Characterization of the Genomics and Antigenicity of a Naturally Attenuated Gammacoronavirus Infectious Bronchitis Virus Strain in the Genotype GVI-1 Lineage

Abstract

Infectious bronchitis virus (IBV) causes a highly contagious disease in chickens. The prevalence of GVI-1 is increasing; however, the genomic characteristics and antigenic properties of this genotype strain remain insufficiently characterized. In this study, the genome characteristics and antigenic properties of a naturally attenuated CK/CH/SC/YC_GVI-1-DK/LMB20210104 (abbreviated as YC_GVI-1) strain were systematically analyzed. YC_GVI-1 occupies a distinct phylogenetic lineage and shares a similarity of 98.2%, the highest nucleotide sequence homology, with the reference strain CK/CH/FJ/202005 (accession number: MW791835.1). This strain was likely originated through a genetic recombination event between two major parental strains, CK/CH/FJ/202005 and CK/CH/GX/HX (accession number: PP817796.1). However, its S protein harbors ten unique amino acid substitutions, compared to the same protein in the other two virulent strains in the same genotype. AlphaFold3-based structural prediction reveals that one of these substitutions, methionine 485 to valine substitution, may induce a conformational change in the adjacent phenylalanine residue at position 431, resulting in a shift in the local secondary structure from β-sheet to random coil. Characterization of its antigenicity showed that this strain induces a strong humoral immune response, with neutralizing antibody titers of 2^6.40 against homologous strain YC_GVI-1 and 2^4.00 against heterologous strain JS96_GI-19. Furthermore, vaccination of chickens with this strain conferred complete protection (100%) against JS96_GI-19. The findings provide novel insights into the molecular evolution and antigenicity of YC_GVI-1, offering key information for improving IBV surveillance and vaccine development.

1. Introduction

Infectious Bronchitis (IB) is an acute, highly contagious respiratory illness triggered by the Infectious Bronchitis Virus (IBV), which presents a significant risk to the poultry production systems. IBV infection mainly damages the respiratory, urinary and reproductive, and digestive systems, may cause death of chicks, reduce feed conversion rate, and decrease egg production and quality of laying hens, seriously threatening the chicken breeding industry. Currently, the primary strategy for controlling IBV infection relies on vaccine-induced immunity [1], and commercially available vaccines are predominantly classified as either live attenuated or inactivated formulations. However, the genome of the IBV is highly prone to genetic mutations, deletions, insertions, and recombination events, leading to the continual emergence of novel genotypes that show variable and often poor cross-protection [2,3,4].

The IBV is the first known member of the gammacoronavirus genus and was first discovered in North Dakota, USA, in 1931 [5]. Its genome is approximately 27.6 kilobases in length and contains a 5′ cap structure and a 3′ polyadenylated (PolyA) tail. As a positive-sense single-stranded RNA (+ssRNA) virus, the IBV initiates translation of its genome immediately after entry. It sequentially encodes non-structural proteins and four major structural proteins, spike (S), envelope (E), membrane (M), and nucleocapsid (N). S protein represents the largest structural protein and plays a central role in viral attachment and entry, as well as in eliciting neutralizing antibodies [6]. Additionally, multiple accessory genes are located within the structural gene-coding regions in the 3′ one-third segment of the genome. While these accessory genes may not encode structural proteins, they are critically involved in the regulation of viral replication and pathogenesis [7].

Based on the complete S1 gene sequences, the IBV was initially classified into six genotypes (GI~GVI), encompassing 32 distinct lineages (GI-1~GI-27, GII-1~GII-2, GIV-1, GV-1, and GVI-1) as reported by Valastro et al. [8]. The most recent classification system based on the full-length S1 further divides the IBV into seven genotypes (GI~GVII) and thirty-three distinct lineages [9]. The prevailing strains in China at present are GI-1, GI-7, GI-13, GI-19, GI-22, GI-28, GVI-1 and some mutant strains [10,11]. The GVI-1 genotype exhibits high transmissibility in chickens. The first genotype GVI isolate in China was from Guangxi Province in 2007 and designated as strain TC07-2 [12]. Subsequently, GVI-1 strains were detected in Japan (2009) [13], South America (2010) [14], and South Korea (2015) [15]. Epidemiological data indicate that the prevalence of GVI-1 increased from approximately 5.62% during 2020–2021 [16] to 6.88 ± 1.84% over the period 2019–2023 [10], demonstrating a rising trend over time. This genotype has emerged as one of the IBV lineages requiring close surveillance and ongoing monitoring, particularly in southern China [17], where its prevalence is significantly higher. The GVI-1clinical isolates primarily affect the respiratory system and are associated with a relatively low mortality rate [12,18]. Certain isolates have also been observed to impact the reproductive system [19]. Highly virulent strains, such as the HX strain, can induce pathological changes including thickening of the proventricular mucosa with hemorrhagic spots, hemorrhages in the thymus and spleen, as well as pale, marble-like kidneys accompanied by uric acid deposition [20].

Currently, the GI-19 genotype (QX-like strain) is the predominant circulating lineage in China. It exhibits a broad geographical distribution, with a prevalence exceeding 50%, and demonstrates an ongoing trend of dissemination and genetic diversification [21], but corresponding vaccines are lacking [22,23]. Furthermore, the GVI-1 strains in China might have independently evolved from a recombination event between the GI-19 strain from Colombia and the CO8089L/CO8091L-like virus [12], and strains in the GVI-1 lineage displayed higher identities with GI-19, ranging from 90.5% to 93.4%, compared to those of other lineages [12]. Therefore, we chose YC_GVI-1 as a representative strain, in this study, to characterize its genomics and immunogenicity and the efficacy against GI-19.

