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Amir H. Noormohammadi,1
Peter Hooper,2
Jagoda Ignjatovic,1,2 and
Glenn F. Browning1*
Department of Veterinary Science, University of Melbourne, Parkville, Victoria 3010,1 CSIRO Livestock Industries, Australian Animal Health Laboratory, Private Bag No. 24, Geelong, Victoria 3220, Australia2
Received 4 August 2007/ Accepted 14 November 2007
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All coronaviruses maintain a set of essential genes, including those that encode the polymerase (Pol), spike (S), small membrane (E), membrane (M), and nucleocapsid (N) proteins, in the invariable order 5'-Pol-S-E-M-N-3' (24). In addition to these essential genes, the genomes of all coronaviruses contain group-specific, or accessory, genes, which encode small proteins of unknown function (24, 35). The 27.6-kb IBV genome contains nine functional genes, four of which encode structural proteins. The polymerase gene, gene 1, occupying approximately two-thirds of the genome at the 5' end, encodes two partially overlapping open reading frames (ORFs), 1a and 1b. Gene 2 consists of one ORF that encodes the spike glycoprotein (S), which is posttranslationally cleaved into the amino-terminal S1 (92-kDa) and the carboxyl-terminal S2 (84-kDa) subunits (24). In the mature virion, S2 associates with S1, anchoring the S1 protein to the membrane to form the multimeric coiled-coil S protein (3). The S1 subunit is involved in virus entry and also contains epitopes for virus-neutralizing and hemagglutination-inhibiting antibodies (5, 22, 29). The S1 sequences from different strains vary significantly, usually by between 2 and 25% at the amino level, whereas the S2 subunit is conserved (24).
Gene 3 contains three ORFs, 3a (174 nucleotides), 3b (195 nucleotides), and 3c (321 nucleotides). ORF 3c encodes the E protein, which is a structural protein required for virion assembly, while 3a and 3b encode nonstructural proteins of unknown function (24). ORFs 3a and 3b have been found in all IBV isolates discovered thus far (26) and also in other group 3 coronaviruses (turkey and pheasant coronaviruses) (6, 7). Recently, it was shown using reverse genetics that neither the 3a nor the 3b protein is essential for IBV replication in vitro and that these proteins can thus be considered to be accessory proteins (17). In a study using plasmid expression, Liu and Inglis (27) suggested that the translation of ORF 3c, which encodes the E protein, is dependent on the upstream sequence elements (3a and 3b), which together may serve as an internal ribosomal entry site. However, the suggestion that these genes were essential for initiating the translation of the E protein has now been challenged by the generation of mutant IBVs lacking 3a and 3b genes (17). Gene 4 contains one ORF encoding the M glycoprotein, which is essential for the production of coronavirus-like particles (12, 24). IBV is unique among members of genus Coronavirus in that it has two ORFs in gene 5 (5a and 5b). Gene 5 is present in all group 3 coronaviruses characterized to date, but it was shown to be dispensable for replication in vitro using reverse genetics (1). Gene 6 has one ORF, encoding the N protein, which, together with the genomic RNA, forms the helical nucleocapsid (12, 24). This protein is highly conserved, differing between different IBV isolates by only 2 to 6% at the amino acid level (37, 38). There is an untranslated region (UTR) located immediately downstream of gene 6, which is thought to be important for the initiation of negative-strand RNA synthesis. This region has been found to be highly conserved among different strains of the same coronavirus (10, 36). In porcine, canine, and feline coronaviruses, the 3' UTR contains at least one ORF, but functional ORFs have not been detected in the 3' UTR of IBV (30).
Coronaviruses replicate via the synthesis of a 5'-coterminal nested set of subgenomic mRNAs. Preceding the coding sequence of each subgenomic mRNA are transcription regulation sequences (TRSs) that lie 5' of the first ORF. The TRSs are conserved in each coronavirus group, and the distance between the TRS and the first ORF is different in each subgenomic mRNA of different coronaviruses.
Antigenic analyses of S, N, and M proteins (20) and sequence analyses of S1 and N genes (30, 31) of Australian IBV strains have identified two genotypically and antigenically distinct groups, tentatively classified as "classical" and "novel" strains. The classical group shared 80.7 to 98.3% identity in the deduced amino acid sequences of their S1 regions, whereas novel strains shared only 53.8 to 61.7% identity with classical strains at the amino acid level in this region (31). Analysis of the N gene confirmed the observations made with S1, that classical and novel strains belonged to phylogenetically distinct groups of viruses. The 3' UTR immediately downstream from the N gene was also distinct, with extensive deletions apparent in the novel strains (30). A longitudinal study of IBV strains in commercial poultry in Australia indicated that these novel IBV strains emerged suddenly at three geographically distant commercial sites. The novel strains persisted at the sites at which they were initially isolated for a period of 3 to 4 years, causing respiratory disease and mortalities and necessitating the introduction of vaccines to control the problem (21).
