Efficient replication of recombinant Enterovirus B types, carrying different P1 genes in the coxsackievirus B5 replicative backbone

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Abstract
Recombination is an important feature in the evolution of the Enterovirus genus. Phylogenetic studies of enteroviruses have revealed that the capsid genomic region (P1) is type specific, while the parts of the genome coding for the non-structural proteins (P2–P3) are species specific. Hence, the genome may be regarded as consisting of two modules that evolve independently. In this study, it was investigated whether the non-structural coding part of the genome in one type could support replication of a virus with a P1 region from another type of the same species. A cassette vector (pCas) containing a full-length cDNA copy of coxsackievirus B5 (CVB5) was used as a replicative backbone. The P1 region of pCas was replaced with the corresponding part from coxsackievirus B3 Nancy (CVB3N), coxsackievirus B6 Schmitt (CVB6S), and echovirus 7 Wallace (E7W), all members of the Enterovirus B species. The replication efficiency after transfection with clone-derived in vitro transcribed RNA was studied and compared with that of pCas. All the recombinant viruses replicated with similar efficiencies and showed threshold cycle (Ct) values, tissue culture infectivity dose 50 %, and plaque-forming unit titers comparable to viruses generated from the pCas construct. In addition to this, a clone without the P1 region was also constructed, and Western Blot and immunofluorescence staining analysis showed that the viral genome could be translated and replicated despite the lack of the structural protein-coding region. To conclude, the replicative backbone of the CVB5 cassette vector supports replication of intraspecies constructs with P1 regions derived from other members of the Enterovirus B species. In addition to this, the replicative backbone can be both translated and replicated without the presence of a P1 region.

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