Infectious bronchitis is a highly contagious and widespread disease of chickens and is a major ongoing problem in all countries with an intensive poultry industry (Sapats et al., 1996; Casais et al., 2003) . The aetiological agent is infectious bronchitis virus (IBV), a member of the family Coronaviridae (Beaudette & Hudson, 1937; Tyrell, 1968) , subfamily Coronavirinae and genus Gammacoronavirus (group 3; Cavanagh et al., 1994) . While IBV has been detected in other avian species, including guinea fowl (Numida meleagridis), partridge (Alectoris sp.), peafowl (Pavo cristato) and the blue-winged teal (Anas sp.), clinically detectable disease has only been observed in chickens (Liu et al., 2005; Cavanagh, 2007; Sun et al., 2007) .
IBV has a positive sense, single-stranded RNA genome approximately 27.6 kb in length (Tannock, 1973; Watkins et al., 1975; Schochetman et al., 1977; Jackwood & de Witt, 2013) with the general organization 5′ untranslated region (UTR) -1 a, ab (or polymerase genes) -S -3 a, b, c (E) -M -4 b, c -5 a, b -N -6b -3′ UTR (Cavanagh, 2007; Ammayappan et al., 2008) . The genes encoding the structural proteins are S (spike glycoprotein), E (envelope glycoprotein), M (membrane glycoprotein) and N (nucleocapsid protein) (Ignjatovic et al., 1997; Cavanagh, 2007) , while the 3a, 3b, 4b, 4c, 5a, 5b and 6b genes encode accessory proteins (Liu et al., 1991; Liu & Inglis, 1992a, b; Cao et al., 2008; Hewson et al., 2011; Bentley et al., 2013) , the functions of which are still unknown (Liu et al., 2008) . The S glycoprotein is a virion surface, rod-shaped protein that is post-translationally cleaved into two subunits, S1 and S2 (Cavanagh, 1981; Stern & Sefton, 1982; Cavanagh, 2007) .
During IBV infection, virions bind to the target cell receptors and release the viral genome into the cytoplasm of the host cell (Ziebuhr, 2005; Knoops et al., 2008) . The polyproteins 1a and 1ab (pp1a and pp1ab) are then translated, and the papain-like (PLP) and 3-chymotrypsin-like (3CL) proteases are released from these polyproteins after an autocatalytic process. These proteases initiate the cleavage in trans of the 15 peptides contained in pp1a/pp1ab. These peptides, probably together with some cellular proteins, form the replication-transcription complex. This initiates the transcription of a series of 3′ nested subgenomic RNAs that are translated into the viral structural and accessory proteins (Gorbalenya et al., 1989; Liu & Brown, 1995; Tibbles et al., 1996; Liu et al., 1998; Ziebuhr et al., 2001; Ziebuhr, 2005) .
In Australia, three genotypic subgroups of IBV have been distinguished (Hewson et al., 2012) . Subgroup 1 or "classical" strains were isolated between 1962 and 1984 and include the subtype B vaccines, which were classified using serological methods (Wadey & Faragher, 1981; Ignjatovic & Galli, 1995) and vaccine-related strains. Subgroup 2 or "novel" strains emerged between 1988 and 1994, while subgroup 3 strains, which emerged around 2002, are recombinants derived from subgroup 1 and 2 strains (Ignjatovic et al., 2006; Hewson et al., 2012) .
Four different IBV strains are included in the commercially attenuated vaccines available for the control of infectious bronchitis in Australia. Three of these, VicS, I (Inghams) and S (Steggles), belong to the same serotype (subtype B), while A3 (Armidale) is serotypically distinct (subtype C; Ignjatovic et al., 2006; Hewson et al., 2012) . Previous studies (Hewson et al., 2012) have demonstrated that the VicS vaccine contains two subpopulations, referred to as VicS-v (the predominant subpopulation in the vaccine) and VicS-del, which has a 40-nucleotide deletion in the 3′ UTR and multiple single-nucleotide differences in the structural protein genes compared with VicS-v (Hewson et al., 2012) . These viral subpopulations differ in their ability to replicate in vitro and in their pathogenicity in vivo. In VicS-v inoculated groups, all the birds exhibit clinical signs, including head shaking and eye scratching/irritation, from day 2 to day 8 post-inoculation, whereas in the VicSdel inoculated group the clinical signs are less severe and only seen between days 6 and 9 post-inoculation. Birds inoculated with VicS-v also have significantly more severe tracheal lesions than those inoculated with VicS-del.
