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. 2026 Jul 16;105(11):107437. doi: 10.1016/j.psj.2026.107437

Oral vitamin D3 supplementation enhances antiviral immunity in chickens challenged with fowl adenovirus serotype 4

Zhanbang Ma a,b,1, Fangfang Wang a,b,1, Bing Wang a,b, Zhihui Zuo a,b, Zongxi Han b, Huixin Li b, Deying Ma a,⁎
PMCID: PMC13416652  PMID: 42486032

Abstract

Vitamin D is increasingly recognized as an important regulator of innate and adaptive immunity, yet its antiviral role in chickens during fowl adenovirus serotype 4 (FAdV-4) infection remains unclear. In this study, we investigated the effects of oral vitamin D3 supplementation on humoral immunity, tissue viral load, and immune-related gene expression in FAdV-4-infected chickens. Ninety 8-day-old specific pathogen-free chickens were randomly divided into two groups (n = 45/group) and fed a basal diet. The experimental group received oral vitamin D3 dissolved in 0.1 mL soybean oil at a dose equivalent to 3000 IU/kg diet daily for 3 weeks, whereas the control group received soybean oil alone. After supplementation, all birds were challenged with FAdV-4. Vitamin D3 supplementation significantly reduced tissue viral loads and increased serum antibody levels compared with the control group. At the transcriptional level, vitamin D3 modulated key innate and adaptive immune markers, including Toll-like receptors (TLRs), avian β-defensins, and cytokines. It downregulated several TLRs and reduced the pro-inflammatory cytokines interleukin (IL)-6 and IL-8, while increasing the antiviral cytokines interferon-γ and IL-1β. Vitamin D3 also increased vitamin D receptor expression, decreased inducible nitric oxide synthase, and reduced the apoptosis regulators B-cell lymphoma-2 (BCL-2) and Bcl2-associated X protein in selected tissues. In conclusion, vitamin D3 supplementation enhanced antiviral defense against FAdV-4 infection, possibly by promoting humoral immunity and modulating innate immune and apoptosis-related responses. These findings provide new insight into the role of vitamin D3 in limiting FAdV-4 infection and support its potential as a nutritional strategy for controlling viral diseases in poultry.

Keywords: Vitamin D3, Fowl adenovirus serotype 4, Chicken, Antiviral immunity, Innate immune response

Introduction

Among viral pathogens affecting poultry, fowl adenovirus serotype 4 (FAdV-4) is of particular concern because of its association with hydropericardium-hepatitis syndrome and severe hepatic lesions characterized by basophilic intranuclear inclusion bodies, vacuolar degeneration, and multifocal hepatocellular necrosis (Jadhao et al., 2003). FAdV-4 outbreaks can result in mortality rates of up to 80%, leading to substantial economic losses in commercial poultry production (Chandra et al., 2000; Mazaheri et al., 1998). Despite its importance, control of FAdV-4 remains challenging, and additional strategies to improve host resistance are needed. This issue is particularly relevant under modern poultry production conditions, where birds are frequently exposed to environmental and physiological stressors that can impair immune function and increase susceptibility to infectious and inflammatory diseases (Shojadoost et al., 2021).

Nutritional factors, including vitamins, play important roles in growth performance, health, and immune competence in poultry, and can substantially influence host responses to infectious agents and experimental antigens (Jose et al., 2017; Pan et al., 2018; El-Senousey et al., 2018). Among these nutrients, vitamin D has received increasing attention because of its established functions in mineral metabolism and its emerging role in immune regulation. Vitamin D3 (cholecalciferol), through its active metabolite 1,25-dihydroxyvitamin D3 [1,25(OH)2D3; calcitriol], exerts a wide range of biological effects. Although its contribution to calcium and phosphorus homeostasis, bone development, and eggshell quality is well established (DeLuca and Schnoes, 1983; Underwood and DeLuca, 1984, DeLuca and, Schnoes, 1983; de Matos, 2007), its role in regulating immune responses in poultry is increasingly being recognized.

