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The molecular characterization of Coxsackievirus B3 Pathogenisis

University of British Columbia

The group B coxsackieviruses (CVBs) cause a variety of clinical manifestations in humans and are often fatal in infants under the age of three months. In the cardiovascular field, CVBs are considered the primary etiologic agent of virus-induced myocarditis. Although often a subclinical illness in adults, myocarditis may lead to chronic dilated cardiomyopathy, arrhythmias and sudden death. Unlike other family members such as poliovirus, there is relatively little understanding of the mechanisms of pathogenesis of coxsackievirus and no vaccine is available. Therefore, the objective of this project was to map genetic regions associated with CVB3 virulence by studying antibody escape mutants which differ in their pathogenesis for heart tissue. In conjunction with previous work from the laboratory, a panel of ten antibody escape mutants was isolated: EM1- EM10. Sequence analysis of the genes encoding the capsid proteins (VP1-VP4) revealed that all but two mutants, EM7 and EM 10, contained a lysine to arginine mutation within the 'puff region' of VP2. In contrast, EM7 and EM10 contained a glutamate to glycine mutation within the 'knob region' of VP3. Both of these regions, the 'puff of VP2 and the 'knob' of VP3, have been shown to be neutralizing and/or immunodominant sites on the capsid surface of other picornaviruses. Complete sequence analysis of EMI and EM 10 revealed further mutations within the viral polymerase gene and the 5' nontranslated region which may be responsible for the phenotypes of these variants. As characterized in previous studies, EMI produces about tenfold lower titres in the hearts of infected mice as compared to the wildtype strain. This inability of EMI to replicate to wildtype levels in the hearts of infected animals does not appear to be the result of an inability to replicate in cardiomyocytes as demonstrated by in vitro analysis. The pathogenic phenotype of EM 10 in A/J mice has also been examined as part of this study. Data from these experiments suggest that EM 10 has a higher level of attenuation than EMI producing 100-1000 fold lower titres in the hearts of infected animals as compared to the wild type CVB3(RK) strain. In an attempt to determine if the lysine to argihine mutation in VP2 was responsible for the reduced myocarditic potential of EMI, this mutation was incorporated into an infectious clone of the wildtype strain. Despite multiple attempts the mutant clones did not produce infectious virus that could be tested for cardiovirulence. In summary this project has identified several loci within the CVB3 genome which may be responsible for the attenuated phenotypes of EMI and EM10. Correlation of such mutations with an alteration in a phenotypic property of interest, such as tropism for heart tissue, will lead to an increased understanding of CVB pathogenesis. This knowledge may be exploited in vaccine development.

Thesis/Dissertation

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2009-06-29

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http://hdl.handle.net/2429/9783

Pathology

University of British Columbia

UBCV

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