Characterization of a recombinant coronavirus infectious bronchitis virus with distinct S1 subunits of spike and nucleocapsid genes and a 3' untranslated region

Abstract

An infectious bronchitis virus (IBV), ck/CH/LZJ/111113, was isolated from a H120-vaccinated chicken which showed disease suspected of IBV infection. Neutralization testing showed that ck/CH/LZJ/111113 was distinct from either the Chinese predominant IBV LX4-type or Mass-type vaccine strains. Phylogenetic analysis confirmed that ck/CH/LZJ/111113 is of the 4/91 type; however, further extensive analyses of full-length genomes identified occurrence of recombination events. Therefore, ck/CH/LZJ/111113 originated from the recombination events between ck/CH/LDL/091022- and 4/91-like strains at three switch sites located upstream of the spike (S) glycoprotein gene, and the 3' ends of S1 and nuceocapsid (N) genes, respectively. The difference of serotypes and tissue tropisms in kidneys between ck/CH/LZJ/111113 and ck/CH/LDL/091022 may have been contributed by the uptake of the S1 gene by a ck/CH/LDL/091022-like virus from a 4/91-like strain. This recombination event took place at the 3' end of the N gene and the 3' untranslated region may account for differences in replication efficiency in tissues of chickens inoculated by the two viruses.

Fig. 1

Relationships among the ck/CH/LZJ/111113, ck/CH/LDL/091022 and reference strains illustrated by a maximum likelihood phylogeny unrooted tree, based on S1 nucleotides 1–1626 (A) and the full-length of genomic sequences (B), respectively.In (A), the strains ck/CH/LZJ/111113, ck/CH/LDL/091022 are shown in bold. The genotypes of 4/91-, LX4- and mass-type strains were shown in pink, blue and red, respectively. In (B), the US and China strains were shown in pink and blue, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 2

Analysis of recombination among IBV ck/CH/LDL/091022, 4/91 and ck/CH/LZJ/111113 strains. (A) Multiple sequence alignments surrounding numbered regions in A. The numbers on the right of each alignment showed the nucleotide positions in genome of each virus. The sequences of ck/CH/LZJ/111113 are listed and the only nucleotides differing from those of ck/CH/LZJ/111113 are depicted. The region where the template switches (switch site) have taken place is underlined. The deleted nucleotides are indicated by -. NA represents unavailable sequence. (B) Percentages of nucleotide sequence identity among ck/CH/LZJ/111113, ck/CH/LDL/091022 and 4/91.Percentages of nucleotide sequence identity of gene fragments before and after the switch sites in S1 and N genes are also indicated.

Fig. 2

Analysis of recombination among IBV ck/CH/LDL/091022, 4/91 and ck/CH/LZJ/111113 strains. (A) Multiple sequence alignments surrounding numbered regions in A. The numbers on the right of each alignment showed the nucleotide positions in genome of each virus. The sequences of ck/CH/LZJ/111113 are listed and the only nucleotides differing from those of ck/CH/LZJ/111113 are depicted. The region where the template switches (switch site) have taken place is underlined. The deleted nucleotides are indicated by -. NA represents unavailable sequence. (B) Percentages of nucleotide sequence identity among ck/CH/LZJ/111113, ck/CH/LDL/091022 and 4/91.Percentages of nucleotide sequence identity of gene fragments before and after the switch sites in S1 and N genes are also indicated.

Fig. 2

Analysis of recombination among IBV ck/CH/LDL/091022, 4/91 and ck/CH/LZJ/111113 strains. (A) Multiple sequence alignments surrounding numbered regions in A. The numbers on the right of each alignment showed the nucleotide positions in genome of each virus. The sequences of ck/CH/LZJ/111113 are listed and the only nucleotides differing from those of ck/CH/LZJ/111113 are depicted. The region where the template switches (switch site) have taken place is underlined. The deleted nucleotides are indicated by -. NA represents unavailable sequence. (B) Percentages of nucleotide sequence identity among ck/CH/LZJ/111113, ck/CH/LDL/091022 and 4/91.Percentages of nucleotide sequence identity of gene fragments before and after the switch sites in S1 and N genes are also indicated.

Fig. 3

Viral genome RNA copies in tissues from chickens inoculated with ck/CH/LZJ/111113 and ck/CH/LDL/091022, respectively. Viral RNA copy number was measured by real-time RT-PCR in tissues from three chickens on days 4 and 7 post-inoculation, respectively. Results represent the mean of three independent experiments with three replicates per experiment and each bar is the mean ± SD. The data were analyzed using SAS (SAS, 1996); *P < 0.05.