Over the last few years, a number of complete genome sequences of many IBVs have been determined and the sequences of more than a hundred are now available in GenBank (National Center for Biotechnology Information, NCBI). However, the complete genome of an IBV isolated in Australia has not been determined. As the pathogenicity and replication of the two IBV subpopulations, VicS-v and VicS-del, in the VicS vaccine, differ in vivo and in vitro under laboratory conditions, the complete genome sequences of these two variants were compared in order to identify potential novel molecular determinants of pathogenicity and replication in IBV.
Virus. The VicS-v and VicS-del strains of IBV, purified previously in our laboratory, were propagated in specific pathogen-free embryonated hen eggs (Australian SPF Services, Woodend, Victoria, Australia), as described previously (Hewson et al., 2009) . After 8-9 days of incubation, the allantoic cavities of the specific pathogen-free embryonated eggs were inoculated with one of the two strains. After 48 h of incubation, the eggs were chilled for 12-18 h at 4°C and the allantoic fluid collected and stored at -80°C.
Viral purification and nucleic acid extraction. The allantoic fluid was clarified as previously described (Lougovskaia et al., 2002) , with slight modifications. The gradients were fractioned into 1 mL volumes, and the fractions containing the highest concentration of virus were determined by PCR using primers and a protocol described previously (Hewson et al., 2009) . After centrifugation at 90,000 Â g for 1 h at 4°C the viral pellets were resuspended in 250-350 µL of Tris-buffered saline (pH 7.4; 0.02-M Tris-HCl, 0.15-M NaCl) and stored at -80°C. Viral RNA was extracted using RNeasy™ extraction kits (Qiagen Pty. Ltd., Chadstone, Victoria, Australia), following the manufacturer's protocol, and then stored at -80°C.
Complete genome sequencing. Complete genome sequencing of the VicS-v and VicS-del strains of IBV was performed using the PGM Ion Torrent™ platform (Life Technologies Australia Pty. Ltd., Mulgrave, Victoria, Australia). RNA was fragmented using RNase III (Life Technologies) and purified using magnetic beads. To construct the libraries, the RNA was reverse-transcribed using SuperScript™ III (Life Technologies). The cDNA was purified and amplified following the manufacturer's protocols and then sequenced using the 314 chip, 200 base sequencing Ion OneTouch™ Kit v2.
All the reads that matched to chicken ribosomal RNA and mitochondrial genome sequences were discarded. For VicS-v, the remaining reads were mapped to the genomes of the Beaudette strain [Genbank accession number (GAN) NC_001451], two vaccine strains from USA (Conn46 1996 and Massachusetts, GAN FJ904716 and GQ504724, respectively) and two strains from China (SAIBK and SC021202, GAN DQ288927 and EU714029, respectively) using Geneious™ version 6.1.4 (Biomatters Ltd., Auckland, New Zealand). Because of the high level of sequence diversity in the S gene, the readings were also mapped to the previously determined sequence of structural protein genes from the VicS vaccine (GAN JN176213). For VicSdel, the readings were mapped to the genome of VicS-v and the previously determined sequence of the structural protein gene region of VicS-del (GAN JN983807). All gaps and ambiguous sequences were corrected using Sanger sequencing and the Big Dye™ Terminator v3.1 sequencing protocol (Life Technologies), following the manufacturer's instructions. as well as three turkey coronavirus (TCoV) sequences and sequences of coronaviruses isolated from a duck and a peafowl in China (GAN EU022525, TCoV-540; EU022526, TCoV-ATCC; GQ427174, TCoV/TX-GL/01; JF705860, Duck CoV; and AY641576, Peafowl, respectively), were used for phylogenetic analyses. The sequences were aligned using Clustal-Omega (Sievers et al., 2011; www.ebi.ac.uk) and the phylogenetic tree constructed using Geneious™ 6.1.4, using the nearest neighbour interchange maximum likelihood heuristic method with 100 bootstrap replications and the general time-reversible (GTR) substitution model, gamma distributed with invariant sites (G+I). The GTR+G+I substitution model was selected as the ideal model for these data using the "Find Best DNA/Protein Model" tool, available in Mega5.2 (Tamura et al., 2011) .