In this study, the complete genome of YC_GVI-1 was sequenced and analyzed to elucidate the origin and evolutionary trajectory of this viral lineage. Furthermore, its molecular characteristics and cross-reactivity with other predominantly circulating strains in serum neutralization assays were examined to assess the immunogenicity of GVI-1 strains and their protective spectrum. Our results demonstrated that YC_GVI-1 is a naturally attenuated strain capable of eliciting a robust humoral immune response and providing partial cross-protection against heterotypic GI-19 genotype. Further evidence indicates that this attenuation may be potentially caused by a methionine to valine substitution at amino acid position 485 (M485V) in the S protein.

2. Materials and Methods

2.1. The Viruses, Animals and Ethics Statements

CK/CH/SC/YC_GVI-1-DK/LMB20210104 (abbreviated as YC_GVI-1, GenBank accession number: PX767077) and CK/CH/JS/TAHY (abbreviated as JS96_GI-19, GenBank accession number: ON260865.1) are strains isolated and preserved by our laboratory. One-day-old (SPF) chickens and SPF chicken embryos were purchased from Xinxing Dahuanong Poultry Egg Co., Ltd. (Yunfu, China) for the immune efficiency assay. This study did not involve endangered or protected species, and it was approved by the Animal Experiments Committee of Zhaoqing Dahuanong Biopharmaceutical Co., Ltd. (LL-G-20250201-01, Zhaoqing, China).

2.2. Virus Purity and Electron Microscopy Examination

YC_GVI-1 was isolated from poultry farms in Sichuan province in 2021. The viral isolates were cultured in 9-day-old SPF chicken embryos. Under aseptic conditions, allantoic fluid was harvested from the infected eggs at 3 days post-inoculation and kept at −80 °C for further use. The allantoic fluid was collected for RNA extraction using TRizol (Invitrogen, Carlsbad, CA, USA), which was subsequently reverse-transcribed into cDNA (CWBIO Biotech Co., Ltd., Beijing, China). Meanwhile, viral DNA in the allantoic fluid was extracted using a Viral DNA Extraction Kit (OMEGA Bio-Tek, Inc., Norcross, GA, USA). Conventional PCR [24] was applied to screen exogenous viral with primer sequences listed in Table 1. The allantoic fluid was centrifuged at 12,000× g for 15 min, and the supernatant was collected and subjected to ultracentrifugation at 30,000× g for 3 h. The white pellet from the bottom of the tube was collected and resuspended in 500 μL of sterile PBS for transmission electron microscopy analysis.

Table 1.

Detection primers for exogenous virus.

Virus	Primer Sequence	Length (bp)
IBV	F:5′-TGTTGGAGAAGTTACTGTTTTTAGTG-3′	1623
	R:5′-TGAGGTATTGGTTAATCTAAC-3′
NDV	F:5′-GACTGTAAGATGGCAAGACGACCAGCTC-3′	670
	R:5′-GGCTGAAGGATCCTCATTC-3′
AIV H5	F:5′-AACTGAGTGTTCATTTTGTGTGAAT-3′	380
	R:5′-AATGCAGACGGAGGAGGAACT-3′
AIV H9	F:5′-CTCCTCACAGACGATAATCC-3′	480
	R:5′-GTACAGTTCTTGTTCTATG-3′
ILTV	F:5′-AGGTTGGCGCTGTATACTTAGC-3′	430
	R:5′-TTGCAAATAGCGTCTGCTCGATTGA-3′
ALV-A	F:5′-GGTAGACCTCAGTAAGAAAC-3′	700
	R:5′-ACACAAAGAGGCCTCTGTAAGGACA-3′
IBDV	F:5′-AGCGATGACGAACCTGCAAG-3′	1300
	R:5′-ACCACCGGCACAGCTATCCT-3′
FAdV-4	F:5′-TTCGCCAAGTCTCAGTACAAT-3′	291
	R:5′-GGAGTGGTGATACAGCAGGTT-3′

2.3. Viral Titer and Growth Curve of the Isolated Strain

YC_GVI-1 was serially diluted tenfold in sterile PBS from 10⁰ to 10⁻⁶, and 0.2 mL of each dilution was inoculated into 9-day-old SPF chicken embryos, with six replicates per dilution. Embryonic mortality and pathological changes were monitored and documented daily, and the 50% egg infectious dose (EID₅₀) was determined using the Reed-Muench statistical method [25]. Due to the low pathogenicity of YC_GVI-1 in chicken embryos, Western blot analysis was employed to validate and adjust the EID₅₀ titration results.

The assay was performed based on a previously described protocol with minor modifications [26]. In brief, equal quantities of total protein were loaded and resolved via SDS-PAGE using the Bio-Rad Mini-Protean Tetra system (Bio-Rad Laboratories, Hercules, CA, USA). Following electrophoretic separation, proteins were transferred onto a 0.2 μm nitrocellulose membrane using the Bio-Rad Trans-Blot system (Bio-Rad Laboratories, Hercules, CA, USA). To prevent non-specific interactions, the membrane was blocked with 5% skim milk in TBST buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20) at room temperature for 1 h. After blocking, the membrane was incubated at 4 °C overnight with primary antibody (Mouse monoclonal antibody) against IBV nucleocapsid protein (anti-IBV N) (XJLab, Zhaoqing, Guangdong, China) at a concentration of 1 mg/mL, diluted in TBST containing 3% (w/v) BSA. The membrane was then washed three times with TBST and probed with a horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (Selleck Chemicals, Beijing, China) at a 1:5000 dilution for 2 h at room temperature. Following three additional TBST washes, protein signals were visualized using the Amersham ImageQuant 800 imaging system (Cytiva, Uppsala, Sweden) according to the manufacturer’s guidelines.

