
==== Front
Poult Sci
Poult Sci
Poultry Science
0032-5791
1525-3171
Elsevier

S0032-5791(24)00835-6
10.1016/j.psj.2024.104256
104256
GENETICS AND MOLECULAR BIOLOGY
Molecular characteristic, evolution, and pathogenicity analysis of avian infectious bronchitis virus isolates associated with QX type in China
Lu Yuanlu *
Zeng Yiran *
Luo Haowei *
Qiao Bingchen *
Meng Qi *
Dai Zijian *
Chen Na *
Zhao Lingcai *
Meng Xianchen †§
Zhang Haitao †
Xia Jun 1065291747@qq.com
‡1
Ping Jihui jihui.ping@njau.edu.cn
⁎2
⁎ MOE Joint International Research Laboratory of Animal Health and Food Safety, Engineering Laboratory of Animal Immunity of Jiangsu Province, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, 210095, China
† Lihua Nanjing Industrial Research Institute Co. Ltd. Nanjing, 213168, China
‡ Key Laboratory for Prevention and Control of Herbivorous Animal Diseases of the Ministry of Agriculture and Rural Affairs & Xinjiang Animal Disease Research Key Laboratory, Xinjiang Academy of Animal Sciences Institute of Veterinary Medicine, 830000, China
§ Key Laboratory of Jiangsu Preventive Veterinary Medicine, Key Laboratory for Avian Preventive Medicine, Ministry of Education, College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu, 225009 China
2 Corresponding author. jihui.ping@njau.edu.cn
1 Co-corresponding author.

28 8 2024
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© 2024 Published by Elsevier Inc. on behalf of Poultry Science Association Inc.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Infectious bronchitis virus (IBV) is one of the major avian pathogens plaguing the global poultry industry. Although vaccination is the primary preventive measure for IBV infection, the emergence of virus variants with mutations and recombination has resulted in IBV circulating globally, presenting a challenge for IB control. Here, we isolated 3 IBV strains (CZ200515, CZ210840, and CZ211063) from suspected sick chickens vaccinated with IBV live attenuated vaccines (H120, 4/91, or QXL87). Phylogenetic analysis of the S1 gene sequence of the spike (S) revealed that the 3 isolates belonged to the QX-type (GI-19 lineage). Whole genome sequencing and recombination analysis indicated that CZ200515 and CZ210840 contained genetic material from 4/91 and Scyz3 (QX-type), possibly due to recombination between the circulating strain and the 4/91 vaccine strain, while no evidence of recombination was found in CZ211063. Pathogenicity analysis in 1-day-old specific pathogen-free (SPF) chickens demonstrated that all 3 isolates caused severe tissue damage and varying degrees of mortality. Virus cross-neutralization assay revealed decreased antigen relatedness between the isolates and the QX-type vaccine strain (QXL87). Amino acid sequence homology analysis of S1 revealed 5%-6.5% variances between the isolates and QXL87. Analysis of the S1 subunit structure revealed that mutations of amino acid residues in the hypervariable region (HVR) and the neutralizing epitope region resulted in antigenic variation in isolates by changing the antigen conformation. Our data indicate antigenicity variances between QX isolates and QXL87 vaccine strains, potentially resulting in immune evasion occurrences. Overall, these results offer crucial insights into the epidemiology and pathogenicity of QX-type IBV, facilitating improved selection and formulation of vaccines for disease management.

Key words

Infectious bronchitis virus
genome recombination
pathogenicity
neutralizing epitope
antigenic variation
==== Body
pmcINTRODUCTION

Infectious bronchitis virus (IBV) is an avian coronavirus that causes infectious bronchitis (IB), an acute and highly contagious respiratory disease in poultry (Cook et al., 2012). IBV primarily infects chickens and targets the respiratory epithelial cells initially, but some strains can also damage the genitourinary and digestive systems, exhibiting extensive tissue tropisms (Cavanagh, 2007). Additionally, IBV infection increases susceptibility to secondary infections in chickens, including bacteria or other pathogens, leading to more serious clinical symptoms (Li et al., 2022). Since its first isolation in 1931, a significant number of IBV variants have been discovered worldwide (Schalk and Hawn, 1931; Valastro et al., 2016). There is little cross-protection between different genotypes or serotypes, which brings great trouble to the prevention and control of IB (Eldemery et al., 2017b).

IBV is an enveloped, single-stranded, positive-sense RNA virus with a genome of about 27.6 kb (Cavanagh, 2005). The 5′ end two-thirds of the genome encodes 2 polyproteins (pp1a and pp1ab) (Boursnell et al., 1987) . The remaining one-third of the 3′ end of the genome encodes at least 4 structural proteins: the nucleoprotein (N), which binds to the RNA to form a helically symmetric nucleocapsid; the membrane proteins (M) surrounding the viral nucleocapsid; the envelope protein (E) embedded in the membrane protein; the pear-shaped spike protein (S), consisting of 2 subunits (S1 and S2) anchored to the membrane protein and extended to form a projection; and 4 accessory proteins (3a, 3b, 5a, and 5b) (Liu et al., 2009) .

Currently, live attenuated or inactivated vaccines are used to control the spread of IBV (Wang et al., 2024). However, the high mutation rate of IBV constantly leads to the emergence of novel strains, making complete control of the disease through vaccinations a challenging task (Cavanagh, 2007). To adapt more effectively to the host under the immune pressure of vaccination, the S1 subunit of the virus needs to mutate to escape host immune surveillance (Shang et al., 2018). Consequently, the substantial variability of the S1 subunit results in antigenic diversity and reduces cross-protection among various IBV strains (Eldemery et al., 2017a). IBV has recently been categorized into 9 genotypes (GⅠ-GⅨ) based on S1 sequence differences, with GⅠ genotypes further divided into 29 lineages (Chen et al., 2017b; Jiang et al., 2017; Ma et al., 2019; Mendoza-González et al., 2022). QX-type (GI-19 lineage) IBV is prevalent worldwide, causing respiratory symptoms and nephritis with a broad tissue tropism (Hou et al., 2020; Khanh et al., 2018).

