Phylogenetic distribution and predominant genotype of the avian infectious bronchitis virus. Mutation analysis
Mutation analysis
S1 genes of the newly strains contain mutations, insertions and deletions, resulting in different lengths of nucleotides. S1 genes of these strains were generated and confirmed from three time sequencing results, contained 1641, 1647, 1650, 1653, 1656, 1659 and 1662 nucleotides, amino acids sequences ranging from 547 (LC strain) to 554 (LC strain). The length differences indicated amino acid insertions and deletions exist among the different strains.
Most variations in the deduced amino acid sequences of Chinese IBVs were observed among residues 63-69, 211-212 and 354-358 (numbering was with reference to S1 sequence of the Mass41 strain).
The precursor protein of S glycoprotein is cleaved into amino-terminal S1 and S2 protein by the protease during viral maturation [9]. In this study, the most common cleavage recognition sites of S1 gene were RRF(S/L) RR (49/80) or HRRRR (28/80) in the China field strains. The exceptional ones included CQ8 (RRTGR), HY52 (RRSKR), and HY2 (RRSKR). The cleavage sites of these two strains containing amino acids K, T, and G, were novel motifs compared to the reference strains, and quite different with the other isolates of the cleavage site.
Phylogenetic analysis of the isolated strains
A phylogenetic tree was constructed from the nucleotides sequences of the S1 glycoprotein genes. The 80 isolates IBV strains were clustered into five distinct genetic groups or genotypes which were considerably heterogeneous, including A2-type (49 newly isolated strains), 4/91-type (9 newly isolated strains), HN08-type (20 newly isolated strains), Gray-type and M41-type. The newly isolated strains mainly belonged to A2-type, 4/91-type and HN08-type branch. The phylogenetic relationship of strains at different times and geographical regions displayed complexity and diversity.
Strains isolated from Hubei, Zhejiang, Jiangsu, Guangdong, Guangxi and Fujian province mainly belonged to the A2 branch, also including other seven published IBV strains from China (QXIBV, CK/CH/LJL/07II, CK/CH/LJS/07IV, CK/CH/LSD/08-12, IBVSX4, LZ05 and LZ07). The isolated strains of Hainan province and a few isolated strains from Guangdong and Fujian province belonged to the HN08 branch, included PSH050513 and CK/CH/LCQ/08II. Group Gray-type was correlative with the American strain (Gray), included other two classical American strains (ARK99 and Holte), one Japanese strain (JP9758), and the exceptional field strain (CQ08). Most of the current vaccine strains (H120, H52, Ma5, M41, W93, 4/91 and 28/86) were belonged to the M41 branch, which including one field strains (NJ). However, the current pandemic strains were mostly 4/91-type, A2-type (QXIBV-type) and HN08-type, indicating that the field IBVs co-circulating in chicken flocks in China were evolutionarily distant from the known vaccine strains.