Abstract
BackgroundCell culture adaptation of very virulent infectious bursal disease virus (vvIBDV) was shown to be mainly associated with the VP2 capsid protein residues 253, 279, and 284. The single mutation A284T proved critical for cell culture tropism, but did not confer efficient virus replication, which at least required one additional mutation, Q253H or D279N. While the double mutation Q253H/A284T was unambiguously shown to confer both efficient replication in cell culture and attenuation in chickens, conflicting results have been reported regarding the replication efficiency of vvIBDV mutants bearing the D279N/A284T double mutation, and no data are hitherto available on their virulence in chickens.FindingsHere we used an in vivo reverse genetics system to assess the impact of the D279N/A284T double mutation on the replication and attenuation of a chimeric IBDV virus, whose polyprotein derived from a non-culturable vvIBDV clinical isolate. We found that the D279N/A284T double mutation did indeed confer efficient replication in chicken embryo fibroblast (CEF) cell culture, but the mutant virus remained highly pathogenic to chickens.ConclusionsThe double mutation D279N/A284T of the VP2 major capsid protein of vvIBDV is sufficient to confer cell culture tropism and replication efficiency, but does not necessarily lead to virus attenuation.
Highlights
Cell culture adaptation of very virulent infectious bursal disease virus was shown to be mainly associated with the VP2 capsid protein residues 253, 279, and 284
In vivo reverse genetics mediated recovery and replication efficiency in chicken embryo fibroblast (CEF) cells of Infectious bursal disease virus (IBDV) specifying the polyprotein sequence of PO7, a Tunisian very virulent infectious bursal disease virus (vvIBDV) strain Using as recipient hosts infectious clones pVAXSA.Rib and pVAXSB.Rib of the cell culture-adapted and attenuated IBDV P2 strain, we attempted to rescue in CEF cells IBDV virus specifying the polyprotein sequence of the Tunisian bursal-derived vvIBDV PO7 strain [35]
We exchanged the P2 segment A sequence extending from the natural BspEI restriction site to the 3′ end ultimate nucleotide with its corresponding sequence of the wild-type Tunisian PO7 vvIBDV strain
Summary
Cell culture adaptation of very virulent infectious bursal disease virus (vvIBDV) was shown to be mainly associated with the VP2 capsid protein residues 253, 279, and 284. The virus causes severe immunodepression in young chicken by destruction of B cells and the bursa of Fabricius [2]. These infected immunodepressed chickens become highly susceptible to secondary infections and have a reduced capacity to respond to vaccination [3,4,5]. Genome segment A (3.2 kbp) contains two overlapping ORFs. The first smallest ORF encodes VP5, a 17-kDa non structural protein, while the second ORF codes for the precursor polyprotein NH2-pVP2-VP4-VP3-COOH [12,13]. This polyprotein is co-translationally self-cleaved, yielding the capsid protein VP2, the viral protease VP4, and the multifunctional scaffolding nucleocapsid VP3 [14,15,16]
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