INTRODUCTION
Avian influenza (AI) is a virus of the genus influenzavirus A in the
Orthomyxoviridae family of viruses [
1]. Based on genetics and disease severity, AI viruses are classified either as highly pathogenic avian influenza (HPAI) or lowly pathogenic AI [
2]. In particular, a high number of deaths were observed in birds caused by HPAI [
3]. Moreover, the influenza A virus subtype H5N1, type of HPAI virus (HPAIV), is a threat to the poultry industry and the economy and remains a potential source of pandemic infection in humans [
4].
Mx dynamin like GTPase (Mx) proteins are members of the dynamin family of large GTPases. They prevent viral RNA replication by inhibiting the trafficking or activity of viral polymerases [
5]. Several previous reports showed that only the asparagine (Asn-AAT) polymorphism at the 631st position triggered antiviral activity, while Mx proteins carrying a serine (Ser-AGT) at that position did not suppress viral growth [
6]. In addition, the major histocompatibility complex (MHC) haplotype can affect the antiviral activity of the host [
7]. Previous research has shown a significant association of the major histocompatibility complex class I antigen BF2 (BF2) haplotype in chicken MHC class I with resistance or susceptibility to a number of pathogens, including Marek virus [
8], and AI virus [
9]. Furthermore, chickens with the
BF2-B13 haplotype had higher mortality than those that have the
BF2-B21 haplotype [
9].
One of the key determinants of the severity and outcome of AI virus infection is the regulation of the host innate immune response [
10]. Cytokines and chemokines play crucial roles in the balance of the immune system. Previous studies in various animals have shown that tissue damage and host death are caused by cytokine and chemokine dysregulation [
11]. In chickens, HPAIV H5N1 induced the excessive expression of cytokines and chemokines in lung tissues [
12]. Furthermore, cytokine-cytokine receptor interactions were significantly increased in Fayoumi and Leghorn chicken lines after infection with HPAIV [
13]. The regulation of cytokines and chemokines has been shown to be important in host defense against HPAIV.
In this study, we used Ri chickens, a local chicken breed in Vietnam, as an experimental animal [
14]. Chickens resistant or susceptible to HPAIV were distinguished by genotyping their
Mx and
BF2 genes. We infected Ri chickens with HPAIV H5N1 and analyzed gene expression patterns in the lung tissue using high-throughput RNA sequencing. In particular, we analyzed the expression of genes related to cytokine-cytokine receptor interactions between resistant and susceptible Ri chickens.
DISCUSSION
In this study, we analyzed the transcriptome profiles of Ri chickens infected with HPAIV H5N1 using RNA sequencing. HPAIV-resistant and HPAIV-susceptible Ri chicken lines were selected based on their Mx and BF2 genotypes and were infected with HPAIV H5N1. RNA sequencing was conducted after infection and 972 DEGs were identified after comparing the transcriptome profiles of lung tissue obtained between resistant and susceptible chickens. KEGG analysis revealed that most of the DEGs were related to cytokine-cytokine receptor interactions.
Avian influenza viral pathogen-associated molecular patterns are recognized by host pattern recognition receptors (PRRs). Viral double-stranded RNA (dsRNA) and cytosine-guanosine oligodeoxynucleotides, which form during the replication of AIV, are recognized by toll-like receptor 3 (TLR3) [
17] and TLR21 [
18], respectively, through adaptor proteins such as TIR-domain-containing adapter-inducing interferon and myeloid differentiation primary response 88 (MyD88), respectively [
19]. These adaptors activate the transcription factor interferon regulatory factor 7 (IRF7) and nuclear factor kappa B (NF-κB) to induce cytokines, chemokines, and IFN-stimulated genes [
20]. High expression levels of PRRs, MyD88, IRF7, and NF-κB were previously observed in H5N1-infected chickens [
12]. Our results showed that the expression levels of TLR3, TLR21, IRF7, and MyD88 were higher in resistant chickens compared to susceptible chickens (
Supplementary Table S1); therefore, we suggest that resistant Ri chickens respond more sensitively to HPAIV H5N1 infection than susceptible Ri chicken lines through increased expression of TLR3, TLR21, IRF7, and MyD88.
