Infectious bursal disease virus’ interferences with host immune cells: what do we know?

ABSTRACT

Infectious bursal disease virus (IBDV) induces one of the most important immunosuppressive diseases in chickens leading to high economic losses due to increased mortality and condemnation rates, secondary infections and the need for antibiotic treatment. Over 400 publications have been listed on PubMed.gov in the last 5 years pointing out the research interest in this disease and the development of improved preventive measures. While B cells are the main target cells of the virus, other immune and non-immune cell populations are also affected, leading to a multifaceted impact on the normally well-orchestrated immune system in IBDV-infected birds. Recent studies clearly revealed the contribution of innate immune cells as well as T cells to a cytokine storm and subsequent death of affected birds in the acute phase of the disease. Transcriptomics identified differential regulation of immune-related genes between different chicken genotypes as well as virus strains, which may be associated with a variable disease outcome. The recent availability of primary B cell culture systems allowed a closer look into virus-host interactions during IBDV infection. The new emerging field of research with transgenic chickens will also open up new opportunities to understand the impact of IBDV on the host under in vivo conditions, which will help to understand the complex virus-host interactions further.

KEYWORDS:

• Infectious bursal disease
• immunosuppression
• innate immunity
• recovery
• T lymphocytes
• B lymphocytes
• receptors
• viral proteins

Introduction

The infectious bursal disease virus was first described in 1962 in Gumboro, Delaware, USA (Cosgrove, 1962; Eterradossi & Saif, 2020) and belongs to the family of Birnaviridae (Dobos et al., 1979). Today, different IBDV strains, antigenic variants and reassortants as well as recombinant viruses can be detected in chicken flocks almost all over the world (reviewed in Brown Jordan et al. (2018); Islam et al. (2021); Deorao et al., 2021; Feng et al., 2021). Recently, a new unified nomenclature was proposed allowing the classification of isolates based on both genome segments, specifically the regions encoding the viral proteins (VP) 2 and VP1 (Michel & Jackwood, 2017; Jackwood et al., 2018; Islam et al., 2021). While the sequence of VP1 is encoded in one open reading frame on segment B of the bisegmented double-stranded RNA (Azad et al., 1985; Morgan et al., 1988), segment A consists of two partially overlapping open reading frames (Müller, Scholtissek, et al., 1979). The smaller ORF encodes VP5 (Mundt et al., 1995), and the larger ORF encodes the polyprotein precursor (p) VP2-VP4-VP3 (Azad et al., 1985; Hudson et al., 1986). Therefore, eight genogroups based on segment A within serotype 1, and one genogroup of serotype 2 have now been confirmed. Based on segment B, five genogroups were identified. Due to currently known possible combinations of the two segments, a total of 15 genogroups with different lineages were determined (Islam et al., 2021). Recently, additional new genogroups were suggested based on novel VP2 sequences identified in Europe and Asia (Wang et al., 2021c; Legnardi et al., 2022). Ongoing monitoring is required to identify newly emerging IBDV strains and variants in the field as continuous selection pressure will lead to immune escape strains and viruses with functional advantages for segment B (Pikula et al., 2021).

Virulence is not always clearly associated with a genogroup (Lu et al., 2015; Jackwood et al., 2018; Islam et al., 2021). Live vaccine virus strains show different degrees of attenuation from mild to intermediate plus. In many countries, very virulent strains circulate (Tammiranta et al., 2018; Ekiri et al., 2021; Jiang et al., 2021), which may lead to high morbidity and mortality rates. Antigenic variants can also be detected and lead to subclinical infections associated with strong immunosuppression (Myint et al., 2021; Thai et al., 2021).

The overall goal in the field is to protect chickens against IBDV through prophylactic strategies, including biosecurity as well as vaccination of parent flocks and progeny (Müller et al., 2012). Current efforts are directed towards the development of more efficient vaccines, which match the circulating field viruses as the emergence of new variants may limit the efficacy of current vaccination approaches (Yang & Ye, 2020; Qiao et al., 2021; Yang et al., 2021). In addition, live attenuated vaccines may induce transient immunosuppression making the birds vulnerable to other invading pathogens (Prandini et al., 2016; Thomrongsuwannakij et al., 2021), and therefore new generation vaccines may provide suitable alternatives (Hein et al., 2021; Qiao et al., 2021). To improve prophylactic measures, the understanding of the immunosuppressive mechanisms of the virus and possible host-associated factors influencing the outcome of infectious bursal disease (IBD) is an important requirement. In this article, we will review the current knowledge of the interaction of IBDV with the immune system of the host leading to different degrees of immunosuppression in affected birds.

