Mechanisms of circovirus immunosuppression and pathogenesis with a focus on porcine circovirus 2: a review

Abstract

Certain pathogens, due to their adverse effects on the immune reaction, aggravate the course of concomitant heterologous infections. Here we summarize mechanisms by which circoviruses, including the most studied porcine circovirus 2, and other mammalian and avian circoviruses, trigger their own replication and confound the hosts’ immune response. At different stages of infection, from latent state to disease induction, these viruses markedly influence the cellular signaling pathways. Circoviruses have been found to interfere with interferon and proinflammatory cytokine producing and responsive pathways. Apoptotic processes, altered cellular transport and constraint of the mitotic phase all support the viral replication. The cytokine imbalance and lymphocyte depletion, thus the impaired immunity, favors invasion of super- or co-infecting agents, which in concert with circoviruses induce illnesses with increased severity. The information summarized in this review point out the diversity of host and viral factors involved in the mechanisms of disease progression during circovirus infections.

Keywords:

• Pig
• porcine
• circovirus
• PCV2
• immunosuppression
• pathogenesis
• review

1. Introduction

The formation of an antiviral state in host cells is the result of sequential steps of variable cascade mechanisms and interactions between molecular networks, which activate the innate immune response as a first step. A key functional component of the innate immunity is the initiation of cytokine expression. Interferons (IFNs) constitute a class of cytokines that are released in response to pathogens in humans and animals (Schneider et al. 2014; Rojas et al. 2021; Walker et al. 2021). IFNs mediate autocrine and paracrine signaling that ultimately contributes to expression of IFN stimulated genes (ISGs). ISGs encode proteins with direct and indirect antiviral effects that inhibit different steps of the viral cell cycle, facilitate antigen recognition and presentation, as well as secretion of distinct classes of cytokines, or stabilize elements of the signaling pathways. IFNs activate immune cells (NK cells, natural killer cells; macrophages) and components of the adaptive immunity (Schneider et al. 2014; Rojas et al. 2021). Another pivotal point is the stimulation and fine regulation of proinflammatory cytokine secretion to activate the adaptive immunity in which transcription factor nuclear factor κB (NF-κB)-mediated signaling has a central role (Driessler et al. 2004; Cao et al. 2006; Ablasser and Hur 2020; Rojas et al. 2021).

IFN molecules as well as the proinflammatory pathways act in concert and ensure feedback to each to restrict viral propagation in and between susceptible cells. To by-pass this fine-tuned network, viruses evolved countermeasures at variable points by targeting and interacting with host-origin molecules (Garcia-Sastre 2017; Rojas et al. 2021). From an immunological view, the general presentation of the virus-host interactions goes beyond the scope of this review. Here we attempt to summarize how porcine circovirus type 2 (PCV2), an immunosuppressive self-replicating DNA virus with minimal genome and coding capacity, disturbs cellular processes in order to diminish the innate antiviral responses of host cells and antagonize the cytokine signaling, and how these actions promote co-infective pathogens to multiply.

2. Antiviral pathways of DNA viruses

The viral nucleic acids and proteins are perceived by variable host pattern recognition receptors (PRRs) depending on the composition of the viral nucleic acid and on the phase of the viral life cycle (Ablasser and Hur 2020; Rojas et al. 2021; Walker et al. 2021). PRRs involve the cell membrane or endosome-associated Toll-like receptors (TLRs), the RIG-I-like receptors (e.g. RIG-I, retinoic acid-inducible gene-I receptor; MDA5, melanoma differentiation-associated gene 5 receptor) and the cytosolic GMP-AMP synthase (cGAS) (Li et al. 2013; Ablasser and Hur 2020; Eaglesham and Kranzusch 2020; Rojas et al. 2021; Walker et al. 2021).

Activation of RIG-I and MDA-5 molecules lead to interaction with the mitochondrial antiviral adaptor protein (MAVS), a protein located in the intracellular membranes of mitochondria, endoplasmatic reticulum (ER) and peroxisome (Ablasser and Hur 2020; Rojas et al. 2021). MAVS activates inhibitor of kappa-B kinase epsilon (IKKε) and TANK binding kinase 1 (TBK1), two enzymes that phosphorylate either interferon regulatory factor (IRF) 3 or IRF7. The homodimers of IRF3 and IRF7 proteins translocate to the nucleus and initiate transcription of Type I (IFN-α and IFN-β) and Type III (IFN-λ) IFNs (Ablasser and Hur 2020; Rojas et al. 2021). Type I and Type III IFNs stimulate pathways leading to an antiviral state of cells (Lee and Ashkar 2018; Rojas et al. 2021; Walker et al. 2021). Type II IFN (IFN-γ) production is predominantly triggered by Type I IFNs (Lee and Ashkar 2018). IFN-γ plays a key role in antigen presentation and macrophage activation, and as an immunomodulatory and proinflammatory protein it contributes to the activation of the adaptive immune system (Lee and Ashkar 2018; Rojas et al. 2021; Walker et al. 2021).

