Randomly assigned to either the control or the infected group were 3-week-old specific pathogen-free (SPF) chicks. Chicks from the infected groups were carefully inoculated intranasally with the IBV-M41 and QX strains. In contrast, the control group was administered PBS to ensure a valid comparison. Both infected and control chicks were sacrificed at 6, 12, 24, and 48 h post-infection (hpi) based on the different experimental requirements. Samples of nasal, trachea, lungs, kidneys, proventriculus, spleen, duodenum, colon, and peripheral blood monocytes (PBMCs) were collected from each group. All tissue samples were obtained for histopathology and mRNA and protein examination. However, PBMC samples were used for flow cytometry analysis.

Histopaque-1077, which has a density of 1.077 g/mL, was acquired from Sigma-Aldrich (St. Louis, MO, USA) to isolate PBMCs from chicken blood. Magnetic beads were used for cell separation with specific antibodies, including anti-FITC, anti-APC, and anti-PE micro-beads. Miltenyi Biotec (Auburn, USA) was the source of all materials, including the MACS assembly, column, and miniMACS starting kits. Mouse anti-chicken antibodies were used for flow cytometry and sourced from Southern Biotech (Birmingham, AL, USA).

Six control and 12 infected chicks were euthanized using intravenous pentobarbital injection, and samples were collected from the nasal passages, trachea, lungs, kidneys, and proventriculus at 12, 24, and 48 hpi. The beak in front of the nostril and the head were cut off. The nasal sections were fixed in Bouin solution for 72 h (Tamura et al., 1998). The nasal blocks from the histological analysis were sectioned according to specific fractions. The tissue blocks were effectively decalcified using a solution of 4% paraformaldehyde (PFA) and 5% formic acid in PBS. The nasal sections underwent a thorough dehydration process using progressively higher concentrations of ethyl alcohol, were cleared with xylene, and were embedded in high-quality paraffin wax. The paraffin-embedded sections of tissues were precisely prepared by cutting them into 4-µm cross-section slices with a Minux S700 microtome machine. The section was carefully mounted on slides and deparaffinized in xylene. It was rehydrated by transitioning through descending grades of ethyl alcohol and underwent IHC staining to provide detailed insights. The IBV-QX positive antigens were effectively detected through IHC staining, utilizing a particular mouse monoclonal primary antibody sourced from Novus Biological Company. The detection process was further enhanced by employing an HRP-conjugated anti-mouse IgG SABC kit from Beyotime. The slides were subsequently mounted using oleoresin of the balsam. The stained sections were examined using the Olympus BX53 microscope in conjunction with the SC180 digital camera system. The quantification of IBV-QX-positive antigens was statistically measured by using ImageJ.

Antigen retrieval was carried out as described, following deparaffinization and rehydration of the paraffin-embedded slides. The slides were kept in a blocking solution (10% PBS, 0.0l g/mL donkey serum (DS), 0.1% BSA, and X-100 Triton). The first antibody was diluted in PBS and 1% DS. The sections were then incubated in the second antibody against IBV-N protein (IB95) (NBP2-31102, NovusBio) for 90 min at room temperature in darkness. Following three washes, conjugated-gold DAPI (P36962, Invitrogen, CA, USA) was dropped and imaged by using a microscope (BS61VS, Olympus).

PBMC cell suspensions were carefully collected from each treatment group. The cells were precisely standardized at 1 × 10⁶ cells, ensuring consistency and accuracy for optimal staining results. For both B and T cells, PBMCs were triple-stained using Bu1, CD4, CD45, and MHC II markers. The antibodies used for flow cytometry included FITC-conjugated mouse anti-chicken Bu1, PE-conjugated mouse anti-chicken CD4, APC-conjugated mouse anti-chicken CD45, and PE-conjugated mouse anti-chicken MHCII, incubated for 30 min at 4°C. PBS was used to wash the cells. The positively stained cells were analyzed using FACS (BD FACS Calibur). FlowJo™ v10.9 (BD Life Sciences, USA) was used to analyze the data.