In this study, the 3' 7.5 kb of the genomes of 17 different Australian IBV strains, including four novel strains, was sequenced and analyzed to compare the genes encoding their structural (S, E, M, and N) and nonstructural (3a, 3b, 5a, and 5b) proteins in order to better understand the evolution of IBV in its natural host. Some phenotypic characteristics of the novel and classical strains were also compared in embryos and in chickens.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Isolation of IBV from field samples and propagation in embryonated eggs. Samples of kidney and trachea were pooled, and 10% (wt/vol) tissue suspensions were made in 0.1% phosphate-buffered saline (PBS) containing 100 U penicillin and 100 µg streptomycin/ml. After 12 h of incubation at 4°C, 200 µl of supernatant was inoculated into the allantoic cavity of 9- to 11-day-old embryos of SPF chickens (SPAFAS; Woodend, Victoria, Australia). Five eggs were used for each sample. The inoculated eggs were incubated at 37°C and candled daily. After 72 h of incubation, the eggs were chilled at 4°C, and allantoic fluids were harvested, pooled, clarified by centrifugation at 3,000 x g, and stored at –70°C.
Virus titration and virus neutralization tests. Virus titers were determined in tracheal organ cultures (TOCs) made from SPF chicken embryos at 19 days of incubation and expressed in median ciliostatic doses (CD50). For each dilution, five rings with 100% of the cilia beating were used. Complete deciliation at 5 days after inoculation in at least three rings indicated that virus replication had taken place. Virus neutralization tests were performed with 100 CD50 of virus and twofold dilution series of sera (starting dilution, 1:20).
Growth of IBV in ovo. Approximately 100 CD50 of IBV strains Q1/76, N1/62, N1/88, Q3/88, V18/91, and V6/92 as allantoic fluid at egg passage level 4 or 5 were inoculated into the allantoic cavities of five 10- to 11-day-old SPF chicken embryos. After incubation for 48 or 72 h at 37°C, eggs were chilled overnight at 4°C, and allantoic fluid was collected, pooled, centrifuged at 3,000 x g for 30 min, and stored at –70°C.
Assessment of IBV growth in TOCs.
Medium was removed from five TOCs with 100% ciliary activity, 100 µl allantoic fluid, diluted with PBS to contain 10 CD50 of virus, was added, and the TOCs were incubated at room temperature to allow virus adsorption. After 1 h, 1 ml of PBS was added, the TOCs were incubated for 5 min, the inoculum was removed, and 1 ml of incubation medium was added (medium 199, containing 100 IU penicillin/ml, 100 µg streptomycin/ml, and 20 mM 
-methyl-D-glycoside). After 3 and 5 days of incubation, medium from the five rings was collected and pooled, fetal calf serum was added to a concentration of 10%, and the medium was stored at –70°C. The titer of virus in the medium was subsequently determined in TOCs as described above.
Assessment of growth and immunogenicity of IBV strains in chickens. SPF chicks (n = 20) housed in positive-pressure isolation units were inoculated intraocularly at 2 weeks of age with approximately 103 CD50 of each strain of IBV. At 3, 5, and 7 days postinoculation (p.i.), four chicks were euthanized, and their tracheas were collected aseptically. Tracheal scrapings were collected in 2 ml of medium 199 supplemented with 5% fetal calf serum, penicillin (100 IU/ml), streptomycin (100 µg/ml), and neomycin (50 µg/ml) and stored at –70°C until testing. Viral titers were determined for individual tracheal scrapings using TOCs as described above. At 6 weeks of age, serum was collected from the remaining chicks (n = 8), the chicks were then reinoculated intraocularly with 2 x 103 CD50 of the same virus, and serum was collected when the chicks were 10 weeks of age.