The expression of proinflammatory cytokines is mostly induced by MAVS via the NF-κB pathway (Driessler et al. 2004; Cao et al. 2006; Ablasser and Hur 2020; Rojas et al. 2021). The inactivated form of the most abundant mammalian NF-κB p65/p50 heterodimer is associated with the IκB (NF-κB inhibitor) inhibitory complex. Phosphorylation of IκB by IκB kinase (IKK), followed by polyubiquitination and degradation, activates NF-κB that acts as transcription factor and transactivate the promoter of the targeted genes through the p65 protein (Driessler et al. 2004; Cao et al. 2006).

The connection of viral DNA with cGAS triggers the catalytic activity of the enzyme. As a result, cGAS synthesizes cyclic GMP-AMP (cGAMP) that activates STING (stimulator of interferon genes) dimers within the ER (Ablasser and Hur 2020; Eaglesham and Kranzusch 2020; Rojas et al. 2021). Oligomerized STINGs translocate from the ER to the Golgi apparatus and recruit TBK1 for downstream signaling. At this point, the processes overlap with pathways connected to RIG-I and MDA5 (Ablasser and Hur 2020; Eaglesham and Kranzusch 2020; Rojas et al. 2021). via TBK1, STING stimulates the NF-κB pathway as well (Ablasser and Hur 2020; Eaglesham and Kranzusch 2020).

Type I IFNs induce the dimerization of IFN receptor 1 and 2 that elicit the IFN response. The receptor complex, and the associated tyrosine kinase (Tyk) 2 and Janus tyrosine kinase (Jak) 1 phosphorylate each other and signal transducer and activator of transcription (STAT) proteins (Rojas et al. 2021). In the canonical IFN response pathway the phosphorylated STAT1 and STAT2 form heterodimers and together with IRF9 constitute the transcription factor ISGF3. ISGF3 translocates to the nucleus, where it interacts with IFN-stimulated response element (ISRE) promoter sequences of ISGs (Lee and Ashkar 2018). After binding to its receptor, IFN-γ activates Jak1/Jak2, and controls the expression of further regulatory elements (i.e. transcription factors) by phosphorylated STAT1 homodimers (Schroder et al. 2004). Beside specific heterodimeric receptors, type III IFNs also uses the interleukin (IL)-10 receptor 2 to induce signal transduction via the JAK/STAT cascade (Stanifer et al. 2020). In addition to the classical ones, a number of distinct pathways are initiated by IFNs with contribution of variable types of STATs, IRFs and kinases (Schroder et al. 2004; Lee and Ashkar 2018; Stanifer et al. 2020).

3. Introduction to circoviruses

Circoviruses (Circovirus genus, Circoviridae family) are small, non-enveloped, icosahedral viruses comprising a circular ssDNA genome of ∼1600–2200 nt in length (Rosario et al. 2017; Feher et al. 2022). The ambisense genome contains two main open reading frames (ORFs) encoding one or more replication-associated proteins (Reps, ORF1) on the viral strand and capsid protein (Cap, ORF2) on the complementary, replicative intermediate DNA strand (Lv et al. 2014; 2015; Rosario et al. 2017). In the absence of virus-encoded DNA polymerase, the replication of circoviruses depends on the cell cycle and is linked to the nucleus; in susceptible mitotic cells, a large amount of nascent virus particles is produced (Tang et al. 2013; Saikumar and Das 2019). Circoviral proteins, including Cap, Rep, ORF3, ORF4 and ORF5 may support viral replication interacting with the viral DNA and regulating cellular proteins (Lv et al. 2014; 2015; Rosario et al. 2017).

A wide variety of circoviruses has been identified in vertebrates and insects, often without association with pathogenic changes or clinical signs (Rosario et al. 2017). However, some avian and mammalian circoviruses are considered of high veterinary importance, which comes from their capacity to induce marked immunosuppression. Beside the direct pathogenic role, the immunosuppressive effect together with co-infecting agents and secondary infections will determine the outcome of the infection caused by these circoviruses (Abadie et al. 2001; Schmidt et al. 2008; Saikumar and Das 2019; Kroeger et al. 2022).

Avian circoviruses, including beak and feather disease virus (BFDV), goose circovirus (GoCV), duck circovirus (DuCV), pigeon circovirus (PiCV), finch circovirus and canary circoviruses, may generate lethargy, weight loss, as well as beak deformation, feathering issues and gastrointestinal disorders (Ritchie et al. 1989; Soike et al. 1999; Todd et al. 2001; Shivaprasad et al. 2004; Raue et al. 2005; Todd et al. 2007; Guo et al. 2011; Circella et al. 2014; Feher et al. 2022). Microscopic lesions associated with avian circoviruses are lymphocyte and macrophage infiltration, atrophy and necrotic areas in the lymphoid and non-lymphoid tissues; however, subclinical infection without clinical signs and lesions may also occur (Abadie et al. 2001; Hong et al. 2018; Wang et al. 2022b). Avian circoviruses are widely distributed in wild and domestic fowl, and are regularly diagnosed together with other avian pathogens. This co-occurrence can worsen the course of infection and makes the etiological role of circoviruses in disease development difficult to determine (Kozdruń et al. 2012; Stenzel and Koncicki 2017; Hong et al. 2018; Kaszab et al. 2020; Sahindokuyucu et al. 2022).

