Prevalence, Genotype Diversity, and Distinct Pathogenicity of 205 Gammacoronavirus Infectious Bronchitis Virus Isolates in China during 2019-2023

Abstract

Gammacoronavirus infectious bronchitis virus (IBV) causes a highly contagious disease in chickens and seriously endangers the poultry industry. The emergence and co-circulation of diverse IBV serotypes and genotypes with distinct pathogenicity worldwide pose a serious challenge to the development of effective intervention measures. In this study, we report the epidemic trends of IBV in China from 2019 to 2023 and a comparative analysis on the antigenic characteristics and pathogenicity of isolates among major prevalent lineages. Phylogenetic and recombination analyses based on the nucleotide sequences of the spike (S) 1 gene clustered a total of 205 isolates into twelve distinct lineages, with GI-19 as a predominant lineage (61.77 ± 4.56%) exhibiting an overall increasing trend over the past five years, and demonstrated that a majority of the variants were derived from gene recombination events. Further characterization of the growth and pathogenic properties of six representative isolates from different lineages classified four out of the six isolates as nephropathogenic types with mortality rates in one-day-old SPF chickens varying from 20-60%, one as a respiratory type with weak virulence, and one as a naturally occurring avirulent strain. Taken together, our findings illuminate the epidemic trends, prevalence, recombination, and pathogenicity of current IBV strains in China, providing key information for further strengthening the surveillance and pathogenicity studies of IBV.

Keywords: IBV; epidemic trend; homologous recombination; pathogenicity.

Figure 1

Geographical distribution and evolution analysis of IBV isolates. (A) Regional distribution of 205 IBV isolates from 2019 to 2023 in China. (B) Evolution analysis of IBV isolates. After conducting a comparative analysis of the S1 sequences between the 205 isolates and reference strains, an evolutionary tree was constructed by the neighbor-joining method using the MEGA7 software, and the resulting evolutionary tree was further beautified utilizing the iTOL online software.

Figure 2

Analysis of proportion and prevalence of different lineages of IBV isolates, as well as the furin motif sequences. (A) Percentages of different lineages of 205 isolates from 2019 to 2023 in China. (B) The annual proportion of different lineages in 2019–2023. (C) Analysis of furin motif sequences in diverse lineages of isolates. The furin motif sequences were aligned using Mega sequencing, saved as Fasta files, and subsequently subjected to analysis using WebLogo 3 online software. The amino acid proportion at a certain position with sequence variation is represented by the relative height of the single amino acid letter.

Figure 3

Analysis of the recombination events in the S1 gene of different variants. Determination of the potential recombination events in 12 isolates of four variant-lineages by the SimPlot analysis. These isolates are: (A) CK CH CQ BS-DK_WXL20200504, (B) CK CH GD SG LH-XQJ_20210222, (C) CK CH JS CZ-SJQY 12482-20201129, (D) CK CH CQ DK-YSN 20200908, (E) CK CH GD QY TN-1B 20230227, (F) CKCH GZ DK20200410, (G) CK CH XJ CJ TK-1 20230111, (H) CK CH GD KPlhnm 2019, (I) CK CH CQ BS DK-LWC 20210913, (J) CK CH XN-XYWS-LJF 2019, (K) CK CH YN KM-DK YJZ20200804, and (L) CK CH GX NN LY-ZDC 20211213. The y-axis represents the ratio of identity within a 200 bp wide sliding window centered on the position plotted, with a 20 bp step size between plots.

Figure 4

Deletion and insertion events in the S1 gene of 17 variants. Megalign analysis was performed to detect gene deletion and insertion in the S1 from 17 variants in 5 variant-lineages, including GI-7-like (A), GI-13-like (B), GI-19-like (C), GI-22-like (D), and GVI-1-like (E).

Figure 5

Homology, growth kinetics, and pathogenicity analyses of six representative isolates. (A) Two software programs, MEGA 7.0 and Megalign, were successively used to analyze the homology of S1 sequences between the vaccine strain H120 and six representative isolates with different lineages. S1-N and S1-A represent homology of nucleic acid and amino acid sequences, respectively. (B) Growth curves of six representative isolates in chicken embryos. Nine-day-old chicken embryos were infected at an EID₅₀ ~10^3, respectively. The copy numbers of IBV in allantoic fluids were determined and calculated by RT-qPCR. (C) Surviving curves of six representative isolates in 1-day-old SPF chickens. Chickens were infected with ~10^5.5 EID₅₀ each of the six representative isolates by the nasal–ocular route, respectively. The number of deaths occurring within 7 days after the challenge was recorded, and a survival curve was generated using the Graphpad Prism 9 program. (D) Examination of pathological lesions in the trachea, lung, and kidney autopsies. At 7 days post-challenge, the trachea, lung, and kidney autopsies of dead chickens or chickens randomly selected from chickens infected with the six representative isolates were examined macroscopically. Blue arrows indicate the pathological lesions observed.

Figure 6

Histopathological examination of the trachea, lungs, and kidneys in chickens infected with each of the six representative isolates. At 7 days post-challenge, the trachea, lung, and kidney autopsies of dead chickens or chickens randomly selected from each experimental group were conducted and examined by microscopy.

Figure 7

Viral loads in the trachea, lung, and kidney tissues from chickens infected with six representative isolates. At 7 days post-challenge, three chickens from each experimental group were randomly selected, the trachea, lung, and kidney tissues were collected, and the viral RNA copy numbers were determined and calculated by RT-qPCR. Significance levels are presented by the p-value (ns, non-significant; * p < 0.05; ** p < 0.01; *** p < 0.001; and **** p < 0.0001). (A) Statistical analysis of the total viral loads in the three tissues within each challenging group and the control group. (B) Statistical analysis of viral load variations in different tissues from chickens challenged with six representative isolates. (C) Statistical analysis of viral load variations in the same organs from chickens challenged with the other five isolates and the predominant lineage strain KP_GI-19, respectively.

Figure 8

Immunohistochemical examination of IBV replication in the trachea, lungs, and kidneys from chickens infected with six representative isolates. At 7 days post-challenge, the trachea, lung, and kidney autopsies of dead chickens or chickens randomly selected were conducted, and lung and kidney sections were immunohistochemically stained with a monoclonal antibody against IBV N protein.