Assessment of pathogenicity of IBV strains. For each IBV strain, groups of 20 2-week-old chicks were inoculated intraocularly with approximately 103 CD50 of virus. At 3, 5, 7, and 10 days p.i., four chicks were euthanized, and their tracheas and the cranial right kidney lobes were removed, placed into 10% buffered formalin, sectioned, stained with hematoxylin and eosin, and examined histologically for lesions. The tracheal lesions scored included cellular infiltration, loss of cilia, squamous metaplasia of surface epithelium, and loss of glands. Renal lesions scored included cellular infiltration, edema, epithelial degeneration, and tubular dysplasia. Lesions were scored as follows: 0 for no lesions, 1+ for mild lesions, 2+ for moderate lesions, 3+ for severe lesions, and 4+ for extensive lesions.
Extraction of RNA. After 72 h of incubation, the eggs were chilled at 4°C, and allantoic fluids were harvested and tested for the presence of IBV using reverse transcription-PCR. Allantoic fluids containing IBV were clarified by centrifugation at 1,100 x g at 4°C for 10 min, and virus was pelleted by centrifugation at 34,000 x g at 4°C for 1 h. The virus pellet was resuspended in sterile PBS at a 1:100 dilution of its original volume and stored at –72°C. Kidney and lung tissues from diseased birds were prepared as 10% to 15% suspensions in Tris-buffered saline by repeated passage through a three-way tap or by grinding with a mortar and pestle. Tracheas were scraped with a scalpel blade, and the tracheal mucosa was then homogenized by passage through a three-way tap in Tris-buffered saline. Purification of RNA was performed using RNeasy kits (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Approximately 50 µl of virus from allantoic fluid or 100 µl of homogenized tissue was used for each extraction, and RNA was eluted in 30 µl of buffer. The extracted RNA was used as a template in a reverse transcription reaction using a method described previously (28).
Synthesis of cDNA. For the synthesis of cDNA, 5 µl of extracted RNA was denatured at 100°C for 1 min, cooled by placing it on ice for 5 min, and then mixed with 20 µl of premix containing 10 µl diethyl-pyrocarbonate-treated water, 0.5 µM oligo(dT), 0.65 U RNAguard, 50 µM each of dATP, dTTP, dGTP, and dCTP, 5 µl of 5x reaction buffer, and 50 U of Moloney murine leukemia virus reverse transcriptase. The reaction mixture was incubated at 42°C for 1 h and subsequently incubated at 100°C for 5 min to inactivate the reverse transcriptase. The resultant cDNA was immediately used for PCR or stored at –70°C for later use.
PCR. For the amplification reaction, two primers, POLY-F1 (5'-GATTGTGCATGGTGGACAATG-3') and UTR-R1 (5'-CTGTACCCTCGATCGTACTC-3'), binding to the 3' end of the polymerase gene (nucleotides 20,070 to 20,090; Beaudette strain; GenBank accession number NC_001451) and the 3' UTR (nucleotides 27,489 to 27,508; Beaudette strain) of the IBV genome, respectively, were used to amplify a 7.5-kb fragment of the IBV genome that contains all the genes for the structural and nonstructural proteins. The PCRs were carried out in 50-µl volumes containing 50 µM each of dATP, dTTP, dGTP, and dCTP, 0.5 µM of each primer, 5 µl of 10x High Fidelity PCR buffer, 2 mM magnesium sulfate, 1.5 U platinum Taq high-fidelity DNA polymerase, and 5 µl cDNA as a template. Amplification was performed using 35 cycles of incubation at 94°C for 30 s, 57°C for 30 s, and 68°C for 8 min, with a final extension step at 68°C for 10 min. The resultant PCR products were separated in a 0.8% agarose gel.
Purification of PCR products. PCR products were purified using a PCR purification kit (Ultra Clean PCR Clean-Up; Mo Bio Laboratories) according to the manufacturer's instructions.
DNA sequencing. The 3' 7.5-kb PCR products from all the Australian IBV strains and field isolates were cloned in pGEM-T using the pGEM-T vector system cloning kit (Promega, Madison, WI). Different primers (13 to 15 in total) were designed and used for sequencing of the 3' 7.5 kb of the genome of the IBV strains. The cloned PCR products were sequenced using the Big Dye Terminator v3.1 cycle sequencing kit (Applied Biosystems), and the reaction products were sent to the Australian Genomic Research Facility, Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia, for analysis on an ABI Prism 3100 genetic analyzer.
Sequence analysis. The sequences of the different genes of the IBV strains were aligned using ClustalW (34). The nucleotide and amino acid sequence identities were calculated using the OldDistance program in the GCG package. Phylogenic trees were inferred from nucleotide sequences using the DNAml program in the PHYLIP package (14). To obtain the most accurate phylogenetic analysis, different transition/transversion rates were assessed, and the transition/transversion rate (1.35) that gave the tree with the greatest maximum likelihood was then used for inferences of all subsequent trees.
