Characterization of an infectious cDNA copy of the genome of a naturally occurring, avirulent coxsackievirus B3 clinical isolate

Abstract

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.

Group B Coxsackievirus Diabetogenic Phenotype Correlates with Replication Efficiency

“…This observation, which obscures a simple definition of a diabetogenic CVB phenotype, is consistent with CVB diabetogenicity being modulated by the host environment. The impact of the host environment on viral replication is well-established: expression of CVB phenotypes are modulated by a variety of influences, including immune status, age, strain, and gender of the mouse host (14,23,25,49,50).To test the hypothesis whether diabetogenicity is a common phenotype, we used a well-characterized, poorly pathogenic CVB3 strain, CVB3/GA (33,55,56), reasoning that rapid T1D onset should not be observed if the virus lacks a putative …”

“…To test the hypothesis whether diabetogenicity is a common phenotype, we used a well-characterized, poorly pathogenic CVB3 strain, CVB3/GA (33,55,56), reasoning that rapid T1D onset should not be observed if the virus lacks a putative specific genetic determinant necessary to trigger T1D onset. The precedent for considering this hypothesis is the case for CVB3-induced myocarditis in mice: the primary structure of domain II in the 5Ј-nontranslated region (NTR) of the viral genome determines a myocarditic phenotype (15,16).…”

“…To test the hypothesis whether diabetogenicity is a common phenotype, we used a well-characterized, poorly pathogenic CVB3 strain, CVB3/GA (33,55,56), reasoning that rapid T1D onset should not be observed if the virus lacks a putative…”

“…Furthermore, CVB3/GA does not induce T1D onset shortly after inoculation of 5 ϫ 10 5 50% tissue culture infective doses (TCID 50 ) into old NOD mice (14), sharply contrasting with another virulent strain, CVB3/28, that induces Ն70% T1D incidence at this dose within 1 to 2 weeks postinoculation (p.i.). All virus stocks used in these experiments were derived from infectious cDNA copies of the genomes (33,55) by transfection of HeLa cells. Virus stocks were collected by ultracentrifugation through 30% (wt/vol) sucrose in 1 M NaCl, 10 mM Tris HCl pH 7.6, 0.1 mM MgCl 2 into a glycerol button and resuspended in 100 mM NaCl, titers were determined on HeLa cells (TCID 50 /ml), and stocks were stored aliquoted at Ϫ75°C.…”

Dysregulation of the Ubiquitin-Proteasome System by Curcumin Suppresses Coxsackievirus B3 Replication

“…Group B3 coxsackievirus (CVB3) is a major human pathogen that causes meningitis and myocarditis (10,14). Despite extensive efforts, no specific and approved treatment has been developed that is effective against CVB3-induced diseases.…”