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. 2026 Feb 10;12(2):e70834. doi: 10.1002/vms3.70834

Chicken Origin Tribasic H9N2 Avian Influenza Virus Induces Potent Early Antiviral Response With Low Pathogenicity in Japanese Quails

Rupaida Akter Shila 1, Ismail Hossain 1, Rokshana Parvin 1, Emdadul Haque Chowdhury 1, Jahan Ara Begum 1,✉
PMCID: PMC12887686  PMID: 41664991

ABSTRACT

Background

Low‐pathogenic avian influenza (LPAI) virus H9N2 has been endemic in Bangladesh since 2006. While the molecular epidemiology and pathogenicity of circulating tribasic H9N2 viruses are well‐documented in chickens, data on pathogenicity in quails remain limited. Given the fact that quails serve as potential mixing vessels for avian influenza viruses, understanding of currently circulating tribasic H9N2 viruses’ pathobiology is crucial.

Objectives

The aim of this work was to observe pathogenicity, including clinicopathological changes, virus shedding, and cytokine expression of the recent circulating tribasic H9N2 virus–infected Japanese quails in Bangladesh.

Methods

A total of 64 quails were randomly assigned into two groups: an infected group (n = 32) and a control group (n = 32). Infected quails (at 4 weeks) received 500 µL of virus (106 EID50/mL) via the oculo‐nasal route. Quails were subsequently monitored for clinicopathological changes, virus shedding, and cytokine expression at various intervals until 70 days of age.

Results

Infected quails exhibited decreased egg production (7%–24%) and reduced weight gain (5%–12%) compared to controls, though no mortality was observed. Gross lesions included congestion and mild‐to‐moderate haemorrhages in the trachea, lungs, intestine, and kidney until 10 days post‐infection (dpi). Histopathology revealed mild tracheitis, pneumonia, slight haemorrhages and degenerative kidney changes at different dpi. The virus replicated prominently in the trachea, lungs, intestine, and kidney up to 5 dpi, with peak shedding via the oropharyngeal route. Following infection, IL‐8, TNF‐α, IFN‐β, and IFN‐γ were expressed in the trachea, lungs, intestine, and lymphoid organs at 2, 5, 10, and 15 dpi. Proinflammatory cytokine TNF‐α was upregulated to the significantly higher levels (p ≤ 0.001) in trachea and lungs at 10 dpi in tribasic H9N2‐infected quails compared to non‐infected control group. Notably, quails exhibited a robust early antiviral response (IFN‐β and IFN‐γ on 2 dpi) against H9N2 infection except for lymphoid tissues regarding IFN‐γ.

Conclusions

This study gives valuable insights into host–pathogen interaction and confirms that the circulating tribasic H9N2 virus remains phenotypically low pathogenic in Japanese quails in Bangladesh but cause long‐term impairment of important productivity parameters (weight gain, laying rates).

Keywords: antiviral response, avian influenza, pathogenicity, quails, tribasic H9N2

This study gives valuable insights into host–pathogen interaction and confirms that the circulating tribasic H9N2 virus remains phenotypically low pathogenic in Japanese quails in Bangladesh but cause long‐term impairment of important productivity parameters (weight gain, laying rates).

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1. Introduction

Avian influenza (AI) viruses pose a significant challenge to the poultry industry in Bangladesh. Both low‐pathogenic avian influenza (LPAIV) H9N2 and highly pathogenic avian influenza (HPAIV) H5N1 are co‐circulating in the poultry population in Bangladesh since 2007. Along with HPAIV, LPAIVs have been regularly identified from commercial and back yard poultry, environment as well as from live bird markets (Jannat et al. 2013; Islam et al. 2018; Parvin et al. 2018; Parvin et al. 2020). The H9N2 LPAI viruses mostly replicate at respiratory tract (Begum et al. 2023) and can cause coughing, sneezing, rales, and excessive lacrimation or sometimes run asymptomatic courses in commercial and backyard poultry, and in wild birds (Kariithi et al. 2020; Kye et al. 2021). It causes significant reduction in egg production (14%–75%) in commercial layers and hatchability in breeder flocks (Keshtkari et al. 2010; Pusch and Suarez 2018) and leads to 50% morbidity and 3% mortality in commercial breeders (Jannat et al. 2013). However, in field, H9N2 infection causes severe respiratory illness and up to 35% mortality in chickens concurrently infected with bacterial and viral pathogens like E. coli, mycoplasma, Newcastle disease, infectious bronchitis, etc. (Gu et al. 2017; Parvin et al. 2019; Samy and Naguib 2018).