Subsequently, a viral inoculum containing approximately 10² EID₅₀ was administered into 9-day-old SPF embryos, with three independent replicates for each experimental group. Following inoculation, the embryos were incubated at 37 °C. Allantoic fluid samples (200 μL) were collected at 0, 12, 24, 48, 72, 96, and 120 h post-infection, for virological analysis. Allantoic fluids were collected at each time point to determine viral titers via EID₅₀ assays and to construct the corresponding growth curve. Concurrently, viral copy numbers were quantified using the same samples. Total nucleic acids were extracted from the collected allantoic fluids using a commercial RNA/DNA extraction kit (Vazyme Biotech Co., Ltd., Nanjing, China). The presence or absence of the virus in the allantoic fluid was confirmed by Western blot analysis, and RT-qPCR was performed at each time point to measure viral copy numbers for subsequent generation of a standard curve for viral load quantification [27].

2.4. Viral Genome Sequencing and Assembly

RNA was extracted from allantoic fluid using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and the concentration and purity of the extracted RNA were evaluated. Qualified RNA samples were rapidly frozen at −80 °C and transported on dry ice to Shanghai Tanpu Biotechnology Co., Ltd. (Shanghai, China) for whole-genome sequencing. The sequencing results were assembled and annotated using SnapGene software v4.2.4 (GSL Biotech. Co., Ltd., Chicago, IL, USA).

2.5. Phylogenetic, Homology and Recombination Analysis

The evolutionary relationship of YC_GVI-1 was analyzed based on the full-genome sequence, and the full-genome sequences of 22 representative reference strains of different genotypes circulating in China were all downloaded from the NCBI database and subsequently aligned using ClustalW (MEGAX64). A phylogenetic tree was constructed using the neighbor-joining method in MEGA X, and the bootstrap values were calculated based on 1000 replicates of the original data. The homology of the full-genome, 1ab, S1, S2, 3a, 3b, E, M, 5a, 5b and N genes of YC_GVI-1 were compared and analyzed with eight representative IBV strains of major prevalent genotypes in China, including H120 (FJ888351.1), TW-1 (KT946798.1), 4/91 (KF377577.1), JS96_GI-19 (ON260865.1), E160_YN (MK644086.1), LDT3-A (KR608272.1), CK/CH/FJ/202005 (MW791835.1), and CK/CH/GX/HX (PP817796.1), using MEGA X and MegAlign (Ver 7.1.0.44) software.

The complete genomic sequences of YC_GVI-1 and twenty-two representative strains were processed using MegAlign (Ver 7.1.0.44) software and stored in MEGA (.meg) file format. To detect possible recombination events across the 23 strains and infer putative parental genotypes, the Recombination Detection Program version 4.0 (RDP 4.0, v4.100) was employed with a p-value cutoff of less than 0.05 and a sliding window length of 30 base pairs. Default parameters were applied in all analyses, which incorporated several recombination detection methods, RDP, BootScan, GeneConv, MaxChi, Chimaera, SiSscan and PhylPro. Putative recombination signals and their corresponding breakpoints were further validated through similarity plot analysis conducted in SimPlot (version 3.5.1), utilizing a 200 bp window and a step increment of 20 bp.

2.6. Comparative Analysis of Sequence and Structural Variations in the S Protein Between YC_GVI-1 and Previously Reported Virulent Strains

The S protein sequence of the YC_GVI-1 strain was aligned with previously reported virulent strains of the same genotype, CK/CH/FJ/202005 and CK/CH/GX/HX, using MEGA X and MegAlign (Ver 7.1.0.44) to identify amino acid residues unique to YC_GVI-1. The three-dimensional structures of the S proteins from YC_GVI-1, CK/CH/FJ/202005, and CK/CH/GX/HX were predicted using AlphaFold3 and visualized using PyMOL v3.1. Structural superposition was subsequently performed using the align function in PyMOL to compare the three-dimensional conformation of YC_GVI-1 S protein with the reference strains, enabling a systematic evaluation of the influence of the identified unique residues on the spatial architecture of the protein.

2.7. Immunization and Serum Antibody Analysis

Twenty-four SPF chickens were allocated to two groups in negative pressure isolators and vaccinated subcutaneously at 1-day-old with 10³ EID₅₀ of YC_GVI-1 (n = 11) and PBS Con (n = 13), respectively. A booster immunization was conducted using the same approach and dose at 10 days old. Serum samples were taken from each group of chickens at 7 and 21 dpi. Total serum immunoglobulin G (IgG) specific for IBV was measured by indirect enzyme-linked immunosorbent assay (ELISA) To date, no commercially available serological assay has been validated for the detection of antibodies against the GVI-1 genotype of infectious bronchitis virus (IBV). Accordingly, we developed a prototype self-developed ELISA using the recombinant S1 subunit protein specific to this genotype.

