Analysis of the complete genomic sequences of two virus subpopulations of the Australian infectious bronchitis virus vaccine VicS

Abstract

Although sequencing of the 3' end of the genome of Australian infectious bronchitis viruses (IBVs) has shown that their structural genes are distinct from those of IBVs found in other countries, their replicase genes have not been analysed. To examine this, the complete genomic sequences of the two subpopulations of the VicS vaccine, VicS-v and VicS-del, were determined. Compared with VicS-v, the more attenuated VicS-del strain had two non-synonymous changes in the non-structural protein 6 (nsp6), a transmembrane (TM) domain that may participate in autocatalytic release of the 3-chymotrypsin-like protease, a polymorphic difference at the end of the S2 gene, which coincided with the body transcription-regulating sequence (B-TRS) of mRNA 3 and a truncated open reading frame for a peptide encoded by gene 4 (4b). These genetic differences could be responsible for the differences between these variants in pathogenicity in vivo, and replication in vitro. Phylogenetic analysis of the whole genome showed that VicS-v and VicS-del did not cluster with strains from other countries, supporting the hypothesis that Australian IBV strains have been evolving independently for some time, and analyses of individual polymerase peptide and S glycoprotein genes suggested a distant common ancestor with no recent recombination. This study suggests the potential role of the TM domain in nsp6, the integrity of the S2 protein and the B-TRS 3, and the putative accessory protein 4b, as well as the 3' untranslated region, in the virulence and replication of IBV and has provided a better understanding of relationships between the Australian vaccine strain of IBV and those used elsewhere.

Figure 1.. Nucleotide sequence alignment of the VicS-v and VicS-del genomes (GAN KF460437 and KF931628). ORF 4b is absent in VicS-del and there is a 40-nucleotide deletion in the 3′ UTR of VicS-del. There are also non-synonymous changes at the end of nsp6 and a SNP at the end of the S glycoprotein sequences (boxed). Non-structural protein 1 (nsp1), present in other members of family Coronaviridae, is absent in IBV. Vertical lines indicate differences in the nucleotide sequences. Dashes indicate deletions in the sequence.

Figure 2.. Unrooted phylogenetic tree of 35 complete genomes of IBV strains isolated in different countries. The nucleotide alignment was performed using Clustal-Omega (Sievers et al., 2011) (http://www.ebi.ac.uk) and the phylogenetic tree inferred in Geneious 6.1.4, using the nearest neighbour interchange (NNI) maximum likelihood heuristic method with 100 bootstrap replications and a general time-reversible (GTR) substitution model, gamma distributed with invariant sites (G+I); a₁ and a₂, subclusters within the USA cluster; a₃, turkey coronavirus (TCoV) cluster; b₁ and b₂, subclusters of the Chinese cluster; c, strains outside the main clusters, including a QX-like strain (Ck/Swe/0658946/10 and Ita/90254/2005). The star highlights VicS-v and VicS-del, and the dots indicate other vaccine strains (H120, H52, Mass 41, Georgia 1998 and Arkansas).

Figure 3.. Phylogenetic trees constructed with the nucleotide sequence alignments of the 3CL, helicase (Hel), papain-like proteinase (PLP) and RNA-dependent RNA polymerase (RdRp) genes inferred using the nearest neighbour interchange (NNI) maximum likelihood heuristic method and 100 bootstrap replications, and a general time-reversible (GTR) substitution model, gamma distributed with invariant sites (G+I); a₁ and a₂ indicate the two USA subclusters, b₁ and b₂ the two Chinese subclusters. The stars mark the position of VicS-v and the dots the position of other vaccine strains (H120, H52, Mass 41, Georgia 1998 and Arkansas).

Figure 4.. Phylogenetic trees constructed with the nucleotide sequence alignments of the S1 glycoprotein genes, and with Geneious 6.1.4, using the nearest neighbour interchange (NNI) maximum likelihood heuristic method with 100 bootstrap replications and the general time-reversible (GTR) substitution model, gamma distributed with invariant sites (G+I): A, tree with TCoV strains removed (TCoV included in B); a, USA cluster; b, Chinese cluster, including QX strains; c, Ibadan strain; d, Georgia and Delaware cluster; e, TCoV strains. The star marks the position of VicS-v and VicS-del, and the dots the position of other vaccine strains.

Figure 5.. Phylogenetic trees constructed with the amino acid sequences alignment of the S1 glycoprotein genes, and with Geneious 6.1.4, using the nearest neighbour interchange (NNI) maximum likelihood heuristic method with 100 bootstrap replications and the general time-reversible (GTR) substitution model, gamma distributed with invariant sites (G+I): A, tree with TCoV strains removed (TCoV included in B); a, USA cluster; b, Chinese cluster; c, Georgia and Delaware cluster; d, TCoV strains. The star marks the position of VicS-v and the dots the position of other vaccine strains.

Figure 6.. Network tree analysis constructed with the nucleotide sequence alignments of the full genome of different IBVs (SplitsTree4): a₁₊₂, USA IBV subclusters, a₃ TCoV subcluster; b₁ and b₂, Chinese subclusters; c₁ and c₂, strains outside the main clusters, including the QX-like strains (Ck/Swe/0658946/10 and Ita/90254/2005). Boxes indicate the likelihood of recombination between strains. The star marks the position of the Australian vaccine strain and the dots the position of other vaccine strains.