Complete nucleotide analysis of the structural genome of the infectious bronchitis virus strain md27 reveals its mosaic nature

Abstract

Infectious bronchitis virus (IBV) causes highly contagious respiratory or urogenital tract diseases in chickens. The Maryland 27(Md27) strain was first isolated in 1976 from diseased chicken flocks in the Delmarva Peninsula region. To understand the genetic diversity and phylogenetic relationship of existing strains with Md27, the complete nucleotide sequence of the 3'end coding region (∼7.2 kb) of Md27 was determined and compared with other IBV strains and coronaviruses. It has the same S-3-M-5-N-3' gene order, as is the case of other IBV strains. The spike gene of Md27 exhibits 97% identity with the SE17 strain. There are deletions at the spike gene, non-coding region between M and 5 genes, and at the 3' untranslated region (UTR), which is different from Ark-like strains. Phylogenetic analysis and sequence alignments demonstrate that Md27 is a chimera containing different gene segments that are most closely related to the SE17, Conn and JMK strains. This current study provides evidence for genomic mutations and intergenic recombination that have taken place in the evolution of IBV strain Md27.

Keywords: infectious bronchitis virus; phylogenetic analysis; recombination; sequencing; structural genes.

Figure 1.

Comparison of 5′ terminus of 3′ untranslated region (UTR) of Md27 with some selected strains of IBV*. Nucleotide deletion of Md27 at 3′UTR is clearly shown. Sequences were compared using Gendoc software. *Nucleotides of Md27 shown are starting from 86^th nucleotide after the stop codon of N gene.

Figure 2.

Comparison of 7.27 kb of the 3′end of the Md27 genome sequence with other IBV strains by an unrooted phylogenetic tree. Sequences are aligned by neighbor-joining method using MEGA4 program. Md27 clustered with ArkDPI, Jilin and Cal99 IBV strains. The scale at the bottom indicates the number of substitution events.

Figure 3.

Phylogenetic analyses of the structural genes of Md27 virus with those of other IBV strains. The Md27 strain had clustered with ArkDPI, HK, Conn, and Cal99 in most of the trees. The tree is constructed from aligned nucleotide sequences of structural genes (S1, S2, spike gene; E, envelope gene; M, matrix gene; N, nucleocapsid gene) by neighbor-joining method with 1000 bootstrap replicates using the MEGA4 program. For each tree, the bootstrap value for each branch is indicated and the scale at the bottom indicates the number of substitution events.

Figure 3.

Phylogenetic analyses of the structural genes of Md27 virus with those of other IBV strains. The Md27 strain had clustered with ArkDPI, HK, Conn, and Cal99 in most of the trees. The tree is constructed from aligned nucleotide sequences of structural genes (S1, S2, spike gene; E, envelope gene; M, matrix gene; N, nucleocapsid gene) by neighbor-joining method with 1000 bootstrap replicates using the MEGA4 program. For each tree, the bootstrap value for each branch is indicated and the scale at the bottom indicates the number of substitution events.

Figure 4.

Schematic representation of the structural genome of Md27. Entire structural genome of Md27 was analyzed for its similarity with other IBV strains. Structural genes and their ORFs are marked by ▾. Putative recombination sites in Md27 genome are denoted by x. Picture is not drawn to scale.