The detailed protocol for the ELISA is as follows: (1) Coating: Purified S1 protein was diluted to 1.0 μg/mL in carbonate–bicarbonate coating buffer (pH 9.6), and 100 μL per well was added to a 96-well high-binding microplate. Each sample concentration was tested in triplicate. Plates were incubated overnight at 4 °C. (2) Blocking: Following incubation, plates were washed three times with PBST (0.05% Tween-20 in phosphate-buffered saline) using an automated plate washer (5 s soak per wash), and residual liquid was removed by gentle tapping on absorbent paper. Subsequently, 200 μL of blocking solution (5% (w/v) skimmed milk powder in PBST) was added to each well, and plates were incubated for 1.5 h at 37 °C. After blocking, plates were washed again as described above. (3) Primary antibody (serum) incubation: Serum samples were diluted 1:100 in dilution buffer (PBST supplemented with 15% (v/v) fetal bovine serum), and 100 μL per well was added. Plates were incubated for 1 h at 37 °C. Following incubation, plates were washed three times with PBST. (4) Secondary antibody incubation: Goat anti-chicken IgG–horseradish peroxidase (HRP) conjugate was diluted 1:5000 in enzyme diluent (PBST containing 1% (w/v) BSA), and 100 μL per well was added. Plates were incubated for 1 h at 37 °C, followed by three PBST washes. (5) Colorimetric detection and data analysis: TMB substrate solution (100 μL per well) was added, and plates were incubated in the dark at 37 °C for 10 min. The reaction was terminated by adding 100 μL of 2 M H₂SO₄ per well. Absorbance at 450 nm (OD₄₅₀) was immediately measured using a microplate reader. The S/P ratio was calculated as the mean OD₄₅₀ of test wells divided by the mean OD₄₅₀ of negative-control wells.

As YC_GVI-1 is a naturally attenuated strain with weak pathogenicity in chicken embryos, a Western blot method was developed to detect the neutralizing antibody response against YC_GVI-1 and the heterologous genotype JS96_GI-19, as follows. Inactivated serum at 56 °C for 30 min was serially diluted by twofold from 2² to 2⁷, and the virus solution was diluted to 100 EID₅₀/0.1 mL with sterile PBS. Each diluted serum was mixed with the virus solution in a 1:1 ratio. After incubating at 37 °C for 1 h, 0.2 mL of the mixture was inoculated into a 9-day-old SPF chicken embryo through the allantoic cavity, with a serum toxicity control included. The chicken embryos were placed in a 37 °C incubator and observed daily for death. After 7 days of culture, the chicken embryos were moved to a 4 °C refrigerator overnight, and the allantoic fluid of each embryo was collected under aseptic conditions. The expression of IBV N protein in the allantoic fluid was verified by Western blot to calculate the neutralizing antibody titer (NT₅₀).

To determine the affinity of immune serum, S1 proteins of YC_GVI-1 and JS96_GI-19, expressed and purified using the CHO cell expression system [28], were coated on ELISA plates at concentrations of 1.0 µg/mL, 0.5 µg/mL, 0.25 µg/mL and 0.125 µg/mL, respectively, and blocked with 2% BSA. The positive immune serum of YC_GVI-1 at 21 dpi was serially diluted by 10-fold from 1000 µg/mL to 0.0001 µg/mL, and 100 µL of each diluted serum was added to each well. After incubating at 37 °C for 1 h, 100 µL of 1:5000 diluted anti-chicken secondary antibody (Solarbio Science & Technology Co., Ltd., Beijing, China) was added to each well, incubated at 37 °C for 1 h, TMB color-developed, and the OD₄₅₀ value measured. Data analysis was performed using GraphPad Prism 8 software. The half-maximal effective concentration (EC₅₀) of the immune serum against S1 proteins of the two different IBV genotypes was calculated according to the formula EC₅₀ (µg/mL) = molar concentration/molecular weight (~150 kDa).

2.8. Challenge Assay

At 28 days post-immunization (dpi), six chickens were randomly selected from each group and inoculated with 10^5.5 EID₅₀ of JS96_GI-19 via the nasal-ocular route. Following challenge, both challenged and control chickens were monitored daily for eight days to assess clinical signs, including tracheal rales, wheezing, nasal discharge, and mortality. At 8 days post-challenge, surviving birds were humanely euthanized, and necropsies were conducted immediately after death. Tracheal and kidney tissue samples from challenged groups were collected and preserved in 10% neutral buffered formalin for subsequent histopathological evaluation. Histopathological lesions were scored using the following criteria: Trachea: 0—no observable lesions; 1—mild ciliary loss in tracheal epithelium; 2—presence of inflammatory cell infiltration; 3—sloughing of tracheal epithelial cells. Lung: 0—normal architecture; 1—slight alveolar hemorrhage and alveolar epithelial exfoliation; 2—widespread hemorrhage and inflammatory infiltration; 3—interstitial proliferation with thickened alveolar septa. Kidney: 0—no pathological changes; 1—renal tubular epithelial cell shedding and minor hemorrhage; 2—inflammatory cell infiltration; 3—urate deposition, tubular degeneration and necrosis affecting renal tubules and collecting ducts.

2.9. Statistical Analysis

Data analysis was performed using the GraphPad software platform, with two-way analysis of variance (ANOVA) applied to assess statistically significant differences between the specified experimental groups and their corresponding control groups. Statistical significance is indicated by p values, where ns represents non-significant differences, * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

3. Results

3.1. Detection of Exogenous Viruses and Characterization of Viral Particle Morphology

We have previously reported the isolation of YC_GVI-1 from poultry farms in Sichuan province, China, in 2021 [10]. Chicken embryos were infected with this isolate, and a PCR test for exogenous viruses was carried out, showing that only the IBV was positive (~1600 bp) (Figure 1A). Other common avian viruses, including Newcastle disease virus (NDV), avian influenza virus subtype H5 (AIV H5) and subtype H9 (AIV H9), infectious laryngotracheitis virus (ILTV), avian leukosis virus subtype A (ALV-A), infectious bursal disease virus (IBDV), and avian adenovirus type 4 (FAdV-4), were all negative (Figure 1A). This confirms the purity of the isolated YC_GVI-1 strain.

Figure 1.

Detection of exogenous viruses and the morphological characteristics of the YC_GVI-1 strain: (A) Identification of exogenous viruses by conventional PCR. (B) Following purification by ultracentrifugation, the viral suspension is subjected to scanning electron microscopy (SEM) for morphological characterization.

