The RT‒PCR results for the IBV virus across various farms revealed that six farms tested positive, whereas the remaining farms tested negative. High positive samples were found on four farms (two broiler farms and two-layer farms), with Ct values ranging from 20 to 24 (Table 1). Additionally, two-layer farms presented low-positive samples, with Ct values of 33 and 34. The farms with positive results used a variety of vaccines, including 4/91, Ma5, Ma5 clone30, and H120, with poultry ages ranging from 4 to 50 weeks, and included both layer (seventeen farms) and broiler (four farms) types. In contrast, the farms with negative results, regardless of their vaccine combinations and poultry ages, did not show detectable levels of IBV.

The Saudi IBV in GenBank was found to be clustered in GI-1, GI-16, GI-13 and GI-23. The four IBV strains genotyped in the present study were related to GI-1 (Ck/SA/Kharj-1/2023 and Ck/SA/Shaqra-4/2023) and GI-23 (Ck/SA/Kharj-2/2023 and Ck/SA/Kharj-3/2023). GI-23 is subclustered into 23.1 and 23.2 (23.2.1, 23.2.2, 23.2.3). The Saudi strains in the current study, GI-23|Ck/SA/Kharj-2/2023 and GI-23|Ck/SA/Kharj-3/2023, were found to be subclustered with GI-23.1, which contains the original Egypt/Beni-Suef/01/Var I strain that was first isolated in 1999. This subcluster also contains MRB02/2016- Iran, Var2-06, which also contains two strains from Saudi Arabia, SA/IH1/12 and IBV/CH/SA/5/2019. Another Saudi strain (IBV/CH/SA/6/2019) was found in the NCBI database and found to be related to GI-23.2.2 together with Egy/Var II. strains and a commercial vaccine strain against Egy Var II (Fig. 1).

Phylogenetic tree of the full-length spike gene from infectious bronchitis virus isolates in Saudi Arabia. This figure illustrates the phylogenetic tree of the full-length spike gene of infectious bronchitis virus (IBV) isolates from Saudi Arabia. The tree was generated through maximum likelihood analysis with 1000 bootstrap replications to ensure robust statistical support. The isolates from the current study are represented in red font, whereas the vaccine strains are shown in blue font

ORF1ab showed that Ck/SA/Shaqra-4/2023, which was classified as GI-1, was clustered with ORF1ab from GI-13, including the 4/91 vaccine strain, whereas Ck/SA/Shaqra-1/2023, which was classified as GI-1, was clustered with other GI-1 strains. Saudi GI-23 strains did not cluster with other GI-23 strains but clustered with strains belonging to GI-26, GI-14, and GI-19 (Fig. 2).

Both the 3ab and E genes showed that GI-23|Ck/SA/Kharj-2/2023 and GI-23|Ck/SA/Kharj-3/2023 gained their 3ab genes from GI-19 FN30414|ITA/90,254/2005 (Fig. 2).

The M gene of Ck/SA/Shaqra-4/2023, which was classified as GI-1, was clustered as a subtree from Saudi strains that belong to GI-23.1, whereas Ck/SA/Karj-1/2023 was clustered with other GI-1 strains. The Egyptian GI-23 strains were clustered in another subtree distant from that of the Saudi GI-23 strains. Moreover, the GI-23|Ck/SA/Kharj-2/2023 5ab gene recombined, and the 5ab gene was obtained from GI13 strains, including the 4/91 vaccine (Fig. 2).

N gene analysis revealed that the GI-23|Ck/SA/Kharj-2/2023 and GI-23|Ck/SA/Kharj-3/2023 genotypes, which are GI-23.1 genotypes, are clustered with GI-13, including the 4/91 vaccine. In contrast, Ck/SA/Kharj-1/2023, the GI-1 genotype, is clustered with other GI strains.

Phylogenetic tree of the full-length ORF 1ab, 3ab, E, M, 5ab, and N genes of infectious bronchitis virus isolates from Saudi Arabia constructed via maximum likelihood with 1000 bootstrap replications. Figure 3 shows the phylogenetic tree based on the full-length sequences of the ORF1ab, 3ab, E, M, 5ab, and N genes of infectious bronchitis virus (IBV) isolates from Saudi Arabia. The trees were constructed via the maximum likelihood method with 1000 bootstrap replications to ensure robust statistical support. Isolates from the current study are shown in red

An amino acid substitution at 9 V to an A was found in the signal peptide in the IBV-EG/1212B-SP1-2012/vaccine in comparison to the Saudi GI-23 strains (CK/SA/Kharj-2/2023 and CK/SA/Kharj-3/2023). The Saudi G1 genotype (CK/SA/Kharj-1/2023 and Ck/SA/Shaqra-4/2023) showed no amino acid substitutions from the H120 vaccine. The cleavage site between S1 and S2 was found to be RRFRR/S for the genotype 1 Saudi strains but RRTRR/S for Saudi genotype 23 (CK/SA/Kharj-2/2023 and GI-23|CK/SA/Kharj-3/2023) (Suppl. 1).

