in 2008, and to analyze the molecular characterization of VP1 gene of the Coxsackievirus B3(CVB3) isolates.

Methods: Stool and cerebrospinal fluid(CSF) specimens were collected from this outbreak for virus isolation with RD and Hep-2 cell. After typing by neutralization test, the VP1 gene of the isolates were amplified by RT-PCR and sequenced. Homologous comparison and phylogenetic analysis were performed.

Results: 35 strains of enteviruse were isolated from 22 stools and 120 CSFs(7 from stools and 28 from CSFs), 34 strains identified as CVB3 and 1 as Echovirus 30(ECHO30) by neutralization test. The nucleotide homologies were 90.5%-100.0% in the partial VP1 gene (381 bp) among 34 CVB3 isolates. Homology comparisons indicated that Shandong strains have the identity of 79.5%-81.6% with the CVB3 prototype strain Nancy. 012/2008TC/SD/CHN and 177/2008TC/SD/CHN showed the highest nucleotides homologies (98.2% and 91.0% respectively) with Fuyang19 strain of Anhui province in 2008 in complete VP1 gene. The phylogenetic tree based on complete VP1 genes showed that all the CVB3 correlated with aseptic meningitis in China recently came from the same evolution linkage and formed a monophyletic cluster.

Conclusion: The causative agent of this outbreak of aseptic meningitis was CVB3. CVB3 circulated in China was genetically different from other countries.

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