Recent studies indicate that vitamin D3 can influence both innate and adaptive immunity through transcriptional regulation of multiple immune-related genes. Its reported immunological effects include modulation of humoral and cell-mediated immune responses, regulation of inflammatory signaling, and broader immunomodulatory activity (Almerighi et al., 2009; Dickie et al., 2010; Hughes et al., 2009; Khoo et al., 2011; Sadeghi et al., 2006). In poultry, vitamin D3 has been shown to enhance immune responses to infectious bursal disease virus in vitro (Jaime et al., 2020), increase nitric oxide production and responsiveness to Toll-like receptor (TLR) ligands in chicken macrophages (Shojadoost et al., 2015, 2021), and upregulate TLR2b, TLR4, cathelicidin1, and cathelicidin B1 expression in the spleen of broilers, together with a predominantly Th2-type cytokine profile (Rodriguez-Lecompte et al., 2016). In addition, vitamin D3 has been implicated in the regulation of pro-inflammatory cytokine production and leukocyte infiltration at sites of inflammation (Helming et al., 2005; Bahar-Shany et al., 2010; Jadhav et al., 2018). These findings suggest that vitamin D3 may be an important nutritional modulator of host defense in chickens.

Because immune competence is critical for both disease resistance and production efficiency, nutritional interventions that support immune function are of considerable interest in poultry science. Given the immunoregulatory properties of vitamin D3, we hypothesized that vitamin D3 supplementation could enhance host antiviral defense against FAdV-4 infection. Therefore, the objective of the present study was to determine whether vitamin D3 supplementation can improve antiviral immunity in specific pathogen-free (SPF) chickens challenged with FAdV-4.

Materials and methods

Ethics statement

All animal experiments were conducted in accordance with the animal welfare guidelines of the World Organisation for Animal Health. All procedures involving animals were reviewed and approved by the Agricultural Animal Care and Use Committee of Heilongjiang Province, China.

Virus strain, vitamin D3, and animals

The virulent FAdV-4 strain HN/151025 (GenBank accession no. KU245540) was isolated in our laboratory in October 2015 from a dead chicken in China, as previously described (Li et al., 2016). Vitamin D3 [1,25(OH)2D3] was purchased from Aladdin (Shanghai, China).

One-day-old specific-pathogen-free (SPF) White Leghorn chickens were obtained from the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Harbin, China). All chickens were housed in separate isolators and fed a basal diet (Table 1) until use.

Table 1.

Ingredients and nutrients content of the basal diet.

Ingredients Percentage (%) Nutrient content Percentage
(%)
Soybean meal 21.00  ME (MJ/Kg) 11.92
Expanded soybean 5.00 Crude protein1 16.73
Corn 59.20 ether extract1 4.15
Corn gluten meal 6.00 Crude fiber1 4.06
Wheat shorts 5.00 Sodium Chloride1 0.30
limestone 1.50 Calcium1 1.25
Calcium hydrogen phosphate 1.20 Total phosphoros1 0.62
Salt 0.30 Lysine 0.80
Adhesive 0.30 Methionine+cysteine 0.80
Lysine (98%) 0.15 Threonine 0.75
Methionine 0.10
Choline chloride 0.10
vitamin premix2 0.05
Mineral premix3 0.10
Total 100.00
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1

measured value.

2

Supplied per kilogram of final diet:vitamin A 8000 IU/kg;vitamin E 10 IU/kg;vitamin D3 2200 IU/kg;vitamin K3 1.6 mg/kg; vitamin B1 1.6 mg/kg; vitamin B2 4.4 mg/kg; vitamin B6 1.6 mg/kg; vitamin B12 0.088 mg/kg; biotin 0.055 mg/kg; Nicotinamide 27.5 mg/kg; d-calcium pantothenate 6.6 mg/kg; folic acid 0.055 mg/kg.

3

Supplied per kilogram of final diet:Fe 44 mg/kg (FeSO4.H2O) ;Cu 4.4 mg/kg (CuSO4.5H2O) ;Mn 68 mg/kg (MnSO4.H2O) ;I 0.9 mg/kg (KI) ;Zn 66 mg/kg (ZnSO4.H2O) ;Se 0.3 mg/kg (Na2SeO3) ..