Virus proteins and their interaction with the host

IBDV is a non-enveloped virus. The icosahedral capsid is single-layered and its diameter is from 50 to 70 nm, differing by virus strain. The single layer of the capsid consists of the two main structural proteins VP2 and VP3 (Dobos et al., 1979; Jackwood et al., 1984). They are processed by VP4, the viral serine-lysine protease (Birghan et al., 2000; Lejal et al., 2000). VP4 cleaves the polyprotein pVP2-VP4-VP3 into single proteins (reviewed in Qin & Zheng, 2017). Additionally, VP4 may suppress the host’s antiviral immune response by interacting with glucocorticoid-induced leucine zipper in a strain-dependent manner (reviewed in Dulwich et al., 2020; Table 1).

Table 1. Overview of the functions of IBDV viral proteins (VP) and their interaction with host factors.

Viral protein (VP)	Function	Interaction with the host		References
		Induction of	Protection from
VP1	Viral RNA-dependent RNA polymerase			Morgan et al. (1988) van den Berg (2000) Felice et al. (2017)
VP2	Outer layer of the capsid	Neutralizing antibodies Apoptosis through Reduction of ORAOV1 Detection by PKR		Böttcher et al. (1997) Fahey et al. (1989) Busnadiego et al. (2012) Qin & Zheng (2017)
VP3	Inner layer of the capsid Ensures viral replication and morphogenesis Builds RNPs with VP1 and dsRNA for replication Activates VP1	Target of TRIM25	Apoptosis Bound to VP2 Bound to IBDVs dsRNA Inhibition of IFN-ß production Autophagy by the PIK3C3-PDPK1	Böttcher et al. (1997) Maraver et al. (2003) Garriga et al. (2007) Luque et al. (2009) Busnadiego et al. (2012) Zhang et al. (2020) Deng et al. (2021) Wang, Yu, et al. (2021)
VP4	Viral serine-lysine protease Cuts Precursor polyprotein pVP2 and releases its endopeptidase		Innate immune response by Inhibition of K48-coupled ubiquitylation of GILZ	Birghan et al. (2000) Qin & Zheng (2017) He et al. (2018) Da Costa et al. (2002)
VP5	Non-structural protein	Late apoptosis for virus release by sequestering with VDAC2 and RACK1	Early apoptosis by activating PI3K/ Akt pathway	Mundt et al. (1995) Qin & Zheng (2017)

Notes: RNP = ribonucleoprotein, ORAOV1 = oral cancer overexpressed 1, PKR = double-stranded RNA-dependent protein kinase, VDAC = voltage-dependent anion channel, RACK1 = receptor of activated protein C kinase 1, TRIM = tripartite motif, IFN = interferon, PIK3C3-PDPK1 = phosphatidylinositol 3 – kinase catalytic subunit type 3 – phosphoinositide dependent protein kinase 1 – mammalian target or rapamycin pathway, GILZ = glucocorticoid-induced leucine zipper, PI3K = phosphatidylinositol 3-kinase.

pVP2 is further divided into VP2 and four small peptides (Da Costa et al., 2002; Irigoyen et al., 2009). These small peptides play different or unknown roles in virus replication and capsid assembly (Chevalier et al., 2005). The outer layer of the viral capsid is composed of VP2 trimers (Böttcher et al., 1997) and, therefore, VP2 is the main target for neutralizing antibodies (reviewed by Fahey et al., 1989). Different amino acid compositions at the variable region of VP2 lead to different antigenic characteristics and epitopes. These can be used to differentiate strains and genogroups (reviewed in Jackwood et al., 2018), and therefore need to be considered for the generation of vaccines (Huo et al., 2019; Li et al., 2020; Wang et al., 2020; Guo et al., 2021). Lazarus et al. (2008) speculated that the variable region for binding neutralizing antibodies and the regions associated with virulence are encoded at different locations within the VP2 sequences.

In in vitro studies, it was shown that VP2 induces apoptosis by the reduction of oral cancer overexpressed 1 (Qin et al., 2017; Qin & Zheng, 2017). VP2 may also be detected by host intracellular sentinels, which leads to apoptosis and virus release (reviewed by Busnadiego et al., 2012; Li et al., 2012). It is only detectable by intracellular host sentinels if it is not connected to VP3 (Busnadiego et al., 2012).

