Characterization of enterovirus isolates from patients with heart muscle disease in a selenium-deficient area of China

An association of enterovirus infection with endemic cardiomyopathy (Keshan disease [KD]) and outbreaks of myocarditis in selenium-deficient rural are as of southwestern China has been established. Enteroviruses have been isol ated from patients with KD or during outbreaks of myocarditis in last two d ecades. Six of these isolates grew readily in cell lines (Vero or HEp-2) an d were investigated by a novel molecular typing method apart from serotypin g and pathogenicity. A neutralization assay identified two isolates from RD as coxsackievirus serotype B2 (CVB2) and two isolates from myocarditis as coxsackievirus serotype B6 (CVB6) but failed to type the remaining two isol ates, also from myocarditis. Direct nucleotide sequencing of reverse transc ription-PCR products amplified from the 5' nontranslated region (5'NTR) of these viruses confirmed that they belong to a phylogenetic cluster consisti ng of coxsackie B-like viruses, including some echovirus serotypes, Sequenc e analysis of the coding region for viral capsid protein VP1 showed that tw o isolates serotyped as CVB2 have the highest amino acid sequence homology with CVB2 and that the remaining four isolates, two CVB6 and the two unknow n serotypes. are most closely related to the sequence of CVB6 Sequences amo ng these isolates varied from 82.3 to 99% in the 5'NTR and from 69 to 99% i n VP1, indicating no cross contamination, The pathogenicity of these viruse s in adult and suckling mice was assessed. None caused pathologic changes i n the hearts of adult MF-1 or SWR mice, although pancreatitis was evident. However, the four CVB6-like viruses caused death in suckling mice, similar to a virulent coxsackievirus group B3 laboratory strain. In conclusion, the sequence data confirm that coxsackievirus group B serotypes are predominan t in the region in which KD is endemic and may be the etiological agents in outbreaks of myocarditis. VP1 genotyping of enteroviruses is accurate and reliable. Animal experiments indicate that isolates may differ in pathogeni city.