title: Characterization of Coxsackie B virus RNA in myocardium from patients with dilated cardiomyopathy by nucleotide sequencing of reverse transcription-nested polymerase chain reaction products.
abstract: This study was performed to detect and characterize the enterovirus present in myocardium of some patients with heart muscle disease by nucleotide sequencing of polymerase chain reaction (PCR) products after amplification with enterovirus group-specific primers. Enterovirus sequences have been detected previously in myocardium of patients with myocarditis or dilated cardiomyopathy and seem causal, although the particular virus serotypes involved have not been identified. In a prospective study of endomyocardial biopsy specimens from 35 consecutive patients with suspected heart muscle disease, enterovirus sequences from the 5' nontranslated region were amplified by reverse transcription-nested PCR using group-specific primers. This region contains both conserved and variable sequence motifs, characteristic of particular enterovirus serotypes. The nucleotide sequences of individual PCR products were determined by cycle sequencing and compared with all known sequences (GenBank/EMBOL), using the GCG software package. Endomyocardial biopsy specimens from 9 of 21 (42.9%) patients with a histologically confirmed diagnosis of dilated cardiomyopathy were positive for enterovirus by PCR, compared with only 1 of 14 (7.1%) patients with other myocardial pathological conditions (Fisher's exact probability=0.0275: odds ratio=9.75; 95% confidence interval=1.31-72.78). The nucleotide sequence of the PCR products differed, indicating no cross-contamination. However, computerized comparison showed that each had greatest homology with the 5' nontranslated region of Coxsackie B virus but contained up to 11% sequence variations compared with the prototype Coxsackie B3 strain Nancy. Parallel investigation of tissue from our mouse model of Coxsackievirus B3-induced myocarditis showed that nucleotide sequence changes are not introduced by reverse transcription or PCR. These data support the link between enteroviral infection and dilated heart muscle disease and suggest that Coxsackie B serotypes are the enteroviruses most frequently involved.
authors.affiliation: Biochemistry Department, Charing Cross and Westminster Medical School, Kings College Hospital, London, United Kingdom.
authors.forename: L C
authors.initials: LC
authors.lastname: Archard
authors.forename: M A
authors.initials: MA
authors.lastname: Khan
authors.forename: B A
authors.initials: BA
authors.lastname: Soteriou
authors.forename: H
authors.initials: H
authors.lastname: Zhang
authors.forename: H J
authors.initials: HJ
authors.lastname: Why
authors.forename: N M
authors.initials: NM
authors.lastname: Robinson
authors.forename: P J
authors.initials: PJ
authors.lastname: Richardson
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D002311:Cardiomyopathy, Dilated / Q000473:pathology / Q000821:virology*; D003384:Coxsackievirus Infections / Q000473:pathology / Q000821:virology*; D017931:DNA Primers / Q000737:chemistry; D029822:Enterovirus B, Human / Q000235:genetics* / Q000302:isolation & purification; D005260:Female; D006321:Heart / Q000821:virology*; D006801:Humans; D008297:Male; D008875:Middle Aged; D009206:Myocardium / Q000473:pathology*; D016133:Polymerase Chain Reaction; D011446:Prospective Studies; D012367:RNA, Viral / Q000032:analysis*; D017423:Sequence Analysis, RNA; D014158:Transcription, Genetic
chemical_list: D017931:DNA Primers; D012367:RNA, Viral