Molecular evolutionary genetic analysis. The relative abundances of synonymous and nonsynonymous substitutions in individual genes were determined using the Molecular Evolutionary Genetics Analysis (MEGA) program (23).
Nucleotide sequence accession numbers. GenBank accession numbers for the sequences obtained in this study are as follows: DQ490205 for Armidale, DQ490206 for N1/62, DQ490207 for N1/88, DQ490208 for N2/75, DQ490209 for Q1/73, DQ490210 for Q1/76, DQ490211 for Q1/99, DQ490212 for Q3/88, DQ490213 for S, DQ490214 for V1/71, DQ490215 for V2/02, DQ490216 for V2/71, DQ490217 for V3/02, DQ490218 for V5/90, DQ490219 for V6/92, DQ490220 for V18/91, and DQ490221 for Vic S.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(a) Gene 3. Gene 3 of the classical strains encoded three ORFs, 3a, 3b, and 3c, of 174, 195, and 321 nucleotides, respectively. The gene 3 region of the novel strains encoded only two ORFs (Fig. 1). The first ORF was 264 nucleotides in length and was present in all four novel strains. No significant similarity between this ORF and other coronavirus accessory genes or any other protein in the GenBank database was found. Therefore, this ORF in the novel IBVs was named X1 because the significance of the putative gene product was uncertain. The second ORF in the novel strains was similar to the 3c (E gene) ORFs of classical IBVs, although it differed in length between the novel strains: 318 nucleotides in N1/88 and Q3/88 and 312 nucleotides in V18/91 and V6/92 (Fig. 1). In the novel IBVs, there was a region between ORFs X1 and 3c (nucleotides 362 to 623) that corresponded in position to ORF 3b of classical strains but did not contain an ORF. No similarity between this region and any sequence in the GenBank database was found.
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The 3a and 3b genes were absent from the novel IBVs (Fig. 1). Sequence analysis of gene 3 (Fig. 1) in the novel IBVs revealed a potential TRS for the 3c gene upstream of its initiation codon. This putative TRS sequence was GTGAACAA in N1/88 and Q3/88 and GTGAGCAG in V18/91 and V6/92 and was located about 124 nucleotides upstream from the start codon (Fig. 1).
(b) Gene 5. The nucleotide and deduced amino acid sequences of gene 5 from 17 Australian IBV strains and the Beaudette strain were compared (Fig. 2). In the classical strains and the Beaudette strain, this gene encoded two ORFs, 5a and 5b, of 198 and 249 nucleotides, respectively. However, the sequences in the gene 5 region in V18/91 and V6/92 (nucleotides 280 to 545) (Fig. 2) did not contain an ORF of any significant length. The novel strains N1/88 and Q3/88, on the other hand, had a single ORF of 246 nucleotides in this region. This ORF was similar, but not identical, to ORF 5b in the classical IBV strains. Thus, the novel strains N1/88 and Q3/88 lacked ORF 5a but had ORF 5b. The predicted TRS for the 5b gene in N1/88 and Q3/88 was AGAAACAA and was located within the M gene sequence 124 nucleotides upstream from the start codon for the 5b protein (Fig. 2), the same distance between the predicted TRS for gene 3c in the novel IBVs and its start codon.
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Molecular evolutionary genetic analysis.
To investigate evolutionary pressures on different IBV genes, tests for neutral evolution, positive selection, and purifying selection were performed. Purifying selection results in selection against nonsynonymous substitutions at the DNA level, while positive selection results in selection in favor of nonsynonymous substitutions. For all tests, the null hypothesis was that synonymous substitution rates (dS) were equal to nonsynonymous substitution rates (dN), while the alternative hypotheses were as follows: dS 
dN (for neutral evolution), dN > dS (for positive selection), and dN < dS (for purifying selection). Tables 3 and 4 show the P values for these tests for all the structural genes for five classical and four novel IBVs. The five classical strains that clustered together phylogenetically were chosen for comparison. This group of strains included two of the three most commonly used vaccine strains in Australia and three field isolates. The tests were not performed for the small nonstructural genes because the sequences of these genes were very similar.
