Evolution of infectious bronchitis virus in Taiwan: characterisation of RNA recombination in the nucleocapsid gene

Abstract

Avian infectious bronchitis virus (IBV) belongs to the Coronaviridae family and causes significant economic loss in Taiwan (TW), even in flocks that have been extensively immunised with Massachusetts (Mass)-serotype vaccines. Phylogenetic analysis of all non-structural and most structural genes shows that TW IBV is genetically distinct from the US strain and more similar to Chinese (CH) IBV. In contrast, the nucleocapsid (N) gene of TW IBV presents phylogenetic incongruence. RNA recombination at the 5' end of the N gene between TW and US IBV is shown to be responsible for this discordance. Surprisingly, the recombinant N gene is found in all of tested TW IBV isolates, suggesting that a recombination event gave origin to a founder lineage. Our data indicate that RNA recombination in the recombinant 5' end of the N gene may have caused the emergence of the current IBV population in Taiwan.

Fig. 1

Phylogenetic relationships among the S1 genes of IBVs. (A) The phylogenetic tree, based on Bayesian inference, shows relationships among the three major groups, Taiwanese (TWI and II), Chinese (CH) and American (US). Supporting values are shown in the form of Bayesian posterior probability/maximum likelihood bootstrap. (B) Bootstrap values, estimated from 1000 replicates of the ME analysis in MEGA 4.0, are given (only values >60 are shown). The scale bar indicates the number of nucleotide replacements per site.

Fig. 2

Phylogenetic trees of genes 1, E, and M of IBVs. The trees were constructed using ME algorithms. Gene 1a (A): the 5′-UTR and full-length sequence of 1a (12,528 bp); 1b (B): nt 18,782–20,459 (1678 bp); the full-length envelope (306 bp) and membrane (678 bp) genes: E (C) and M (D), respectively. Bootstrap values greater than 60% are shown. The scale bar indicates the number of nucleotide replacements per site.

Fig. 3

ME trees of the N gene and protein of IBVs. Both genes (A, C, and E) and their encoded proteins (B, D, and F) are illustrated. full-length, nt 1–1230 (A) and aa 1–409 (B); N-terminus, nt 1–600 (C) and aa 1–200 (D); C-terminus, nt 601–1230 (E) and aa 201–409 (F). The scale bars correspond to the number of nucleotide replacements per site.

Fig. 4

Bayesian and ML unrooted trees of the N gene of IBVs. These phylogenetic reconstructions are based on BI analysis in MrBayes 3.1.2 (A, C, and E) and ML analysis in GARLI v0.951 (B, D, and F). Full-length, nt 1–1230 (A and B); N-terminus, nt 1–600 (C and D); C-terminus, nt 601–1230 (E and F). The scale bars correspond to the number of nucleotide replacements per site.

Fig. 5

Recombination and putative breakpoints within genes 5 and N of TW IBV. (A) For the RDP/Bootscan analysis, TW2575/98 was used as the query strain for potential recombination detection. TW2296/95 (solid line) and CU-T2 (dashed line) were used as parental sequences; CH-LX4 was as an outlier sequence. The black region represents a potential crossover within nt 278–757 of the queried sequences. The arrow indicates the suggested breakpoint at nt 278, proposed by both the Bootscan and GARD programs. The N gene, starting with the underlined ATG, is shaded (A). Alignments of the first 150 nt (B) and the deduced aa sequences (C) of N are illustrated. The CH IBVs in panel B and C are shaded to highlight their discrepancy.

Fig. 6

Alignment of the N-termini of IBVs with N-terminal domain secondary structures. The regions of numbered β-sheets and the α-turn (α) are deduced from the N-terminal domain stereography of the Beaudette strain.

Supplementary Fig. S1

A phylogenetic relationship among the S1 genes of 74 IBVs. The tree was constructed by ME algorithms. Bootstrap values, estimated from 1000 replicates of the ME method, were conducted in MEGA4. TW strains in the US clade are shown in bold and CH strains in the US clade are shown in italic. The scale bar indicates the number of nucleotide replacement per site.

Supplementary Fig. S2

A phylogenetic tree of the full-length N genes of 58 IBVs. The tree was constructed by ME algorithms. Bootstrap values, estimated from 1000 replicates of the ME method, were conducted in MEGA4. TW strains in the US clade are shown in bold and CH strains in the US clade are shown in italic. The bar indicates the number of nucleotide replacement per site.