Identification And Complete Genomic Sequence Analysis Of Pathogen Of An Unknown Outbreak Of Aseptic Meningitis

A large scale meningitis outbreak occurred in Yancheng Prefecture, lieing in the north part of Jiangsu Province in China, from January to July in 2003. A total of 1681 cases were notified to be involved in this outbreak, with 99% of the patients being children less than 15 years old. According to clinical manifestations and the germiculture results for CSF (cerebrospinal fluid) from patients occurred in this outbreak, aseptic meningitis was the initial diagnoses for these patients.The epidemiological distribution of this outbreak was summed from corresponding data collected by questionnaire survey. To identify the etiological agent of this outbreak, 204 CSF from 204 patients and 4 fecal specimens from other 4 cases were collected, and then subjected to cell culture for virus isolation. After the first period of isolation on two kinds of cell-lines (Vero, HEp-2), no positive result could be obtained from those 204 CSF specimens. For this reason, two other cell-lines originated from human embryo (MRC-5, 293) were included sequentially to isolate possible pathogen in CSF and fecal samples. 18 of 66 CSF specimens cultured on MRC-5 presented an enterovirus (EV)-like CPE (cytopathic effect). Such a phenomenon was observed on MRC-5 cultures for fecal samples available in this study as well. While there was no positive results got from 293 cell-line. Microscope was used to understand morphological properties for these isolates. And EV general primer-mediated RT-PCR based on highly conserved 5'UTR in enteroviral genome was performed, from which identification of Genus Enterovirus for all isolates was proved to be true.Evidences for serotype specific identification were obtained from standard neutralization assays and genotyping based on VP1 sequences. Using KMB enteroviral anti-sera pool and tens of monovalent antibodies not included by KMB, serotype of echovirus 30 was determined for all 22 isolates in this study. At the sametime, genotyping based on sequencing complete VPl encoding genes of 4 randomly selected isolates confirmed the definite type of echovirus 30 for them. Thus, it is reasonable to infer that echovirus 30 should be responsible for the outbreak of aseptic meningitis we talked above. This is the first report on probable pathogen for this outbreak since it occurred. Isolates in this study were named as Echo30-FDJS03.Sequence analysis found a 10% difference of VPl at least between FDJS03 isolates and other global E30 strains at nucleotide sequence level. Phylogenetic analysis based on complete sequences of VPl was employed to further characterize the FDJS03 isolates. In comparison, high homology was observed among 4 FDJS03 sequences, which clustered them together so closely to form a relatively lonely lineage. It is not difficult to differentiate them from other sequences in phylogenetic tree based on VPl, although some epidemic strains isolated from Europe and North America in 1970s and 1980s showed closer relationships to FDJS03 isolates than others. According to this result, a conclusion that a relatively distinct lineage of echovirus 30 was the most probable pathogen of this outbreak could be arrived at.To achieve better understanding on molecular characteristics of this pathogen, the complete genomic sequence was determined for FDJS03₈4, being a representative isolate from this outbreak, and the sequential analyses based on this sequence were carried out as well, with the aid of bioinformatical methods. Primer walking was the sequencing strategy in this study. Based on this principle, primers designed targeting conserved regions in enteroviral genomes were used to amplify 7 overlapping fragments to cover the whole genome. Either direct sequencing or cloning sequencing was performed on these PCR products, depending on their profiles on 1% agarose gels. The full-length genomic sequence of FDJS0384 was then obtained from the assembly of these fragments. This is the first full-length genomic sequence report of an epidemic echovirus 30 strain since the initial complete sequence publication of its prototype strain, Bastianni in 1999. It has been submitted to GenBank and the accession number is AY948442.