NCI-N87 and DF1 cells were successfully sourced from Oricell Bio and ATCC. DF1 cells were cultured in Dulbecco’s Modified Eagle Medium and supplemented with 10% fetal bovine serum (FBS). The NCI-N87 cells were seeded at 4 × 10⁵ cells/cm² in high-quality RPMI-1640 medium (Invitrogen), supplemented with 5% FBS and 1% penicillin–streptomycin (PenStrep; Gibco, Invitrogen).

NCI-N87 cells were seeded in 12-well plates for 24 h, followed by infection with IBV-QX strain for 2 and 24 h and siRNAs’ or their inhibitors’ (200 nM) transfection via Lipofectamine 2000 transfection reagents (Invitrogen). At 24 h post-transfection, cell cultures were harvested to assess gene silencing efficiency.

Total RNA was successfully extracted from both in vivo and in vitro samples utilizing TRIzol (Invitrogen) and following the manufacturer’s precise protocol (Shah et al., 2025). This robust method ensured high-quality RNA isolation, essential for reliable downstream analyses. The RNA concentration was measured using a Nanodrop spectrophotometer. Complementary DNA (cDNA) was synthesized from RNA using reverse transcription with the HiScript™ qRT SuperMix kit from (Vazyme Biotech Co., Ltd) according to the manufacturer’s manual. Then, qRT-PCR was manipulated by using the SYBR qPCR master Mix kit (Vazyme Biotech Co., Ltd.) on a real-time PCR System ForeQuant F4/F6. All of the amplification primers utilized in this study are listed in Table 1 . The relative gene expression level was normalized according to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. Each experiment and sample were independently repeated three times and run in technical triplicate. The relative expression was determined using 2^^-ΔΔCt.

The concentrations of IL-1β (PI305), IL-6 (PI330), TNF-α (PT518), IL-4 (PI618), and IL-10 (PI528) in NCI-N87 cells were quantified using human enzyme-linked immunosorbent assay (ELISA) kits (Beyotime Biotechnology, China) following the manufacturer’s protocols. All experiments were performed independently three times in triplicate. Data were normalized according to the manufacturer’s protocols.

Western blot (WB) analysis was carried out following the established procedures described in prior studies (Shah et al., 2025). Total protein from the tissues and cell lines was extracted using radioimmunoprecipitation assay (RIPA). A protein assay kit (Boster, China) with bicinchoninic acid (BCA) and protease inhibitors (Merck, USA) was used for quantification. Proteins were separated from the gel by SDS-PAGE and transferred to PVDF paper. After an hour of blocking with skimmed milk, a paper was then incubated with primary antibodies against beta actin (β-actin), (AC026, ABclonal), IBV-N protein (3BN1, Hytest), Argonaute2 (BS70734, Bioworld), Dicer (BS70707, Bioworld), Exportin5 (BS8443, Bioworld), Drosha (BS90429, Bioworld), IL-1β (A16288, ABclonal), IL-6 (EM1701-45, Huabio), TNF-α (11948T, CST), IL4 (ER1706-55, Huabio), and IL-10 (701122, Thermo Fischer Scientific) overnight at 4°C. The enzyme was linked with a secondary antibody at 1-h incubation, and the ECL (enhanced chemiluminescence) kit from Millipore was used to visualize the protein bands. Protein band intensities were quantified and analyzed using ImageJ software (v1.53, National Institutes of Health, USA). Band densities were normalized to β-actin to calculate the relative protein levels. All experiments were performed three times independently.

A statistical analysis approach using Student’s t-test and one-way analysis of variance (ANOVA), from the procedure of comparing means, was employed to calculate the significant differences among the groups. Results are expressed as means ± standard deviation (SD), and statistical significance was defined as p <0.05. The following symbols denote statistical significance in the figures: ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, and ns—nonsignificant. Unless otherwise specified, data from three independent experiments were analyzed.