Phylogenetic trees were also constructed for individual peptides encoded by the polymerase genes, the main proteinase, also referred to as 3CL, the helicase (Hel), the PLP and the RNA-dependent RNA polymerase (RdRp), and also for the S1 glycoprotein. The sequences were aligned with ClustalW (Geneious™ 6.1.4) and the phylogenetic trees constructed as described above. In these phylogenetic trees, only the VicS-v sequence was included (except for the S1 glycoprotein tree, where both VicS-v and VicSdel were included), as the VicS-v and VicS-del sequences were highly similar (99.8%), and VicS-v is the main component of the VicS vaccine (Hewson et al., 2012) .
To assess the extent of potential recombination between viruses, a network tree was constructed using the same complete genome alignments as described above and SplitsTree 4.13.1 (Huson & Bryant, 2006) . The distances were calculated with the uncorrected P-method and the network constructed with Neighbor-Net using 1000 bootstrap replicates.
The sequences of the complete genomes of VicS-v and VicS-del have been submitted to GenBank under accession numbers KF460437 and KF931628, respectively.
Sequencing and coverage. For VicS-v and VicS-del, the Ion-Torrent 314 chip generated 52.9 and 130.3 Mb of data, comprising 503,622 and 480,037 reads, respectively, with mean read lengths of 102.9 and 124.2 nucleotides, respectively. After removal of the host-matched reads, a total of 466,235 and 456,359 reads were retained (92.57% and 95.06%), respectively. For VicS-v, a total of 212,507 readings (45.6%) were matched with the complete genome sequence of the Beaudette strain. The mean depth of coverage was 716 fold. A total of 98,124 reads could be mapped to the structural protein region, yielding a mean depth of coverage of 1200 fold and a minimum depth of 92 fold. For VicS-del, 285,759 reads (62.6%) were matched with the complete genome sequence of the VicS-v strain. The mean depth of coverage was 760 fold. A total of 95,802 reads could be mapped to the structural protein region, yielding a mean depth of coverage of 925 fold and a minimum depth of 126 fold.
The VicS-v and VicS-del genomes were 27,610 and 27,567 nucleotides in length, with a G + C content of 38.2% and 37.8%, respectively. Vics-v had the typical IBV genome organization (5′ UTR -1a/1ab -S -3 a, b, c (E)-4 a, b -M -5 a, b -N -3′ UTR; Stadler et al., 2003; Cavanagh, 2007; Thor et al., 2011) , while VicS-del had a truncated 4b open reading frame (ORF).
Comparisons of the complete genomes of VicS-v and the more attenuated VicS-del ( Figure 1 ) revealed several differences, which are summarized in Table 1 . The most significant of these differences found in VicS-del were the absence of a glycine (Gly) in two Gly-Gly motifs in VicSdel and two insertions-deletions (indels) in the nucleotide sequence of the non-structural protein 6 (nsp6) gene, which would lead to a change in 11 amino acids. This frameshift introduced two positively charged amino acids, changing the region from predominantly hydrophobic, and resulted in the loss of a third Gly-Gly motif. There was also a single-nucleotide polymorphism (SNP) in the S gene at nucleotide 3475, close to the 3′ end of the gene, with approximately equal numbers of VicS-v genomes having either a G or a U, while in VicS-del most genomes had a U in this position. Finally, a frameshift mutation resulting in the truncation of ORF 4b and a 40-nucleotide deletion in the 3′ UTR region were recognized in the genome of VicS-del.
