Characterization of an Infectious Cdna Copy of the Genome of a Naturally Occurring, Avirulent Coxsackievirus B3 Clinical Isolate

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 59 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. INTRODUCTION Group B coxsackieviruses (CVB1–6) are enteroviruses in the species Human enterovirus B (HEV-B), family Picorna-viridae (Pallansch & Roos, 2001). The CVB genome comprises a positive-stranded RNA that is 7400 nt long; the single ORF encodes a 2200 aa polyprotein that is processed during translation by two viral proteases. The ORF is flanked by 59 and 39 non-translated regions (NTRs), which encode no proteins, but are highly structured RNA sequences that are important for virus replication (Jackson & Kaminski, 1995; Liu et al., 1999). CVB cause diverse human diseases, including myocarditis, pancreatitis, aseptic meningitis and possibly type 1 diabetes (Bowles et al. CVB3 strains can be myocarditic (inducing both myo-carditis and pancreatitis in mice) or pancreovirulent (inducing only pancreatitis) (Tracy et al., 2000). Assays of diverse CVB3 strains in mice, isolated between the early 3These authors contributed equally to this work. 1950s and the late 1990s, suggest that avirulent strains are rare (Tracy et al., 2000; S. Tracy, unpublished data). It is unknown whether this is due to a sampling bias, as most CVB strains are isolated from suspected cases of viral disease and thus would be expected to express a virulent phenotype, or …