Another crucial mechanism for the emergence of novel strains of IBV is genome recombination, a fundamental evolutionary process (Worobey and Holmes, 1999). Because of the discontinuous RNA synthesis process in coronavirus transcription, the viral replicase switches from the donor template to the acceptor template, leading to RNA recombination among different strains and the generation of novel recombinant viruses (Yount et al., 2006; Zúñiga et al., 2010). Therefore, co-infection of the same cell by different strains is essential for recombination (Trang et al., 2024). When exposed to serotype-mismatched vaccines, the vaccine may not offer effective protection against circulating strains, enabling both the vaccine strains and circulating strains to replicate in the same host (Gong et al., 2022). This further increases the genetic diversity and complexity of IBV

Here, we successfully isolated 3 QX-type strains from vaccinated chickens (H120, 4/91, or QXL87). The QX-type isolates exhibit 93.5% to 95.0% homology in the S1 amino acid sequence with QXL87, indicating that the virus is evolving continuously under the pressure of vaccine-induced immunity. The variation in S1 amino acid residues is probably the primary reason why vaccines do not provide complete protection against circulating strains. In this case, incorrect vaccine utilization could cause recombination between circulating and vaccine strains, leading to the emergence of CZ200515 and CZ210840 strains, thereby increasing the genetic diversity of the virus. Pathogenicity studies revealed that the 3 isolates caused varying degrees of mortality and tissue damage in chickens. The virus cross-neutralization assay revealed decreased antigen relatedness between the isolates and QXL87, which was attributed to variations in the neutralizing epitope of the S1 subunit. This may be the key to the escape immune protection of the circulating strains. It is not clear whether the variant strains can better adapt to the host and alter the pathogenicity of the virus, but mutations and recombination undoubtedly enhance genetic diversity and complexity, facilitating virus evolution, thereby complicating the prevention and control of IB. In conclusion, our study demonstrates that the QX-type isolates have evolved into different serum subtypes, resulting in decreased antigen relatedness with QX vaccine strains. This evolutionary divergence may be the primary factor contributing to the incomplete protection provided by vaccines against circulating strains. Understanding these antigenic differences is essential for the development of more effective vaccines and for formulating strategies to combat the ongoing challenge of viral evolution in the field.

MATERIALS AND METHODS

Sample Collection and Virus Isolation

Throat swab and cloacal swab samples were collected from suspected cases in intensive chicken farms in Jiangsu Province, China, from 2020 to 2021. All chickens received vaccinations with IBV live attenuated H120 (FJ888351), 4/91 (KF377577), or QX (MH743141) vaccines. The vaccines were provided by QYH Biotech Co., Ltd. (Nanjing, China). The samples were initially propagated in the allantoic cavities of 9-day-old specific pathogen-free (SPF) embryos (Jinan SAIS Poultry Co. Ltd., China). Subsequently, the HiScript II One Step RT-PCR Kit (P612-01, Vazyme, China) was used to confirm IBV infection, while tests for avian influenza virus (AIV), Newcastle disease virus (NDV), and infectious bursal disease virus (IBDV) in the allantoic fluid all showed negative results. The allantoic fluid was purified through 3 rounds of limiting dilutions in 9-day-old SPF embryos (Guo et al., 2023). IBV isolates were confirmed by reverse transcription polymerase chain reaction (RT-PCR) and embryonic lesions. The virus was then inoculated into 9-day-old SPF embryos, and the 50% embryo infectious dose (EID50) was calculated using the formula by Reed and Muench (Reed and Muench, 1938).

Viral Growth Kinetics Analysis

To evaluate the replication ability of isolates in vitro, 100 EID50 of IBV isolates were inoculated into 9-day-old SPF embryos, respectively. Allantoic fluid (500 μL) was collected from each embryo at 12, 24, 36, 48, 60, and 72 h postinfection (hpi), followed by RNA was extracted using the RNA isolater Total RNA Extraction Reagent (R401-01, Vazyme, China) according to the manufacturer's instructions. cDNAs was generated using a random primer and the HiScript II 1st Strand cDNA Synthesis Kit (R211-01, Vazyme, China). Real-time fluorescence quantitative PCR (RT-qPCR) was performed using the AceQ qPCR SYBR Green Master Mix (Q111-02, Vazyme, China) to quantify viral load, as described previously (Callison et al., 2006) with minor modifications. Briefly, the RT-qPCR primers were designed based on the genome sequence of the reference strains: IBV F391 (5′-GCTTTTGAGCCTAGCGTT-3′) and IBV R533 (5′-GCCATGTTGTCACTGTCTATTG-3′). The standard curve equation was y = −3.315x+39.704 (R2 = 0.9991). All experiments were performed in triplicate, and the copy number of each virus was calculated based on the standard curve.

Genome and S1 Gene Sequencing

As described previously, viral RNA extraction and cDNA synthesis were conducted for IBV isolates. BioEdit software was used to compare and analyze the complete genomes of vaccine strains H120, 4/91, and GI-19 reference strain Scyz3 (JF732903) (GI-19 vaccine strain QXL87 lacked complete gene sequence information). Oligo7 was employed to design fragment primers, with the corresponding primers listed in Supplementary Table S1. Using viral cDNAs as a template, whole genome DNA fragments were amplified through polymerase chain reaction (PCR) with fragment primers. Then, the PCR products were purified using the Biospin Gel Extraction Kit (BSC02M1, BioFlux, China). The PCR products were TA cloned using the Mighty TA-Cloning Reagent Set for PrimeSTAR® kit (6019, Takara, China), and the positive samples were sequenced by Sanger at General Biol Corporation (Anhui, China) (Sanger et al., 1977). PCR products that could not be cloned by TA were directly sequenced by Sanger. The S1 gene is contained in a fragment of the genome. The 5′- and 3′-terminal sequences of the isolates' genomes were determined using a SMARTer RACE 5′/3′ Kit (634858, TaKaRa, China). All sequencing results were assembled using DNAstar's SeqMan II software based on the above reference sequences (Huang et al., 2020).