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Comparisons of growth in embryos and growth and immunogenicity in chicks. As strains N1/88 and Q3/88 were found to lack the 3a, 3b, and 5a genes and strainsV18/91 and V6/92 were found to lack these genes as well as gene 5b, we assessed the growth rates of these strains in embryos and in chickens. The classical strains Q1/76 and N1/62, which possess all four of these genes, were included for comparison. In embryonated eggs, TOCs, and infected chicks, all four novel strains had slower growth rates and produced less virus than the two classical strains (Table 5). The novel strains were also poorly immunogenic (Table 5). The virus-neutralizing antibody titers against homologous virus after a single inoculation and after a second inoculation were markedly lower in chicks infected with N1/88, Q3/88, V18/91, or V6/92 (titers of 80 to 160 after the second inoculation) than in chicks inoculated with Q1/76 or N1/62 (titers of 2,560 after the second inoculation).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Comparison of sequences of essential genes in IBV, in particular the S1 gene, has been commonly used to determine IBV strain relatedness and for elucidation of IBV evolution. In this study, comparisons of the entire 3' 7.5 kb of the genome, a region that includes all the essential and accessory genes except that for the polymerase, demonstrated the value of this approach for the analysis of IBV strains and revealed major differences between IBV strains. The absence of the 3a, 3b, 5a, and 5b accessory genes has not been reported for any other naturally occurring IBV, and in experimentally constructed variants of IBV, only the gene 3 or the gene 5 accessory gene has been deleted. Thus, this study has confirmed that gene 3 (with the exception of the E gene) and gene 5 are indeed accessory genes, as previously suggested (1, 17).
Sequence analysis of genes 3 and 5 of several IBV strains, and also of turkey and pheasant coronaviruses, has indicated that their nucleotide sequences, locations, and consensus TRSs are highly conserved (6, 7, 25). This level of conservation has led to the suggestion that the presence of genes 3 and 5 can be taken as a distinguishing feature of group 3 coronaviruses, reflecting the close evolutionary relationship of all members (25). Members of coronavirus groups 1 and 2 also possess accessory genes, the size and location of which are group specific (16, 24, 35). Recently, it was demonstrated that the location and the number of accessory genes also differ in bat coronaviruses (33). Although the functions of accessory gene products have not been fully elucidated, to date no naturally occurring coronavirus that does not have a full complement of accessory genes has been found, although a derivative of strain Beaudette that contains a truncated 3b gene was obtained after at least 35 passages in embryonated chicken eggs (32). Recombinant IBVs that lack ORFs 3a and 3b, or in which the expression of a complete gene 5 is interrupted, have been generated recently using reverse genetics (1, 17). These recombinants replicated in cell culture, as did the parent virus, suggesting that genes 3 and 5 are accessory genes. Our study confirms this observation and extends it by showing that both genes can be absent simultaneously and that the viruses lacking these genes can replicate in all the substrates in which IBV grows: in embryonated eggs, in chickens, and in tracheal explants. However, in contrast to the studies that examined recombinant IBVs (1, 17), we found that all of the novel IBVs had a reduced capacity to replicate compared to that of a classical IBV with a full complement of ORFs within genes 3 and 5.
Our study has further shown that genes 3 and 5 may not be determinants of pathogenicity, at least for the tracheal mucosa. While the novel strains did not induce renal lesion changes, it is difficult to attribute this to the lack of accessory genes alone, as strains of IBV that are more similar to the classical Australian strains also fail to cause renal lesions (19). IBV tissue tropism is determined by the S1 gene (2). Since the S1 genes of classical and novel IBVs differ significantly (31), it is just as likely that the difference in tissue tropism between the classical and novel strains is a result of the absence of receptor binding sites in S1. Using reverse genetics, Haijema et al. (16) deleted the nonstructural genes 3a, 3b, and 3c or 7a and 7b in feline coronavirus and showed that these deletions resulted in viruses that grew well in cell culture and were immunogenic, inducing high levels of neutralizing antibodies in cats, but did not induce any clinical signs. However, in our study, the immunogenicity of the novel strains was markedly reduced compared with that of the classical strains. It is not clear if this reduced immunogenicity was the result of less efficient replication (possibly because of the lack of accessory genes) or the lack of tropism for kidney tissue and the restriction of viral replication to the tracheal mucosa. However, the phenotypic characteristics of the novel IBVs cannot be unequivocally ascribed to the lack of accessory proteins unless comparisons are made using isogenic viruses.
The novel viruses had a novel ORF, designated X1, in the gene 3 region. The size of this ORF, 264 nucleotides, was significantly greater than the size of either ORF 3a (174 nucleotides) or 3b (195 nucleotides), and it has no sequence similarity to any other coronaviral structural or nonstructural gene, or any other gene in the database, including the novel ORF recently identified in bat coronaviruses. However, this ORF was conserved in all novel IBVs and had its own TRS, which differed from that of the 3a gene in the classical strains, but was located almost the same distance (24 nucleotides) upstream of the initiation codon for gene 3 as the TRS for gene 3 (23 nucleotides) in other group 3 coronaviruses (25).