Molecular characterisation of the circulating H9N2 LPAI viruses in Bangladesh revealed continuous circulation of clade G5.7 (G1‐like or h9.4.1) and also exhibited mutations that favour interspecies transmission (Parvin et al. 2014; Fusaro et al. 2024). In birds, haemagglutinin endoproteolytic cleavage site (HACS) motif is highly connected with the pathogenicity of avian influenza virus (AIV) subtypes. Since the first outbreak in 2006, Bangladeshi H9N2 viruses expressed a dibasic cleavage site (PAKSSR*GLF) in the HA protein (Parvin et al. 2014, Parvin et al. 2018). However, the currently circulating strains expressed a tribasic (PAKSKR*GLF) HA cleavage site motif (Shanmuganatham et al. 2013; Parvin et al. 2014; Parvin et al. 2018; Parvin et al. 2020, Begum et al. 2023). Changes in cleavage site influences viral replication, stability, pathogenicity and transmission in poultry species (Parvin et al. 2020; Zhang et al. 2021). Thus, this tribasic H9N2 virus has gained significant concern in recent years. Nowadays, quail farming is a thriving industry due to its economic importance as a commercially farmed species because of its excellent yields of meat with a distinctive flavour, its modest body size, and minimal maintenance costs. Up to four generation per year are produced (Islam et al. 2018) in Bangladesh. Despite its economic importance, quail farming faces significant challenges from H9N2 avian influenza. In Bangladesh, vaccination and biosecurity measures are the primary strategies to combat H9N2 infection. While inactivated vaccines are occasionally used in broiler and layer chickens, often around 7–10 days of age, no or less standardised vaccination program exists for quails (Islam et al. 2025). Quails are more prone to H9N2 virus isolated from wild aquatic birds. Furthermore, co‐infections (E. coli, Avibacterium paragallinerum, etc.), environmental stressors (poor ventilation, overcrowding, dust, fluctuating temperature, etc.) and immunosuppression are the complicating factors for H9N2 infection to cause high mortalities in quails. Quails are considered as a potential ‘mixing vessel’ for influenza viruses as their respiratory tract contains both avian (SAα 2,3‐gal) and human (SAα 2,6‐gal) type receptors. This allows quails to be infected by diverse strains, facilitating viral reassortment (Perez et al. 2003; Wan and Perez 2006). Several studies revealed that molecular evolution (through either mutation or antigenic drift) in the HA gene (mono‐, dibasic to polybasic HACS) is sufficient to start effective replication and transmission in quail even without showing overt clinical signs (Perez et al. 2003; Uno et al. 2012; Seiler et al. 2018). The pathogenicity of mono‐ and tribasic H9N2 virus–infected commercial chickens and ducks has been extensively explored (Reemers et al. 2010; Nang et al. 2011; Subtain et al. 2011; Wang et al. 2016; Begum et al. 2023; Hossain et al. 2024). Furthermore, the pathogenicity of circulating tribasic H9N2 virus in both experimentally and naturally infected chickens is well documented (Parvin et al. 2018; Begum et al. 2023; Hossain et al. 2024); however, little information is available about its pathogenicity in quails. Therefore, a detailed pathogenicity study of tribasic H9N2 is required for understanding the host–pathogens interactions in quails. In this experiment we aimed to assess the clinicopathological changes, viral distribution and cytokines expression of experimentally infected Japanese quails with the tribasic H9N2 A/chicken/Bangladesh/LT56/2021 (H9N2) virus isolated from chickens.

2. Materials and Methods

2.1. Ethic Statement

Regarding the usage and care of quails, the authors confirm that all relevant institutional and national rules were followed. The study was conducted strictly in compliance with the Bangladesh Agricultural University, Mymensingh Ethical Standard of Research Committee authorisations. Ethical Standard of Research Committee reviewed and approved the methodology and procedures of this experiment (7/BAURES/ESRC/VET‐16/22).

2.2. Experimental Pathogenesis Study of H9N2 AIVs

2.2.1. Virus Titration and EID50 Calculation

A/Chicken/Bangladesh/LT56/2021 (H9N2) field isolate was selected for experimental pathogenesis study in Japanese quails as it contains tribasic HACS motifs in its HA protein. The virus isolate was retrieved from the virus repository of the Department of Pathology, BAU, Mymensingh, which was originally isolated from a natural outbreak in commercial poultry farm with the history of respiratory distress in 2020. The H9N2 virus was further propagated in 10‐day‐old embryonated chicken eggs through the allantoic cavity and incubated at 37°C for 96 h. Allantoic fluid (AF) was harvested and subjected to haemagglutination (HA) test using 1% chicken red blood cells following standard procedures (WOAH 2021). RT‐qPCR was performed to confirm the presence of virus in HA positive allantoic fluids using an established protocol (Begum et al. 2023). After performing sequential 10‐fold dilutions in PBS and culture in 10‐day‐old embryonated chicken eggs, the embryo infectious dose (EID50) was calculated by using standard protocols of the Reed‐Muench method (Reed and Muench 1938).