Sequence Comparison and Phylogenetic Analysis

Genome sequences and S1 protein coding sequences of IBV strains were retrieved from NCBI GenBank (https://www.ncbi.nlm.nih.gov/). A total of 79 S1 gene sequences from 7 genotypes (GI-GVII) and partial genome sequences were selected as references, with relevant reference sequences information displayed in Supplementary Table S2. All sequences were aligned and manually adjusted using the BioEdit software (Hall et al., 2011). Phylogenetic analysis of the S1 gene sequence was performed by 1,000 bootstrap replicates using the neighbor-joining method with MEGA version 7.0 software.

Recombination Analysis

The potential recombination events in the S gene and complete genome sequences of isolates were analyzed using Recombination detection program 4 (RDP4, version 4.94) (Martin et al., 2015). A total of 7 methods implemented in RDP4 were applied, including RDP, GENECONV, 3Seq, Chimaer, SiScan, MaxChi, and LARD (Holmes et al., 1999). Recombination detected by at least 3 of the 7 methods with a P ≤ 10-12 was considered true recombination (Sabir et al., 2016; Wang and Hou, 2023). Then, potential recombination events and breakpoints were further verified through BootScan analysis with the SimPlot software (version 3.5.1) (Kolb et al., 2017). To demonstrate the reorganization event, the vaccine strains H120, 4/91, QX-type reference strain Sczy3, and isolated strain sequences were individually disconnected at the breakpoints determined by RDP4 and SimPlot software. Then, sequences were aligned using the BioEdit software, and evolutionary trees were reconstructed by MEGA 7.0 to determine similarities among the fragments (Huang et al., 2020).

Pathogenicity of IBV Isolates in 1-Day-Old SPF Chicken

Eighty 1-day-old SPF chickens (Jinan SAIS Poultry Co. Ltd., China) were randomly divided into 4 groups, each containing 20 chickens, and placed in isolators with food and water. The chickens in each group were infected with 105 EID50 IBV isolates CZ200515, CZ210840, and CZ211063 via eye drop and intranasal routes, respectively, with the day of infection recorded as 0 d postinfection (dpi). A negative control group received the same dose of phosphate buffered saline (PBS). Clinical symptoms and mortality were monitored and recorded daily for 14 d. At 3, 6, 9, and 14 dpi, 2 chickens from each group were randomly selected for euthanasia and autopsy (if any chickens had died, they were also autopsied on the same day). Gross lesions were observed, and tissue samples from the trachea, lungs, and kidneys were collected for viral load detection. Throat swabs and cloacal swabs were collected at 6 and 12 dpi, and viral shedding in each group was detected.

Histopathological Examination

At 6 dpi, tissue samples from the trachea, lung, and kidney were collected and fixed in 10% neutral formalin for 48 h at room temperature. The fixed samples underwent standard processing, were embedded in paraffin wax, and sliced into 5 μm-thick sections. The sections were then stained with hematoxylin and eosin (H&E) for observation under light microscopy (TS100, Nikon, Japan).

Tissue Viral Load and Viral Shedding Determination

The Total RNA Extraction Reagent was utilized for extracting total RNA from collected tissue and swab samples. Random primer and the HiScript II 1st Strand cDNA Synthesis Kit were employed to generate cDNAs from 1 µg of total RNA per sample. RT-qPCR was conducted using the AceQ qPCR SYBR Green Master Mix to quantify tissue viral load and viral shedding, as described previously. All experiments were performed in triplicate, and the copy number of virus was calculated based on the standard curve.

Virus Cross-Neutralization Assay

The blood from each group of surviving chickens was collected at 21 dpi, and the anti-IBV serum was then isolated. To evaluate the antigen relatedness between IBV isolates and QXL87, virus cross-neutralization assays were performed (anti-QXL87 serum was supplied by Lihua Nanjing Industrial Research Institute Co., Ltd., Nanjing, China). Briefly, serum samples were inactivated at 56°C for 30 min and then serially diluted 2-fold with PBS. Simultaneously, the virus was diluted to 200 EID50 using PBS. The diluted serum was mixed with an equal volume of virus and incubated at 37°C for 1 h. Subsequently, 0.2 mL of the mixture was inoculated into the allantoic cavities of 9-day-old SPF chicken embryos. After 6 d, the embryonic lesions were observed and counted, and the neutralization titers of each group were calculated using the Reed and Muench methods (Reed and Muench, 1938). The virus cross-neutralization R-value for each strain was calculated as previously described (Yan et al., 2017). The difference in antigenicity (serotype) between 2 specific strains was defined as follows: an R-value above 70% indicates identical antigenicity between the 2 viruses tested, an R-value between 33 and 70% indicates a minor subtype difference, an R-value between 11% and 32% indicates a major subtype difference, and an R-value below 11% reflects a different serotype (Gravendyck et al., 1996; You et al., 2023).

S1 Amino Acid Residue Mutation and Antigenic Variation Analysis

To compare amino acid residue differences in the hypervariable region (HVR) and the neutralizing epitope region, the online software ESPript3.0 (ESPript 3.x / ENDscript 2.x (ibcp.fr)) was used to display the alignment results between the isolates and QXL87 S1 subunit amino acid sequences. Visual analyses were performed using PyMol (Molecular Graphics System, Version 2.0 Schrödinger, LLC, https://pymol.org/2/) to analyze the impact of mutations in the HVR and the neutralizing epitope region of the S1 subunit (Xu et al., 2019; Zou et al., 2015). The protein model was created with SWISS-MODEL (https://swissmodel.expasy.org/) using PDB file 6CV0, which represents the Cryo-EM structure of the IBV-S protein (Shang et al., 2018). Subsequently, the protein structure was compared using PyMol with the program's setting tool.

