Delmarva (DMV/1639) Infectious Bronchitis Virus (IBV) Variants Isolated in Eastern Canada Show Evidence of Recombination

Abstract

Infectious bronchitis virus (IBV) infection in chickens can lead to an economically important disease, namely, infectious bronchitis (IB). New IBV variants are continuously emerging, which complicates vaccination-based IB control. In this study, five IBVs were isolated from clinical samples submitted to a diagnostic laboratory in Ontario, Canada, and subjected to detailed molecular characterization. Analysis of the spike (S)1 gene showed that these five IBVs were highly related to the Delmarva (DMV/1639) strain (~97.0% nucleotide sequence similarity) that was firstly isolated from an IB outbreak in the Delmarva peninsula, United States of America (USA), in 2011. However, the complete genomic sequence analysis showed a 93.5-93.7% similarity with the Connecticut (Conn) vaccine strain, suggesting that Conn-like viruses contributed to the evolution of the five Canadian IBV/DMV isolates. A SimPlot analysis of the complete genomic sequence showed evidence of recombination for at least three different IBV strains, including a Conn vaccine-like strain, a 4/91 vaccine-like strain, and one strain that is yet-unidentified. The unidentified strain may have contributed the genomic regions of the S, 3, and membrane (M) genes of the five Canadian IBV/DMV isolates. The study outcomes add to the existing knowledge about involvement of recombination in IBV evolution.

Keywords: Canada; Infectious bronchitis virus (IBV); chicken; complete genome sequence; phylogenetic analysis; recombination.

Figure 1

Randomized accelerated maximum likelihood (RAxML) phylogenetic tree based on the complete nucleotide sequences of S1 genes of our five IBV isolates and 86 reference sequences. All genotypes of representative IBV strains are indicated on the right side. Our five IBV isolates are marked in red color.

Figure 2

Sequence alignment of open reading frame (ORF) 6b from our five IBV isolates. The deletion in gene 6b of isolate IBV/Ck/Can/18-048192T results in a truncated 6b protein. Sequences of isolates IBV/Ck/Can/17-035614, IBV/Ck/Can/17-036989, IBV/Ck/Can/18-048430, and IBV/Ck/Can/18-049707, as representatives of the normal 6b gene, were compared. The ATGs with a single underline in boldface are the start codons of 6b gene. TGA and TAGs in boxes are the termination codons of 6b gene in IBV/Ck/Can/18-048192T and the other four isolates, respectively. A 13-nucleotide sequence was deleted in isolate IBV/Ck/Can/18-048192T (represented as –).

Figure 3

Alignment of the complete genome sequences of our five IBV isolates, the Connecticut vaccine, and 4/91 vaccine strains, which was performed using MAFFT. Nucleotide sequence disagreements at the indicated positions are shown in black, while nucleotide sequence agreements are shown in gray.

Figure 4

Alignment of the complete genome sequences of isolate GA9977/2019, the Connecticut vaccine, and 4/91 vaccine strains, which was performed using MAFFT. Nucleotide sequence disagreements at indicated positions are shown in black, while nucleotide sequence agreements are shown in gray.

Figure 5

(a) Recombination analysis by SimPlot of the complete genome sequences of IBV/Ck/Can/18-049707, the Connecticut vaccine, and the 4/91 vaccine strains. Breakpoints are indicated by the vertical red lines. The SimPlot was created using a window size of 1000 nt and a step size of 50 nt. Isolate IBV/Ck/Can/18-049707 was used as the query sequence. (b) Percentages of nucleotide sequence identity among our five IBV isolates, the Connecticut vaccine, the 4/91 vaccine, and H120 (as a representative of the Massachusetts type, the second major live vaccine available in Canada) according to the genome fragments indicated by SimPlot. Genome positions were determined according to isolate IBV/Ck/Can/18-049707 in the alignment. (c) Phylogenetic analysis of genome positions 1–1911, 1912–3735, 3736–12043, 12044–20120, 20121–25100, and 25,101–3′ end of the complete genome among our five IBV isolates, Connecticut vaccine, 4/91 vaccine, Arkansas DPI (GQ504720), H120 (FJ888351), JMK (GU393338), Iowa 97 (GU393337), Delaware 072 (Gu393332) and ck/CH/LDL/05II (kx364298). The trees were constructed using the maximum likelihood method in PhyML. Our five IBV isolates are marked in red color.

Figure 5

(a) Recombination analysis by SimPlot of the complete genome sequences of IBV/Ck/Can/18-049707, the Connecticut vaccine, and the 4/91 vaccine strains. Breakpoints are indicated by the vertical red lines. The SimPlot was created using a window size of 1000 nt and a step size of 50 nt. Isolate IBV/Ck/Can/18-049707 was used as the query sequence. (b) Percentages of nucleotide sequence identity among our five IBV isolates, the Connecticut vaccine, the 4/91 vaccine, and H120 (as a representative of the Massachusetts type, the second major live vaccine available in Canada) according to the genome fragments indicated by SimPlot. Genome positions were determined according to isolate IBV/Ck/Can/18-049707 in the alignment. (c) Phylogenetic analysis of genome positions 1–1911, 1912–3735, 3736–12043, 12044–20120, 20121–25100, and 25,101–3′ end of the complete genome among our five IBV isolates, Connecticut vaccine, 4/91 vaccine, Arkansas DPI (GQ504720), H120 (FJ888351), JMK (GU393338), Iowa 97 (GU393337), Delaware 072 (Gu393332) and ck/CH/LDL/05II (kx364298). The trees were constructed using the maximum likelihood method in PhyML. Our five IBV isolates are marked in red color.