Phylogenetic Analysis Of Enteroviruses And Study On The Methods For Early Laboratory Diagnosis

Enteroviruses, associated with a wide range of human diseases,have manyserotypes and a high mutation rate due to the lack of proofreding activity duringgenome replication. In the last phase of poliovirus eradication, 57 non-polioenterovirus(NPEV) isolates from AFP cases were diagnosed and identified byconventional antibody neutralization test and neucleotide sequence.Conventional method could only identify 54.39% of the isolates, while theneucleotide sequence could identify all the isolates. Two strains(FJ96-71 andFJ02-47) were found to be new serotypes, and the cause of an asepitic encephalitisepidemic was rapidly identified by the molecular method. We can conclude that themolecular method offers the dual advantages of rapidity and the ability to detectmutative strains and previously undescribed serotypes which are difficult andimpossible to type by convential method. The technique also is useful to determinewhether viruses isolated is the cause of an epidemic.The strain FJ96-71 purified by plaquing was identified to be a novel serotypeby morphologic characteristic , cytopathic effect(CPE), antigenic properties and thecomplete genome analysis. Recently the serotype is named as enterovirus 75. Theantibody to the virus was prepared for further use.Many serotypes of NPEV, including ECV6, ECV11, ECV13 and ECV30 whichwere common serotypes associated to severe diseases such as aseptic encephalitisin some countries and ECV19 causing local aseptic encephalitis epidemic, circulatedin Fujian province.VP1 is the most external immunodominant of the capsid proteins, andcorrelated well with the serotype in enterovirus. Phylogenetic analysis on VP1sequence or its partial sequence showed that the sequence divergence was highbetween the Fujian isolates and reference strains. Significant sequence divergencewas found in some intraserotypes among Fujian isolates, which indicates that thereare different transmit chains for epidemic causing by some viral serotypes. Therefore,genetic typing is a useful method for tracing source of the virual infection. The common epitope region of enteroviruses characterized by previous studieswas amplified from Fujian isolates. The PCR products were cloned into pET30aor/and pET42a expression vector and transformed into E.coli BL21(DE3). Theantigenicity of the recombinant protein was identified by Western blotting with thecommercial enterovirus group-specific MAb 5-D8/1, human serum with positivepolio antibody and several sheep monotype sera representing enteroviruses group A,B and D. Rabbit polyclonal antibody and monoclonal antibody to the recombinantantigen were prepared. And the antibodies immunoreactivity with enteroviruses wereevaluated by indirect immunoflurescence assay (IFA) in comparison with MAb5-D8/1. By detecting the 29 strains (27 serotypes) of Fujian isolates whose sequencedivergence was high, it was concluded that the positive rate of MAb 5-D8/1 was10%-40% lower than the antibodies against the recombinant protein. The resultsuggests that the protein expressed based on Fujian isolates might be useful andpractical.