At present, four species of porcine circoviruses (PCVs) are distinguished. PCV1 was discovered nearly 50 years ago in a porcine kidney cell culture, PK-15, and it is considered an apathogenic virus (Tischer et al. 1974; Iizuka et al. 1989). On the contrary, PCV2 induces diseases in their host species (Iizuka et al. 1989; Nayar et al. 1997; Saikumar and Das 2019). PCV2 infection remains subclinical, or manifests in variable forms commonly referred to as PCV-associated diseases (PCVAD). PCVAD involves post-weaning multisystemic wasting syndrome (PMWS), porcine respiratory disease complex, porcine dermatitis and nephropathy syndrome and reproductive and enteric disease forms (Iizuka et al. 1989; Nayar et al. 1997; Allan et al. 1998; Ellis et al. 1998; Saikumar and Das 2019).

PCV2 causes a wide-range of clinical signs depending on the condition of the host. The PCV2-asociated immunosuppression is characterized by lymphopenia, lymphoid cell (B- and T-cell) depletion and altered cytokine production (Darwich et al. 2003b; Saikumar and Das 2019). PCV2 is most often diagnosed with concurrent pathogens, including classical swine fever virus (CSFV), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV) and porcine parvovirus (PPV) (Iizuka et al. 1989; Saikumar and Das 2019). Due to the interference with the host immune system, PCV2 and concurrent porcine pathogens could provoke more severe disease together than those separately (Rovira et al. 2002; Kim et al. 2006; Kekarainen et al. 2008b; Shi et al. 2010; Sinha et al. 2011; Gao et al. 2014a; Ouyang et al. 2019; Opriessnig et al. 2020).

The most recently identified PCV species, PCV3 and PCV4, have been also linked to variable diseases. PCV3 is commonly detected in swine with acute porcine dermatitis and nephropathy syndrome-like signs, reproductive failures, cardiac diseases and multisystemic inflammation (Phan et al. 2016; Palinski et al. 2017). Histological findings include dermatitis and epidermitis, necrotizing vasculitis, pneumonia, glomerulonephritis, granulomatous lymphadenitis, myocarditis and cardiac arteriolitis (Phan et al. 2016; Palinski et al. 2017; Jiang et al. 2020; Sirisereewan et al. 2022; Yang et al. 2022). Lymphocyte and macrophage infiltration is usually observed in the affected tissues (Phan et al. 2016; Kim et al. 2018; Jiang et al. 2020). PCV4 is known from similar diseases as described for PCV2 and PCV3, but it can be the consequence of the often diagnosed co-infection of PCVs and other pathogens (Wang et al. 2022a). Some reports suggest that PCV4 might be pathogenic alone; the virus recovered from a cloned genome induced inflammation and deformities in the spleen, lung, kidney, liver and lymph nodes of inoculated piglets (Niu et al. 2022).

PCV1, PCV2 and PCV3 are widely distributed in pigs and wild boars. Furthermore, PCV2 has also been identified in cattle, dog, deer, chamois, mouse, as well as arthropods (such as ticks and mosquitoes) (Iizuka et al. 1989; Cao et al. 2018; Saikumar and Das 2019; Yang et al. 2019; Zhang et al. 2020a; Amoroso et al. 2021; Cui et al. 2022; Fanelli et al. 2022; Hu et al. 2022; Igriczi et al. 2022; Kroeger et al. 2022; Luka et al. 2022; Vargas-Bermudez et al. 2022). Reports about the less prevalent PCV4 in swine, wild boars and cattle originate from Southeast Asia, China and South Korea (Wu et al. 2022; Xu et al. 2022; Wang et al. 2022a).

Canine circovirus (CaCV) is also characterized as a widely distributed pathogenic mammalian circovirus that may cause hemorrhagic gastrointestinal and respiratory diseases of dogs (Anderson et al. 2017; Dankaona et al. 2022). As for avian and porcine circoviruses, co-infections contribute probably to the development or exacerbation of CaCV related diseases (Anderson et al. 2017; Dankaona et al. 2022).

The lack of cell culture systems is detrimental to the exploration of the pathomechanisms for the vast majority of circoviruses. However, PCV1, PCV2 and PCV3 can be propagated both in lymphoid and non-lymphoid cell cultures, a finding that permits insight into molecular aspects of virus-host interactions (Tu et al. 2015; Kroeger et al. 2022).

4. Target cells of circoviruses

The primary sites of active replication remain to be clarified for many circoviruses (Abadie et al. 2001; Schmidt et al. 2008; Steiner et al. 2008; Guo et al. 2011; Robino et al. 2014; Hong et al. 2018; Kroeger et al. 2022). Viral components of PCV2 were identified in monocytic cells and other blood cells, in lymphoid tissues, as well as in endothelial cells and epithelial cells of different organs (such as liver, lung, kidney) (Rosell et al. 1999; Darwich et al. 2004). Pig fetuses were shown to carry PCV2 in a latent form in the medulla of thymus and other lymphoid organs (Dong et al. 2015; Sydler et al. 2016). The virus causes NK-, T- and B-cell depletion in PCVAD, in part, by direct destruction of the immune cells and by other, currently not fully understood mechanisms (Rosell et al. 1999; Darwich et al. 2004).