Experimental design

To determine the optimal dose of vitamin D3, 60 SPF chickens at 8 days of age were randomly assigned to 3 groups (n = 20 per group). Birds were fed diets (Table 1) supplemented with vitamin D3 at 1,000, 3,000, or 5,000 IU/kg diet until 28 days of age. On day 28, all birds were inoculated with 0.1 mL of the FAdV-4 HN/151025 strain at a dose of log105.31 50% tissue culture infective dose (TCID50). At 3, 7, 14, and 21 days post-inoculation (dpi), 5 birds from each group were humanely euthanized, and the trachea, lungs, kidneys, spleen, cecal tonsils, and bursa of Fabricius were collected from each bird (Xu et al., 2015). Viral loads in tissue samples were determined by RT-qPCR as described in the section entitled “Detection of FAdV-specific antibodies and viral load.” Based on these results, supplementation with 3,000 IU vitamin D3/kg diet significantly reduced viral loads in tissues from FAdV-4-infected chickens compared with the other supplementation levels.

For the main experiment, ninety SPF chickens at 8 days of age were stratified by body weight to minimize variation, housed in separate isolators, and randomly allocated to 2 groups (n = 45 per group). All birds were fed the basal diet (Table 1) formulated to meet the nutrient requirements recommended by the National Research Council (NRC, 1994). Feed and water were provided ad libitum throughout the experiment.

Based on the dose-optimization results, birds in the experimental group received vitamin D3 dissolved in 0.1 mL soybean oil at a dose equivalent to 3,000 IU/kg diet once daily by oral administration (vitamin D group), whereas birds in the control group received the same volume of soybean oil without vitamin D3 until 28 days of age. Body weight was recorded on days 8 and 28, and feed intake and mortality were monitored throughout the experimental period.

Viral challenge and sample collection

On day 28, all chickens were orally inoculated with 0.1 mL of the FAdV-4 HN/151025 strain at a dose of log105.31 TCID50 per bird. After challenge, 10 birds from each group were individually identified and monitored for clinical signs. Serum samples were collected from these birds at 3, 7, 14, and 21 dpi. At 0, 3, and 7 dpi, 10 birds from each group were humanely euthanized, and the heart, liver, spleen, kidneys, and bursa of Fabricius were collected (Xu et al., 2015). Tissue samples were rinsed with cold sterile saline, snap-frozen in liquid nitrogen, and stored at −70°C until analysis. The experiment was terminated at 21 dpi, and all remaining samples were stored at −70°C until further use.

Detection of FAdV-4-specific antibodies

FAdV-specific antibodies were measured in triplicate using a FAdV BioChek ELISA kit (BioChek USA Corporation, Scarborough, ME, USA) according to the manufacturer’s instructions. The mean sample-to-positive (S/P) ratio was calculated as previously described (Li et al., 2016).

Quantitative real-time PCR analysis of gene expression and viral load

Total RNA was extracted from tissues using RNAiso Plus reagent (Takara Bio, Shiga, Japan) as previously described (Li et al., 2015). A one-step PrimeScript real-time RT-PCR kit (Takara Biotechnology, Dalian, China) was used to quantify mRNA expression levels as described previously (Li et al., 2015). 18S ribosomal RNA (18S rRNA) served as house keeping gene in this study. The genes analyzed included 18S rRNA; TLR1-5, TLR7, TLR15, and TLR21; avian β-defensins (AvBD1-14); cytokines (interferon-γ (IFN-γ), interleukin (IL)-1β, IL-6, and IL-8); vitamin D receptor (VDR); inducible nitric oxide synthase (iNOS); B-cell lymphoma-2 (Bcl-2), Bcl2-associated X protein (BAX). The FAdV-4 genome load in chicken tissues was also quantified by real-time RT-PCR as previously described (Li et al., 2016). All primers used in this study were adopted from previous reports (Li et al., 2015). Gene expression was normalized to 18S rRNA and expressed as the ratio of the vitamin D3 group to the control group.