2.2.2. Experimental Design

A total of 64 commercial Japanese quails were obtained at 14 days of age from a commercial quail farm in Bangladesh. From 14 to 28 days of age, quails were housed in a wire‐mesh cage measuring 91 × 91 × 152 cm under standard hygienic conditions with proper ventilation and a controlled environmental temperature. During this acclimatisation period, the quails had ad libitum access to a commercial quail grower feed (typical intake approximately 20–25 g/bird/day) and clean drinking water available through bell trough drinkers. At 21 days, blood samples were collected randomly from quails to check the level of maternally derived antibodies before infection. At 4 weeks of age, the quails were redistributed into two identical wire‐mesh cages (n = 32 quails/cage), each measuring 91 × 91 × 152 cm; one case designated for infected quails (n = 32) and one for control group (n = 32). The infected group received 500 µL of 106 EID50/mL of A/Chicken/LT56/2021 (H9N2) virus isolate via intranasal (250 µL) and conjunctival (250 µL) routes. Control groups received an equivalent volume (500 µL) of phosphate buffer saline (PBS) via the same routes (Figure 1). Quail chicks were monitored daily for clinical signs throughout the experimental period. Body weight of the quails was recorded every 5 days following H9N2 virus infection while egg production was recorded from 41 days to 70 days of age. At 2, 5, 10, and 15 dpi, 3 quails from both of the infected (n = 3) and control (n = 3) groups were humanely euthanised by cervical dislocation performed by a veterinarian. Prior to sacrifice, oropharyngeal and cloacal swabs were collected from all birds in 1 mL PBS containing antibiotics (Gentamycin @ 500 µg/mL) and stored at –80°C until subsequent analysis. Blood samples were collected from the infected birds at 0 (pre‐infection), 7, 14, and 21 dpi to assess immune response. Tissue samples including trachea, lungs, intestine, and lymphoid tissue (pooled spleen and bursa) from both infected and control quails were collected for evaluation of cytokine expression.

FIGURE 1.

FIGURE 1

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Experimental design of chicken origin tribasic H9N2 infection in Japanese quails using A/chicken/Bangladesh/LT56/2021 (LT_56) isolates of Bangladesh. *IN and CO: intranasal and conjunctival; EID50: embryo infectious dose 50; dpi: days post‐infection.

2.2.3. Clinical and Pathological Investigation

Quails were examined twice daily for altered behavioural or clinical signs. Necropsy of sacrificed quails was conducted, and gross pathological changes were recorded. At necropsy, pieces of trachea, lungs, intestine and kidneys were collected in 10% neutral buffered formalin for histopathological examination and a separate piece from each was placed into a separate sterile Eppendorf tube for detection of virus and cytokine gene expression. Lymphoid organs (spleen, and bursa) were collected, pooled into a tube separately for each bird and stored at –80°C to analyse the cytokine genes expression. The fixed tissue samples were processed, embedded in paraffin, sectioned and stained with haematoxylin and eosin (H&E) stain following standard procedure (Luna 1968). The stained slides were examined under light microscope (Olympus BX43 equipped with an EP50 Olympus camera).

2.2.4. Viral Shedding and Distribution

RT‐qPCR was conducted to detect H9N2 virus shedding in the oropharyngeal and cloacal swabs, as well as virus distribution in different tissues (trachea, lungs, intestine, and kidney) of infected quails at 2, 5, 10, and 15 dpi. Swabs and tissue suspensions were prepared in 2 mL of PBS supplemented with Gentamycin (10 mg/mL). Each tissue sample was individually homogenised using a fresh stainless‐steel bead for lysis with a Tissue Lyser instrument (Qiagen, Hilden, Germany). RNA extraction was performed by using the commercially available PureLink RNA Mini Kit (Thermo Scientific, Waltham, Massachusetts USA). The RNA was quantified using the Nanodrop One machine (Thermo Scientific, USA) and subjected to RT‐qPCR using H9 and N2 subtype specific primers using the Luna universal probe one‐step RT‐qPCR kit (Biolabs, New England) as per manufacturer's instructions. Briefly, 12.5 µL of reaction mixture was prepared containing the following: 6 µL master mix, 1 µL forward primer (10 pmol/µL), 1 µL reverse primer (10 pmol/µL), 0.5 µL enzyme mix, 1.5 µL RNase‐free water, and 2.5 µL of extracted RNA. The reaction was performed in QuantStudio Real‐Time PCR (qPCR) system (Applied Biosystem, Thermo Fisher Scientific, USA) using the following thermal profile: reverse transcription at 55°C for 2 min, initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C, 15 s and annealing at 60°C, 1 min. The quantity of viruses was semi‐quantified using the cycle threshold (Ct) value following the method of TaqMan‐based RT‐qPCR with a H9, N2 gene target (Table S1). A Ct value of <40 was considered as a threshold for positive results (Begum et al. 2023), reflecting a small quantity of viral RNA compared to the positive control.