To address the least transmission of IBV strains in the nasal mucosa, inoculation of IBV-QX to chickens intranasally led to the viral antigens in internal organs. At 12 hpi, IBV-QX-positive antigens are found in the epithelial cells of the nasal orifices, and at 24 hpi, these antigens then transmit into the nasal-associated lymphoid tissues ( Figure 1A ). Complete immunofluorescence images are shown in Supplementary Figure S1 . At 24 hpi, IBV-QX strain RNA replicates highly in the trachea, kidney, and proventriculus ( Figure 1B ), although M41 strain copies are seen highly in the trachea and lungs ( Figure 1B ). IBV-QX proteins exhibit a robust expression in the proventriculus, whereas M41 demonstrates significant presence in the trachea at 24 hpi ( Figures 1C, D ). This marked differential expression underscores the distinct pathogenic mechanisms associated with these viral strains. IBV-QX primarily targets the respiratory tract. Its ability to infect nasal lymphocytes facilitates systemic spread and potentially affects the gastric mucosa through ingestion or viremia, highlighting its broader tropism. This could explain how the virus affects multiple tissues or organs, potentially contributing to broader pathogenesis. Further studies on viral entry mechanisms in these tissues could reveal novel therapeutic targets.

A gammacoronavirus primarily affects chickens, causing infections at least in the respiratory system, renal system, and stomach. At 12 hpi, viral stains appear in trachea pseudostratified ciliated cells and inflammatory cell infiltration ( Figure 2A ). At 24 hpi, desquamation of cells occurs ( Figure 2A ). In the lungs, it accumulates in the capsular mesothelial and connective tissue at 12 hpi. At 24 hpi, it is highly expressed at the alveolar epithelium ( Figure 2B ). Transmission occurs in the kidney at 12 hpi in proximal and distal convoluted tubules (PCT and DCT) at 12 hpi. At 24 hpi, it accumulates in the regions and edges of the renal cortex and renal corpuscles ( Figure 2C ). Previous results demonstrate that QX strain replication occurs in almost all organ tissues, but in the stomach, they are very high because this strain invades mucus-secreting surfaces. At 12 hpi, it is highly expressed in mucus-secreting epithelial cells at the epithelial lining, while at 24 hpi it accumulates highly in mucosal ridges and lamina propria ( Figure 2D ). Here we revealed that IBV-QX affects respiratory lymphocytes and enters the bloodstream, where it transmits and replicates throughout the body by targeting its destination.

Immune B- and T-lymphocytes play a crucial role in eliminating viral infection. We identified B- and T-lymphocytes in PBMC, at least in the infection of chicks with IBV-QX. The reduction in B-lymphocytes (expressing Bu-1) and T-lymphocytes (expressing CD4 and CD45) resulted in viral infections at various time points ( Figures 2E–G ). However, the B-lymphocytes expressing MHC-II levels increase following an intranasal infection with IBV-QX ( Figure 2H ). These results altogether show that alterations in immune lymphocytes occur within 24 h due to high transmission and replication of IBV-QX.

IBV-QX binds to sialic-acid-containing α-2,3-linked receptors on the trachea and kidney. The entire deletion of sialylated mucins from tracheal mucosa and kidney tissue completely inhibits the binding of both receptor binding domain (RBD) proteins. This finding underscores the critical role of sialic acids on host tissues in facilitating QX-RBD binding (Bouwman et al., 2020). After binding, the virus enters via endocytosis or membrane fusion, depending on host protease activity. The virus replicates highly intracellularly, causing cell death and inflammation. Here we found that IBV-QX might interact with known receptors in chicken systemic organs such as the trachea, lungs, kidney, and proventriculus. Aminopeptidase (APN) in (kidneys), AGO2 in (trachea, lungs, kidneys, and proventriculus), ACE2 in (trachea, lungs, and kidneys), and Dicer in (lungs and proventriculus) in a time-dependent manner are all considerably upregulated ( Figures 3A–D ). Our findings revealed that IBV-QX nucleocapsid (N) protein interacts with ACE2, AGO2, and Dicer, blocking pre-miRNA nuclear export and processing, thereby suppressing miRNA-mediated antiviral responses. IBV-QX may disrupt this pathway, impairing pre-miRNA transport.