VP3 forms the inner surface of the viral capsid (Böttcher et al., 1997). Additionally, VP3 interacts with VP1 for morphogenesis and viral replication (reviewed in Maraver et al., 2003, Table 1). VP3 binds not only to VP2 but also to single-stranded and double-stranded (ds) RNA as well as single-stranded DNA (Kochan et al., 2003). Therefore, it may not only interfere with VP2- but also double-stranded RNA-dependent protein kinase (PKR)-induced apoptosis, the latter being activated through dsRNA (reviewed in Cubas-Gaona et al. (2018)). In cell culture, VP3 also inhibits autophagy (Zhang et al., 2020) and protects viral replication by inhibition of melanoma differentiation-associated gene 5-dependent interferon (IFN)-ß production through binding to the deSUMOlyated apoptosis inhibitor 5 (Deng et al., 2021).

VP1 represents the RNA-dependent RNA polymerase of IBDV (Müller, Scholtissek, et al., 1979; Morgan et al., 1988). It is not only important for mRNA synthesis and viral replication (Felice et al., 2017) but also for virus encapsidation (reviewed in van den Berg, 2000). Recent studies demonstrated that VP1 also contributes to the virulence of an IBDV strain and, therefore, may contribute to the genetic shift of the virus and the evolution of new variants with variable virulence (Deorao et al., 2021).

VP5 is a non-structural protein of IBDV with structural similarities to host Toll-like receptor 3 (Mundt et al., 1995; Ganguly & Rastogi, 2018; Table 1). It accumulates at the plasma membrane of IBDV-infected cells (Mendez et al., 2015). By binding to the p85α subunit of phosphatidylinositol 3-kinase, followed by activation of NF-κB and subsequent inhibition of the caspase cascade, VP5 mediates the inhibition of early apoptosis (Wei et al., 2011). In the early phase of virus infection, this step is important to ensure replication (Liu & Vakharia, 2006). On the other hand, VP5 can also induce apoptosis at later stages of replication to allow virus release. It is suggested that VP5 binds to the voltage-dependent anion channel 2 and receptor of activated protein C kinase 1 in the mitochondria in host cells, which leads to the activation of the caspase-activated apoptosis (Liu & Vakharia, 2006; Li et al., 2012; Lin et al., 2015). Comparing recombinant viruses expressing or not expressing the VP5 polypeptide revealed that this virus protein is required for non-lytic release of the virus from infected cells, which additionally enhances the dissemination speed between cells (Mendez et al., 2017). Therefore, VP5 can be considered a virulence factor, supported by the fact, that VP5-knockouts show strong attenuation of the virus (Yao et al., 1998).

IBDV replication

Various virus receptors and receptor complexes were suggested to be involved in the recognition and attachment of IBDV (Table 2). Most of these tentative receptors were identified in vitro with only a few in in vivo studies (Nieper & Muller, 1996; Ogawa et al., 1998; Lin et al., 2007; Delgui et al., 2009; Luo et al., 2010; Liu et al., 2020, 2022). It was speculated that different virus strains might use different receptor complex compositions for attachment. How IBDV enters host cells is still not fully clear; while in vitro investigations suggested endocytosis (Yip et al., 2012), more recent studies provided evidence for macropinocytosis (Gimenez et al., 2015; Ye et al., 2017).

Table 2. Host receptors for binding IBDV.

Receptors	Receptor identification in IBDV-infection				Reference
	In vitro	In vivo
		BF	Thymus	Spleen
CD44*	DT40 (A) 293T (M) DF-1 (A)	X	X		Liu et al. (2022)
CD74**	DT40 (A) 293T (M) HeLa (M)	X			Liu et al. (2020)
λ light chain of IgM	DT40 (A)				Luo et al. (2010)
α4β1 integrin	BALB/c 3T3 (M)				Delgui et al. (2009)
cHsp90	DF-1 (A)				Lin et al. (2007)
N-glycosylated protein	LSCC-BK3 (A)				Ogawa et al. (1998)
40 kDa and 46 kDa protein	CEF (A)	X	X	X	Nieper & Muller (1996)

* Widely spread transmembrane glycoprotein on lymphocytes and non-lymphocytes.

** Invariant chain of major histocompatibility complex class II.

(A) = avian derived cell line; (M) = mammalian derived cell line; X = detected.