The numbering of the amino acid sequences was based on the S1 sequence of IBV strain H120 and included signal sequences (18 amino acids, MLVTPLLLVT LLCALCSA). Amino acids 38, 43, 63, and 68 are critical for receptor binding. D 38 to S (GI-23|CK/SA/Kharj-2/2023, GI-23|CK/SA/Kharj-3/2023) was detected, whereas D 38 to T was detected in IBV-EG/1212B-SP1-2012|Vaccine (Suppl. 1). H43 was conserved in all the Saudi strains screened in the present study. G 63 to D was detected in GI-23|CK/SA/Kharj-2/2023, GI-23|CK/SA/Kharj-3/2023), whereas deletion of this residue was detected in the IBV-EG/1212B-SP1-2012|Vaccine (Supp. 1). Moreover, an I to A 68 amino acid substitution was detected in GI-23|CK/SA/Kharj-2/2023, GI-23|CK/SA/Kharj-3/2023) and the IBV-EG/1212B-SP1-2012|Vaccine (Fig. 3, Suppl. 1). GI-23|Ck/SA/Kharj-2/2023 and GI-1|Ck/KSA/Shaqra-1/2023 did not show any amino acid substitutions in HVR-I from the H120 or Ma5 vaccine. Among the 3 HVRs, only the H117Q aa substitution was detected in the GI-1|Ck/KSA/Shaqra-1/2023 Saudi GI-1 strain in comparison with the H120 vaccine. In contrast, 54 V to L, 57 S to A, Q63 to D, and S77 to T were detected in the Ck/KSA/Kharj_3/2023 and Ck/KSA/Kharj_2/2023 from the IS/1949/06 and Egy Var 2 vaccines. S63 deletion was also detected in both GI-23 vaccines (Fig. 3).

Amino acid sequences of hypervariable regions (HVRs) HVR-I, HVR-II, and HVR-III of the spike protein from Saudi strains of infectious bronchitis virus compared with reference and vaccine strains. This figure illustrates the deduced amino acid sequences of the hypervariable regions (HVRs) HVR-I, HVR-II, and HVR-III of the spike protein from infectious bronchitis virus (IBV) isolates collected in Saudi Arabia. The sequences are compared to those of the reference and vaccine strains

In HVR-II, S105 to T, H/S 119 to N, V121S, S130 to H, and G131N/Y and deletion of the 142 aa residue were detected in IBV-EG/1212B-SP1-2012|vaccine|Var2 in comparison to Saudi GI-23 strains (Ck/KSA/Kharj-2/2023 and Ck/KSA/Kharj-3/2023). In HVR-III, T279Y, S/L285H, T288N, N292H, N295S and I296L were detected in the currently available vaccine of GI-23 (IBV-EG/1212B-SP1-2012|Vaccine||Var2). I609V amino acid substitution was detected in 3/4 of the Saudi strains, including GI-1: Ck/SA/Shaqra-4/2023 and genotypes GI-23|CK/SA/Kharj-2/2023 and GI-23|CK/SA/Kharj-3/2023 (Fig. 3). The sequence variation between Saudi IBVs is summarized in Suppl. 1. The 3D modelling of the S1 protein from different Saudi strains in the current study, along with closely related commercial vaccines, showed marked difference between the G1 and G23 strains. The HVR-1 to HVR-3 regions showed complete identity between PP840344|CK/SA/Kharj-1/2023|G1 and H120, while only H to Q117 was detected in PP840347|CK/SA/Shaqra-1/2023|G1 (Fig. 4a). Figure 4 also illustrates the amino acid differences between H120 and the G23 genotypes of Saudi strains, as well as the currently available commercial vaccine (KU979007|IBV-EG/1212B-SP1-2012). It highlights the amino acid substitutions in HVR-I to HVR-3 of G23 vaccine variant II compared to the Saudi strains of G23 (Figs. 3 and 4). Notably, significant 3D structural changes were observed in the S1 protein of the commercial G23 vaccine compared to the Saudi G23 strains.

