Enteroviral evolution: interspecies recombination and implications for picornavirus research.
PhD thesis, University of Glasgow.
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The original aim of the project was to determine whether a non-poliovirus HEV could evolve to use the poliovirus receptor (PVR). A variety of methods were used to exploit aspects of the evolutionary capacity of viruses to achieve this goal. Although the aim was not attained, the investigation of recombinants between different HEVs yielded interesting results, which were pursued further. Two approaches were developed: in vitro generation of recombinant viruses and phenotypic analysis of such chimeras and the selection for recombinant viruses in vitro.
In vitro generation of reciprocal recombinants between the structural and the non-structural coding region of coxsackievirus A21 (CVA21) and poliovirus type 3 (PV3) was initiated. Transfected and passaged chimeras did not produce infectious virions.
Immunofluorescence analysis of VP1 protein expression suggested that the recombinants were not acytopathic. A series of assays were then carried out to investigate the nature of the defect. HeLa S10 translation/transcription reactions of the in vitro generated recombinants expressed the correct protein-processing pattern suggesting efficient processing occurred in vitro. Replication assays demonstrated that the chimeras were replication competent. Trans-encapsidation experiments were then carried out and preliminary results strongly suggested that the defect could lie at the packaging level.
Selection of recombinants in vivo, without predetermining the crossover sites, was also conducted. Under the conditions used, recombinant between CVA21 and PV3 impaired genomes and echovirus 7 (EV7) and PV3 impaired genomes proved to be unsuccessful. Characterisation of the impaired parental genomes used for the experiment needs to be carried out. However, recent reports of recombinants between Sabin polioviruses and HEV-C confirm the possibility of such a recombination event occurring and emphasize concerns regarding the success of the polio eradication program.
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