Scanning electron microscopy observations revealed that the isolated strain possesses typical morphological characteristics of coronaviruses (Figure 1B). The virus particles are spherical with a diameter of ~200 nm, and the surface is covered with distinct spherical protrusions (red arrows), with relatively wide intervals between the protrusions (yellow arrows) (Figure 1B). In some virions, part of these protrusions had fallen off (blue arrows), probably due to the mechanical damage during the purification process by ultracentrifugation (Figure 1B).

3.2. Viral Titer and Growth Curve

Characterization of growth and pathogenic properties of the isolate in chicken embryos was then conducted. At seven days post-inoculation, five out of six chicken embryos in groups inoculated with 10⁰ and 10⁻¹ virus exhibited marked embryonic dwarfing (red arrows), while one embryo in the 10⁻¹ group displayed significant hemorrhagic lesions (blue arrows) (Figure 2A). No apparent pathological changes were observed in the remaining groups, indicating that this strain indeed exhibits relatively low pathogenicity in chicken embryos (Figure 2A). The EID₅₀ titer was approximately 10^2.0 (Figure 2A). Western blot analysis further confirmed that the EID₅₀ titer was at 10^2.23 (Figure 2B).

Figure 2.

Determination of virus titer and growth curve: (A) The allantoic fluid collected from YC_GVI-1 virus-infected chicken embryos is diluted 10-fold with PBS. The diluted viral solutions from 10² to 10⁶ are inoculated into 9-day-old SPF chicken embryos at a dose of 200 µL per embryo. The embryos are continuously cultured and observed for 7 days, and the EID₅₀ is calculated using the Reed–Muench method. (B) The allantoic fluid collected from embryos infected as in (A) is subjected to Western blot analysis to confirm the presence or absence of viral replication and subsequently used to refine the EID₅₀. (C) The YC_GVI-1 viral suspension is diluted to a titer of 10² and inoculated into 9-day-old SPF chicken embryos at a volume of 200 µL per embryo, with three independent replicates for each experimental group. Allantoic fluids are harvested at 0, 12, 24, 48, 72, 96, and 120 h post-infection, respectively. The EID₅₀ is determined for each time point, and a viral growth curve is constructed. (D) Allantoic fluids are collected as described in (C), viral copy numbers at each time point are quantified using fluorescence-based quantitative RT-PCR, and a viral growth curve is generated.

Characterization of the growth kinetics showed consistent EID₅₀ and viral copy number (Figure 2C). The EID₅₀ and virus titer continuously increased from 0 to 72 h post-inoculation, reaching peak EID₅₀ of 3.65 ± 0.28 and viral copy number of 10.04 ± 0.56, respectively, at 72 h post-inoculation (Figure 2C). The viral titer remained relatively stable from 72 to 120 h post-inoculation (Figure 2C). These results further demonstrated that YC_GVI-1 is a naturally attenuated IBV isolate.

3.3. Viral Genome Sequencing and Analysis

The complete genome sequence of the YC_GVI-1 isolate was determined by Illumina NovaSeq 6000 PE150 (Illumina, Inc, SanDiego, CA, USA) sequencing. The results showed that the average sequencing depth (Avg depth) is 10,523.88, and the read alignment coverage reaches 100%. The genome circle plot presents the GC content, GC skew of the assembled genome, and the annotated coding sequences (CDS) (Figure 3A). Moving outward, the GC skew and GC content are displayed, which visually reflect the genomic structural features of each contig. The outermost circle marks the specific positions of the annotated CDS in the genome, with those on the positive strand indicated in a clockwise direction (Figure 3A). The complete genome obtained through assembly is 27,750 bp in length and contains 9 open-reading frames (ORFs), encoding 1a, 1b, S, 3a, 3b, E, M, 5a, 5b, and N proteins, respectively. The sizes and genomic positions of the nine open reading frames (ORFs) are presented in Figure 3B.

Figure 3.

Whole genome sequencing, genome assembly and reading frame annotation: (A) The whole-genome sequencing of YC_GVI-1 is performed using the Illumina NovaSeq 6000 platform with paired-end 150 bp (PE150) sequencing chemistry. (B) The complete genome of YC_GVI-1 is assembled using the reference sequence as a guide, and open reading frames (ORFs) within each viral protein-coding region and subgenomic RNAs (sgRNAs) are systematically annotated.

3.4. Phylogenetic and Recombination Analyses

Phylogenetic analysis based on the full-genome sequence was conducted using MEGA X. YC_GVI-1 strain was classified in the GVI-1 genotype and was most closely related to strains CK/CH/FJ/202005 and CK/CH/TJ1904 (accession number: MW815494.1) but located in an independent sub-branch (Figure 4A). Homology analysis revealed that the nucleotide sequence identities between this isolate and eight representative strains of the major prevalent genotypes in China (GI-1, GI-7, GI-13, GI-19, GI-22, and GI-28) ranged from 84.3% to 93.4% in the full genome and from 63.4% to 66.7% in the S1 gene. Among them, YC_GVI-1 exhibits the highest sequence identity with the previously reported strain CK/CH/FJ/202005 from China. The nucleotide sequence homologies of the complete genome and individual genes, including 1ab, S1, S2, 3a, 3b, E, M, 5a, 5b, and N, were 98.2%, 97.9%, 98.5%, 98.6%, 97.8%, 100%, 99.0%, 99.5%, 97.5%, 100%, and 99.5%, respectively (Figure 4B).

Figure 4.