Statistical Analysis

Data was analyzed using GraphPad Prism version 7.0 (GraphPad, La Jolla, CA, USA). Statistically significant differences were evaluated by performing a 2-way ANOVA analysis of variance (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P <0.0001). The data are presented in the figures as means ± the standard deviation (SD).

RESULTS

Virus Isolation and Identification

Three IBV strains were successfully isolated from suspected cases in intensive farms that had been vaccinated with live attenuated IBV vaccines during the period from 2020 to 2021 (Figure 1A). They were named CK/CH/JS/CZ200515 (abbreviation CZ200515, GenBank ID: PP438798); CK/CH/JS/CZ210840 (abbreviation CZ210840, GenBank ID: PP438799); and CK/CH/JS/CZ211063 (abbreviation CZ211063, GenBank ID: PP438800), respectively, these IBV isolates may be associated with immune escape. To study the biological characteristics of the isolates, they were inoculated into the allantoic cavities of 9-day-old SPF chicken embryos. After 6 d, all embryos showed typical embryonic lesions such as stunting or embryo dwarfing (Figure 1B). To determine the virus titer of the isolates, the allantoic fluid containing the virus was serially diluted 10-fold in PBS and inoculated into the allantoic cavities of 9-day-old SPF chicken embryos, respectively. After 6 d, EID50 was calculated using Reed and Muench methods by observing the embryonic lesions. The results showed that the CZ211063 strain had a higher virus titer than CZ200515 and CZ210840 (P < 0.01 and P < 0.05) (Figure 1C). To compare the growth kinetics of the isolates, allantoic fluid of 100 EID50 was inoculated into the allantoic cavities of 9-day-old SPF chicken embryos and harvested at 12, 24, 36, 48, 60, and 72 hpi. The virus titer at each time point was determined by RT-qPCR, and a growth curve was plotted. The results revealed that CZ200515 and CZ210840 reached the highest virus titers at 48 hpi, while CZ211063 reached its peak at 36 hpi, higher than CZ200515 and CZ210840 (P < 0.01 and P <0.001) (Figure 1D). The CZ211063 strain exhibits a higher replication capacity in chicken embryos, potentially impacting its virulence and transmissibility in poultry populations.Figure 1 Characteristics of IBV isolates. (A) The throat swab and cloacal swab samples were inoculated into the allantoic cavities of 9-day-old SPF chicken embryos for reproduction, followed by testing for infectious bronchitis virus (IBV), avian influenza virus (AIV), Newcastle disease virus (NDV), and infectious bursal disease virus (IBDV) in allantoic fluid using RT-PCR; 1, 2, and 3 represent CZ200515, CZ210840, and CZ211063, respectively. (B) The 3 isolates were inoculated into the allantoic cavities of 9-day-old SPF chicken embryos, and after 6 d, embryo lesions (stunting or embryo dwarfing) were observed. (C) Allantoic fluid containing the virus was continuously diluted 10-fold in PBS and inoculated into the allantoic cavities of 9-day-old SPF chicken embryos. EID50 was calculated after 6 d by observing embryonic lesions using the Reed and Muench methods. (D) The number of viral RNA copies of the virus was detected by RT-qPCR at 12, 24, 36, 48, 60, and 72 hpi after the virus was inoculated into the allantoic cavities of 9-day-old SPF chicken embryos. Error bars indicate the means ± SD.

Figure 1

Phylogenetic Analysis of the S1 Gene Sequence

To analyze the genetic characteristics of the S1 gene sequence from 3 isolates, the S1 gene was sequenced. The genetic evolution of the S1 gene in 3 isolates and 79 reference strains was analyzed using MEGA7.0 software. The results indicated that the 3 isolates were classified as QX-type but exhibited a noticeable evolutionary divergence. This divergence suggests that despite sharing a common lineage, the isolates have undergone genetic changes that set them apart from the vaccine strain (Figure 2). This observation is crucial in understanding the genetic drift and potential implications on vaccine efficacy and virus evolution in the field. The S1 amino acid homology of the isolates and QXL87 was 95.0% (CZ200515), 94.1% (CZ210840), and 93.5% (CZ211063), respectively (Supplementary Table S3). These accumulating mutations may affect the antigenic properties and potentially the pathogenicity of these strains.Figure 2 Phylogenetic analysis of the S1 gene sequence. The phylogenetic tree of the S1 gene sequence was created using MEGA7.0 software with 79 S1 gene sequences from 7 different genotypes (GI-GVII) as references. The isolates CZ200515, CZ210840, and CZ211063 were highlighted with black dots, whereas the vaccine strains H120, 4/91, and QXL87 were highlighted with black triangles.