2.2.5. Quantification of Innate Immune Response Genes

The RNA extracted from various tissue samples (trachea, lungs, intestine, kidney, pooled lymphoid organs particularly spleen, and bursa) at 2, 5, 10, and 15 dpi was used for cDNA synthesis with the Revert‐Aid First Strand cDNA synthesis kit (Thermo ScientificTM, USA) following the manufacturer's instructions. After the measurement of the concentration of the synthesised cDNA, it was diluted at a ratio of 1:10 using nuclease‐free water. Afterward, the diluted cDNA was subjected to qPCR to assess the relative expression of IL‐8, TNF‐α, INF‐β, and INF‐γ cytokines, using GAPDH as housekeeping gene. The analysis was based on changes in SYBR Green fluorescence after amplification with specific primer sets (Table S1) following the designated thermal profile: initial denaturation at 95°C for 10 min followed by 45 cycles of denaturation at 95°C for 15 s, annealing at 50°C–53°C for 1 min and extension at 72°C for 1 min. The reaction mixture of 10 µL were prepared containing nuclease‐free water (1 µL), 5 pmol of each primer (1 µL), Luna Universal qPCR Master Mix (5 µL), and 100 ng of cDNA (2 µL) using Luna Universal qPCR Master Mix (New England Biolabs, Inc.). One cycle for the dissociation curve for all reactions was added and the melting curve was analysed.

2.2.6. Serology

To confirm seroconversion, blood was collected from jugular vein of randomly selected quails at 0 (pre‐infection), 7, 14, and 21 dpi. The samples were centrifuged at 5000 rpm for 5 min to obtain clear straw‐coloured sera and stored at –20°C until used. Haemagglutination inhibition (HI) assays were performed following (WOAH 2022) standard procedure to detect H9‐specific antibodies using 4 haemagglutination units (4HA) of the homologous H9N2 antigens.

2.3. Statistical Analyses

Statistical analysis was performed using GraphPad Prism 8, USA. Using GAPDH as the reference gene, the fold change was calculated using the –ΔΔCt method to normalise the target gene expression level to control quail birds. Data was analysed by two‐way ANOVA followed by Tukey's multiple comparison test. Fold change values of three replicates per gene were used for analysis. The standard deviation of the mean was determined and a p‐value ≤0.05 was considered statistically significant.

3. Results

3.1. Clinical Signs, Gross and Histopathological Findings

No mortality and clinical signs were observed in quails of the infected and control groups throughout the experimental periods of 70 days. Infected quails showed reduced feed intake during the first few days following infection, although quantitative feed intake was not measured. By gross pathology, infected birds exhibited mild tracheal haemorrhages along the rings at 2, 5, and 10 dpi, mild‐to‐moderate lung congestion from 2 to 15 dpi and mild‐to‐severe kidney congestion and haemorrhages from 2 to 10 dpi. No gross pathological lesions were observed in quails from the control group (Figure S1). Histopathologically, infected quails exhibited mild tracheitis at 5 and 10 dpi, progressing to moderate tracheitis with mononuclear infiltration at 15 dpi. Mild pneumonia with haemorrhages was observed at 2 dpi, which was followed by moderate‐to‐severe pneumonia with haemorrhages at 5–15 dpi. The kidneys exhibited mild haemorrhages and congestion at 2 dpi, progressing to moderate congestion and haemorrhage in inter‐tubular space at 5 and 10 dpi. However, the kidneys appeared normal at 15 dpi. The intestine remained normal, showing no lesions throughout the 2 to 15 dpi period (Figure 2).

FIGURE 2.

FIGURE 2

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Morphopathological changes of trachea (a–e), lung (f–j), intestine (k–o), and kidney (p–t) of both controlled and tribasic H9N2 virus–infected Japanese quails. Tracheitis with mild infiltration of inflammatory cells at 5 dpi (c) and tracheitis characterised by moderate infiltration of mononuclear cells at 10 and 15 dpi (d, e). Pneumonia characterised by congestion and haemorrhages was observed at 2 dpi (g) followed by moderate pneumonia characterised by congestion (h, i) and haemorrhages within the bronchus (j) at 5 to 15 dpi. No significant changes were observed in the intestine (k–o). Mild congestion and haemorrhages were observed in kidney at 2 dpi (black arrow head, q) followed by moderate congestion and haemorrhages at 5 and 10 dpi (black arrow head, r, s); however, kidneys of infected quails at 15 dpi showed no significant changes (t). ‘+’ mild; ‘++’ moderate; H&E stain, 50 µm.