Analysis of the YC_GVI-1 genome sequence: (A) Evolution analysis of YC_GVI-1 and representative strains of different genotypes. After conducting a comparative analysis of the whole genome sequences between the YC_GVI-1 and 22 reference strains, an evolutionary tree is constructed by the neighbor-joining method using the MEGA7 software, and the resulting evolutionary tree is further beautified utilizing the iTOL v7 online software. (B) The nucleotide sequence homology analysis of YC_GVI-1 and 8 representative strains of different genotypes. After conducting a comparative analysis of the whole genome, 1ab, S1, S2, 3a, 3b, E, M, 5a, 5b and N genes between the YC_GVI-1 and reference strains, a homology analysis is constructed by the neighbor-joining method using the MEGA7 and MegAlign software. S protein is cleaved by host proteases to generate S1 and S2 subunits. (C) Analysis of the recombination events in YC_GVI-1. Determination of the potential recombination events in the YC_GVI-1 and 22 reference strains by the RDP4 analysis. (D) The potential recombinant strains in the YC_GVI-1 identified by RDP4 were further validated and analyzed using SimPlot analysis.

Two recombination events were identified by RDP4 analysis: one between CK/CH/FJ/202005 and CK/CH/SX/2204 (accession number: OQ189491.1) and another between CK/CH/GX/HX and ck/CH/LSD/111218 (accession number: KX364300.1). The corresponding average p-values for these potential recombination events were 5.090 × 10⁻²⁶ and 1.586 × 10⁻⁹, respectively (Figure 4C).

The potential recombinant strains in the YC_GVI-1 identified by RDP4 were further validated and analyzed using SimPlot analysis. The results indicated that YC_GVI-1 may be a recombinant virus, with CK/CH/FJ/202005 and CK/CH/SX/2204, or alternatively CK/CH/GX/HX and ck/CH/LSD/111218, serving as the major and minor parental strains, respectively. The recombination regions are 24,281~26,441 bp and 19,881~25,641 bp, respectively (Figure 4D).

3.5. Further Analysis of the Amino Acid Substitutions in YC_GVI-1 S Protein That May Affect the 3D Structure as Well as the Antigenicity of the Protein

Compared with the S proteins of the more virulent strains in the same genotype previously reported in China, YC_GVI-1 S protein contains 10 unique amino acid substitutions, specifically V39L, A109V, G119R, K131E, L185S, F389L, and V485M in the S1 subunit, and L942I, A957V, and V982I in the S2 subunit (Figure 5A). These distinct residues may be the potential molecular determinants for the reduced virulence of YC_GVI-1.

Figure 5.

Analysis of amino acid differences in the S protein between YC_GVI-1 and the two reported highly virulent strains and prediction of protein structure: (A) Analysis of amino acid differences in the S protein between YC_GVI-1 and the two reported highly virulent strains (CK/CH/FJ/202005 and CK/CH/GX/HX) using the MEGA7 and MegAlign software. (B–D) The 3D structure prediction of the S protein of YC_GVI-1 and the two highly virulent strains (CK/CH/FJ/202005 and CK/CH/GX/HX) is conducted. The S proteins of the three strains are predicted using AlphaFold3 and then visualized with PyMOL. The three distinct colors represent the three identical subunits of the S trimer. (E,F) The V485M mutation in the YC_GVI-1 strain induces a conformational change in the adjacent phenylalanine residue at position 431, transforming it from a β-sheet to a random coil. The 3D structures are aligned and analyzed using the align function in PyMOL.

3.6. The Humoral Immune Response Induced by YC_GVI-1 and Cross-Reaction Against the Heterologous Strain JS96_GI-19

To evaluate the immunogenicity of YC_GVI-1, 1-day-old SPF chickens were inoculated with 10² EID₅₀. A booster immunization was administered 10 days post-primary inoculation using an identical dose. Blood samples were collected, and sera were separated at 7 and 21 dpi, respectively, and analyzed by indirect ELISA (Figure 6A). It revealed that the anti-IBV IgG antibody response in immunized chicks became positive by 7 dpi, indicating a short immune window period (Figure 6A). Following the booster immunization, significantly higher levels of antibody titers were reached, compared to those observed after primary immunization (p = 0.0134) (Figure 6B).

Figure 6.

Immunization/challenge protocols and assessment of the immune serum efficacy. (A) Immunization and viral challenge protocols. (B) Determination of the immune serum titer is performed using an in-house indirect ELISA. Serum samples were are collected on days 7 and 21 post-immunization, and titers are assessed based on S/P values, with higher S/P values indicating higher antibody titers. * and **** represents p < 0.05 and p < 0.0001, respectively. (C) The neutralizing titers of immune serum against homologous and heterologous viruses are assessed using Western blot analysis. Serum samples collected 21 days post-immunization are pooled, heat-inactivated, and then incubated with 100 EID₅₀ of YC_GVI-1 or JS96_GI-19 virus at 37 °C for 1 h. The virus-serum mixtures are inoculated into 9-day-old SPF chicken embryos. After 7 days of incubation, allantoic fluids are harvested and analyzed by Western blot to determine the presence of neutralizing antibodies. (D) The neutralizing titer curve of immune serum is generated using the Log2-transformed serum dilution as the x-axis and the virus inhibition rate as the y-axis, enabling quantitative determination of neutralizing titers. (E,F) The affinity of the immune serum for the S1 proteins of homologous (E) and heterologous (F) viruses is evaluated by Western blotting. The IBV S1 protein is coated on ELISA plates at concentrations of 1.0 µg/mL, 0.5 µg/mL, 0.25 µg/mL and 0.125 µg/mL. The antibody-positive serum is diluted 10-fold from 1000 µg/mL to 0.0001 µg/mL, and 100 µL is added to each well. The plates are incubated at 37 °C for 1 h. The secondary antibody is then incubated, and the OD₄₅₀ value is determined after TMB color development. The affinity curve is plotted with the logarithm of the serum antibody concentration (µg/mL) from −4 to 4 (equivalent to 0.0001 µg/mL~1000 µg/mL) on the x-axis and the OD₄₅₀ value on the y-axis.