Figure 2

Recombination Analysis

The potential recombination events in the whole genome and S gene sequence of IBV isolates were analyzed using RDP4 software. Among the 3 isolates, CZ200515 and CZ210840 exhibited potential recombination, while CZ211063 showed no recombination in the whole genome (Supplementary Table S4). Recombination differences increase the genetic diversity and complexity of viruses. No recombination event was found in the S gene sequence (results were not shown). CZ200515 was formed from the recombination of 4/91 and Scyz3, with recombination breakpoints at 12954 nt (nonstructural protein12 [NSP12] region encoded by Open reading frame 1ab [ORF1ab]) and 25883 nt (transcription regulatory sequences [TRS] region before the N protein coding sequence). CZ210840 was formed from the recombination of 4/91 and Scyz3 with a breakpoint at 26133 nt (within the N protein coding sequence region). CZ211063 showed no recombination breakpoints. The inconsistency in recombination breakpoints between CZ200515 and CZ210840 underscores the complexity of genetic recombination. To further understand the recombination characteristics, H120, 4/91, and Sczy3 (QX-type) were chosen as reference strains, and recombination was visualized using BootScan analysis of SimPlot software (Figures 3A–C). The genome sequences of the isolated and reference strains were divided into 3 parts at the recombination breakpoints: 1 to 12,954 nt (coding ORF1a), 12,955 to 25,883 nt (coding ORF1ab+S+E+M), and 25,884 to 27,681 nt (coding N). This segmentation enabled a more nuanced understanding of the genetic contributions from the parent strains. The phylogenetic tree was reconstructed using the neighbor-joining method of MEGA software. BootScan and phylogenetic analysis revealed that CZ200515 had 4/91 as the major parent and obtained the ORF1ab+S+E+M gene coding sequence (12,955–25,883 nt) from Scyz3. CZ210840 had Scyz3 as the major parent and acquired the N gene coding sequence (26,133–27,681 nt) from 4/91 (Figure 3D–F). In addition, we found that the 4/91 vaccine strain appears to have a higher recombination rate, either as a major or minor parent. This finding has significant implications for understanding the genetic stability and evolutionary potential of vaccine strains in the context of circulating field strains.Figure 3 Recombination analysis. Recombination characteristics of complete genome sequences of isolates. (A–C) IBV genome structure model map and BootScan recombination analysis. The colored lines represent different virus strains: red for the 4/91 (KF377577) vaccine strain; green for the Sczy3 (JF732903) strain; blue for the H120 (FJ888351) vaccine strain. (A) The CZ200515 strain resulted from the recombination of 4/91 and Scyz3 strains. The black arrows indicate 2 probable recombination breakpoints at 12954nt and 25883nt, respectively. (B) The CZ210840 strain resulted from the recombination of 4/91 and Scyz3 strains. The black arrows indicate the probable recombination breakpoint at 26133nt. (C) No potential recombination events were detected in the CZ211063 strain. (D–F) Phylogenetic analysis of regions divided based on potential recombination breakpoints. The IBV genome is segmented into 3 regions: 1-12954 (encoding ORF1a) (D), 12955-25883 (encoding ORF1ab+S+E+M) (E), and 25884 to 27681 (encoding N) (F). The phylogenetic evolutionary tree was reconstructed using MEGA software through the neighbor-joining method, with black dots representing the isolates.

Figure 3

Pathogenicity of IBV Isolates in 1-Day-Old SPF Chicken

To evaluate the pathogenicity of IBV isolates, 20 1-day-old SPF chickens per group were infected with 105 EID50 of CZ200515, CZ210840, and CZ211063 via eye drop and intranasal routes. A control group received PBS inoculation for comparison. Clinical symptoms were monitored, and mortality was recorded from 1 to 14 dpi. Following infection with the isolates, all chickens in each group exhibited symptoms such as lethargy, dyspnea, sneezing, and tracheal rales. These results highlight the potential impact of isolates on the health and welfare of poultry. Mortality rates varied among the isolates: 40% (CZ200515), 50% (CZ210840), and 80% (CZ211063) (Figure 4A), while the PBS control group showed no symptoms or mortality. Viral shedding levels in the throat and cloacal were high at 6 and 12 dpi in the group infected with isolates, with CZ211063 showing higher shedding than CZ200515 and CZ210840 (P < 0.05 or P < 0.001) (Figures 4B and C). This higher shedding rate suggests a potentially greater transmissibility of the CZ211063 strain. Viral load in the trachea, lungs, and kidneys of the infected group peaked at 6 dpi and gradually declined thereafter, though still detectable at 14 dpi. Additionally, CZ211063 showed a higher viral load than CZ200515 and CZ210840 (P < 0.05 or P < 0.001) (Figures 4D–F). These results indicated that the isolates had a strong affinity for the respiratory and urinary systems and were capable of sustained virus shedding through the throat and cloaca. These isolates possess a robust pathogenic potential that could pose significant challenges in poultry health management.Figure 4 Survival curve, viral shedding, and viral loads in different tissues. (A) The survival curve shows the percentage of survival in each group over the 14-dpi observation period. Viral shedding in the throat (B) and cloacal (C) of SPF chickens in each group was measured using RT-qPCR after being infected with isolates. (D-F) Viral loads in different tissues of SPF chickens following infection with isolates were measured by RT-qPCR, including trachea (D), lung (E), and kidney (F). Error bars indicate the means ± SD.

Figure 4

Gross Lesions in the Trachea, Lung, and Kidney

Based on autopsy observations at 6 dpi, except for the PBS group, each group infected with isolates exhibited severe tissue damage, underscoring the intense inflammatory response triggered by the virus. The trachea of all groups exhibited severe tracheal hemorrhage, with a small amount of mucoid exudate present in the trachea of the CZ210840 group. In contrast, the CZ211063 group exhibited a more severe exudation, indicating the potential for this strain to induce a more aggressive pathological process. The lungs exhibited marked congestion and edema in all infected groups, along with severe necrosis of the lungs in the CZ211063 group and a milder necrosis symptom in the CZ210840 group. The kidneys of all groups exhibited a typical "flower-spotted kidney" appearance due to swelling and white urate deposits. In conclusion, group CZ211063 was more pathogenic to chickens than the other 2 groups (Figure 5A).Figure 5 Gross lesions and histopathologic lesions in the trachea, lung and kidney. (A) Chickens were euthanized at 6 dpi, and trachea, lung, and kidney autopsies in each group were shown, respectively. The black triangles in the figure indicate each representative lesion area. No gross lesions were observed in the PBS control group. (B) Chickens were euthanized at 6 dpi. Figures show representative histopathological slides of the trachea, lung, and kidney. The figures were stained with hematoxylin and eosin. In the trachea tissues, black arrows indicate mucosal thickening; mucosal epithelial cell ciliary shedding and infiltration of inflammatory cells. In the lung tissues, black arrows indicate capillary stenosis and infiltration of inflammatory cells; black triangles indicate hemorrhage. In the kidney tissues, black arrows indicate infiltration of inflammatory cells; hollow triangles indicate necrosis in the epithelial cells of renal tubules; hollow arrows indicate glomerular atrophy.