An in vitro study demonstrates that IBV-QX replicates highly in the gastric mucosa. To validate our in vivo findings, we chose the gastric epithelial cell line along with the DF-1 cell line and transfected it with the IBV-QX strain. IBV-QX replication in DF-1 cells decreases after 12 hpi, whereas it peaks at 12 hpi ( Figure 4A ). However, the QX strain replicates highly in NCI-N87 cells in a time-dependent manner. This study aims to understand the cellular pathways implicated in IBV-QX replication, specifically targeting the miRNA biogenesis pathway to investigate its role in regulating the host immune response during viral replication. We target the miRNA biogenesis pathway to check whether it regulates the host immune response in viral replication. In NCI-N87 gastric cells, we first employ inhibitors to silence (si-miRNAs) host transcription factors like Drosha, Dicer, AGO2, and XPO5 for 24 h. Then, we transfected the cells with QX strain for 24 hpi. By using miRNA inhibitors, the mRNA replication of QX strain increases almost once in Drosha, AGO2, and XPO5 and twice in Dicer compared to the blank ( Figures 4B–F ). Here only the cells transfected with QX decrease the mRNA levels of miRNA biogenesis transcription factors in a time-dependent manner, except Dicer ( Figures 4G–J ). QX transfection inhibits the protein synthesis of these biogenesis factors (Drosha, Dicer, AGO2, and XPO5) in gastric cells at 24 hpi ( Figures 4K–O ). To validate these results, we also checked the protein expressions at 2 hpi. We found the protein levels of Dicer ( Figure 4M ) and Drosha ( Figure 4O ) to increase, whereas AGO2 ( Figure 4L ) and XPO5 ( Figure 4N ) decrease slightly. Here we use inhibitors by silencing miRNA biogenesis factors in cells to compare our findings. Specific studies on IBV-QX are limited; it likely disrupts miRNA biogenesis similarly to other coronaviruses by interfering with the processing, exporting, or functioning of miRNAs to promote viral survival. Further research is needed to pinpoint the exact mechanisms. These findings altogether highlight that QX inhibits the synthesis of key components of the miRNA biogenesis pathway and ultimately replicates highly in gastric cells. Our findings suggest that the IBV-QX strain may be potentially involved in cross-species transmission.

We evaluated the inflammatory and anti-inflammatory cytokines in NCI-N87 cells infected with IBV-QX at 2 and 24 hpi. Likewise, we also use a Dicer inhibitor to silence their synthesis in gastric cells. Moreover, our ELISA analysis confirmed that the levels of three key pro-inflammatory cytokines (IL-1β, IL6, and TNF-α) are activated ( Figures 5A–C ), and the anti-inflammatory cytokines (IL-4 and IL-10) are suppressed ( Figures 5D, E ) with QX transfection at 2 and 24 hpi. Similarly, these pro-inflammatory cytokines increase ( Figures 5A–C ), and the anti-inflammatory cytokines decrease ( Figures 5D, E ) when the miRNA biogenesis key transcription component Dicer is silenced in gastric cells at 24 hpi. Similarly, the mRNA levels of IL-1β, IL6, and TNF-α increase in gastric cells transfected with QX strain at 24 hpi and silenced with Dicer as shown in Supplementary Figures S2A–C . Furthermore, we validate these cytokine protein expressions with WB, which are parallel to the ELISA and mRNA findings. At 24 hpi, QX transfection and Dicer inhibition activate the levels of inflammatory cytokines in gastric cells ( Figures 5F–I ), whereas the levels of anti-inflammatory molecules’ protein decrease with QX transfection and the inhibition of the Dicer synthesis in NCI-N87 cells at 24 h ( Figures 5F, J, K ). Consequently, these results conclude that the IBV-QX strain highly replicates in gastric cells by inhibiting the miRNA biogenesis pathway, leading to the activation of inflammatory and suppression of anti-inflammatory cytokines, respectively.