IBDV needs to enter endosomes to release ribonucleoprotein (RNP) complexes (reviewed in Gimenez et al., 2021) consisting of the IBDV dsRNA, VP1 and VP3. In cell culture, the RNP is stabilized by voltage-dependent anion channel 1 (Han et al., 2017), protecting the complex from host sentinel proteins (Valli et al., 2012; Gimenez et al., 2021). But RNPs can also be recognized by tripartite motif 25, which is involved in a host antiviral feedback mechanism. Tripartite motif 25 targets Lys854 of VP3 and leads to ubiquitination and degradation (Wang, Yu, et al., 2021).

VP3 can bind to endosomal phosphatidylinositol-3-phophate on early endosomal membranes (Gimenez et al., 2015, 2021). This step is probably needed for the RNPs to travel and bind to the Golgi complex allowing RNPs to enter (Gimenez et al., 2022). In addition, VP3 induces a conformational change in VP1 to take off the steric blockade of the active part. Subsequently, VP1, the viral RNA polymerase, starts the replication (Garriga et al., 2007). Chicken eukaryotic translation elongation factor 1α may support VP1 polymerase activity of very virulent IBDV strains (Yang et al., 2020). Virus replication is promoted through the function of the Rab1b – Golgi-specific BFA resistance factor 1 – ADP ribosylation factor 1 axis. While the Golgi complex is rearranged, its secretory functionality is not affected (Gimenez et al., 2022). During replication and transcription, chicken heat shock protein 70 may interact with the dsRNA of IBDV (Meng et al., 2021). The viral assembly also takes place in the Golgi complex (Delgui et al., 2013; Gimenez et al., 2021). IBDV is subsequently released by the induction of apoptosis as described above (reviewed in Qin & Zheng, 2017; Li & Zheng, 2020).

Pathogenesis and impact of the host genotype

After oral infection, IBDV can be detected in the gut, especially in the caecum, and at 4 hours post-infection (hpi), the virus appears in the caecal tonsils. IBDV may infect macrophages and lymphocytes of the gut-associated lymphoid tissue (GALT) along the whole intestine without damaging the enterocytes. IBDV-positive macrophages and lymphocytes enter the bloodstream and reach the liver via the portal vein by 5 hpi (reviewed in Eterradossi & Saif, 2020). Kupffer cells may contribute to the control of the virus in the liver (Müller, Käufer, et al., 1979). If virus-positive cells enter the bloodstream, they are spread to other lymphoid tissues including the main target organ, the bursa of Fabricius (BF) by 11 hpi (reviewed in Eterradossi & Saif, 2020). The pathogenesis might slightly vary if the route of infection is changed, as after intranasal infection virus-positive cells may initially also be found in the follicle-associated epithelium and nasal-associated lymphoid tissue of the nasal cavity before virus spread to lymphoid tissues through the blood or intestinal route (Shah et al., 2021).

IgM-bearing B lymphocytes are the main target cells of IBDV, and massive virus replication leads to the destruction of these cells. A second viraemia may occur (Müller, Käufer, et al., 1979), and further lymphoid organs are affected, including the thymus and the spleen (Müller, Käufer, et al., 1979; Tanimura et al., 1995; Vervelde & Davison, 1997; Rautenschlein et al., 2007). Viral particles are also found in other cell types, such as in epithelial reticular cells in the thymus and in monocytes in bone marrow, but there is probably no replication in these cells (Tanimura et al., 1995). Recent studies demonstrated changes in the expression pattern of various antiviral factors, and cytokines associated with macrophage and T-cell activity early after IBDV-infection (Chen et al., 2022). Also, intestinal villi structure and mast cell activity, as well as mucous production, may be modified (Wang, Liu, et al., 2009; Li et al., 2018). Subsequently, IBDV-infection associates with changes in the gut microbiota composition (Li et al., 2018). Depending on the virulence of the virus, recovery will start at 7–10 days post-infection (dpi).

There are differences in the severity of IBD between different chicken breeds and lines (Dobner et al., 2020), which may be associated with variable immune reactions. IBDV-infected layer-type chickens often show more severe clinical signs than meat-type chickens (Nielsen et al., 1998; Aricibasi et al., 2010; Silva et al., 2016), possibly due to a stronger systemic innate immune response leading to a “cytokine storm” and death. Meat-type birds showed a higher magnitude of T cell reactions in the BF, which may correlate with better control of viral replication and faster recovery (Aricibasi et al., 2010). The association of a fast and vigorous innate immune response in some genotypes with clinical disease and bursa lesions was also confirmed by transcriptome analysis (Farhanah et al., 2018; Mohd Isa et al., 2020; Asfor et al., 2021). In the BF of more susceptible chicken lines, Asfor et al. (2021) demonstrated an up-regulation of pathways involved in inflammation, cytoskeletal regulation by Rho GTPases, nicotinic acetylcholine receptor signalling, and Wnt signalling compared to other genotypes less susceptible to clinical disease.