3.2. Distribution and Shedding of H9N2 Viruses Following H9N2 Infection

The shedding and distribution of H9N2 viruses in secretions and tissue samples were evaluated by RT‐qPCR targeting H9 and N2 gene segments. The H9N2 viral RNA was detected in the trachea, lungs, intestine, and kidney tissues of infected quails from 2 to 10 dpi. The highest viral load was observed at 2 dpi followed by a constant decrease until 10 dpi in all aforementioned tissues examined (Figure 3). Viral RNA was also detected in oropharyngeal and cloacal swabs from 2 to 10 dpi. Overall, viral shedding was consistently higher in oropharyngeal swabs than in cloacal swabs at all time points. However, no viral RNA was detected in any tissues and swabs at 15 dpi.

FIGURE 3.

FIGURE 3

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Viral loads in different organs and secretions of quails infected with H9N2 (A/Chicken/Bangladesh/LT56/2021) virus, the viral load in trachea (a), lungs (b), intestine (c), kidney (d), and shedding through the oropharyngeal and cloacal routes (e), Days post‐infection and cycle threshold are plotted on the X‐axis and Y‐axis, respectively. The graphs showed the CT values generated at RT‐qPCR targeting the H9 gene of the H9N2 virus. The samples that showed a value of < 40 were considered positive.

3.3. Expression of Cytokines in Respiratory, Gastrointestinal, and Lymphoid Tissues

3.3.1. Cytokine Expression in Trachea

In the trachea, IL‐8 remained at a minimum level between 2 dpi and 15 dpi with no significant differences. In contrast, the expression of TNF‐α was stable at 2 and 5 dpi (2.45 ± 0.48 and 2.45 ± 0.57, respectively) but exhibited a significant peak at 10 dpi (5.30 ± 0.18; p < 0.001), which declined to minimal levels at 15 dpi (1.50 ± 0.36) (Figure 4a, Table S2). The antiviral cytokines IFN‐β expression was elevated significantly at 2 dpi (6.26 ± 0.50; p < 0.001), followed by a progressive decline at 5 dpi (3.76 ± 0.33; p < 0.001), and 10 dpi (2.39 ± 0.54). Likewise, more pronounced IFN‐γ expression was noticed at 2 dpi (16.37 ± 0.87; p < 0.001) which gradually declined at 5 dpi (6.19 ± 0.78; p < 0.001) and 10 dpi (2.94 ± 0.39; p < 0.001). After 10 dpi, no discernible IFN‐γ expression was observed (Figure 4a; Table S2).

FIGURE 4.

FIGURE 4

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Expression of proinflammatory (IL‐8 and TNF‐α) and antiviral (IFN‐β and IFN‐γ) cytokines in the trachea (a), lungs (b), intestine, (c), and lymphoid organs (d) in tribasic H9N2 LPAI virus–infected quails. Expression levels were normalised to the housekeeping gene GAPDH, which was validated for stable expression across groups (infected and control groups). Bar diagram indicates the relative expression of IL‐8, TNF‐α, IFN‐β, and IFN‐γ cytokines compared to GAPDH. Error bars represent the mean ± SD. Star indicates level of significance as ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05.

3.3.2. Cytokine Expression in Lungs

Infected quails expressed IL‐8, TNF‐α, IFN‐β, and IFN‐γ in the lung at all time points tested (Figure 4b). However, IL8 expression was consistently minimal at every observed time points with no significant differences. In contrast, TNF‐α showed a significantly higher peak at 10 dpi (37.04 ± 1.89) and remained elevated till 15 dpi (19.57 ± 0.51) compared to earlier time points. The antiviral cytokines IFN‐β and IFN‐γ levels increased at all observed times (Figure 4b). IFN‐β was markedly elevated at early time points, with mean fold‐changes of 8.28 ± 0.26 at 2 dpi (p < 0.001) and 5.85 ± 0.17 at 5 dpi (ns). Thereafter, IFN‐β levels declined to 10 dpi (3.27 ± 0.55) and 15 dpi (3.11 ± 0.54), with no statistically significant differences at these times. Similarly, IFN‐γ exhibited a comparable early response, peaked at 2 dpi (7.40 ± 0.52; p < 0.001) and remained significantly elevated at 5 dpi (5.70 ± 0.56; p < 0.001), followed by lower expression at 10 dpi (1.71 ± 0.39) and 15 dpi (0.78 ± 0.22), where no significant differences were observed (Table S2).

3.3.3. Cytokine Expression in Intestine

All cytokines (IL‐8, TNF‐α, IFN‐β and IFN‐γ) exhibited increased expression in intestinal tissues between 2 and 15 dpi (Figure 4c). Both proinflammatory cytokines IL‐8 and TNF‐α expressions were significantly higher at 2 dpi (7.4 ± 1.14; p ≤ 0.01) and 5 dpi (5.37 ± 0.57; p ≤ 0.01), respectively (Figure 4c) followed by progressive decline until 15 dpi (Figure 4c; Table S2). The anti‐viral cytokine IFN‐β exhibited a strong and highly significant early expression at 2 dpi (13.75 ± 0.42; p < 0.001), followed by a gradual but significant decline at 5 dpi (9.73 ± 0.37; p < 0.001) and 10 dpi (8.02 ± 0.15; p < 0.001) (Figure 4c; Table S2). In contrast, IFN‐γ expression started at 2 dpi (2.59±0.42), with a significant peak at 10 dpi (24.37±0.49; p < 0.001). The expression declined thereafter at 15 dpi (5.81 ± 0.27; p < 0.001) (Figure 4c; Table S2). These findings indicate that both IFN‐β and IFN‐γ contribute to a sustained antiviral response in the intestine.

