Genomic characterization of a proventriculitis-associated infectious bronchitis coronavirus.

Transmissible proventriculitis associated with infectious bronchitis virus (IBV) was at first seen in eastern China in mid-1995, and is now endemic in China. Herein, the complete genome sequence of a proventiculitis-associated infectious bronchitis coronavirus (ZJ971) was sequenced and analyzed. Compared with the genome of the vaccine strain H120, ZJ971 had 54 nucleotide substitutions and a deletion in the 3'-UTR. The substitutions were in the regions of nsp2-nsp5, nsp7, nsp12, nsp13, nsp15, S and N genes, and the untranslating region. The results indicated that ZJ971 could be a variant of IBV strain H120.

Proventriculitis has been seen in chickens throughout the world since the early 1970s [1]. However, the causative agent has not yet been definitively established. Both infectious and dietary agents have been implicated as contributing causes [1]. Some studies have reported infectious bronchitis virus (IBV) to be a candidate etiological agent [2, 3]. In mid-1995, there was an outbreak of disease in the eastern China characterized by a swollen proventriculus and since then this disease has become endemic in China. Several proventriculitis-associated IBV strains have been isolated and analyzed [4-7], but the full genomes of these viruses have not yet been determined. Here, we report the complete genome sequence of the proventriculitis-associated IBV isolate ZJ971 [4, 8].

The entire genome of ZJ971 is 27,627 nucleotides (nt) in length (GenBank accession No. EU714028), with an A + T content of 61.8%, excluding the polyA tail, and has the same genomic organization as the classical IBV strain Beaudette [9]. Its 5′-UTR is 528 (1–528) nt in length. Gene 1 (polymerase gene) consists of 19,836 nt, encompassing ORF 1a and ORF 1b. ORF 1a is 11,802 nt in length (529–12,330) and encodes a polyprotein of 3933 amino acids (aa; polyprotein 1a, pp1a), while ORF 1b is 7,959 nt in length (12,405–20,363), and encodes a protein of 2652 aa (pp1b). Gene 2 (spike, S) of ZJ971 is 3,489 nt in length (20,314–23,802), and encodes a protein of 1162 aa. There is a 50 nt overlap between the 3′-end of ORF 1b and the 5′-end of the S gene. Gene 3 is 678 nt in length, and contains three ORF: 3a (23,802–23,975), 3b (23,975–24,169), and the envelope (E) protein (3c, 24,150–24,479). The length of gene 4 (membrane protein, M) is 678 nt (24,451–25,128), with a 29 nt overlap between the 3′-end of the E gene and the 5′-end of the M gene. There is a non-coding region of 359 nt between the 3′-end of the M gene and gene 5. Gene 5 contains ORF 5a (25,488–25,685) and 5b (25,682–25,930). Gene 6 (25,873–27,102) encodes the N protein (409 aa). There is a 58 nt overlap between the 3′-end of 5b and the 5′-end of the nucleocapsid (N) gene. The 3′-UTR of ZJ971 is 525 nt long, 205 nt and 20 nt longer than those of strains M41 (AY851295) and Beaudette (NC_001451), respectively.

The complete genomic sequence of ZJ971 is very similar to that of the vaccine strain H120 (FJ888351), which is widely used in China, with 99.8% sequence identity, and only 54 nucleotide substitutions and a deletion of three bases in the 3′-UTR detected in ZJ971. The 54 nucleotide substitutions were distributed throughout the genome, two in 5′-UTR, 35 in gene 1, 14 in the S gene, two in the N gene, and one in the 3′-UTR. The 35 nucleotide substitutions in gene 1 resulted in 19 amino acids changes, with six in non-structural protein (nsp) 2 (198T → A, 322G → D, 331S → P, 358A → T, 475A → V, and 501 V → I), three in nsp3 (1126 N → D, 1489 N → D, and 1910R → K), three in nsp4 (2348I → M, 2498 N → T, and 2562R → K), two in nsp5 (2772G → V and 2879A → T), two in nsp7 (3368E → K and 3418I → V), one in nsp12 (4488P → Q), one in nsp13 (4919P → S), and one in nsp15 (6129I → M). The 14 nucleotide substitutions in S gene resulted in nine aa substitutions, including seven in the S1 subunit (19A → V, 52 I → V, 118G → V, 122I → L, 130S → F, 355E → Q, and 440T → I) and two in the S2 subunit (1047S → F and 1136R → K). The two nucleotide substituions in the N gene resulted in one amino acid change (22S → P). There are eight epitope clusters within the S glycoprotein of IBV strain D207, located between amino acid residues 24–61 (S1D), 132–149 (S1E), 291–398 (S1C/A/B), 497–543 (S1F), 548-574(S2G), and 1095-1118(S2H) [10, 11]. Three of the nine aa difference between the S proteins of H120 and ZJ971 were located in these epitope clusters (52 I → V, 130S → F, and 355E → Q).

These findings suggest ZJ971 could be a variant of IBV strain H120, with mutations distributed throughout the genome.