Sequence analysis of enteroviruses isolated from patients with myocardial diseases

Objective To characterize the gene sequences of six enteroviral isolates from patients with myocarditis and Keshan disease in selenium deficiency area of Yunnan province and one isolate from patients with myocarditis in Shanghai. Methods The 5′ nontranslated regional sequences and partial VP4 sequences of isolates (serotype: coxsackievirus A9, B2, B3, B5, B6, B6 and one unidentified) were amplified by reverse transcription polymerase chain reaction with enterovirus group specific primers. The nucleotide sequences of individual PCR products were determined by cycle sequencing and compared with known enteroviral sequences (GenBank/EMBOL) using the AssemblyLIGN software package. Results The 710bp fragment sequences from nucleotide position 40 to 750 of 5′ terminal sequences of the isolate (coxsackievirus A9, B2, B3, B3, B5, B6 and B6) were determined and sequencing of the PCR product from isolate with serotype unknown revealed the presence of multiple sequence bands, indicating multiple enteroviruses. Compute comparison showed that coxsackievirus isolate A9, B2, B3 and B6 had 14.51% 16.48% sequence variations compared with published coxsackievirus A9, B2, B3 and B6, respectively, suggesting that intratypic genetic divergence of 5′ nontranslated region among the coxsackieviruses are not difference from intertypic genetic divergence among group B coxsackieviruses, while B5 showed up to 34.08% variations compared with published coxsackievirus B5. Variations at 234(C T) and 690(A C) have been found from 2 isolates of B3 from Selenium deficiency area of Yunnan province in comparison with the prototype coxsackie B3 strain Nancy. The variation at 234 (C T) needs further study. Conclusions The study demonstrates that it is difficult to determine the genetic serotyping of the viruses by sequencing of 5′ nontranslated region. The described method can be applied to the epidemiologic investigation of enteroviral infections.