3.3.4. Cytokine Expression in Pooled Lymphoid Organs

Very minimal expression of IL‐8 and TNF‐α was observed in the lymphoid organs of H9N2‐infected quails throughout all time points (Figure 4d; Table S2). IFN‐β expression started from 5 dpi (0.15±0.11; ns) and persisted up to 15 dpi (0.82±0.09; ns) but significantly increased at 10 dpi (9.79±1.59; p <0.001) (Figure 4d; Table S2). This expression pattern may indicate that IFN‐β expressed at later in the lymphoid tissues in response to H9N2 viruses. In contrast, the infected quails did not express IFN‐γ in their lymphoid organs (Figure 4d).

3.4. Body Weight and Egg Production

Following H9N2 infection, infected quails experienced a substantial reduction in body weight compared to the control group. The maximum observed weight loss of 5%–12% was recorded up to 30 days post‐infection (58 days of age). Despite a period of recovery, the infected group exhibited a persistent 6% reduction in body weight relative to the control group that continued through the end of the experiment (68 days of age) (Figure 5a; Table S3). Infected quails exhibited reduction in egg production (24%) at the onset of lay (41–45 days of age) despite producing morphologically normal eggs weighing between 9 and 12 g. A consistent recovery trend was observed thereafter, with the production deficit gradually decreasing from 17% (46–50 days) to a final deficit of 7% by 66–70 days of age. Throughout the experiment, the overall reduction in egg production of the infected group ranged from 7% to 24% compared to controls (Figure 5b; Table S4).

FIGURE 5.

FIGURE 5

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Body weight and egg production of quails both in control and H9N2‐infected groups (a), where the X‐axis represents the period from before infection to 40 days post‐infection, and the Y‐axis shows body weight in gm. Egg production of quails in the control and infected groups (b), where the X‐axis represents the age of quails (days) and the Y‐axis shows the total number of eggs. Both body weight and egg production were consistently lower in the H9N2 LPAI virus–infected group compared to the non‐infected control group.

3.5. Seroconversion

Due to unavailability of specific pathogen‐free (SPF) quails in Bangladesh, commercially sourced 2‐week‐old quail chicks were used in this study. Before infection, all quails were considered susceptible to AIV. Thus, the quail chicks were reared in strict isolation under biosecurity protocols. Serological screening conducted before H9N2 infection, confirmed that majority of the quails had low, non‐protective levels of antibodies (≤3 HI units). However, following H9N2 infection, the infected quails developed a significant increase in protective antibody levels by 7, 14, and 21 days post‐infection (dpi) (Figure 6).

FIGURE 6.

FIGURE 6

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Seroconversion of infected quails receiving 106 EID50/mL of H9N2 LPAI virus. The HI titre of birds is plotted on the Y‐axis, and days post‐infection is plotted on the X‐axis, respectively. The level of significance is calculated using one‐way ANOVA with Tukey's multiple comparisons test and is denoted by an asterisk mark. ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05.

4. Discussion

Low‐pathogenic avian influenza (LPAI) H9N2 virus has been a persistent problem in Bangladesh since 2006, affecting both commercial and backyard poultry. The pathogenicity of AIV is influenced by the structure of the haemagglutinin cleavage sites (HACS). Low‐pathogenic AIV (LPAIV) typically possesses mono‐ or dibasic HACS. However, since 2011 H9N2 strains with a tribasic HACS have been circulating in Bangladesh. Although the pathogenicity of circulating tribasic H9N2 viruses is well documented in commercial chickens, their effects in quails are poorly characterised. This study investigated the pathogenicity, viral distribution, and cytokine responses (IL‐8, TNF‐α, IFN‐β, IFN‐γ) in Japanese quails infected with a tribasic H9N2 virus.

