The use of animals in this study was approved by the South China Agricultural University Committee for Animal Experiments [approval ID: SYXK (Guangdong) 2019-0136]. All study procedures and animal care activities were conducted following the national and institutional guidelines for the care and use of laboratory animals.

Between Sept 2020 and July 2021, several chick flocks, having developed upper respiratory symptoms, including coughing, wheezing, rales, and nasal discharge, were suspected of being infected with IBV. A total of 910 oropharyngeal and cloacal mixed swab samples were collected from the suspected chick flocks from eight provinces in mainland China (Fujian, Guangdong, Guangxi, Yunnan, Hunan, Hubei, Jiangsu, and Zhejiang). The swab specimens were stored in 500 μL of 1 mM phosphate-buffered saline (PBS) containing 200 U/mL of penicillin and 200 μg/mL of streptomycin, and vortexed for 20 min to prepare the virus solution for testing. The total RNA was extracted by using the RaPure Viral RNA/DNA Kit (Magen, Guangdong, China). The viral RNA was measured by quantitative reverse transcription PCR (qRT-PCR) according to the following protocol (10) using the HiScript® II One Step qRT-PCR Probe Kit (Vazyme, Nanjing, China), with the primer pairs designed to target the gene RNA nucleotide sequence (Accession: AF093793.1). The reaction conditions were as follows: 50°C for 5 min, 95°C for 30 s, and 45 cycles of 95°C for 5 s and 60°C for 34 s on the Applied Biosystems™ 7500 (Thermofisher, Beijing, China). The positive judgment value for this assay is between 15 and 35 cycle thresholds (CT) per 2 μL RNA template. The sequences of primers and probes used were as follows: IBV-F (5′-CCTCTAAGGGCTTTTGAG-3′), IBV-R (5′-GTCACTGTCTATTGTATGTC-3′), and IBV-P (5′-FAMCACCACCAGAACCTGTCACCTC BHQ1-3′).

Special pathogen free (SPF) embryonated eggs were purchased from Guangdong DHN Poultry and Egg Products Co. Ltd. (Yunfu, Guangdong, China). After the positive swab samples were vortexed shortly and centrifuged at 750 × g for 5 min at 4°C, the supernatants (0.2 mL) were collected and serially diluted 10-fold from 10⁻¹ to 10⁻³ in PBS, which inoculate into 9-day-old SPF chicken embryonated eggs respectively with 0.2 mL by the allantoic cavity route. Infectious allantoic fluid was harvested from the embryos at 2–3 days post-inoculation and the viral RNA was determined as previously described. Next, specific primer pairs targeting the complete IBV S1 gene were designed for reverse transcription polymerase chain reaction (RT-PCR). The thermal cycling (Bio-Rad, UK) parameters were as follows: 94°C for 1 min, and 30 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 1.5 min. The complete S1 gene, ~1,640 bp, was amplified using the PrimeScript™ RT-PCR Kit (Takara, Japan), and its products were separated on agarose gel and purified using a QIAquick Gel Extraction Kit (Qiagen, USA). The purified PCR products were cloned into a pMD™19-T vector (Takara, Japan) and then sequenced by BGI Genomics Co., Ltd. The sequences of the primers used were as follows: S1-F (5′-AACTGAACAAAAGACCGACT-3′), S1-R (5′-CAAAACCTGCCATAACTAACA-3′) (11).

The sequencing was performed using DNAStar Lasergene software (DNAStar, Madison, WI, USA). Additional publicly available complete S1 gene sequences, obtained from GenBank, were aligned by the MEGA (version 7.0) ClustalW algorithm and the Test Neighbor-Joining Tree for designing the IBV S1 phylogenetic tree. A total of 1,000 bootstraps were calculated. Finally, the tree was visualized using Interactive Tree Of Life (iTOL) web-based tool (http://itol.embl.de/). All of the reference complete IBV S1 sequences representing all currently recognized types and subtypes used in the present study are listed in the Supplementary Material. Furthermore, the potential recombination events assessed by the Recombination Detection Program 4.0 (RDP 4.0, version 4.96) with strong P-values (<10–20), were further investigated by similarity plot and bootscan analyses implemented in Simplot 3.5.1.

