Adult human enteroviral heart disease is often associated with the detection of enteroviral RNA in cardiac muscle tissue in the absence of infectious virus. Passage of coxsackievirus B3 (CVB3) in adult murine cardiomyocytes produced CVB3 that was noncytolytic in HeLa cells. Detectable but noncytopathic CVB3 was also isolated from hearts of mice inoculated with CVB3. Sequence analysis revealed five classes of CVB3 genomes with 5 termini containing 7, 12, 17, 30, and 49 nucleotide deletions. Structural changes (assayed by chemical modification) in cloned, terminally deleted 5-nontranslated regions were confined to the cloverleaf domain and localized within the region of the deletion, leaving key functional elements of the RNA intact. Transfection of CVB3 cDNA clones with the 5-terminal deletions into HeLa cells generated noncytolytic virus (CVB3/TD) which was neutralized by anti-CVB3 serum. Encapsidated negative-strand viral RNA was detected using CsCl-purified CVB3/TD virions, although no negative-strand virion RNA was detected in similarly treated parental CVB3 virions. The viral protein VPg was detected on CVB3/TD virion RNA molecules which terminate in 5 CG or 5 AG. Detection of viral RNA in mouse hearts from 1 week to over 5 months postinoculation with CVB3/TD demonstrated that CVB3/TD virus strains replicate and persist in vivo. These studies describe a naturally occurring genomic alteration to an enteroviral genome associated with long-term viral persistence.The six serotypes of the group B coxsackieviruses (CVB1-6) are enteroviruses (Picornaviridae, species HEV-B) (53). The CVB genome is a single-stranded RNA molecule, 7,400 nucleotides (nt) in length, that is encapsidated within an icosahedral shell (74). The 11 viral proteins (83) are encoded by a single open reading frame which is flanked on the 5Ј and 3Ј termini by nontranslated regions (NTRs) (20). The CVB induce numerous human illnesses, including inflammatory heart disease, pancreatitis, and aseptic meningitis and may also trigger the onset of type 1 diabetes (5,31,72,81,82). The CVB were recognized as causes of human heart disease shortly after their description early in the 1950s (26, 27) and remain the enteroviruses most commonly associated with human cardiomyopathies on the grounds of isolation and serology (5,6,34,62,88). Meta-analysis shows an association between enteroviruses and myocarditis in 23% of cases (7), although this association is more variable in cases of dilated cardiomyopathy, a serious disease that often leads to a failing heart (61). Mouse models of CVB-induced pancreatitis (94, 110), myocarditis (45,52,110), myositis (104, 105), and rapid-onset type 1 diabetes (31) which facilitate the study of these diseases have been developed.Enterovirus infections are generally considered to be acute events, with symptoms and virus titers peaking within a few days postinoculation (p.i.) and with virus being cleared by the adaptive immune response (17). However, enterovirus infections can persist under conditions of immunodeficiency (48,51,...

Coxsackievirus B3 (CVB3) generates 5-terminally deleted genomes (TDs) during replication in murine hearts. We show here that CVB3 populations with TDs can also be generated within two to three passages of CVB3 in primary, but not immortalized, cell cultures. Deletions of less than 49 nucleotides increase in size during passage, while 5 TDs of 49 nucleotides appear to be the maximum deletion size. The cellular environment of contact-inhibited primary cell cultures or the myocardium in vivo is sufficient for the selection of 5 TDs over undeleted genomes.The six serotypes of the group B coxsackieviruses (CVB1 to CVB6) (human enteroviruses, Picornaviridae) (18,22) are small, nonenveloped, positive-strand RNA viruses. The enterovirus RNA genome, which is flanked by 5Ј and 3Ј nontranslated regions (NTR), is translated upon infection of the host cell (4) and then serves as the template for synthesis of complementary negativestrand RNA, which in turn templates progeny positive-strand genomic RNAs. Human enteroviruses cause acute myocarditis and are etiologically linked as causes of dilated cardiomyopathy, a disease that can proceed to heart failure (2,3,11,20,22). Enteroviral RNA persistence in adult hearts with myocarditis or dilated cardiomyopathy in the absence of detectable infectious virus has been a common observation (reviewed in references 3, 11, and 12) but remained without adequate explanation until we demonstrated that, during replication in isolated murine cardiomyocytes and in hearts of infected mice, CVB3 can naturally delete sequence information at the genomic 5Ј terminus (13). This work showed that, weeks following the inoculation of mice with CVB3, mouse myocardium in which no cytolytic virus was evident was able to transmit virus to cell cultures, which, in turn, showed no cytopathic effects (CPE) despite the detection of CVB3 RNA. Genomic viral RNA lacked native 5Ј termini, demonstrating deletions ranging from 7 to 49 nucleotides (nt) from the 5Ј genomic terminus; these novel, naturally occurring CVB3 strains with 5Ј-terminally deleted (TD) viral genomes were termed CVB3TD. The cloning and placement of naturally occurring deletions into an infectious CVB3 cDNA clone revealed that, while the resultant viruses are replication competent, CVB3TD replication is very slow, resulting in no observable CPE on HeLa indicator monolayers. In addition, purified CVB3TD virions encapsidate both positive-and negative-strand RNA in a strand ratio similar to that of total RNA from CVB3TD-infected cell cultures. Because CPE in CVB3TD-infected cell cultures are not measurable, a method was developed to titer infectious CVB3TD using real-time quantitative reverse transcription-PCR (RT-PCR) analysis of the viral RNA content in CVB3TD preparations (13). It was not surprising to note deviations from wild-type enteroviral replication, as the region in the 5Ј NTR that is progressively deleted (the cloverleaf RNA structure termed domain I) is also an essential region for viral replication (1,17,19). Having generated CVB3TD stra...

Group B coxsackieviruses (CVB) cause numerous diseases, including myocarditis, pancreatitis, aseptic meningitis and possibly type 1 diabetes. To date, infectious cDNA copies of CVB type 3 (CVB3) genomes have all been derived from pathogenic virus strains. An infectious cDNA copy of the well-characterized, non-pathogenic CVB3 strain GA genome was cloned in order to facilitate mapping of the CVB genes that influence expression of a virulence phenotype. Comparison of the sequence of the parental CVB3/GA population, derived by direct RT-PCR-mediated sequence analysis, to that of the infectious CVB3/GA progeny genome demonstrated that an authentic copy was cloned; numerous differences were observed in coding and non-coding sequences relative to other CVB3 strains. Progeny CVB3/GA replicated similarly to the parental strain in three different cell cultures and was avirulent when inoculated into mice, causing neither pancreatitis nor myocarditis. Inoculation of mice with CVB3/GA protected mice completely against myocarditis and pancreatitis induced by cardiovirulent CVB3 challenge. The secondary structure predicted for the CVB3/GA domain II, a region within the 5′ non-translated region that is implicated as a key site affecting the expression of a cardiovirulent phenotype, differs from those predicted for cardiovirulent and pancreovirulent CVB3 strains. This is the first report characterizing a cloned CVB3 genome from an avirulent strain.

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