Evidence indicate that PCV2 primarily infects monocytic cells, including monocytes, macrophages, dendritic cell (DC) precursors, myeloid DCs and plasmacytoid DCs (pDC). These cells are not the main virus factories but promote dissemination of the virus into variable tissues (Figure 1) (Gilpin et al. 2003; Vincent et al. 2003; 2005; Chang et al. 2006; Darwich and Mateu 2012). PCV2 binds heparan sulfate, chondroitin sulfate B and dermatan sulfate receptors and enters the monocytic cells via clathrin-mediated endocytosis, where the virus persists in latency (Misinzo et al. 2005; Vincent et al. 2005; Misinzo et al. 2006; Wei et al. 2018; Huang et al. 2021). The amino acid characteristics and distribution of the Cap has an impact on the PCV2 binding to the receptors, thus the uptake of distinct virus strains shows differences (Wei et al. 2019; Huang et al. 2021). In contrast to monocytic cells, actin- and small GTPase-regulated clathrin- and caveolin-independent pathways support PCV2 internalization and replication in swine kidney and testicle epithelial cells, while the virus enters T-lymphoblasts by various ways (Misinzo et al. 2009; Wei et al. 2019). PCV3 invades PK-15 cells through clathrin- and dynamin-2-mediated endocytosis (Stenzel et al. 2019).

Figure 1. Putative course of circovirus infection through the example of porcine circovirus 2 (PCV2). PCV2 infects primarily monocytic cells that aid dissemination of the virus throughout the body, allowing infection of multiple cell types. The impaired immune functions, provoked by the virus, together with IFNs elicited by co-infective agents, promote initiation of PCV2 replication in permissive cells in the early phase of infection. Cap (capsid), Rep (replication-associated protein) and open reading frame (ORF) 4 proteins contribute to activation of virus replication, as well as maintenance of anti-apoptotic state. In the late stage of infection excessive PCV2 replication is associated with apoptotic processes that is supported by upregulation of Cap, Rep, ORF3 and ORF5 proteins of the virus. Enhancement of immunosuppression favors replication of other pathogens that may lead to development of severe diseases.

In cultured DCs, PCV2 alone does not influence cell differentiation, antigen processing and presentation, or proinflammatory cytokine production (Vincent et al. 2005). The virus does not affect the IFN-α/tumor necrosis factor (TNF)-α induced DC maturation, and major histocompatibility complex class II (MHCII) antigen presenting protein and CD80/86 T-cell co-stimulatory molecule expression. However, the virus inhibits IFN-α and TNF-α expression induced by distinct swine pathogens (such as PRV, CSFV and transmissible gastroenteritis virus or TGEV) or by ligands in pDCs that impairs DC maturation, immune cell (NK, T and B lymphocyte) activation and direct antiviral effect (Vincent et al. 2005; 2007; Ablasser and Hur 2020). PCV2 inhibits pathogen recognition and, with the dissemination into a wide range of organs, it facilitates the deterioration of tissues against other pathogens (Figure 1) (Vincent et al. 2005; 2007).

PCV2 affects the differentiation of immune cells indirectly through cytokine changes elicited from DCs. PCV2 infection and PCV2/PRRSV co-infection of DCs induce transforming growth factor beta 1 (TGF-β)-dependent regulatory T-cell (Treg) development. A similar mechanism was observed in experiments with IPEC-J2 intestinal porcine enterocytes (Cecere et al. 2012). In these cells, the activation of NF-κB increased the transforming growth factor (TGF)-β level that stimulated CD4+ T-cell differentiation into Treg cells via extracellular signal-regulated kinase (ERK) phosphorylation (Liu et al. 2022). The increased amount of Treg cell leads to reduced responsiveness of the host immune system and to chronic infections (Maizels and Smith 2011; Liu et al. 2022).

PCV3 antigen/nucleic acid is commonly identified in lymph nodes, intestines, cardiac cells, various internal organs and in the dermis, as well as in inflammatory cells (presumably in macrophages) of the infected pigs (Phan et al. 2016; Palinski et al. 2017; Deim et al. 2019; Kroeger et al. 2022). Similarly, lymphoid cells and macrophages tested positive for CaCV nucleic acid by in situ hybridization (Dankaona et al. 2022). However, the role of these cells in the viral life cycle and in the pathogenesis is still in question (Phan et al. 2016; Palinski et al. 2017; Kim et al. 2018).