Figure 5

Histopathologic Lesions in the Trachea, Lung, and Kidney

Histopathological results revealed that all groups infected with IBV isolates exhibited severe lesions in the trachea, lung, and kidney. The trachea tissues showed mucosal thickening, indicating a robust inflammatory response, shedding of mucosal epithelial cells, and infiltration of inflammatory cells, highlighting the disruptive impact of the virus on the structural integrity of the tracheal lining. The lung tissues exhibited capillary dilatation, hemorrhage, thickening of the pulmonary septa, and infiltration of inflammatory cells, indicating a systemic inflammatory response and tissue damage that could potentially affect respiratory function. Additionally, the kidney tissues demonstrated inflammatory cell infiltration, glomerular atrophy, and necrosis in the renal tubular epithelial cells, indicating a risk of severe renal injury and impaired function. However, no lesions were observed in any tissues of the PBS control groups (Figure 5B). In conclusion, the histopathological findings emphasize the multi-organ pathogenicity of the IBV isolates, particularly affecting the respiratory and urinary systems.

Virus Cross-Neutralization Assay Between IBV Isolates and QXL87

Antigen relatedness among IBV isolates and the QXL87 was evaluated through a virus cross-neutralization assay. The neutralization titers and R-values are detailed in Table 1. All IBV isolates exhibited R-values exceeding 11%, indicating that they belonged to the same serotype as QXL87. The R-values between CZ200515, CZ210840, and QXL87 were 39.47% and 36.70%, respectively, indicating a minor subtype difference. However, a major subtype difference was indicated by the R values of 27.72% between CZ211063 and QXL87, indicating a more substantial divergence in antigenic properties. This variance could potentially influence the effectiveness of the QXL87 vaccine against the CZ211063 strain. The homology of S1 amino acid residues between IBV isolates and the QXL87 ranged from 93.5% to 95%, showing a positive correlation with the R-value (higher S1 homology leading to greater R-value). This correlation suggests that higher S1 homology leads to a greater R-value, reinforcing the idea that antigenic relatedness is closely tied to the sequence similarity in the S1 amino acid residues. These results indicate that the QX-type isolates have evolved into different serum subtypes, resulting in decreased antigen relatedness with QX vaccine strains. This discrepancy may be the primary factor contributing to the incomplete protection provided by vaccines against circulating strains.Table 1 Virus cross-neutralization between isolates and the QXL87 vaccine strain.

Table 1IBV
Strain	Serum titera	Antigen relatedness
value (R)
(%) to QXL87b	
QXL87	CZ200515	CZ210840	CZ211063	
QXL87	28.32	27.37	27.00	26.59	100%	
CZ200515	26.84	28.00	—	—	39.47%	
CZ210840	27.00	—	28.50	—	36.70%	
CZ211063	26.60	—	—	28.57	27.72%	
a Titers were obtained in reciprocal virus neutralization tests (diluted serum, constant virus).

b R values were calculated according to a previously described method (Gravendyck et al., 1996; You et al., 2023). The criteria used to classify antigenic relatedness were as follows: >70%, antigenic identity; 33 to 70%, minor subtype difference; 11 to 32%, major subtype difference; and <11%, no relatedness (distinct serotype).

Multiple Sequence Alignment and Homologous Modeling Analysis of the S1 Subunit

Multiple sequence alignments of the IBV-S1 among isolates and QXL87 showed differences in amino acid residue sequences, uncovering the molecular basis of antigenic variation. Various mutations were identified in the hypervariable regions (HVR) and neutralizing epitope regions based on previous research (Figure 6A). Analysis of the S1 subunit structure among isolates and QXL87 revealed that the amino acid residue mutations in the neutralizing epitope region of the isolates were mainly concentrated in the S1 N-terminal domain (S1-NTD, 21-240aa) and S1 C-terminal domain (S1-CTD, 272-417aa) domains (Figures 6B and C). In the 21-27aa region of S1-NTD (within epitope D), CZ200515 exhibited point mutations of Pro23Phe and Thr26Asn; CZ210840 exhibited point mutations of Pro23Ser and Thr26Asn; and CZ211063 exhibited point mutations of Pro23Ser, Asn25Lys, and Thr26Asn; all resulting in conformational changes in epitope D, potentially leading to antigenic variation (Figure 6D). In the 116-123aa region of S1-NTD (within epitope E and HVR Ⅱ), CZ200515 exhibited a point mutation of Ser120Thr; while CZ210840 and CZ211063 exhibited a point mutation of Ser120Ala; all resulting in conformational changes in epitope E, potentially causing antigenic variation (Figure 6E). The S1-CTD domain contains a β-sheet structure, and the Ala353Thr mutation in CZ200515 caused conformational changes in the β-sheet structure (399-402aa region), independent of the neutralizing epitopes. Similarly, the Ala353Thr and Ile402Val mutations in CZ210840 and CZ211063 led to conformational changes in the β-sheet structure as well as in the 371-375aa region, all resulting in conformational changes in epitopes B/C/A, potentially causing antigenic variation (Figure 6F). These sites directly or indirectly impact the conformation of neutralizing epitopes and could be crucial for neutralizing epitopes of the subunit. Isolated strains variation potentially leads to decreased antigen relatedness with QXL87 and virus escape from immune surveillance. In summary, the identified mutations in the S1 subunit of the isolates directly or indirectly impact the conformation of neutralizing epitopes. These alterations could be crucial for the antigenic properties of the S1 subunit. The variation in the S1 subunit of the isolates decreased the antigenic similarity with the QXL87 vaccine strain. Furthermore, these variations might enable the virus to evade immune surveillance, highlighting the need for a vigilant approach in vaccine development and disease monitoring.Figure 6 Multiple sequence alignment and structural analysis of the S1 subunit. (A) The difference in the S1 amino acid sequence between the IBV isolates and QXL87 was visualized using the ESPript program. The regions of S1 neutralizing epitopes were highlighted in previous studies, with the neutralizing epitope sequences marked with black frames and the HVR sequences marked with blue lines. Structural analysis of mutation sites in S1 neutralizing epitopes. (B) Diagram of IBV S1. S1-NTD is cyan; the subdomain is yellow (including SD1 and SD2); and S1-CTD is green. (C) S1 single structure. The structure is colored to match (A), and mutation sites in the neutralizing epitope region are shown as red spheres. (D) Conformational change in the 21-27aa region (within epitope D) of S1-NTD. (E) Conformational change in the 116-123aa region of S1-NTD (within epitope E). (F) Conformational change in the β-sheet structure of the S1-CTD domain, with the amino acid sequence of the β-sheet structure below. (D–F) Green for QXL87, blue for CZ200515, magenta for CZ210840, gray for CZ211063. Conformational changes are indicated in red, and mutations in amino acid residues are highlighted by stick structures. All models were created using SWISS-MODEL and visualized with PyMol using PDB: 6CV0 as the crystal structure mode of the IBV S protein.