In this study, infected quails exhibited no obvious clinical signs and mortality, identical to H9N2‐infected chickens and other quail species (Begum et al. 2023; Seiler et al. 2018). However, they experienced subclinical production losses, including 5%–12% decrease in body weight, and 7%–24% reduction in egg production, consistent with the known impact of LPAIV H9N2 (Keshtkari et al. 2010). Despite the absence of clinical signs, mild‐to‐moderate pathological lesions were evident in the respiratory organs (trachea and lungs) and kidneys of infected quails at different time points. In the study, infected quails exhibited slight haemorrhages in trachea and mild‐to‐moderate congestion and haemorrhages in lung up to 10 dpi. Similar findings have been reported in Sonali and broiler chickens following tribasic H9N2 virus infection (Begum et al. 2023). In this study, kidneys of infected quails appeared haemorrhagic which were not evident in Sonali and broiler chickens with tribasic H9N2 AIV (Begum et al. 2023). It may indicate that the tribasic H9N2 virus gaining properties to replicate in other visceral organs of quails. Microscopically, the infected quails displayed mild‐to‐moderate tracheitis and variable pneumonic lesions in lungs that are comparable to the tribasic H9N2‐infected Sonali and broiler chickens (Begum et al. 2023). Notably, the infected quails developed mild‐to‐moderate renal congestion, haemorrhages and tubular degeneration, which were absent in tribasic H9N2‐infected Sonali and broiler chickens (Begum et al. 2023). Conversely, such renal pathology has been documented in commercial chickens intravenously infected with H9N2 strains (Subtain et al. 2011). This inconsistency might be attributed to variations in the specific H9 virus subtype used, route of inoculation and breed and health status of the experimental birds. Moreover, the absence of gross lesions in the reproductive organs suggests that the notable decline in egg production is probably due to systemic effects of the infection such as prolonged malabsorption of nutrients available for egg production (Kapic et al. 2021) and sustained immune‐mediated suppression of reproduction due to elevated pro‐inflammatory cytokines (e.g., TNF‐α, IFN‐γ) detected in this study (Iwasa et al. 2017). These mechanisms may profoundly suppress reproductive output without causing overt structural damage to the reproductive organs.

The replication of low‐pathogenic H9N2 avian influenza viruses is typically confined to the respiratory and intestinal epithelial cells expressing trypsin‐like proteases (Böttcher‐Friebertshäuser et al. 2013). In this study, viral RNA was detected in the trachea, lungs, intestine and notably, the kidneys of the infected quails from 2 to 10 dpi, correlating with the observed gross lesions. Detection of viral RNA in kidneys of quails, in contrast to its absence in tribasic H9N2 infected chickens and monobasic H9N2 infected other quail species (Seiler et al. 2018; Begum et al. 2023) provides evidence for expanded tissue tropism. This renal tropism implies that the tribasic H9N2 strain may be evolved and cleaved by proteases present in renal tissues, thereby facilitating systemic spread. This observation is consistent with other reports (Awadin et al. 2018; Zhang et al. 2020), indicating that certain H9N2 lineages are evolving to replicate in a wider range of visceral organs. Furthermore, predominant oropharyngeal shedding, a pattern consistent across poultry species (Kye et al. 2021; Begum et al. 2023; Yang et al. 2024), highlights the respiratory tract as the primary route for viral transmission. The highest viral load was seen at 2 and 5 dpi, which gradually started to decline from 10 dpi, and eventually disappeared after 15 dpi. These indicate a self‐limiting H9N2 infection course in Japanese quails.

Viral pathogenicity depends on the viral replication rate and the ability of the viral strain to induce proinflammatory cytokines for limiting viral dissemination (Wang et al. 2016; Evseev and Magor 2019; Spencer et al. 2020). In our study, proinflammatory cytokine IL‐8, a potent recruiter of immune cells to sites of infection, was minimal. Conversely, TNF‐α, a key mediator of systemic inflammation, exhibited a delayed peak at 10 dpi and persisted over time, indicating a sustained but delayed strong inflammatory response which is inconsistent with the viral load in the respective organs (between 2 and 5 dpi). However, the expression pattern is slightly different in both monobasic and tribasic H9N2‐infected SPF and commercial chickens, respectively (Nang et al. 2011; Hossain et al. 2024). This sustained inflammatory response might cause tissue damage and production losses without effectively controlling early viral replication.

H9N2 virus is highly sensitive to the antiviral effects of IFNs (Westenius et al. 2014). In this study, antiviral cytokines including IFN‐β and IFN‐γ were significantly upregulated early within the respiratory and intestinal tracts, and lymphoid organs (spleen and bursa), critical for establishing an antiviral state in neighbouring cells and therefore, limiting H9N2 viral spread (Wang et al. 2016; Hossain et al. 2024) and preventing severe disease, despite the observed tissue damage (Yang et al. 2023).

Collectively, the circulating tribasic H9N2 virus produces mild‐to‐moderate clinico‐pathological changes along with progressive decline in viral loads after 10 dpi and a strong early antiviral response in Japanese quails that provide the invaluable insights into the host–pathogen interaction. The ability of quails to shed the virus for a considerable period (up to 10 dpi) with no overt clinical signs makes them efficient reservoirs within live bird markets and mixed‐species farms. The expanded tropism of this tribasic H9N2 virus to the kidneys in quails may represent the potential evolutionary step towards increased its virulence. Hence, the adaptability and silent spread of AIVs in quails present a constant risk of spillovers to other poultry species that are more prone to AIVs, thus underscoring the need for enhanced surveillance across all poultry species.

