Complete genomic sequence analysis of infectious bronchitis virus Ark DPI strain and its evolution by recombination

Abstract

An infectious bronchitis virus Arkansas DPI (Ark DPI) virulent strain was sequenced, analyzed and compared with many different IBV strains and coronaviruses. The genome of Ark DPI consists of 27,620 nucleotides, excluding poly (A) tail, and comprises ten open reading frames. Comparative sequence analysis of Ark DPI with other IBV strains shows striking similarity to the Conn, Gray, JMK, and Ark 99, which were circulating during that time period. Furthermore, comparison of the Ark genome with other coronaviruses demonstrates a close relationship to turkey coronavirus. Among non-structural genes, the 5'untranslated region (UTR), 3C-like proteinase (3CLpro) and the polymerase (RdRp) sequences are 100% identical to the Gray strain. Among structural genes, S1 has 97% identity with Ark 99; S2 has 100% identity with JMK and 96% to Conn; 3b 99%, and 3C to N is 100% identical to Conn strain. Possible recombination sites were found at the intergenic region of spike gene, 3'end of S1 and 3a gene. Independent recombination events may have occurred in the entire genome of Ark DPI, involving four different IBV strains, suggesting that genomic RNA recombination may occur in any part of the genome at number of sites. Hence, we speculate that the Ark DPI strain originated from the Conn strain, but diverged and evolved independently by point mutations and recombination between field strains.

Figure 1

Classical Genome Organization of IBV-Ark DPI. The genome of Ark DPI is 27,620 nt long, excluding poly (A) tract. Middle: ten genes and its ORFs. Ribosomal frameshift and position of transcriptional regulatory sequences (TRS) of each gene is indicated. Top: putative domains of ORF1a/1b polyprotein: nsp-non-structural protein, Ac-acidic domain, X-unknown domain X, PL1- papain like proteinase1, PL2-papain like proteinase 2; Y-unknown domain Y; HD-hydrophobic domain, 3CL-3C-like proteinase, G-Growth factor like protein, RdRp-RNA dependent RNA polymerase, Hel-helicase, ExoN-exoribonuclease, Ne-nidoviral uridylate-specific endoribonuclease, MT- 2'-O-ribose methyltransferase. Bottom: details of spike protein; SP-signal peptide, RRSRR/S- spike protein cleavage site between 544 and 545aa, TM-transmembrane domain of spike protein.

Figure 2

Phylogenetic tree analysis of complete Ark DPI genome sequence with other IBV strains. Phylogenetic tree analysis was conducted by neighbor-joining method using bootstrap analysis (100 replications). The scale at the bottom indicates the number of substitution events.

Figure 3

Schematic presentation of the structural region of Ark DPI genome. Entire genome of Ark DPI was analyzed for its similarity with other IBV strains. Top panel: 5'UTR & ORF1. Shadowed regions were used for comparative analysis. 5'UTR-5'-untranslated region; PL1-papain like proteinase1; M^pro-main or 3C-like proteinase; RdRp-RNA-dependent-RNA polymerase. Bottom panel: ORF2 to 3'UTR. Structural genes and their ORFs are marked by (●). Conserved sequence TGTGTTGATTATAAT in S1 gene is shown; ◆ denotes plausible recombination site in Ark DPI.