Figure 6

DISCUSSION

Although vaccination is the primary preventive measure for IBV infection, the frequent recombination and mutation of the virus lead to the continuous emergence of novel strains, presenting a challenge for IB control (Sjaak de Wit et al., 2011; Wang and Hou, 2023). In suspected cases of vaccinated chicken flocks with IBV live attenuated vaccines (H120, QX, or 4/91), we isolated 3 IBV strains. All isolates belong to the QX-type, highly pathogenic to SPF chickens, with morbidity rates of 100% and mortality rates of 40% (CZ200515), 50% (CZ210840), and 80% (CZ211063). These strains can cause respiratory and urinary system lesions. The group infected with the CZ211063 strain consistently exhibited higher viral shedding and loads at all detection time points postinfection, aligning with the higher mortality rate observed in this group. Even in the late stages of viral infection, surviving chickens continue to shed the virus, and virus detection remains possible in the tissues of surviving chickens at 14 dpi, indicating a prolonged infection period for the 3 isolates. The growth kinetics of the 3 isolates were evaluated in SPF chicken embryos, with CZ211063 reaching the highest virus titer at 36 hpi, while CZ200515 and CZ210840 peaked at 48 hpi. Compared to the other 2 IBV isolates, CZ211063 demonstrates a faster replication capability in vitro, potentially associated with its higher mortality rate, viral shedding, and tissue virus load.

QX-type genotype IBV is a prominent lineage globally, especially in China (de Wit et al., 2018; Meng et al., 2024; Zhao et al., 2017). Within the GI-19 lineage, 6 sub-lineages have been identified due to minor variations in the S1 amino acid residues (Lee et al., 2021; Xu et al., 2018). Our isolates are classified as QX-type genotype strains, and genetic evolution analysis has revealed that 3 IBV isolates had a certain evolutionary distance from the QXL87. The homologies of the S1 amino acids between the isolates and QXL87 were 95.0% (CZ200515), 94.1% (CZ210840), and 93.5% (CZ211063). Previous studies have indicated that a 2-3% difference in S1 amino acid sequence led to the development of distinct serotype strains (Cavanagh et al., 1992); 3 serotypes were identified of QX-type isolates with above 5% variation in S1 (Meng et al., 2024). To evaluate the antigen relatedness between the isolates and QXL87, cross-neutralization assays were carried out. These experiments indicated decreased antigen relatedness between isolates and QXL87 but no discernible serotype variance (R > 11%). The reduced cross-neutralization activity between the isolates and QXL87 potentially explains why the vaccine cannot provide effective protection against the circulating strains.

Multiple sequence alignments of the IBV isolates and the QXL87 S1 showed differences in the HVR (HVR I, HVR II, and HVR III) and neutralizing epitope regions. Previous studies have indicated that alterations in the HVR did not impact the pathogenicity and serotype of IBV. However, as the HVR variation increased, there was a decrease in antigen relatedness (Shan et al., 2018). Consequently, the decrease in R-values between the IBV isolates and QXL87 was associated with the HVR disparity. Analysis of the S1 subunit structure revealed that mutations of amino acid residues in the neutralizing epitope regions were predominantly clustered in the S1–NTD and S1–CTD domains. In the 21–27aa region of S1–NTD, all isolates exhibited mutations leading to conformational changes in epitope D, potentially causing antigenic variation. Mutations at site 120 in the 116–123aa region of S1–NTD resulted in conformational changes in epitope E, potentially causing antigenic variation. Furthermore, within the β-sheet structure of S1–CTD, the Ala353Thr and Ile402Val mutations in CZ210840 and CZ211063 induced conformational changes in the β-sheet structure, all leading to conformational changes in epitopes B/C/A, potentially causing antigenic variation. These specific sites may have a critical role in the conformation of neutralizing epitopes, antigen relatedness, and the ability of the virus to escape immune surveillance. Additionally, 3 linear epitopes were identified in the S2 subunit at 669-685aa, 686-697aa, and 692-703aa (Andoh et al., 2018), exhibiting antigenicity but lacking the capacity to induce neutralizing antibodies against IBV, potentially contributing to the generation of IBV-specific antibodies.

S1 attaches virus particles to the host cell membrane by interacting with sialic acid, triggering infection (Liu et al., 2024a). A recent study identified S1 N-terminal amino acid (aa) residues 19 to 227 (209 aa total) of IBV strains SD (GI-19), as the minimal sialic acid binding domain (SABD), highlighting its role in attenuation, nephrotropism, and serotypes (You et al., 2023). Multiple sequence alignment revealed mutations in various amino acid residues within the SABD region among the isolates. These mutations could directly contribute to reduced cross-neutralization activity between isolates and QXL87. The impact of these mutations on attenuation and nephrotropism remains uncertain and requires further investigation. The S1 subunit of IBV is a heavily glycosylated protein that can impact various characteristics of the virus (Parsons et al., 2019). A recent study suggests that spike N354 glycosylation augments SARS-CoV-2 fitness for human adaptation through structural plasticity (Liu et al., 2024b). This glycosylation modification can enhance the virus's ability to evade the immune system. Importantly, glycosylation can reduce the immunogenicity of the virus, weaken immune imprinting of the host, and facilitate reinfection (Liu et al., 2024b). Here, we did not observe an increase in glycosylation of the S1 subunit in the isolates, indicating that the presumed immune evasion is not linked to S1 subunit glycosylation but may be a direct result of amino acid residue mutations in the S1 subunit.