NT₅₀ of serum against homotypic YC_GVI-1 and the heterotypic JS96_GI-19 were evaluated. The results demonstrated that the immune serum exhibited significantly higher neutralizing activity against the homotypic YC_GVI-1 than the heterotypic JS96_GI-19 (Figure 6C). By generating the neutralization curves based on virus inhibition rates at different serum dilutions, the neutralizing titers were calculated. The NT₅₀ values of the immune serum against the homotypic YC_GVI-1 and the heterotypic JS96_GI-19 were 2^6.40 and 2^4.00, respectively (Figure 6D). The neutralizing activity against the homotypic strain was 5.27 times that of the heterotypic strain, and the difference was statistically significant.

The indirect ELISA demonstrated that the immune serum exhibited significantly higher affinity for the YC_GVI-1 S1 protein compared to the counterpart S protein from the heterotypic JS96_GI-19. The binding affinities to the S1 proteins of YC_GVI-1 and JS96_GI-19 were (1.54 ± 1.53) × 10⁻⁸ and (6.77 ± 2.42) × 10⁻⁸, respectively, ~4.30-fold higher for the homotypic S1 protein. (Figure 6E,F). These results are consistent with those obtained from the neutralization assay.

3.7. Cross-Protection Against Challenges with Heterotypic Virulent Strains JS96_GI-19 in Chickens Immunized with YC_GVI-1

The cross-protective efficacy of YC_GVI-1 against challenges with a heterotypic virulent strain JS96_GI-19 was evaluated by infection of six immunized chickens in each group at 28 dpi. Within 8 days post-challenge with ~10^5.5 EID₅₀ of JS96_GI-19, chickens in the YC_GVI-1-immunized group showed no obvious clinical symptoms, whereas three (50%) in the control group exhibited mortality (Figure 7A). The dead and surviving chickens in each experimental group were euthanized and necropsied, showing no obvious macroscopic lesions in the lungs. However, mild punctate hemorrhages (yellow arrow) were observed in the tracheae of chickens in the YC_GVI-1 group (1/6), and tracheal ring hemorrhage (3/6) (red arrows), tracheal sticky secretions (2/6) (blue arrows), and severe punctate hemorrhage (1/6) (yellow arrow) were noted in the PBS control group (Figure 7B). Notably, the kidneys of the three dead chickens in the PBS group all presented typical “mottled kidney” lesions (Figure 7B). No visible pathological changes were observed in the remaining surviving chickens in the YC_GVI-1 group and the PBS group (Figure 7B).

Figure 7.

Immune protection efficacy against JS96_GI-19: (A) Survival curves of six representative viral isolates were generated using 1-day-old SPF chickens. Each group of chickens is inoculated via the nasal-ocular route with approximately ~10^5.5 EID₅₀ of JS96_GI-19. The number of mortalities occurring within 8 days post-challenge is recorded, and survival curves were plotted using the Graphpad Prism 9 program. The survival curves of YC_GVI-1 and PBS Con are represented by red and blue lines, respectively. (B) Examination of pathological lesions in the lung and kidney autopsies. At 8 days post-challenge with JS96_GI-19, trachea, lung and kidney autopsy of all surviving chickens from each experimental group. (C) Histopathological examination of the trachea, lungs, and kidneys in chickens challenged with JS96_GI-19. At 8 days post-challenge, the trachea, lung, and kidney autopsies from each experimental group are conducted and examined by microscopy. The pathological tissue change scores of the trachea, lungs, and kidneys in each immune-challenged group are statistically analyzed using GraphPad Prism 9 program, at 8 days post-challenge.

Further evaluation of the tissue damage by routine HE sections confirmed that no significant differences were observed in the pathological scores of three tissues between the PBS group and the three immunized groups. However, the tracheas of all chickens in PBS group and three chickens in the YC_GVI-1 group exhibited mucosal epithelial cell shedding and inflammatory cell infiltration (Figure 7C). Although not statistically significant, the pathological scores in the PBS group were higher than those in the immunized groups (Figure 7C). In the PBS group, mild congestion was detected in the lungs of one chicken each. In contrast, the lungs of all chickens in the YC_GVI-1 group appeared normal. Notably, four chickens showed urate deposition and/or inflammatory cell infiltration in the kidneys in the PBS group, whereas three chickens in the immunized groups exhibited inflammatory cell infiltration (Figure 7C).

4. Discussion

Most GVI-1 clinical isolates predominantly target the respiratory system and are associated with a relatively low mortality rate [18]. Compared to these clinical isolates with higher virulence, YC_GVI-1 isolate is less virulent. Its EID₅₀ value is significantly lower than that of the reported virulent GVI-1 strain HX (10^6.038 EID₅₀/mL) [20] and even lower than those of the reported attenuated GVI-1 strains LHB/110615 (10^5.5 EID₅₀/mL) and I0916/16 (10^5.2 EID₅₀/mL) [13]. However, the growth characteristics of YC_GVI-1 demonstrate robust proliferative capacity, with viral copy numbers reaching a maximum of ~10¹⁰ copies/mL. Consistent with a previously reported study [10], these findings confirm that YC_GVI-1 is a naturally attenuated strain, with robust growth and proliferative capacity in chicken embryos. These characteristics would make it a potential candidate vaccine strain.