PiCV, DuCV, GoCV, and BFDV infections are also characterized with lymphocyte depletion, and with marked atrophy and high apoptotic cell rate in the lymphoid tissues of the bursa of Fabricius, thymus, spleen and bone marrow (Raue et al. 2005; Schmidt et al. 2008; Guo et al. 2011; Robino et al. 2014; Huang et al. 2017b; Hong et al. 2018). Circoviruses and virus-associated inclusion bodies are often detected in multiple tissues of the avian hosts, which is most pronounced in lymohoid tissues, chiefly in the bursa of Fabricius. Since inclusion bodies first appear in macrophages of this organ, PiCV is suggested to have a primary bursal tropism (Abadie et al. 2001; Schmidt et al. 2008). During progression of the infection, bursal atrophy and lymphocyte depletion can be observed, and mononuclear cells loaded with inclusion bodies infiltrate other lymphoid organs or tissues that begins to deteriorate (Abadie et al. 2001; Stenzel et al. 2020). In the late stage of PiCV infection the host is affected by secondary and opportunistic infections as a likely consequence of suppressed immune functions (Abadie et al. 2001).

As the above-mentioned data imply, the main targets of circoviruses are monocytic cells. After dissemination and invasion of permissive cells, circoviruses obtain excessive replication and immune impairment that predisposes the host to be invaded by pathogens, advocating disease development.

5. Immunomodulatory elements of circoviruses

The genomic DNA of circoviruses has immunomodulatory effects in the infected cells. As demonstrated for UV-treated PCV2 in pDCs, the viral genomic DNA influences the IFN production independent of viral replication, probably by unmethylated CpG motifs (Hasslung et al. 2003; Vincent et al. 2007; Wikstrom et al. 2007; Kekarainen et al. 2008a). CpG-ODNs (cytosine-phosphorothioate-guanine synthetic oligodeoxynucleotides), that are often used to study DNA motifs, act on the IFN expression via TLR dependent (e.g. TLR9) and independent pathways (Vincent et al. 2007; Wikstrom et al. 2007; Hansoongnern et al. 2022). The fact that incorporated tandem CpG-ODNs increase the PCV2 virus-like particle (VLP) vaccine efficacy underlines the importance of CpG motifs in modulation of cellular signals (Hansoongnern et al. 2022). In addition to the DNA sequence, the secondary and tertiary structure of the circular genomic DNA markedly influence the final action on the immune reaction (Wikstrom et al. 2007; Kekarainen et al. 2008a; 2008b; Balmelli et al. 2011). The purified replicative dsDNA of PCV2 was found to induce cytoskeletal rearrangements via the impairment of actin polymerization, a mechanism that inhibits endocytosis, secretion of cytokines as well as antigen presentation in DCs (Balmelli et al. 2011).

In addition to the viral DNA, virus-encoded proteins also modulate the cellular immune mechanisms. PCV2 Cap-gC1qR (receptor of the globular head of complement component 1q, also called C1QBP or p32) interaction up- or downregulates variable signaling pathways early after the virus enters the cells (Choi et al. 2015; Du et al. 2016). In PK-15 cells, PCV2 activates the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) pathway by replication independent manner, probably at the step of virus attachment/cell entry that support viral replication, translation, and maintenance of an anti-apoptotic state via the cleavage of poly-ADP ribose polymerase (PARP) and caspase-3 (Figure 2) (Wei et al. 2012). Furthermore, HSP70, a cellular chaperon also inhibits caspase-3 at this stage of infection in monocytic cells (Liu et al. 2013). PCV2 Cap interacts with nucleosome assembly protein 1-like 4 (NAP1L4) protein; the silencing of NAP1L4 results in the upregulation of Cap and Rep expression and enhances IFN-β expression via cGAS activation, a process that promotes virus replication (Wang et al. 2020).

Figure 2. Anti-apoptotic and pre-apoptotic processes in porcine circovirus 2 (PCV2) infection. In the early phase of infection, PCV2 activates the PI3K and the NF-κB pathways, and upregulates HSP70 and PCV2 open reading frame (ORF) 4 expression that inhibit pre-apoptotic processes and support virus replication. In the late phase the excessive PCV2 replication contribute to expression of the ORF3 encoded protein, and to activation of NF-κB, p38-MAPK and JNK1/2 pathways. Moreover, the viral protein of ORF5 induces endoplasmic reticulum stress (ERS). These together may further enhance virus replication, and trigger autophagy and apoptotic processes in the infected cells.

The viral Cap modulates the cellular transport processes that trigger virus replication. PCV2 Cap augments the phagocytic activity of macrophages via stabilization of cytoplasmic gC1qR and activation of PI3K (Choi et al. 2015). The gC1qR-Cap interaction promotes the phosphorylation of protein kinase C (PKC)-δ and recruitment of p-PKC-δ that lead to the rearrangement of nuclear lamina and facilitate the viral egress in cultured cells. At later phase of infection, Jun N-terminal protein kinase (JNK) and ERK signaling further enhance these processes (Wang et al. 2019). PCV2 Cap binds and supports nuclear transport of the virus binding dynein thus inducing α-tubulin acetylation (Cao et al. 2015). With downregulation of porcine makorin RING finger protein 1 Cap avoids its own degradation and triggers PCV2 replication via induction of stress mechanisms and apoptosis (Wang et al. 2018; Zhang et al. 2019). Both Cap and Rep antagonize the immune response at several points that was investigated in cell culture based overexpression experiments (see next sections) (Du et al. 2016; 2018; Wu et al. 2019).