In light of the phenomenon that current vaccines do not provide complete protection against the circulating strains, previous studies have proposed that multiple vaccine immunizations based on different lineages can be implemented to broaden the range of protection, following the so-called "protective type" concept (Jordan, 2017; Legnardi et al., 2019). However, administering 2 or more live attenuated vaccines with different antigens significantly raises the risk of recombination between the vaccine and field strains (Cook et al., 1999; Terregino et al., 2008). CZ200515 and CZ210840 are closely related to 4/91 and Scyz3, potentially resulting from recombination between field strains and the 4/91 vaccine strain. Therefore, the emergence of CZ200515 and CZ210840 might be associated with the misuse of the 4/91 vaccine.

Recombination is a vital part of normal coronavirus replication, crucial for generating subgenomic RNAs (sgRNAs) and associated with the emergence of novel strains (Gribble et al., 2021). During sgRNAs synthesis, a set of highly homologous cis-acting elements called TRS are essential. The conservation of the TRS region across different CoV strains implies that template switching of RNA-dependent RNA polymerase (RdRp) could result in the emergence of novel recombination variants, facilitating high recombination rates of coronaviruses (Niu et al., 2023). While CZ200515 and CZ210840 strains both resulted from the recombination of 4/91 and Scyz3, their recombination patterns and breakpoints varied. The CZ200515 strain was formed with 4/91 as the major parental strain and Scyz3 as the minor parental strain, with recombination breakpoints at 12954 nt (NSP12 region encoded by ORF1ab) and 25883 nt (TRS region before the N protein coding sequence). The CZ210840 strain was formed with Scyz3 as the major parental strain and 4/91 as the minor parental strain, with a single recombination breakpoint at 26133nt (within the N protein coding sequence region). Different recombination types and inconsistent recombination sites indicate that coronavirus recombination is not exclusively limited to the TRS region. The recombination breakpoints of the CZ200515 strain in the ORF1ab region appear to be unrelated to the TRS region. Similar studies, such as the recombination analysis of PDCoV, have indicated frequent recombination in the ORF1ab region (He et al., 2020). Recombinant IBV strains with a ck/CH/LJL/140901-like backbone and an S2 fragment from a 4/91-like virus have been identified within the S protein-coding gene (Jiang et al., 2018). Genetic recombination events of the S1 gene of IBV isolates were also reported (Meng et al., 2024). None of these reorganization events occurred in the TRS region. Recent studies propose that the coronavirus proofreading ribonuclease (NSP14-ExoN) is essential in promoting extensive viral recombination (Gribble et al., 2021), potentially explaining why recombination is not limited to the TRS region.

In addition, we found that the 4/91 vaccine strain exhibits a higher recombination rate, whether as a major or minor parent strain. Previous research has indicated that the 4/91 vaccine can promote recombination with other strains (Chen et al., 2024; Feng et al., 2018; Yang et al., 2024), consistent with the findings of this research. The N protein coding genes of CZ200515 and CZ210840 both originate from the 4/91 vaccine strain, but their recombination breakpoints differ (CZ200515 breakpoint before the initiation codon AUG, CZ210840 breakpoint after the initiation codon AUG), further indicating that TRS is not the sole determining factor influencing virus recombination. The CZ200515 strain formed with 4/91 as the major parental strain and the QX strain encoding ORF1ab+S+E+M genes as the minor parental strain, exhibits high pathogenicity and kidney tropism. This suggests a correlation between this recombination region and virus virulence to some extent, with kidney tropism associated with the S gene. Previous studies have indicated that the 1ab region may influence the pathogenicity of the virus (Chen et al., 2024; Thor et al., 2011), potentially elucidating the higher virulence of CZ200515 compared to the major parent 4/91 strain despite the lack of direct evidence, necessitating further investigation. Coronavirus recombination has been associated with increased spread and severe disease, leading to vaccine failure in multiple livestock coronaviruses (Chen et al., 2017a). Therefore, given the continuous emergence of novel recombinant IBV strains, the ability to counteract virus recombination is a critical consideration for vaccine development.

CONCLUSION

In conclusion, the study indicates that QX-type IBV is prevalent in China with a high isolation rate, leading to severe clinical symptoms and significant economic losses in the poultry industry. QX strains have evolved into various sub-lineages, and current vaccines do not provide complete protection against circulating strains. The reduced cross-protection between the vaccine and circulating strains, along with the simultaneous use of multiple live attenuated vaccines, results in the coexistence of the vaccine and circulating strains in the same host, increasing the risk of virus recombination. Recombination events contribute to the genetic diversity of the virus and may also result in the phenomenon of enhanced virulence, causing vaccine immunization failure. Amino acid residue mutations in the S1 subunit result in antigenic variation by changing the conformation of neutralizing epitopes, which is crucial for the virus to escape host immune surveillance. Therefore, conducting real-time molecular epidemiological investigations on IBV prevalence and monitoring the transmission and genetic evolution of IBV strains are essential. This approach aids in developing prompt and accurate prevention and control strategies for IBV, guiding vaccine usage, and facilitating adaptive vaccine development.

DISCLOSURES

The authors declare no financial or commercial conflict of interest.

Appendix Supplementary materials

Image, application 1

ACKNOWLEDGMENTS

This study was supported by the Xinjiang Uygur Autonomous Region Major Science and Technology Project—Xinjiang Animal Disease Prevention and Control System Quality Improvement Project (2023A02007) and "Tianchi Talent" Introduction Program.

Ethical approval: All of the animal experiments were conducted in accordance with the regulations of the administration of affairs concerning experimental animals and approved by the Nanjing Agricultural University Experimental Animal Welfare Ethics Committee with the approval ID: NJAU.No20220706148.

Supplementary material associated with this article can be found in the online version at doi:10.1016/j.psj.2024.104256.
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