The SNP in the S gene generated two non-synonymous codons, GAA (glutamate) or UAA (a premature stop codon), nine codons prior to the usual S gene stop codon. This SNP also lies within the body transcription (B-TRS) for mRNA 3, which encodes the accessory proteins 3a and 3b, and the structural protein E. In VicS-v, 48.4% of the reads (595/ 1222) contained the codon GAA and 51% contained the UAA stop codon. In VicS-del, 5.5% of the reads contained the GAA codon and 93.6% the UAA codon (Table 1) .
A second Gly-Gly motif, which has been described as a PLP cleavage site (Ziebuhr, 2005) , was identified in the VicS-v sequence between nucleotides 7306 and 7311.
Nucleotide alignment and phylogenetic analyses. Phylogenetic analysis assigned most of the genomes into two main clusters (Figure 2 ). The first major cluster (a) included predominantly USA strains, except for the Ck/Ch/ LDL/97I (China) and peafowl (China) strains. The peafowl (China) isolate lay within the Beaudette subcluster and has been described previously as very similar to a Mass 41 strain isolated in 1985 (Liu et al., 2005) . The USA cluster can be subdivided into the Mass 41 vaccine, Beaudette and Holte subclusters. The second cluster (b) contained only strains of Chinese origin, including a strain isolated from a duck, and these were subdivided in two subclusters (b1 and b2 in Figure  2 ). TCoV strains lay in a separate cluster, closely related to USA IBV strains. There was a high level of similarity between the Ita/90254/2005 (Italy) and Ck/Swe/0658946/10 (Sweden) strains, which have been described previously as European QX-like strains, a Chinese origin genotype widely distributed in Europe (Ducatez et al., 2009; Abro et al., 2012) . The strains Ck/Ch/LSD/05I (China) and Tw2575/98 (Taiwan) also shared a cluster. The VicS-v and VicS-del strains (Australia) form their own cluster, while Ibadan (Nigeria) and SNU8067 (South Korea) strains did not fall into any cluster.
In the phylogenetic trees of the 3CL, Hel, PLP and RdRp genes (Figure 3) , the Chinese and USA clusters were conserved. However, VicS-v grouped within the Chinese cluster in the 3CL and PLP gene phylogenetic trees and within the USA cluster in the Hel and RdRp gene phylogenetic trees.
In the S glycoprotein gene trees ( Figure 4 ) the isolates fell into distinct Chinese and USA clusters. In these trees, VicS-v, VicS-del and Ibadan lay closer to the USA cluster, while the South Korean, Taiwanese and European strains were positioned closer to the Chinese cluster. The Georgia 1998 and Delaware 072 strains were distinct from all other strains, and were located between the IBV and TCoV groups. A phylogenetic tree inferred using the amino acid sequences had the same cluster organization ( Figure 5 ).
Network tree analysis. In the analysis shown in Figure 6 , the boxes indicate the likelihood of recombination events between strains of IBV. The tree has the same cluster organization seen in the phylogenetic tree using whole genome sequences (Figure 2 ). The VicS-v and VicS-del branch splits from all clusters close to the centre of the tree, suggesting limited and historically distant recombination with other strains.
Although there has been considerable investigation of IBV and of coronaviruses in general, the determinants of their virulence and their capacity to replicate in vivo remain uncertain. As recent studies have suggested that the virulence determinants may not be located in the structural protein region of some IBVs (Armesto et al., 2009) , it can be inferred that these determinants may be encoded within the 1a and 1ab polyproteins. Therefore, studies of complete genome sequences could provide useful information about the position and characteristics of potential virulence determinants.
In this study, the genome sequences of the strains VicS-v and VicS-del were completed. The 1a and 1ab polyproteins of IBV are post-translationally cleaved by virus-encoded proteinases into 15 peptides (nsp2 to nsp16). Nsp1, which is found in all other coronaviruses, is absent in IBV, but nsp2 is considerably larger (Liu et al., 1998; Snijder et al., 2003; Stadler et al., 2003; Gorbalenya et al., 2004; Ziebuhr, 2005; Fang et al., 2007; Sawicki et al., 2007; Carbajo-Lozoya et al., 2012) . In order to determine the location of the 15 peptides encoded by the ORFs in the 1a and 1ab polyproteins, we used the annotations of the Beaudette strain (GAN NC_001451). We complemented these annotations with those of Ziebuhr (2005) . The locations of these peptides within ORFs 1a and 1ab and their first and last amino acid residues are summarized in Table 2 .