A total of 30 ten-day-old SPF chickens were purchased from Guangdong DHN Poultry and Egg Products Co. Ltd. (Yunfu, Guangdong, China) and housed in the isolators and randomly divided into four groups. The GZ14 strain (TW-type) and strain CK/CH/GD/JR07-7 (QX-type) was isolated and saved in the laboratory. Subsequently, we inoculated the chicks intranasally with 10⁶ EID₅₀ per 0.2 mL of GZ14 strains, CK/CH/GD/JR07-7 strains and CK/CH/JS/YC10-3 isolates respectively and used the same volume of sterile allantoic fluid for the control group. Postmortem was carried out 7 dpi, and the kidney and air sac lesions of the chickens were scored as previously described (12–14) (Tables 1, 2).

The gross lesion scores of kidney and air arc were analyzed using the one-way ANOVA followed by Tukey's multiple comparison tests. A p-value of <0.05 was considered a statistically significant difference (^*), p < 0.01 was considered a moderately significant difference (^**), and p < 0.001 was considered a highly significant difference (^***).

Previous investigations have suggested that IBV will induce respiratory system and kidney disease in chickens (15, 16). In the present study, we collected swabs from chick flocks with suspected respiratory virus infections from eight provinces and analyzed them using RT-qPCR. In the examined flocks showing respiratory symptoms, 17.6% (160 out of 910) were infected with IBV. The most abundant strains were detected in the province of Hebei (Figure 1A). A total of 160 IBV strains were isolated and comparative analysis of the S1 gene revealed that QX-type, TW-type, 4/91-type, HN08-type, TC07-2-type, and LDT3-type exhibited IBV positive rates of 58.15, 25, 8.12, 1.86, 5.62, and 1.25% (Table 3), respectively. Among them, the QX-type was isolated in 73.3% of chicks infected in Fujian, 78.9% in Guangxi, 92.5% in Guangdong, 50% in Yunnan, 58.6% in Hunan, 63.2% in Jiangsu, and 57.1% in Zhejiang. In addition, TW-type threatened 63.6% of infected chicks in the province of Hubei, and Mass-type was not discovered (Figure 1B).

The nucleotide sequence of the complete S1 gene of isolates was aligned to relevant sequences that represent different IBV genotypes available in GenBank using MEGA 7.0. According to neighbor-joining tree phylogenetic analysis (Figure 2).

To reveal possible recombination events, the complete S1 genes of 20 reference strains were used as putative parent strains and recombination detection was conducted with RDP 4.0. Using at least five detection methods, the results suggest that four isolates' S1 genes are possibly recombinant strains, with p-values of ≤ 1 × 10⁻⁶ (Table 4). As shown in Figure 3, strain CK/CH/JS/YC10-2 appears to be recombinant with strain YX10 as the major parent and TW2575-98 as the minor parent, with recombination breakpoints mapping to positions 747 (beginning breakpoint) and 1673 (ending breakpoint). Strain CK/CH/JS/YC10-3 appears to be recombinant with strain YX10 as the major parent and TW2575-98 as the minor parent, with recombination breakpoints mapping to positions 757 (beginning breakpoint) and 1674 (ending breakpoint). Strain CK/CH/GX/ZS04-3 appears to be recombinant with strain TW2575-98 as the major parent and CK/CH/JS/YC10-3 as the minor parent, with recombination breakpoints mapping to positions 1252 (beginning breakpoint) and 1672 (ending breakpoint). Strain CK/CH/HN/LY04-5 appears to be recombinant with strain D90 as the major parent and CK/CH/JS/LYG11 as the minor parent, with recombination breakpoints mapping to positions 88 (beginning breakpoint) and 948 (ending breakpoint).

Based on the viral recombination results, the CK/CH/JS/YC10-3 isolate, which was formed by the major parent strain YX10 (QX-type) and the minor parent strain TW2575-98 (TW-type), was used for the exposure tests.

Postmortem, all of the birds exposed to strain GZ14 showed obvious lesions in the kidneys, in which they were mainly preformed nephritis, as well as different degrees of air sac turbidness. In contrast, the birds with strain CK/CH/GD/JR07-7 or the CK/CH/JS/YC10-3 isolate generally showed swollen kidneys and obviously thickened air sacs covered with yellow white caseous exudate. No clinical symptoms were observed in the chickens of the control group (Figure 4A). In addition, gross lesions in the air sacs and kidneys were scored and recorded (Figure 4B).