Statistical analysis

Data are presented as means ± standard error of the mean (SEM). Statistical analyses were performed using IBM SPSS Statistics 19.0 (IBM Corp., Chicago, IL, USA). Data were compared among groups at each time point using one-way ANOVA followed by Tukey's post hoc test, with the individual chicken as the experimental unit. Differences were considered significant at P < 0.05.

Results

Growth performance and clinical signs

Vitamin D3 supplementation had little effect on body weight gain, feed intake, mortality, or clinical signs in chicks infected with FAdV-4 (data not shown).

Viral replication

In the control group, the highest viral load was detected in the liver at 7 dpi. Notably, viral loads were significantly higher in the heart at 3 dpi, in the kidney and bursa of Fabricius at 7 dpi in chickens that did not receive vitamin D3 supplementation than in those that did (P < 0.05) (Fig. 1). These results indicate that vitamin D3 supplementation reduced viral replication in selected tissues following FAdV-4 infection.

Fig. 1.

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Effects of oral vitamin D supplementation on viral loads in tissues of fowl adenovirus serotype 4 (FAdV-4)-infected chickens. Viral RNA copy numbers in the tissues of SPF chickens were measured by RT-qPCR at 0, 3 and 7 dpi. The results are expressed as mean ± SEM of three independent experiments with three replicates per experiment. *P < 0.05. Statistical analysis was performed using IBM SPSS 19.0 statistical software.

Serum FAdV-specific IgG

Anti-FAdV antibodies were first detected at 7 dpi, and antibody levels reached their peak at 14 dpi in both groups (Fig. 2). Notably, IgG levels were significantly higher in the vitamin D3-supplemented group than in the control group from 7 to 21 dpi (P < 0.05).

Fig. 2.

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Effects of oral vitamin D supplementation on serum antibody levels of FAdV-4-infected chickens. Serum samples were collected to measure FAdV-4-specific IgG serum levels in SPF chickens infected with the pathogenic FAdV-4 HN/151025 strain. FAdV-4-specific IgG serum levels were assayed in triplicate using a commercial ELISA kit. Ten chickens were tested in each group at 3, 7, 14, and 21 days post-inoculation (dpi). Dashes show the S/P ratios, calculated as described in the Materials and Methods section. Serum samples with S/P ratios equal to or above the dashes were considered positive. Values are presented as mean ± SEM. *P < 0.05. The data were analyzed using IBM SPSS 19.0 statistical software.

Expression of immune-related genes

Overall, the expression of several TLR genes was lower in tissues from vitamin D3-supplemented chicks than in those from the control group. As shown in Fig. 3, vitamin D3 supplementation significantly reduced the expression of TLR1 in the liver at 7 dpi, TLR2 in the liver at 7 dpi and in the kidney at 3 dpi, TLR4 in all examined tissues at 3 dpi, TLR5 in the liver at 7 dpi, TLR7 in all tissues except the liver at 3 dpi, and TLR15 in the liver at 7 dpi, compared with the control group (P < 0.05). No significant differences were observed for the other TLR genes at any time point in any tissue examined (data not shown).

Fig. 3.

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Effects of oral vitamin D supplementation on the mRNA expression levels of Toll-like receptors (TLRs) in tissues of FAdV-4-infected chickens. The cDNA copy numbers of TLR3 and TLR7 in the tissues of five SPF chickens from the vitamin D and control groups were measured by RT-qPCR at 0, 3 and 7 dpi. The mRNA expression levels of TLRs were normalized to 18S rRNA. Fold change values of target genes were calculated as the ratio of their expression levels in the vitamin D group to those in the control group. The results are presented as the mean ± SEM of three independent experiments with three replicates per experiment. *P < 0.05). The data were analyzed using IBM SPSS 19.0 statistical software.