The data on the impact of the MHC haplotype on IBDV pathogenesis are controversial (Hudson et al., 2002). There seems to be an association between the MHC haplotype and the quality of the anti-IBDV immune response (Hudson et al., 2002), which was mainly shown in vaccination experiments (Juul-Madsen et al., 2006; Butter et al., 2013).

B cells in IBDV immunopathogenesis and immunosuppression

The most susceptible age for IBD is between 3 and 6 weeks of age, when the main target organ, the BF, reaches its peak of development, and B cell differentiation has advanced (Zhao et al., 2016, Figure 1). The non-specific clinical signs during the acute phase of IBD include distress, ruffled feathers, anorexia and depression, possibly associated with diarrhoea and dehydration (reviewed in Ingrao et al., 2013). The extent of clinical signs may vary between infecting strains; the more virulent the strains, the higher the risk for the development of clinical disease and death (reviewed in Eterradossi & Saif, 2020; Lupini et al., 2020; Wang, Huang, et al., 2021). The acute phase of the clinical disease is accompanied by a macroscopically visible bursal oedema followed by bursa atrophy due to the destruction of the bursal follicles (reviewed in Eterradossi & Saif, 2020). Haemorrhages may occur and are speculated to be due to the cytokine storm (reviewed in Ingrao et al., 2013). Changes in the B cell genomic methylation and loss of genome integrity during the infection process may contribute to B cell death (Ciccone et al., 2017). Further lesions may occur, such as mottling of the spleen, and are described elsewhere in more detail (Mahgoub et al., 2012; Eterradossi & Saif, 2020). Older birds, or birds infected with less virulent strains, may show a fast, microscopically but not macroscopically full recovery of the bursa within only a few weeks (Kim et al., 1999; Rautenschlein et al., 2003; Felföldi et al., 2021).

Figure 1. Schematic overview of key immunological events in the bursa of Fabricius within the context of IBDV infection up to the time of beginning recovery. Created with BioRender.com

The loss of developing B lymphocytes associates with immunosuppression, which leads to an enhanced susceptibility for secondary infections and a poor vaccine response (reviewed in Naqi et al., 2001; Hashemzade et al., 2019; Eterradossi & Saif, 2020). If surviving birds are infected very early in life, they may suffer lifelong immunosuppression due to incomplete recovery of the B cell compartment (reviewed in Kim et al., 1999; Ingrao et al., 2013). Interestingly, the humoral immune response against IBDV is not affected, and birds develop high titres of circulating anti-IBDV antibodies. They may already be detected between 5 and 7 dpi and may contribute to viral clearance (Aricibasi et al., 2010) via the various antibody-mediated mechanisms, including opsonization, antibody-dependent cell-mediated or complement-dependent cytotoxicity.

Felföldi et al. (2021) suggested that the secretory glycoproteins released by the secretory dendritic cells (BSDC), which are located in the medulla of the bursa follicles, may allow first IBDV binding to the BSDCs, and later also to B cells. The infected BSDCs may transform into macrophage-like cells, which migrate into the cortex, where inflammation is induced. Therefore, IBDV infection inhibits the glycoprotein release, which leads to B cell apoptosis (Oláh et al., 2022). Bursal epithelial cells, especially basal cells, accumulate large numbers of IBDV particles (Shah et al., 2021). Depending on the virulence of the IBDV strain, cystic cavities develop in the follicular medulla, and fibroplasia is observed microscopically. In the bursal recovery phase, B cells repopulate the bursal follicles (Vervelde & Davison, 1997; Kim et al., 1999; Withers et al., 2005, Figure 1). This recovery process may depend on the number of remaining BSDC, as IBDV-mediated exhaustion of its precursors prevents the establishment of the necessary microenviroment for B cell maturation and/or survival (Oláh et al., 2022). Recent studies suggested the involvement of C-X-C motif chemokine (CXC) ligand 12 and CXC ligand 13, which are highly expressed in the bursa of IBDV-infected chickens, in the recruitment of immature B cells into the bursa mesenchyme. CXC receptor 5 /CXC receptor 13 interaction may subsequently induce the B cell migration into the follicles (Shah et al., 2021). Withers et al. (2005) described two types of recovering bursal follicles: large fully rebuilt follicles and small underdeveloped follicles, which lack the normal anatomic architecture. Chickens with mostly underdeveloped follicles mounted a lower antibody response to secondary pathogens than chickens with mostly recovered follicles. Nevertheless, both types produced lower antibody levels than IBDV uninfected birds (Vervelde & Davison, 1997; Withers et al., 2005). In contrast, Kim et al. (1999) showed that chickens infected at 1-week post-hatch, returned to an almost normal humoral immune response between 7 and 12 weeks post-infection, depending on the virulence of the infecting strain. Inflammatory foci were observed in the recovering bursa with high numbers of T cells, a few B lymphocytes and γδT cells, and a low number of diffusely distributed macrophages as well as aggregates of CD40+ cells in the foci centre (Withers et al., 2005). The recovery process was shortened if fibrotic lesions were less pronounced and the expression of transforming growth factor β and the matrix metalloproteinase 9 was reduced (Yu, Xu, et al., 2021).