As for PCV2, the Cap of PCV3 intersects variable cellular signaling pathways. The Cap-G3BP1 (GTPase-activating protein-(SH3 domain)-binding protein 1) interaction inhibits viral DNA recognition by cGAS that hampers IFN-β production in HEK-293T cells (Zhang et al. 2020c). The Cap of both PCV2 and PCV3 interact with nucleolar phosphoprotein nucleophosmin-1 that contributes to virus replication (Zhou et al. 2020; Song et al. 2021). PCV3 Cap, but not Rep, was described to induce IL-6 and TNF-α expression via the NF-κB pathway, possibly through the activation of RIG-I/MDA5, as well as TLR-MyD88 pathways in HEK-293T cells (Liu et al. 2021). In IFN-β treated PK-15 and HEK-293T cells PCV3 Cap inhibits transcription activation of ISRE, but phosphorylation and heterodimerization of STAT1 and STAT2 are not affected. Although Cap binds karyopheryn alpha (KPNA) 1, involved in nuclear protein import, this interaction does not inhibit the translocation of the STAT complex to nucleus. It is suggested that PCV3 Cap interacts with the transactivation domain of STAT2 that prevents ISGF3 binding to ISRE (Shen et al. 2020).

Although the protein encoded by ORF5 is a nonessential factor in respect to PCV2 replication, it supports the virus propagation by induction of prolonged S-phase and arresting of cell proliferation in alveolar macrophage cells with the downregulation of transmembrane glycoprotein NMB and cyclin A expression (Tang et al. 2013; Lv et al. 2015; Guo et al. 2018). On the other hand, ORF5 protein, localized in the ER, enhances the replication of PCV2 by inducing ER associated stress that leads to autophagy, protein kinase RNA-like endoplasmic reticulum kinase (PERK)-mediated unfolded protein response (UPR), and ultimately to apoptosis contributed by Ca²⁺ and reactive oxygen species (ROS) (Lv et al. 2015; Zhou et al. 2016; Lv et al. 2020). These processes activate the NF-κB pathway in PK-15 cells by increasing IκBα phosphorylation and degradation, and p65 nuclear translocation (Figure 2) (Wei et al. 2008; Zhang et al. 2013). This signaling seems to be necessary for excessive replication and viral protein expression, and for the enhancement of proinflammatory processes, thus disease progression. Likewise, expression of proinflammatory proteins (IL-6, IL-8 and COX-2) can be induced by the NF-κB pathway in porcine alveolar macrophages (PAMs) (Lv et al. 2015). The ORF5-associated triggering of autophagy and apoptosis proceed via the phosphorylation of PERK and eukaryotic initiation factor 2a (eIF2α) that activate activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP, an UPR component) in PK-15 cells (Figure 2). Furthermore, it mediates the phosphorylation of adenosine monophosphates-activated protein kinase and the activation of ERK1/2 that downregulates the cell survival supporting and autophagy regulator mammalian target of rapamycin (mTOR) pathway (Figure 2) (Zhu et al. 2012; Zhou et al. 2016; Lv et al. 2020). The mTOR is a target molecule of PCV3 as well; PCV3 Cap supports autophagosome formation and induces autophagy via inhibition of mTOR phosphorylation in HEK293T cells (Geng et al. 2020). Both Cap and Rep of PCV2 utilize the PERK-eIF2α-ATF4-CHOP axis for upregulation of ER oxidoreductase and ROS generation that promote nucleocytoplasmic migration of high-mobility group box 1 protein (HMGB1) in PK-15 cells. The decreased retention of nuclear HMGB1 releases the HMGB1-bound viral DNA, allowing higher virus replication rate (Sun et al. 2021).

The expression and stabilization of p53 may have a central role in the above-mentioned processes (Liu et al. 2005; 2006; Wei et al. 2009; Pan et al. 2018; Deng et al. 2022). The PERK-ROS axis promotes p53 upregulation in PCV2 inoculated PAMs and the accumulated phosphorylated p53 leads to cell cycle arrest at S-phase and enhances viral replication. It remains a question whether this phenomenon is associated with ORF5 (Deng et al. 2022). In other experimental design, the prolongation of the S-phase has been retrieved in PAMs by ORF5 (Lv et al. 2015; Zhou et al. 2016; Lv et al. 2020). In addition, the excessive viral replication and translation downregulate HSP70 and upregulate the phospholipase C/inositol 1,4,5-trisphosphate receptor pathways leading to apoptotic processes through p53 and caspase-3 activation, as well as Ca²⁺-release-mediated ER stress (Figure 2) (Pan et al. 2018; Wang et al. 2021a).