Significant changes in AvBD expression were also observed in several tissues (Fig. 4). Chickens supplemented with vitamin D3 showed significantly increased AvBD1 transcription in the heart and kidney at 7 dpi compared with the control group (P < 0.05). In contrast, the expression of AvBD4 and AvBD8 in the liver at 7 dpi was significantly lower in the vitamin D3 group than in the control group (P < 0.05). Likewise, AvBD6 expression was significantly decreased in all examined tissues except the bursa of Fabricius at 3 dpi in the vitamin D3 group (P < 0.05). No significant differences were detected in the expression of the other AvBD genes between groups at any time point (data not shown).

Fig. 4.

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Effects of oral vitamin D supplementation on the mRNA expression levels of avian β-defensins (AvBDs) in tissues of FAdV-4-infected chickens. The cDNA copy numbers of AvBDs in the tissues of five SPF chickens from the vitamin D and control groups were measured by RT-qPCR at 0, 3 and 7 dpi. AvBD mRNA expression levels were normalized to 18S rRNA. Fold change values of target genes were calculated as the ratio of their expression levels in the vitamin D group to those in the control group. The results are presented as the mean ± SEM of three independent experiments with three replicates per experiment. *P < 0.05. The data were analyzed using IBM SPSS 19.0 statistical software.

For cytokine expression, IFN-γ expression was significantly higher in the liver at 3 dpi in chickens receiving vitamin D3 than in the control group (P < 0.05). Similarly, IL-1β expression was significantly increased in all examined tissues except the spleen at 3 dpi in the vitamin D3 group compared with the control group (P < 0.05). In contrast, IL-6 and IL-8 expression levels in the liver at 7 dpi were significantly higher in the control group than in the vitamin D3 group (P < 0.05) (Fig. 5).

Fig. 5.

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Effects of oral vitamin D supplementation on the mRNA expression levels of cytokines in tissues of FAdV-4-infected chickens. The cDNA copy numbers of cytokines in the tissues of five SPF chickens from the vitamin D and control groups were measured by RT-qPCR at 0, 3 and 7 dpi. Cytokine mRNA expression levels were normalized to 18S rRNA. Fold change values of target genes were calculated as the ratio of their expression levels in the vitamin D group to those in the control group. The results are presented as the mean ± SEM of three independent experiments with three replicates per experiment. *P < 0.05. The data were analyzed using IBM SPSS 19.0 statistical software.

VDR expression was significantly higher in the heart and liver at 3 dpi in the vitamin D3-supplemented group than in the control group (P < 0.05). By contrast, iNOS expression was significantly higher in the heart, liver, and spleen at 3 dpi in the control group than in the vitamin D3 group (P < 0.05) (Fig. 6).

Fig. 6.

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Effects of oral vitamin D supplementation on the mRNA expression levels of VDR and iNOS in tissues of FAdV-4-infected chickens. The cDNA copy numbers of VDR and iNOS in the tissues of five SPF chickens from the vitamin D and control groups were measured by RT-qPCR at 0, 3 and 7 dpi. The mRNA expression levels of VDR and iNOS were normalized to 18S rRNA. Fold change values of target genes were calculated as the ratio of their expression levels in the vitamin D group to those in the control group. The results are presented the mean ± SEM of three independent experiments with three replicates per experiment. *P < 0.05. The data were analyzed using IBM SPSS 19.0 statistical software.

The expression of BAX was significantly higher in the liver and spleen at 3 dpi in the control group than in the vitamin D3 group (P < 0.05). Similarly, BCL-2 expression was significantly higher in all examined tissues at 3 dpi in the control group than in the vitamin D3 group (P < 0.05) (Fig. 7).

Fig. 7.

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Effects of oral vitamin D supplementation on the mRNA expression levels of BAX and Bcl-2 in tissues of FAdV-4-infected chickens. The cDNA copy numbers of BAX and Bcl-2 in the tissues of five SPF chickens from the vitamin D and control groups were measured by RT-PCR at 0, 3 and 7 dpi. The expression levels of BAX and Bcl-2 were normalized to 18S rRNA. Fold change values of target genes were calculated as the ratio of their expression levels in the vitamin D group to those in the control group. The results are presented as the mean ± SEM of three independent experiments with three replicates per experiment. *P < 0.05. The data were analyzed using IBM SPSS 19.0 statistical software.