IgA+ B cells may increase while IgM+ B cell populations decrease in the bursa follicles (Shah et al., 2021), suggesting an acceleration of IgA secretion in the bursa of IBDV-infected birds. A recently established primary B-cell model allowed elucidation of the gene expression profiles in IBDV-infected B cells (Dulwich et al., 2017). It was demonstrated that both an attenuated strain and a very virulent strain down-regulated the expression of key genes in B-cell activation and signalling, including tumour necrosis factor superfamily member 13B, CD72 and Grb2-related adaptor protein. On the other hand, the extent of upregulation of antiviral type I IFNs and IFN-stimulated genes in these IBDV-infected B cell cultures was associated with viral virulence (Dulwich et al., 2017). Pretreatment of cell cultures with type I IFN induced downregulation of the dsRNA-dependent protein kinase, which may suppress subsequent IBDV infection. The addition of IFN to infected cultures exacerbated apoptosis, suggesting a significant role of the IBDV genomic dsRNA in the apoptotic response and pathogenesis (Cubas-Gaona et al., 2018). Other studies confirmed that the type I IFN pathway is not a sufficient antiviral defence mechanism in IBDV-infected chickens to control virus infection (Shah et al., 2021).

Depending on the virulence of the infecting strain, B cells in other lymphoid tissues may also be affected, contributing to the suppression of the humoral branch of the chicken immune system. IBDV infection may also account for a transient suppression of B lymphocytes in the spleen, blood (Shah et al., 2021) and the GALT within the caecal tonsils. Interestingly, the B cells at these locations may recover faster than in the BF (Li et al., 2018).

T cells in IBDV-immunopathogenesis and immunosuppression

T lymphocytes are commonly considered not to be susceptible to virus infection but play a significant role in the immunopathogenesis. Mahgoub et al. (2012) also demonstrated viral antigen closely associated with CD8αα+ TCR2+ T cells. Chemokine (C–C motif) ligand 19, which is involved in immunoregulatory and inflammatory processes, is upregulated in the BF of IBDV-infected birds and attracts T cells from blood and lymph vessels to infiltrate into the BF (Tanimura & Sharma, 1997; Wang et al., 2019, Figure 1) and caecal tonsils (Mahgoub et al., 2012). The majority of infiltrating T cells are TCR2+ (Tanimura & Sharma, 1997). CD4+ as well as CD8+ T cells may infiltrate the BF starting from 1-4 dpi, depending on the replication pattern of the virus (Kim et al., 2000; Withers et al., 2005; Aricibasi et al., 2010), while reports vary about the quantity. Some studies observed more CD8+ (Tanimura & Sharma, 1997; Dobner et al., 2020) than CD4+ T cells and others more CD4+ T cells in the BF (Ruan et al., 2020). This is influenced by the bird’s genotype as well as the IBDV strain (Tanimura & Sharma, 1997; Aricibasi et al., 2010; Tippenhauer et al., 2013). T cells express IFN-γ (Tanimura & Sharma, 1997; Rauw et al., 2007) and activate macrophages. Macrophages are also stimulated by the p38 mitogen-activated protein kinases or NF-κB pathway, which subsequently leads to a cytokine storm (Khatri & Sharma, 2006).