In the later phases of infection, PCV2 stimulates p38-mitogen-activated protein kinase (MAPK) and JNK1/2 signal transductions linked to viral replication processes. p38-MAPK and JNK1/2 pathways have a role in the transcription and translation of viral proteins, release of progeny viruses, whilst triggering apoptosis (Figure 2) (Wei et al. 2009). Similarly to ORF5, ORF3 is nonessential with regard to virus replication, but it is considered an important element of PCV2 pathogenesis that triggers apoptosis of the infected cells and thus virus proliferation (Liu et al. 2005; 2006). p38-MAPK and JNK1/2 support the stabilization of p53 that mediates PCV2 ORF3 protein induced apoptosis (Liu et al. 2005; 2006; Wei et al. 2009). In contrast, ORF4 was found to be an anti-apoptotic viral protein. It inhibits the virus replication in early phase of infection and decreases the expression of the pre-apoptotic ORF3 protein (Figure 2) (He et al. 2013; Gao et al. 2014b).

The promotion of apoptotic processes was also described for avian circoviruses, but few details are known about the viral immunomodulatory elements (Raue et al. 2005; Schmidt et al. 2008; Guo et al. 2011; Robino et al. 2014; Huang et al. 2017b; Hong et al. 2018). Like the homologous protein of PCV2, the product of DuCV ORF3 was found to contribute to the viral pathogenesis via activation of apoptosis (Xiang et al. 2012; Lv et al. 2014; 2015; Rosario et al. 2017). Apoptosis was linked to viral protein expression in BFDV affected cells as revealed by TUNEL assay and large caspase-1 positive cell rate in BFDV infected parrots (Robino et al. 2014). The load of PiCV seems to correlate with the disease severity; the virus inhibits the humoral immunity and induces apoptosis of B lymphocytes (Stenzel et al. 2020).

Taken together, the circovirus nucleic acid, as well as the structural and non-structural proteins work in concert with each other to take the control over the cells and to determine the course of infection.

6. Cytokine changes in circovirus infection

In order to evaluate the immune response to PCV2 infection, a number of studies investigated the changes of cytokine level in sera, tissues or isolated blood cells of healthy naïve and vaccinated pigs, and in pigs diagnosed with subclinical infections or PCVAD. Furthermore, the modulatory effect of PCV2 has been frequently studied in relation to co-infection with other pathogens or vaccination. A huge variety of experimental design has been implemented leading to diverse, sometimes controversial, results that are occasionally hard to interpret.

Cytokine response to PCV2, PCV2 DNA, CpG-ODNs and viral proteins in blood cells were intensively examined, often giving dissimilar data in peripheral blood mononuclear cells (PBMC) and monocytic cell cultures. In PBMC of PRV naïve pigs PCV2 induced negligible amount of IFN-α, while most of CpG-ODNs used for PCV2 DNA analysis triggered its production. In PBMC of PRV-immunized animals, PRV-induced IFN-α production was inhibited by PCV2, but not by CpG-ODNs (Wikstrom et al. 2007; Kekarainen et al. 2008a; 2008b). Regarding IFN-γ, neither PCV2 nor CpG-ODNs provoked alone the production of this cytokine, but a decrease of PRV induced IFN-γ mRNA could be detected in PBMCs of PRV/PCV2 co-infected pigs (Gao et al. 2014a). In the PBMCs of PRV immunized pigs, PCV2 and most of the CpG-ODNs did not stimulate the production of IFN-γ and IL-2, while significant downregulation of the PRV induced IFN-γ and IL-2 could be seen for both PCV2 and the CpG-ODNs (Kekarainen et al. 2008a; 2008b; Gao et al. 2014a). Decreased levels of IFN-γ mRNA have been also detected in PBMC of PRRSV/PCV2 co-infected piglets compared to the control groups (Shi et al. 2010). IL-10 production is influenced also by PCV2. The complete virus, but not CpG-ODNs, induced high level of this cytokine in PBMC of both naïve and PRV immunized animals. In this mixed cell population, the monocytic cell fraction may be the source of IL-10 (Kekarainen et al. 2008a; 2008b; Gao et al. 2014a). As IL-10 inhibitory experiments revealed, IL-10 can negatively affect IL-12 and IFN-γ levels in PBMCs of PRV immunized animals during PRV recall, while IL-2 inhibition seemed to be independent of the IL-10 activation (Kekarainen et al. 2008a; 2008b; Ablasser and Hur 2020). VLPs did not provoke or influence significantly the expression of these cytokines in PBMCs (Kekarainen et al. 2008a).

In BMDCs (bone-marrow-derived dendritic cells), PCV2 and rep derived CpG-ODNs (but not PCV2 VLP) inhibited IFN-α secretion during PRV challenge (Kekarainen et al. 2008a; 2008b). Furthermore, PCV2 has been proved to downregulate IFN production induced by CpG-ODNs, CSFV and TGEV in monocytic cells (Vincent et al. 2007; Kekarainen et al. 2008a; Li et al. 2018; Ablasser and Hur 2020). The measurement of IL-12 revealed that PCV2 and VLPs (but not the used CpG-ODNs) induce its production in BMDCs. However, the upregulation of IL-10 expression, an important feature of PCV2, reduced IL-12 secretion (Kekarainen et al. 2008a; 2008b). Overall, the variation seen in cytokine production of blood cells seems to depend on composition of the tested cell population, on the stage of infection, on the vaccination status or the presence of co-infective agents. In general, PCV2 alone does not increase significantly the expression of IFNs, but decreases the IFN levels induced by other agents, while IL-10 and IL-12 expression could be directly influenced by PCV2. VLPs and the variably constructed CpG-ODNs act in a different way on cytokine expression, if compared to the complete virus.