Discussion

This is, to our knowledge, the first systematic evaluation of the immunoregulatory and antiviral effects of vitamin D3 against FAdV-4 infection in chickens. Vitamin D3 is widely used as a feed additive in poultry production and is well recognized for its role in calcium and phosphorus homeostasis. Increasing evidence, however, indicates that it also exerts immunomodulatory and antiviral effects. In the present study, vitamin D3 supplementation reduced FAdV-4 loads in several tissues, particularly in the heart, kidney, and bursa of Fabricius, and this was accompanied by marked changes in immune-related gene expression. These findings suggest that vitamin D3 may enhance host resistance to FAdV-4 infection and highlight its potential as a nutritional strategy to strengthen antiviral defense in chickens. This interpretation is consistent with recent evidence showing that vitamin D3 suppresses infectious bronchitis virus replication in vitro (Wang et al., 2025), and extends previous reports describing antiviral activity against dengue virus, hepatitis C virus, SARS-CoV-2, and rotavirus (Gal-Tanamy et al., 2011; Lin et al., 2018; Maloney, 2023; Puerta-Guardo et al., 2012; Tian et al., 2016).

In addition to its effects on viral load, vitamin D3 enhanced the humoral immune response to infection. Nutritional modulation of vaccine- or infection-induced antibody responses has been widely investigated, and vitamin D3 has emerged as a particularly relevant factor because of its ability to regulate immune function and enhance antibody production (Jaime et al., 2020; Cesur et al., 2023; Wang et al., 2025). In the present study, vitamin D3 supplementation significantly increased IgG levels in FAdV-4-infected chicks, supporting the view that vitamin D3 may promote a more competent humoral immune status. From a practical perspective, these findings suggest that maintaining adequate vitamin D3 status before pathogen exposure may be more beneficial than intervention after infection. Nevertheless, the mechanisms by which vitamin D3 enhances antiviral humoral immunity remain unclear and warrant further investigation.

This study presents novel findings demonstrating that the immunological effects of vitamin D3 were not limited to antibody responses. TLRs are central mediators of innate immune recognition, and previous studies have shown that FAdV-4 infection activates both cytokine-related pathways and TLR signaling, with increased expression of TLR2, TLR3, TLR4, and TLR15 in infected tissues (Li et al., 2018; Zhang et al., 2018; Zhao et al., 2020). Moreover, TLR expression has been positively correlated with viral load in certain tissues, suggesting that excessive activation of these receptors may contribute to tissue injury during infection (Zhao et al., 2020). In the present study, vitamin D3 supplementation reduced TLR expression in some tissues after FAdV-4 challenge. Together with previous observations in IBV-infected chicken embryo fibroblasts (Wang et al., 2025), these results suggest that vitamin D3 may temper excessive innate immune activation and thereby limit harmful inflammatory responses.

This study revealed that vitamin D3 selectively regulates AvBD expression in a subtype- and tissue-specific manner during FAdV-4 infection. AvBDs are important effector molecules of innate immunity and may bridge innate and adaptive responses (Cuperus et al., 2013; Garcia-Valtanen et al., 2014). Viral infection is known to induce AvBD expression in avian species, including during FAdV-4 infection (Zhao et al., 2020). In the present study, vitamin D3 supplementation downregulated AvBD4, AvBD6, and AvBD8, while upregulating AvBD1 in some tissues. This selective regulation suggests that vitamin D3 does not simply amplify or suppress defensin responses globally, but rather reshapes them in a targeted manner. Such differential regulation may help optimize antimicrobial peptide responses while avoiding excessive inflammatory activation. Similar findings have been reported in IBV-infected CEFs, in which vitamin D modulated defensin expression in a subtype-specific manner (Wang et al., 2025).