Structural comparison of the S1 protein from different Saudi strains with closely related vaccine strains. (a) S1 protein of the H120 vaccine strain (G1), (b) PP840344|CK/SA/Kharj-1/2023 (G1), and (c) PP840347|CK/SA/Shaqra-1/2023 (G1), representing Saudi strains related to the G1 genotype. (d) S1 protein of the commercial Egyptian G23 strain (KU979007|IBV-EG/1212B-SP1-2012), (e) PP840345|CK/SA/Kharj-2/2023 (G23), and (f) PP840346|CK/SA/Kharj-3/2023 (G23), representing Saudi G23 strains. The amino acid differences in the highly variable regions (HVR-I to HVR-III) between the H120 vaccine strain and the G23 Saudi strains are highlighted. The variations in amino acids in the G23 vaccine strain (d) compared to the Saudi G23 strains (e, f) are also marked. Circles indicating structural differences in the loops of the 3D models

Our analysis identified multiple recombination breakpoints with varying parentages and detection methods in ORF1ab (Table 2). For GI-1|Ck/KSA/Shaqra-1/2023, recombination events were detected in the nsp3 (papain-like protease (PLpro)) protein at three breakthrough positions, as were nsp4, nsp5 (3 C-like protease (3CLpro)), nsp6, and nsp10. GI-1|Ck/KSA/Kharj-1/2023 showed recombination events in nsp3-nsp5 and nsp3. It also shared a similar recombination event at two positions in nsp3 with GI-1|Ck/KSA/Shaqra-1/2023. GI-23|Ck/SA/Kharj-2/2023 showed recombination events in nsp3 at four positions and showed recombination events at nsp2 (a replicase product important for proofreading viral replication) and nsp12 (an RNA-dependent RNA polymerase (Pol/RdRp)). GI-23|Ck/SA/Kharj-3/2023 showed similar recombination events as GI-23|Ck/SA/Kharj-2/2023 at 3 positions of nsp3 and a single position of nsp12.

For breakpoint positions 419 to 2254 (nsp2 and nsp3), the recombinant sequence GI-23|Ck/SA/Kharj-2/2023 was found to have recombined with Ck/KSA/Kharj-3/2023, with H120, 4/91, and Ma5 as major parents, detected via RDP, GENECONV, Bootscan, Maxchi, Chimaera, SiSscan, and 3Seq. Similarly, breakpoints from 4244 to 5200 and 6115–6476, both located in nsp3, revealed recombination involving GI-23|Ck/SA/Kharj-2/2023 and GI-23|Ck/SA/Kharj-3/2023 with distinct minor and major parents, including NGA/A116E7/2006, 4/91, and IBV/ck/EGY-Monuf/NR725/16. Additional recombination events were detected at various positions, such as nsp12 (13756 to 14064), nsp3 (5593 to 5964), and nsp3-nsp5 (4286 to 8252), with diverse recombinant sequences and parental strains, including GI-1|Ck/KSA/Kharj-1/2023 and GI-1|Ck/KSA/Shaqra-1/2023, and parents, such as MF421319| UY/09/CA/01 and KR231009| B1648.

Notably, breakpoints of GI-1|Ck/KSA/Shaqra-1/2023 at nsp5-nsp6 (8794–9888) with H120 and Ma5 as major parents and nsp2-nsp16 (19876–791) highlighted recombination involving GI-1|Ck/KSA/Shaqra-1/2023 with a combination of minor and major parents detected by multiple methods, including RDP, GENECONV, and Chimaera (Table 2). In addition, the deduced amino acid sequence of nsp3 also revealed 133’ SAGAGECDTA’ 142 246‘EKSV’249, 295’AEE ’297 insertions in GI-23.1 Saudi strains (Kharj 2, and 3) from GI.19|FN430414|ITA/90254/2005 (Suppl. 2).

The detection of recombination events within various ORFs of Saudi IBV strains revealed distinct patterns of genetic exchange involving multiple strains and parent sequences. For the S gene, a recombination event spanning positions 828 to 4198 was identified involving GI-23|Ck/SA/Kharj-2/2023 and GI-23|Ck/SA/Kharj-3/2023, with KU238176| D888/2/4/08_IR and JX173489| Eg/CLEVB-1/IBV/012 as major and minor parents, respectively. This event was detected via various methods, including GENECONV, Bootscan, Maxchi, Chimaera, SiSscan, and 3Seq (Table 3).

The M gene of GI-1|Ck/KSA/Shaqra-1/2023 had two recombination events, with GI-23| Ck/KSA/Kharj-2/2023 and H120 being the major parents for the two recombination events. The N gene GI-23|Ck/SA/Kharj-3/2023 underwent two recombination events, with MF421319| UY/09/CA/01 and B164 being the major parents for the two recombination events. GI-23|Ck/SA/Kharj-2/2023 exhibited recombination events, with KY805846|IBV/Ck/EG/CU/4/2014 as the major parent (Table 3).

In 3ab, FN430414| ITA/90254/2005 was the major parent of the recombination event involving GI-23|Ck/SA/Kharj-2/2023 and GI-23|Ck/SA/Kharj-3/2023. Moreover, at 5ab, ON419883|AR/13/BA/A255 was the major parent of the recombination event detected at GI-23|Ck/SA/Kharj-3/2023 (Table 3).