Based on whole-genome sequence analyses, the YC_GVI-1 strain was identified as belonging to the GVI-1 genotype, exhibiting the highest nucleotide sequence homology with CK/CH/FJ/202005, yet distinct from other previously reported GVI-1 genotypes in China. This genetic divergence suggests that YC_GVI-1 may represent a novel variant within the GVI-1 lineage. Recombination has played a significant role in the evolution of the GVI-1 lineage [28,29,30], and most of the recombination events have been detected in the S gene [31]. Further analysis in this study indicates that YC_GVI-1 is likely originated from recombination events between strain CK/CH/FJ/202005 and CK/CH/SX/2204, or alternatively CK/CH/GX/HX and ck/CH/LSD/111218. The 5′-UTR and the entire ORF1ab region of the YC_GVI-1 genome show high similarity to LX4, whereas the S gene and ORF3 exhibit relatively low sequence identity, which was consistent with prior studies [12]. In addition, compared with other strains of different genotypes, YC_GVI-1 has a relatively high homology with GI-19, with the whole genome sequence homology reaching up to 92.1%. These findings suggested that the GVI-1 strains in China may be independently derived from recombination events that occurred between GI-19 strains and other circulating genotypes.

Coronavirus S glycoprotein forms a homo-trimeric structure and serves as the primary immunogenic component responsible for eliciting neutralizing antibody responses [6]. Additionally, it plays critical roles in mediating viral pathogenicity in vivo and determining cell tropism under in vitro conditions [32]. YC_GVI-1 does not induce observable clinical signs or post-mortem pathological lesions, and exhibits significantly reduced virulence compared to previously reported isolates CK/CH/FJ/202005 and CK/CH/GX/HX. Comparative analysis of the amino acid sequences and 3D structures revealed that the YC_GVI-1 S protein harbors 10 distinct amino acid substitutions relative to the two reference strains. The 3D structures of the S protein from YC_GVI-1, CK/CH/FJ/202005 and CK/CH/GX/HX were predicted using AlphaFold3 software, and the prediction results were visualized and analyzed using PyMOL software. The overall 3D structures of the three S proteins are highly similar, but differences exist in their local structures (Figure 5B,D). The ten unique amino acid substitutions in the YC_GVI-1 S protein all render impact on the N-terminal flexible region, as shown in Figure 5B,D. Among them, the M485V mutation induces a conformational change in the adjacent phenylalanine residue at position 431, transitioning from a β-sheet to a random coil (Figure 5E,F). Notably, the V485M substitution induces substantial alterations in the protein’s secondary structure, indicating that this residue may serve as a critical determinant underlying the differences in virulence among these strains.

Immunization of chickens with the attenuated YC_GVI-1 strain induces significant humoral immunity. Interestingly, cross-reaction and affinity determination showed that the NT₅₀ and EC₅₀ of YC_GVI-1 immune serum against JS96_GI-19 were 10⁴ and (6.77 ± 2.42) × 10⁻⁸, respectively, indicating a high cross-reactivity. The neutralizing titer was also significantly higher than that of the existing GVI-1 (CK/CH/LN/2301) against GI-19 (<2^2.0) [30], suggesting that the YC_GVI-1 attenuated strain has a relatively more potent cross-protection efficacy against JS96_GI-19. Moreover, the protection rate against challenge with a heterotypic virulent strain is as high as 100%. These results demonstrate that the YC_GVI-1 attenuated strain can not only induce significant humoral immunity against homotypic virus, but also shows high cross-protective efficacy against JS96 strain, a heterologous GI-19 strain. This cross-protection against a GI-19 strain would, meanwhile, support that YC_GVI-1 might have been generated through a genetic recombination event involving the two parental strains CK/CH/GX/HX and ck/CH/LSD/111218.

5. Conclusions

In summary, YC_GVI-1 is a naturally attenuated strain with a genome length of 27,750 bp. It is likely generated through genetic recombination between CK/CH/FJ/202005 and CK/CH/SX/2204, or alternatively CK/CH/GX/HX and ck/CH/LSD/111218 strains, representing a novel sub-lineage. This strain exhibits a relatively short immune window period, induces a robust humoral immune response, and provides effective cross-protection against the heterologous virulent GI-19 strain, demonstrating YC_GVI-1 as a promising candidate vaccine strain that can be used to develop a broad-spectrum attenuated live vaccine against the IBV in GVI-1 and GI-19 genotypes. This paper represents one of the key future directions for the development of a vaccine with a broad-protection spectrum against two different lineages of the IBV.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/v18020191/s1.

Author Contributions

Conceptualization, R.C., D.L. and T.X.; methodology, D.L. and T.X.; software, T.X., Y.L. and S.S.; validation, S.W., H.L. and T.X.; formal analysis, S.W., M.J. and F.X.; investigation, T.X.; resources, R.C.; data curation, T.X., S.W., H.F., Z.Y. and T.X.; writing—original draft preparation, T.X.; writing—review and editing, D.L.; visualization, T.X.; supervision, R.C.; project administration, R.C.; and funding acquisition, R.C. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

The animal study protocol was approved by the Animal Experiments Committee of Zhaoqing Dahuanong Biopharmaceutical Co., Ltd. (protocol code: LL-G-20250201-01; approval date: 1 February 2025).

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.

Conflicts of Interest

Ruiai Chen is affiliated with Zhaoqing Dahuanong Biopharmaceutical Co., Ltd. and related entities, including serving as Chief Scientist at Zhaoqing Dahua Agricultural Biological Products Co., Ltd. The animal experiments were conducted at the GCP Laboratory Animal Facility of Zhaoqing Dahuanong Biopharmaceutical Co., Ltd., which also issued the corresponding animal ethics approval. The remaining authors declare no conflict of interests.

Funding Statement

This work was partially supported by Science and Technology Plan projects funded by self-raised funds of Guangdong Provincial Laboratory (XJGDL2022002), and by Zhaoqing Branch Center of Guangdong Laboratory for Lingnan Modern Agricultural Science and Technology (XJ2025KJ008; P20211154-0301).