As discussed above, VicS-v exhibits higher rates of replication in vitro and causes more severe clinical signs and tracheal lesions in vivo than VicS-del. As the genomes of these two viruses have very high levels of nucleotide sequence identity, it can be inferred that any differences in their genomic sequences may influence these differences in virulence and replication.
As described previously, Gly-Gly is the cleavage motif for PLP (Lim et al., 2000; Ziebuhr, 2005) . VicS-del lacks three Gly-Gly motifs seen in VicS-v that are located in the middle of nsp2, nsp6 and nsp14. These motifs may result in additional proteolytic processing of these three nsps, and the absence of them in VicS-del could alter their function. Nsp6 and nsp14 are reported to contain transmembrane (TM) and exonuclease domains (Ziebuhr, 2005) , while the function of nsp2 remains undetermined. Highly conserved Gly-Gly motifs were also found between nucleotides 2044 and 2049 (within nsp2), 3667 and 3672, 4426 and 4431 (within nsp3), 9199 and 9204, and 9391 and 9396 (within nsp5) in both VicS-v and VicS-del and in other IBVs. These motifs may also be further processed by PLP, although the previously characterized PLP cleavage sites (between nsp2 and 3, and nsp3 and 4) have an extended motif of Lys-Ala-Gly-Gly, which has been identified in all the complete genome sequences analysed in this study; this extended motif is not seen at these additional Gly-Gly sites. Positions were based on 1ab from IBV Beaudette strain (accession number NC_001451), and modified according to Ziebuhr (2005) , with position 1 the methionine at the beginning of ORF 1a and 1ab. b Based on Phillips et al. (2012) . The mutations in nsp6 of VicS-del (Table 1 ) may have implications for the replication of this virus, which would be consistent with data from previous in vivo studies (Hewson et al., 2012) . During infection 3CL, the main protease, is auto-catalytically released (Ziebuhr et al., 2001; Ziebuhr, 2005) . There may be an interaction between the 3CL (nsp5), the TM2 (nsp4) and the TM3 (nsp6) hydrophobic domains (Ziebuhr, 2005) , which are also referred to as MP1 and MP2 (Tibbles et al., 1996) , during this proteolytic process. The importance of TM3/MP2, the transmembrane domain downstream of the 3CL proteinase, in the autocatalytic processing of 3CL has been suggested by in vitro assays (Tibbles et al., 1996) . In this experiment, the further processing of a 66-kDa immature protein product into smaller, post-translationally cleaved products of 59, 42, 35 and 24 kDa (with the 35-kDa peptide the size predicted for the fully processed form of 3CL protease) did not occur if these two hydrophobic domains were not associated with microsomal membranes. Changes in TM3/ MP2, such as those detected in VicS-del, could affect the membrane association of this domain and thus the conformation of the protein, and the autocatalytic release of 3CL, which is essential for the initial stages of replication (Ziebuhr, 2005) .
The SNP detected at the end of the S gene is also notable. The difference in the proportions of each of the nonsynonymous codons between the VicS-v and VicS-del populations, with a greater prevalence of the truncation phenotype in the VicS-del population, suggests that truncation of the S2 protein may be related to the difference in virulence between these strains. The carboxyl terminal end of S2 anchors the S protein to the virion and also assists in membrane fusion with target cells (Cavanagh, 2007) . Alternatively, the differing B-TRS sequences, CUGAA-CAA and CUUAACAA, which have both been identified previously in IBV (Mondal & Cardona, 2007; Woo et al., 2010) , may differ in their efficiency in regulating replication and transcription and thus may influence production of mRNA 3, which encodes the E protein. In the Middle East respiratory syndrome coronavirus the deletion of the E protein results in a replication-competent, propagationdefective virus (Almazán et al., 2013) . A nine amino acid truncation, resulting from the nucleotide change from U to G, has been described previously in an M41 strain variant, M41H (Kusters et al., 1989) . While these alternative codons have also been found in other IBV strains, the methods used for sequence determination would have precluded identification of differing subpopulations within these strains, as cloned, single cDNA molecules were used for sequencing.
