Phylogenetic analysis and development of an immunofluorescence assay for untypeable strains of coxsackievirus B3.

BACKGROUND/PURPOSE In recent years, coxsackievirus B3 (CV-B3) has been determined as a dominant enterovirus serotype that may cause severe complications in patients. Since 2008 in Taiwan, some enterovirus isolates have been regarded as untypeable [by employing commercial immunofluorescence assay (IFA) kits]. In 2012, the number of isolates increased. Genetic sequence analysis further confirmed that CV-B3 was present in most of the untypeable viruses. METHODS Isolates of CV-B3 were collected for basic local alignment search tool (BLAST) analysis and for phylogenetic analyses, based on VP1 gene sequences. In addition, the Taiwan Centers for Disease Control (Taiwan CDC) developed an in-house indirect IFA using polyclonal antibodies (e.g., rabbit antisera) for diagnosis. The sensitivity and specificity were both evaluated by testing 61 reference enteroviruses and 307 local enteroviruses that were isolated between 1998 and 2010. RESULTS Based on the results of the BLAST and phylogenetic analyses, five main genogroups (i.e., GI-GV) were classified and the reference strains in Taiwan in previous years were primarily clustered in the GV-A subgenogroup. However, the 15 CV-B3 isolates recently analyzed in this study were classified in four different groups: GIII, GIV, GV-A, and GV-B. Among these 15 isolates, all 10 isolates in the GV-B group were initially reported as untypeable nonpolio enteroviruses when using commercial kits. The conditions of the in-house indirect IFA were optimized by checkerboard titration, thereby resulting in a sensitivity of 100% and a specificity of 98.5%. CONCLUSION This is the first report describing the phylogenetic relatedness of recent CV-B3 strains in Taiwan. An indirect IFA kit was developed by the Taiwan CDC for detecting CV-B3 viruses that are untypeable by commercial IFA kits.