Pro-inflammatory cytokines cause inflammation and recruit other immune cells to the site of infection [
21]. High expression levels of pro-inflammatory cytokines and chemokines have been found in humans and duck infected with H5N1 [
10,
11]. Similarly, high expression levels of pro-inflammatory cytokines and chemokines, including IL-1β, IL-6, IL-8, IL-18, CCL4, CCL17, and IFN-γ, were reported in chickens infected with H5N1 [
12]. A significant increase in the levels of cytokines and chemokines exacerbates the inflammatory response, leading to apoptosis, multi-organ failure, and host death [
22]. However, HPAIV infection was lethal in mice lacking tumor necrosis factor and IL-1 receptors and inhibition of the cytokine response does not protect mice against H5N1 influenza infection [
23]. In this study, expression levels of pro-inflammatory cytokines and chemokines such as IL-1β, IL-6, IL-8, IL-18, CCL4, CCL17, IFN-γ, and receptors were upregulated in resistant chickens, compared to susceptible chickens, after HPAIV H5N1 infection (
Figure 6). However, the induction of cytokines and chemokines did not cause Ri chickens to die during experimentation. Therefore, we suggest that cytokine and chemokine induction is necessary to protect the host against HPAIV infection.
Type I interferons (IFN-α and IFN-β) trigger the expression of IFN-stimulated genes. IFNs and IFN-stimulated genes can inhibit viral replication by blocking virus entry into the host cells, binding to viral RNA to stop translation, and regulating host antiviral responses [
24]. IFN-γ directly or indirectly inhibits viral replication by strictly regulating the production of nitric oxide or interfering with the onset of the RNase L pathway [
25]. Moreover, several studies have shown that IFN-stimulated genes have antiviral activity [
26,
27]. The
Mx gene inhibits the trafficking and activity of viral polymerases [
5]. Viperin (RSAD2) inhibits newly synthesized influenza virions [
28]. The CCL19, CCL21, and CCR7 chemokine axes are homeostatic [
29]. Chicken interferon-inducible 2′-5′-oligoadenylate synthase-like (OASL) and RNase L restrict both viral and cellular RNA, preventing viral genome replication [
30]. Wild-type duck OASL inhibits the replication of a variety of RNA viruses
in vitro, including the influenza virus [
25]. Protein kinase R (EIF2AK2) inhibits the translation of viral mRNAs, including those from influenza A viruses [
26]. Interferon-induced proteins of the tetratricopeptide repeats (IFIT) protein family sequester viral nucleic acids [
31]. Furthermore, the clinical results were ameliorated in chIFIT5-transgenic chickens after treatment with HPAIV and Newcastle disease virus [
27]. As our results showed, although IFN-α and IFN-β were not found in the DEGs, we observed a higher expression of IFN-γ and IFN-stimulated genes (
Mx,
CCL19,
OASL,
RSAD2,
EIF2AK2, and
IFIT5) in the resistant Ri chickens compared to the susceptible ones (
Supplementary Table S1). Therefore, we suggest that the antiviral response of resistant chickens is higher than that of susceptible chickens.
In summary, by using RNA sequencing and qRT-PCR, we evaluated the differential expression of genes associated with cytokine-cytokine receptor interactions from the lung tissue of resistant and susceptible H5N1-infected Ri chickens. Interestingly, the expression of PRRs, cytokines, chemokines, and IFN-stimulated genes in resistant chickens were higher than those in susceptible chickens. These results suggest that resistant Ri chickens show a higher antiviral activity compared to susceptible Ri chickens, and this antiviral activity may be related to the expression of antiviral genes. Therefore, we propose that more studies on Ri chickens be done to compare their resistance and susceptibility to HPAIV infections. With this, there will be more studies to support advancing the development of chicken lines with disease resistance genes.