It is not possible to determine whether the accessory genes were never present in the novel strains or whether they were deleted during their circulation in commercial poultry or some other host. All the novel strains appeared suddenly and simultaneously in three different locations in Australia, suggesting their introduction from an as-yet-unidentified source into commercial poultry. If deletions occurred following their introduction into commercial poultry, it is difficult to explain how very similar deletions occurred simultaneously in poultry reared at distant locations. The introduction of a progenitor virus that already lacked the genes would thus seem more likely. However, as some variants retained one of the four accessory proteins, and as there was already substantial variation between the protein sequences of some of the novel IBVs in 1988, it could be argued that either there were three (or more) independent introductions of the novel IBVs into commercial poultry in Australia or the introduction of a single variant (possibly initially associated with another avian species) occurred well before 1988.
Phylogenetic analysis of sequences of each of the structural genes suggested the same phylogenetic relationships between the classical and novel strains, indicating that all the structural genes of the novel IBVs evolved in parallel. Therefore, it seems that the origin of the novel strains involved neither mutation from, nor recombination with, classical strains.
In IBV, the spike protein, and especially the S1 part, interacts with host cell surface receptors and is thus a target for protective antibody responses, with resultant selection for amino acid changes. Therefore, this protein would be expected to be under continuous positive selection. Molecular evolutionary analysis of the structural genes of two vaccine strains and three closely related field isolates showed that only the S1 gene of these viruses was under positive selection, as might be expected if the field isolates were reisolates of vaccine strains that had been subject to immune selection during circulation in poultry flocks. In contrast, evolutionary analysis of the novel IBVs detected evidence of purifying selection in all the structural genes. The sudden appearance of these novel IBVs suggests that these viruses were introduced into chickens from another species and that the purifying selection observed may have been a consequence of their subsequent adaptation to chickens.
The origin of the novel IBVs has not yet been determined. The most likely explanation is that they were introduced into commercial poultry from another avian species. After their introduction, probably in 1988, when they were first detected, N1/88- and Q3/88-like strains circulated for a period of 4 years. Since the introduction of two vaccines based on strains N1/88 and Q3/88, the novel strains have not been detected. The disappearance of the novel strains in commercial poultry is not unexpected; most IBV variants persist only transiently, particularly if they are targeted by vaccination. A factor contributing to the disappearance of the novel strains could have been their reduced capacity for replication in chickens. In addition, the novel IBVs may have disappeared from the poultry scene because the deletions of the accessory genes are strongly attenuating, as was demonstrated in animal studies of murine hepatitis virus A59 and feline infectious peritonitis virus (11, 16). Hence, these viruses may have lost the competition with the "fitter" classical viruses.
Comparative sequence analyses of the S, E, M, and N structural proteins of three groups of coronaviruses, aimed at a revision of the taxonomy of the Coronaviridae, found that frequency distributions of the identities derived from comparisons of groups 1 and 2 generated two main clusters based on all the structural proteins for each group (15). Pairwise identities within group 3 (IBV and turkey coronavirus) were high, and three proteins, S, E, and M, were closely clustered. However, comparisons of N protein sequences revealed two clearly separated clusters, with the second cluster resulting from comparisons involving the three novel Australian IBV strains N1/88, Q3/88, and V18/91 (15). The results from our study, which has added the sequences of the S, M, and E proteins of N1/88, Q3/88, and V18/91 to the analyses, has confirmed that the group 3 coronaviruses contain two main clusters and reinforce suggestions for revision of Coronavirus taxonomy, possibly requiring the classification of these novel IBVs as distinct types of coronavirus.
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ACKNOWLEDGMENTS |
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We thank Philip Markham and Sen-Lin Tang for assistance with methods.
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FOOTNOTES |
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Published ahead of print on 28 November 2007.
Present address: Faculty of Veterinary Medicine, Urmia University, Urmia, West Azarbaijan, Iran.
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REFERENCES |
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| J. Bacteriol. | Mol. Cell. Biol. | Microbiol. Mol. Biol. Rev. |
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| Clin. Vaccine Immunol. | ALL ASM JOURNALS |
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, ORF X1; 
, 3a gene; 
, 3b gene; 
, 3c gene.
, 5a gene; 
, 5b gene.