The ORFs 4b and 4c have been described in IBV (Hewson et al., 2011 (Hewson et al., , 2012 Thor et al., 2011; Phillips et al., 2012; Bentley et al., 2013) , as well as in TCoV (Cao et al., 2008) , and have been synthesized in vitro (Bentley et al., 2013) , but the products of these ORFs are yet to be identified in infected cells, and their function remains unknown. Both these ORFs could be identified in the VicS-v genome, but in VicS-del, 4b was divided into two smaller ORFs due to a singlenucleotide deletion.
The VicS vaccine is known to induce some clinical signs after vaccination. Hewson et al. (2012) showed that VicS-v caused more severe vaccine reactions than VicS-del, had a higher replication rate in the trachea, kidneys and caecal tonsils, and induced more severe clinical signs. The putative 4b and 4c proteins have two interesting features: their high content of positively charged amino acids (Lys and Arg) and the presence of phosphorylation motifs, suggesting a possible role in the packaging of the genome, which may influence the rate of viral replication and therefore pathogenicity.
Phylogenetic analyses showed that the Australian strains of IBV are distinct from strains isolated elsewhere in the world and that the most commonly used Australian vaccine strain is very distinct from other IBV vaccine strains across the entire genome. This divergence has been shown previously in the structural genes (Yu et al., 2001; Bochkov et al., 2007) , but this is the first study to show that the divergence can be seen consistently throughout the genome. However, most of the fully sequenced IBV genomes are of strains with an origin in the USA or China, as only a few QX-type and QX-like strains (which now predominate in Europe) have been fully sequenced, and this bias could have influenced the topology of the tree. These European strains were represented in this tree by strains from Italy (Ita/90254/2005) and Sweden (Ck/ Swe/0658946/10). The phylogenetic tree constructed with the nucleotide sequences of the S1 protein ( Figure 4 ) includes more QX-type and QX-like strains, including Ck/ UK/KG3P/10 and Ck/UK/3355/09 (UK), Ck/Ger/GB1835/ 10 (Germany), Ck/Swe/0658946/10 and Ck/Swe/082066/ 10 (Sweden), Ita/90254/2005 (Italy) and Ck/Sp/170/09 and La/Sp/116/09 (Spain). Phylogenetic trees of individual ORFs (Figures 3, 4 and 5) indicate that different regions of the VicS-v genome varied in their similarity to the two major clusters, suggesting historical recombination events. However, this seems very unlikely to have happened recently, based on the results from the network tree analysis ( Figure 6 ). VicS-v diverged and evolved as a distinct cluster, with the length of the VicS-v branches, especially in the 3CL, Hel and RdRp phylogenetic trees, suggesting more rapid evolution than most other strains.
In conclusion, full genome analysis of VicS-v and VicSdel has shown that the Australian IBV vaccine VicS has been evolving independently of strains elsewhere in the world for some time. The differences in the replication rate and pathogenicity of these two viral subpopulations may be attributable to the mutation in the TM3 domain in nsp6 and the SNP found at the end of the S glycoprotein gene, described in this study for the first time. These newly described differences are additional to the previously described frameshift/truncation of the ORF 4b and the 40nucleotide deletion in the 3′ UTR in VicS-del (Hewson et al., 2012) . Further analyses of the complete genome sequences of other Australian IBV strains may reveal additional information about relationships between these distinct vaccine strains and IBVs within the USA and Chinese clusters. Additional investigation of the effect of the differences between the VicS-v and VicS-del strains described in this study may shed more light on the replication and pathogenicity of IBV and may also indicate whether the naturally attenuated strain VicS-del and other strains with similar mutations have potential as novel vaccine candidates for the control of IBV infections.