In the acute phase of IBD, T cells control viral replication. This is possibly mediated through the release of proinflammatory cytokines, e.g. interleukin (IL)-2 and IFN-γ, which induce apoptosis and other cell-death pathways. In addition, cytotoxic T cells induce lysis of infected cells and uninfected bystander cells (Kim et al., 2000; Rautenschlein et al., 2002). The CD8+ cells putatively use the Fas-FasLigand and/ or perforin-granzyme A cytolytic pathway to destroy cells (Rauf et al., 2011, 2012).

It was speculated that some resident bursal CD4+ T cells might stimulate B cells to produce antibodies against IBDV before the infection of the B lymphocyte occurs, but the main antibody production was suggested to take place beyond the BF (Tanimura & Sharma, 1997). After this rapid infiltration of T cells, numbers decline again. T cells may interfere with tissue recovery in the chronic phase (Kim et al., 2000; Rautenschlein et al., 2002). Dobner et al. (2020) demonstrated that meat-type birds showed a faster bursal recovery with an earlier decrease in IL-10 mRNA expression and reduction in the number of T cells in the BF than layer-type birds (Dobner et al., 2020). Layer-type chickens had overall higher levels of IL-10 and CD4+ T cells, which could be regulatory T cells, than the broilers. On the other hand, the meat-type birds had higher numbers of CD8+ T cells, which may be involved in an earlier viral clearance and, thereafter, earlier recovery (Dobner et al., 2020). Genotype influences were also confirmed by Mohd Isa et al. (2020), who demonstrated variable regulation of T-cell immunity associated genes between birds developing more severe compared to birds with less bursa lesions. The expression levels of, for example, CD86 and CTLA4 as well as Th1-associated cytokines varied, and, most strikingly, the expression of IL12B was observed only in the more susceptible line, compared to the expression of IL15R, which was only detected in the more resistant birds (Mohd Isa et al., 2020). More resistant birds also showed lower levels of B cell genomic modification after IBDV infection. This reduced the numbers of infiltrating T cells and reduced expression levels of CD40L and FasL. Therefore, less immunopathology is observed in more resistant birds compared to more susceptible birds (Ciccone et al., 2017). Intrabursal T cells of IBDV-infected birds were demonstrated to inhibit the mitogenic response of splenocytes, also indicating immunomodulatory activities of this cell population (Kim & Sharma, 2000).

Also, the GALT undergoes changes in the T cell compartment after IBDV infection. Recent in vivo studies demonstrated a decrease of CD4+ cells in the caecal tonsils of infected birds, while CD8+ T cells were not affected. Interestingly, the T cell subsets were differentially modulated in different lymphoid tissues. While in the caecal tonsils IFN-β and CTLA-4 mRNA expression was upregulated, in the BF the changes in the IFN-γ, IL-10 and T cell checkpoint receptor LAG-3 mRNA expression were more prominent (Ruan et al., 2020).

Innate immune reactions contributing to IBDV immunopathogenesis and immunosuppression

The interactions between the host’s innate immune system and invading IBDV are complex and not fully elucidated yet. The differential activation of pattern recognition receptors was suggested to initiate the expression of antiviral and inflammatory cytokines in various cells of the innate immune systems but also other host cells (Yu et al., 2019). Heterophils are the first line of defence, and they are detected already within hours after virus inoculation but wane beyond the acute phase of the disease. In vitro studies demonstrated alteration of their functions after IBDV infection, which contributes to the overall immunosuppression due to the loss of phagocytic activity (Lam, 1998).

Macrophages were identified as possible target cells for IBDV (Khatri & Sharma, 2007; Lee et al., 2015). Their numbers change, therefore, not only in the bursa of Fabricius but also in the spleen during the infection (Palmquist et al 2006; Aricibasi et al., 2010). Some authors described a transient increase in the relative proportion of these cells in the BF, which may later on also be due to the loss of B cells (Khatri et al., 2005; Aricibasi et al. 2010). Virus and virus particles are detectable in the cytoplasm of macrophages (reviewed in Khatri & Sharma, 2007). Activated macrophages release proinflammatory cytokines, e.g. IL-6, inducible nitric oxide synthase, IL-1ß and tumour necrosis factor α (Khatri et al., 2005; Eldaghayes et al., 2006; Rautenschlein et al., 2007, Figure 1) to destroy virus-infected cells by apoptosis, cytotoxicity and phagocytosis. This strong inflammatory response may contribute to the cytokine storm.

