Phylogenetic analysis of partial S1 and N gene sequences of infectious bronchitis virus isolates from Italy revealed genetic diversity and recombination

Abstract

A total of ten infectious bronchitis virus (IBV) isolates collected from commercial chickens in Italy in 1999 were characterized by RT-PCR and sequencing of the S1 and N genes. Phylogenetic analysis based on partial S1 gene sequences showed that five field viruses clustered together with 793/B-type strains, having 91.3-98.5% nucleotide identity within the group, and one isolate had very close sequence relationship (94.6% identity) with 624/I strain. These two IBV types have been identified in Italy previously. The other three variant isolates formed novel genotype detected recently in many countries of Western Europe. For one of these variant viruses, Italy-02, which afterwards became the prototype strain, the entire S1 gene was sequenced to confirm its originality. In contrast, phylogenetic analysis of more conserved partial N gene sequences, comprising 1-300 nucleotides, revealed different clustering. Thus, three variant IBVs of novel Italy-02 genotype, which had 96.7-99.2% S1 gene nucleotide identity with each other, belonged to three separate subgroups based on N gene sequences. 624/I-type isolate Italy-06 together with Italy-03, which was undetectable using S1 gene primers, shared 97.7% and 99.3% identity, respectively, in N gene region with vaccine strain H120. Only one of the 793/B-type isolates, Italy-10, clustered with the 793/B strain sharing 99.3% partial N gene identity, whereas the other four isolates were genetically distant from them (only 87.7-89.7% identity) and formed separate homogenous subgroup. The results demonstrated that both mutations and recombination events could contribute to the genetic diversity of the Italian isolates.

Fig. 1

A schematic representation of the IBV genome structure and locations of primers used for RT-PCR and sequencing of segments of the S1 and N genes. The dashed lines and numbers above them indicate the size of the PCR products (bp, base pairs). The bent arrows represent locations of primers

Fig. 2

Phylogenetic relationships of Italian IBV isolates and selected reference strains based on the partial S1 gene sequences (a) comprising 166–549 bp (numbering corresponds to H120 vaccine strain [M21970]) and partial N gene sequences (b) comprising first 300 bp beginning from AUG start codon. The rooted trees were generated by MegAlign 5.00 program from the Clustal W multiple sequence alignment (DNAStar Inc., Madison, WI). IBV genotypes that Italian isolates belong to are designated by braces. Italian isolates are shown in bold type. Vaccine strain H120 is boxed