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A biological mutant of coxsackievirus B3 and its pathogenesis in mice

University of British Columbia

An antibody escape-mutant of coxsackievirus B3 / CVB3 (RK strain) was isolated using a neutralising monoclonal antibody against CVB3. This escape-mutant EM1 was then compared with the wt+ strain CVB3 (RK) both in vitro and for its pathogenesis in mice. In vitro EM1 was found to be temperature sensitive at 40°C and less stable on prolonged incubation at 37°C than CVB3 (RK). Also EM1 was slightly growth-restricted in Vero cells, giving yields 2 to 5 fold lower than the parental strain and also has a small plaque phenotype. A similar reduction in viral replication was found in the macrophage cell-line, J774A.1. However growth of EM1 in the Wehi 231 B-cell-line was reduced 10- 20 fold while both EM1 and wt+ were totally restricted in the T-cell-line, EL-4. In vivo. EM1 was found to be less myocarditic while producing equivalent amount of pathological damage in pancreas and liver. Correlating with this equivalent amounts of virus were detected both by plaque assay of tissue homogenates, and in situ hybridization in all tissues apart from spleen and heart. In particular in heart tissue, the ability of the EM1 to replicate and cause damage was much less than the wt+(RK) strain, i.e., EM1 was less cardiovirulent than CVB3 (RK). Sequence analysis of the NTR region and P1 structural gene regions of CVB3 (RK) and EM1 strains following RT/PCR of genomic RNA identified several mutations in EM1 including a single nucleotide change in the E loop of the NTR region and several mutations in P1. The most significant changes appear to be in VP1 where 4 point mutations and a deletion/insertion were identified. One of these is silent but the others are associated with amino acid changes including T to H and F to V substitutions in the BD region of VP1 structure. These mutations most likely explain the lack of reactivity with neutralising MAb and also the reduced heat stability of EM1.

Thesis/Dissertation

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2009-03-13

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http://hdl.handle.net/2429/6010

Pathology

University of British Columbia

UBCV

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