Molecular epidemiology and evolution of avian infectious bronchitis virus in Spain over a fourteen-year period

Abstract

An in-depth molecular study of infectious bronchitis viruses (IBV) with particular interest in evolutionary aspects of IBV in Spain was carried out in the present study based on the S1 gene molecular characterization of twenty-six Spanish strains isolated over a fourteen-year period. Four genotypes were identified based on S1 gene sequence analyses and phylogenetic studies. A drastic virus population shift was demonstrated along time and the novel Italy 02 serotype was shown to have displaced the previous predominant serotype 4/91 in the field. Detailed analyses of synonymous to non-synonymous ratio of the S1 gene sequences of this new serotype Italy 02 suggested positive selection pressures might have contributed to the successful establishment of Italy 02 serotype in our country. In addition, differences on the fitness abilities of new emergent genotypes were indicated. Furthermore, intergenic sequences (IGs)-like motifs within S1 gene sequences of IBV isolates were suggested to enhance the recombination abilities of certain serotypes.

Fig. 1

Phylogenetic tree constructed by a maximum likelihood method (Tree-Puzzle 5.2). Nucleotide sequences of the complete S1 gene of Spanish field isolates investigated in this study (in bold letters) and reference IBV strains obtained from GenBank database are included in the comparison.

Fig. 2

Entropy rates and non-synonymous and synonymous difference (d_N − d_S) plotted per each amino acid position for the first 150 amino acids of the S1 gene. Both values were calculated using an alignment that included all Spanish field isolates of Italy 02 genotype.

Fig. 3

Phylogenetic analysis of putative recombinant breakpoint located at position 586 of Italy 02 and Genotype IV isolates. All twenty-six Spanish IBV field isolates were included in the comparison. Trees were generated by the Maximum Parsimony method with 1000 bootstraps in the Mega 3 package. 3a. Phylogenetic tree obtained when the S1 region from nt 1 to 586 in the alignment was used. Italy 02 and Genotype IV (in bold letters) isolates were clustered together in the same phylogenetic group. 3b. Phylogenetic tree obtained when the S1 region from nt 586 to 1623 in the alignment was used. Genotype IV isolates (in bold letters) were grouped with Spanish 4/91 cluster 1 isolates, whereas Spanish Italy 02 isolates formed an own phylogenetic cluster.

Fig. 4

Phylogenetic analysis of putative recombinant breakpoint located at position 700 of Spanish 4791 cluster 2 isolates. All twenty-six Spanish IBV field isolates were included in the comparison. Trees were generated by the Maximum Parsimony method with 1000 bootstraps in the Mega 3 package. 4a. Phylogenetic tree obtained when the S1 region from nt 1 to 700 in the alignment was used. Spanish 4/91 cluster 2 isolates (in bold letters) were grouped with the remaining Spanish 4/91 isolates. 4b. Phylogenetic tree obtained when the S1 region from nt 700 to 1623 in the alignment was used. Spanish 4/91 cluster 2 isolates (in bold letters) were clustered in an own phylogenetic branch closely related with Spanish Italy 02 isolates.