5. Conclusion

Our findings highlight the low‐pathogenic nature of the currently circulating tribasic H9N2 virus in Japanese quails, despite its tropism to respiratory and gastrointestinal tracts, and kidneys. The observed clinicopathological changes and robust early antiviral cytokines expression (IFN‐β and IFN‐γ) provide valuable insights into the host–pathogen interactions. Regular surveillance and pathogenicity studies of circulating AIVs remain crucial to monitoring their transmission dynamics and evolutionary trends across different host species.

Author Contributions

Rupaida Akter Shila: writing – original draft, software, methodology, and formal analysis. Ismail Hossain: methodology and formal analysis. Rokshana Parvin: methodology, and writing – review & editing. Emdadul Haque Chowdhury: writing – review & editing and validation. Jahan Ara Begum: conceptualisation, writing – review & editing, validation, and supervision.

Funding

This research was jointly funded by Bangladesh Agricultural University, project no. 2020/963/BAU and University Grant Commission (UGC), Project No. 2023/11/UGC.

Ethics Statement

Regarding the usage and care of quails, the authors confirm that all relevant institutional and national rules were followed. The study was conducted strictly in compliance with the Bangladesh Agricultural University, Mymensingh Ethical Standard of Research Committee authorisations. The Ethical Standard of the Research Committee reviewed and approved the methodology and procedures of this experiment (7/BAURES/ESRC/VET‐16/22).

Conflicts of Interest

The authors confirm no financial or personal conflicts of interest that could have influenced this study.

Supporting information

Figure S1: Gross pathological changes in trachea (a–e), lung (f–j), intestine (k–o), and kidney (p–t) of Japanese quails infected with tribasic H9N2 virus. Mild haemorrhages observed at 2 dpi (b) followed by moderate haemorrhages were observed at 5 and 10 dpi (c, d). In lungs mild‐to‐moderate congestion and haemorrhages from 2 to 15 dpi (g–j); however, no visible gross changes were observed in intestine of infected quails (k–o). Kidneys showed moderate congestion and haemorrhage from 2 to 10 dpi (q–s) followed by apparently healthy kidney at 15 dpi. ‘+’ mild; ‘++’ moderate; ‘+++’ severe.

VMS3-12-e70834-s002.jpg (914.8KB, jpg)

Table S1: List of primers used for detection of H9N2 and cytokines expression in different organs in the experimental study.

Table S2: The complete numerical dataset, including mean fold change ± SD, minimum and maximum observed values, and statistical comparisons of cytokines expressed in different organs of tribasic H9N2‐infected quails.

Table S3: Daily observational data on body weight of quails used in this study.

Table S4: Daily observational data on egg production of quails used in this study.

VMS3-12-e70834-s001.docx (38.2KB, docx)

Acknowledgement

We express our gratitude to Md. Shafiqul Islam, Department of Pathology for his technical assistance.

Shila, R. A. , Hossain I., Parvin R., Chowdhury E. H., and Begum J. A.. 2026. “Chicken Origin Tribasic H9N2 Avian Influenza Virus Induces Potent Early Antiviral Response With Low Pathogenicity in Japanese Quails.” Veterinary Medicine and Science 12, no. 2: e70834. 10.1002/vms3.70834

Data Availability Statement

The data will be available from authors upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1: Gross pathological changes in trachea (a–e), lung (f–j), intestine (k–o), and kidney (p–t) of Japanese quails infected with tribasic H9N2 virus. Mild haemorrhages observed at 2 dpi (b) followed by moderate haemorrhages were observed at 5 and 10 dpi (c, d). In lungs mild‐to‐moderate congestion and haemorrhages from 2 to 15 dpi (g–j); however, no visible gross changes were observed in intestine of infected quails (k–o). Kidneys showed moderate congestion and haemorrhage from 2 to 10 dpi (q–s) followed by apparently healthy kidney at 15 dpi. ‘+’ mild; ‘++’ moderate; ‘+++’ severe.

VMS3-12-e70834-s002.jpg (914.8KB, jpg)

Table S1: List of primers used for detection of H9N2 and cytokines expression in different organs in the experimental study.

Table S2: The complete numerical dataset, including mean fold change ± SD, minimum and maximum observed values, and statistical comparisons of cytokines expressed in different organs of tribasic H9N2‐infected quails.

Table S3: Daily observational data on body weight of quails used in this study.

Table S4: Daily observational data on egg production of quails used in this study.

VMS3-12-e70834-s001.docx (38.2KB, docx)

Data Availability Statement

The data will be available from authors upon reasonable request.

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