| Two field virus in Anhui province were isolated from commercial broiler chickens vaccinated with live Mass viral strain (H120). These isolates were identified by series experiments of biological character involving pathogenicity, dwarfing of chicken embryos, interference of NDV—clone-30 propagation, haemagglutination test and electron microscopy. All facts of experiments proved that the two virus strain is avian infectious bronchitis nephritis virus. Lastly they were designated as AH1-99 strain and AH2-03 strain respectively. In order to trace the origin and evolution of the two field strains, we have obtained the sequences of S1, S2, M and N genes by RT-PCR, cloning, sequencing. Using the software of Dnastar and referencing to others IBV strains' published sequences in Genbank, we have analyzed molecular characteristics of those structural genes and proteins and constructed phylogenic trees of S1, S2, M, N genes . S1 gene is composed of 1611-1617 nucleotides coding 537-539 amino acids. Sequence analysis revealed that the S1 gene is far more variable than other genes and the highly variable regions locate at the N-terminal and C-terminal of it. Six nucleotides GGGTCT were inserted in the site of 358-363 in AH2-03 strain S1 gene. Interestingly, there is one EcoR I RE site in S1 gene and three Hae III RE sites can be found. In the two strains S1 protein exist 16-18 potential glycosylation sites, and the quantity and location of these potential glycosylation sites varied. Compared with other IBV strains S1 protein, the epitope at 438-444aa disappeared while at 440-457aa presented a strong antigenicity site. According to phylogenic trees of S1 gene, the two isolates fell into different subgroups. AH1-99 strain is closely related to standard IBV strains of H52, M41, Beaudette and most of domestic isolates and the identity within this subgroup is 91.7%-92.2%, while AH2-03 strain is alienated to those standard strains.The size of S2 gene is 1881 nucleotides and the size of S2 protein is 625 amino acids owing to two stop codons in it. S2 is more conservative than S1 gene. Sequence comparison showed that six nucleotides GCGACT inserted at the position of 895-900 and six nucleotides ATTACT deleted at the site of 1441-1446. The pitch of the amino acids locating between 560 and 600 is probably associated with the viral membrane.10-11 potential glycosylation sites present in the S2 proteins of the isolates, and the quantity and location of these potential glycosylation sites changed. A high degree of similarity (96.2%) was observed between the two isolates S2 genes and they formed an alone gene group in S2 gene phylogenic tree. The extent of nucleotides homology between the two field strains and referencing strains is from 70.3% to 88.9%.M protein comprises 225-226 amino acids and two potential glycosylation sites. present in it. Sequence analysis indicated that M gene of the two strains is the most conservative in the four structural genes. Three nucleotides GGA were inserted in the position of 16-18nt in AH2-03 strain M gene. The identity of nucleotides compared with others is between 87.2% and 95.6%. The evolution of AH1-99 strain M gene is parallel to that of S1 gene. In the phylogenic tree of IBV M genes, AH1-99 strain is in the gene cluster that including H120, M41, Beaudette, and majority of domestic field stains. Though AH2-03 strain shared 93.4% consistent with AH1-99 strain, they weren't in the same gene group. Both AH2-03 strain and a few of inland IBV strains such as SAIB14, GD6-98 were in another subgroup.Sequence analysis suggested that no insertion and deletion of nucleotides in N gene. It contains1230 nucleotides which codes 409 amino acids. The homology of nucleotides with other IBV strains is from 85.2% to 97.6%. Majority of amino acid residues of N protein are hydrophilic. It is abundant with basic amino acids which locate in three conservative regions:63-85aa, 198-249aa, and 330-368aa. Compared with IBV reference strains of M41 and Beaudette, the quantity and location of phosphorylation sit...
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