This study demonstrates that vitamin D3 exerts a dual immunomodulatory role during FAdV-4 infection. In the present study, vitamin D3 supplementation increased IL-1β and IFN-γ expression after infection, suggesting enhanced activation of protective antiviral responses. This is in agreement with Sharma et al. (2024), who reported increased IL-1β and IFN-γ expression in vitamin d-treated, coccidia-infected laying hens. Both cytokines are closely associated with activation of host defense mechanisms and may contribute to resistance against FAdV-4 infection. At the same time, vitamin D3 reduced IL-6 and IL-8 expression compared with untreated controls. Because these cytokines are commonly associated with excessive inflammation and tissue damage during FAdV-4 infection (Li et al., 2018; Zhang et al., 2018; Zhao et al., 2020), their suppression suggests that vitamin D3 may restrain detrimental inflammatory amplification. Similar anti-inflammatory effects of vitamin D have been reported in H9N2-infected A549 cells and IBV-infected CEFs (Gui et al., 2017; Wang et al., 2025). Taken together, these findings indicate that vitamin D3 may enhance antiviral immunity while simultaneously limiting excessive inflammatory cytokine production, thereby reducing the risk of immunopathology.

This study provides new evidence that vitamin D3 upregulates VDR expression in the heart and liver during FAdV-4 infection. VDR is a key mediator of vitamin D signaling and regulates multiple immune components, including macrophages, TLRs, cytokines, and antimicrobial peptides (Shojadoost et al., 2015; Arababadi et al., 2018; Aggeletopoulou et al., 2022; Zhang et al., 2016). In the present study, VDR mRNA expression was increased in the heart and liver of vitamin D3-supplemented chickens, suggesting that the immunoregulatory effects of vitamin D3 during FAdV-4 infection may be mediated, at least in part, through activation of the VDR pathway.

Our previous work showed that FAdV-4 infection upregulated iNOS expression and that iNOS levels were positively correlated with viral genome load in infected tissues (Zhao et al., 2020). In the present study, vitamin D3 supplementation further decreased iNOS expression in some tissues. Although the biological significance of this finding remains unclear, it suggests that vitamin D3 may also influence nitric oxide-related antiviral or inflammatory pathways during FAdV-4 infection. The molecular basis of this response requires further study.

Finally, vitamin D3 supplementation altered the expression of apoptosis-related genes. Specifically, BAX and BCL-2 expression levels were reduced in some tissues of FAdV-4-infected chickens receiving vitamin D3. Because BAX and BCL-2 are key regulators of mitochondria-mediated apoptosis, and mitochondrial injury promotes cytochrome c release and caspase-3 activation (Ola et al., 2011; Pistritto et al., 2016), these findings suggest that vitamin D3 may alleviate mitochondrial damage and thereby modulate apoptosis during infection. Although the precise relationship between these transcriptional changes and tissue protection remains to be clarified, the data support a role for vitamin D3 in limiting cellular injury associated with FAdV-4 infection.

In conclusion, oral vitamin D3 supplementation strengthened antiviral defense against FAdV-4 infection in SPF chickens in three main ways. First, it reduced viral load in several tissues. Second, it enhanced humoral immunity and modulated the expression of immune-related genes. Third, it limited excessive inflammatory responses and apoptosis-related cellular injury. These findings support vitamin D3 as a promising nutritional strategy for controlling viral disease in poultry.

CRediT authorship contribution statement

Zhanbang Ma: Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Fangfang Wang: Project administration, Methodology, Investigation, Formal analysis, Data curation. Bing Wang: Methodology, Investigation, Data curation. Zhihui Zuo: Methodology, Investigation. Zongxi Han: Validation, Methodology. Huixin Li: Methodology, Investigation. Deying Ma: Writing – review & editing, Writing – original draft, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization.

Disclosures

The authors declare that they have no conflict of interest and competing interests.

Acknowledgements

This work was supported by a grant from the Science Fund for Central guided Local Science and Technology Innovation Program (ZY04JD03-4 and ZY04JD03-3) and a China Agriculture Research System grant (CARS-40-K18).

Footnotes

The appropriate scientific section for the paper is Immunology, Health and Disease.

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