There is limited and controversial information on the impact of natural killer (NK) cells on IBDV infection (Sharma & Lee, 1983). Infection with an IBDV vaccine or a very virulent strain of in vitro cultures of intraepithelial lymphocyte NK cells led to either up- or down-regulation of the expression of various surface antigens as well as NK-lysin, respectively (Jahromi et al., 2018). A transcriptome analysis indicated that as early as 3 dpi, genes were differentially regulated in the intraepithelial lymphocyte NK cells of the duodenum of IBDV-inoculated compared to virus-free birds. Some genes were associated with the recruitment of NK cells to the infected area and some with the downregulation of the antiviral activity of NK cells, suggesting a contribution to the compromised local immunity in the gut (Boo et al., 2020).

Anti-inflammatory compounds such as dexamethasone may protect IBDV-infected birds from severe clinical disease development emphasizing the role of inflammatory immune reaction in IBDV-immunosuppression (Shin et al., 2021).

Besides immune cell populations, other cell types may contribute to the innate immune response and/or immunopathogenesis of IBDV. Recently, melanocytes were identified to be susceptible to IBDV infection, and upregulated Toll-like receptor signalling pathways as well as antiviral factors were observed after in vitro infection (Han et al., 2021).

Interestingly, new studies also indicate that IBDV may interact with factors of the innate immune system to evade host immunity. The PTEN, a dual-specificity phosphatase, is involved in various survival strategies of the host (Worby & Dixon, 2014). During the early phase after IBDV infection, PTEN is downregulated, allowing the virus to replicate (Yu, Li, et al., 2021). Another innate host factor supporting virus replication is optineurin. IBDV infection induces endogenous optineurin expression, which subsequently suppresses the MDA5-mediated IFNβ production, allowing more efficient virus replication (Li et al., 2021). On the other hand, the pryin domain-containing 3 inflammasome activation was shown to interfere with IBDV replication in cell culture through the induction of pro-inflammatory cytokines and can therefore be suggested as an antiviral mechanism of the innate immune system (He et al., 2021).

Consequences of immunosuppression on intervention strategies

Not only IBDV may circulate in the field. Other pathogens, including Marek’s disease virus, chicken infectious anaemia virus and non-infectious factors including mycotoxins, may additionally challenge the immune system and exacerbate the IBDV-induced immunosuppression (reviewed by Hoerr, 2010; Gimeno & Schat, 2018). Depending on the combination of immunosuppressive factors, different branches of the immune system are compromised (Gimeno & Schat, 2018).

Just recently, it was shown that co-infection of variant IBDV with fowl adenoviruses promoted the pathogenicity of fowl adenoviruses and enhanced virus shedding (Xu et al., 2021). Similar observations were made with IBDV in combination with other pathogens infecting different host target tissues within the host (for example, Gallardo et al., 2012; Kulappu Arachchige et al., 2021). But the destruction of target cells and/or induction of the antiviral state may also affect co-infecting pathogens; these mechanisms are non-specific, and culminate in antigenic effects (Jackwood, 2011). IBDV-induced immunosuppression is therefore based on compromised systemic innate and acquired immune mechanisms as well as reduced local, specifically mucosal-associated immunity demonstrating the complexity of IBD (Wang, Zhou, et al., 2009; Gallardo et al., 2012; Li et al., 2018; Xu et al., 2021).

Therefore, vaccination programmes have to be carefully adjusted to the field situation. If IBDV-infected birds are immunocompromised, protection will be difficult to achieve.

Conclusions and outlook

The recent availability of transgenic chicken models will help to investigate research questions in the in vivo context, which will help to also advance our knowledge on IBD (Sid & Schusser, 2018). Further understanding of the immunopathogenic mechanisms of IBDV will help to improve current vaccination strategies in the field. Multivalent recombinant vaccines are increasing, conquering the market with different types of recombinant viruses (Qiao et al., 2021), of which some are suitable for the in ovo application route (Hein et al., 2021). Also, other alternative approaches to attenuated vaccine strains or inactivated full-antigen vaccines are under investigation, such as plant-based subunit vaccines (Marusic et al., 2021; Yang et al., 2021). This vaccine research has to be supported by the identification of new adjuvants to further direct and enhance the specific immunity (Ebrahimi et al., 2019; Sachan et al., 2019; Lu et al., 2020).

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by the German Research Foundation DFG [Deutsche Forschungsgemeinschaft] (RA 767/10-1) within the context of the Research Unit “ImmunoChick – Unravelling the avian immune response in the context of infection”.