Numerous studies investigated the cytokine response to PCV2 infection without focusing on a certain structural element of the virus. Although IFN-γ downregulation has been usually noted in monocytic cells, in PBMCs or in internal organs both in subclinical and PCVAD cases, an increased IFN-γ expression was occasionally observed (Darwich et al. 2003a; 2003b; Li et al. 2022). In PCV2 inoculated gnotobiotic pigs the immune response showed marked variances with regard to the neutralizing antibody titer and IFN-γ expression of PBMCs even within an experiment. Neutralizing antibody and increased IFN-γ mRNA levels were shown in some cases without detectable PCV2, while lower neutralizing antibody and indifferent IFN-γ mRNA levels with variable PCV2 titers were seen in others (Meerts et al. 2005b). The IFN-γ mRNA level may only slightly change in animals vaccinated against PCV2 (Borghetti et al. 2013).

PCV2 and concomitant infectious agents may interfere with expression of multiple cytokines, including those responsible for T-cell differentiation and homeostasis, such as IL-2 and IL-4, as well as the proinflammatory cytokines IL-6 and IL-12. These cytokines are typically downregulated by PCV2, but increased levels have been detected as well depending on the type of examined cells and tissues, and the stage of infection (Sipos et al. 2004; Li et al. 2022). Elevated levels of TNF-α and IL-8 were commonly measured during PCV2 infection (Darwich et al. 2003a; 2003b; Chang et al. 2006; Darwich et al. 2008). However, in healthy pigs and severe PCVAD lower levels of IL-8 (a chemoattractant of blood cells) have been found than in animals with moderate signs and lower PCV2 titer (Darwich et al. 2003a; 2003b; Borghetti et al. 2013; Lv et al. 2015). Increased TNF-α, IL-8 and monocyte chemotactic protein-1 levels were observed in PCV2 infected alveolar macrophages with simultaneous reduction of the microbicidal capacity and phagocytic activity (Chang et al. 2006). Higher TNF-α levels were detected in alveolar macrophages in co-infection with PPV and PCV2, and in concavalin A stimulated PBMCs of PRRSV-PCV2 co-infected pigs compared to the controls (Kim et al. 2006; Shi et al. 2010). In in the same time, decreased TNF-α expression was measured in PBMC of PRRSV-PCV2 co-infected pigs and in PCV2-PRV inoculated PK-15 cell culture (Tu et al. 2015; Li et al. 2022). Elevated IL-1β expression has been measured in PMWS and in co-infections of PCV2 with PRRSV or PRV, but the level of this cytokine was shown to depend on the phase of infection similarly to IL-8 (Darwich et al. 2003b; Sipos et al. 2004; Borghetti et al. 2013; Tu et al. 2015; Li et al. 2022). These alterations, that may be different in variable tissues and disease status, disturb the overall cytokine expression and hamper the microbe clearance, but attract macrophages and other target cells to the site of infection in order to support cell survival and the spread of PCV2 (Chang et al. 2006). The impaired immunological reactivity exposes the host to secondary or opportunistic infections (Darwich et al. 2003a; 2003b; Chang et al. 2006; Kim et al. 2006; Shi et al. 2010).

The regulation of cytokine levels relies on the cooperation of cytokines and cellular factors. As an example, suppressor of cytokine signaling family 3 (SOCS3) protein has been identified as a regulator of cytokine signaling that functions in the negative feedback to proinflammatory cytokine production. TNF-α and IL-6 induce removal of IκBα bound to NF-κB, then NF-κB activation initiate gene expression of proinflammatory cytokines (Yasukawa et al. 2003; Hayden and Ghosh 2014). In PCV2 challenge, PBMCs of piglets with subclinical infections showed an increased SOCS3 level, and inhibited IκBα degradation and NF-κB signaling was detected. Likewise, SOCS3 level was elevated after PCV2 inoculation of PK-15 cells. SOC3 interacted with STAT3 (mediator of IL-6 signaling) and TNF-receptor-associated factor 2 (mediator of TNF-α signaling), inhibiting IL-6 and TNF-α-mediated IκBα degradation and NF-κB activation (Zhu et al. 2016). It was concluded that anti-inflammatory reactions result in failure of immune response to PCV2, supporting the change of PCV2 infection from the latent to an active state, while upregulated proinflammatory cytokine production promotes progression of PCV2 infection (Zhu et al. 2016).

PCV1 and PCV2 have been investigated often together in order to compare the cellular processes activated during the infections. Although, some cytokine response could be measured in PCV1 inoculated animals and cell cultures, these changes are not significant when compared to controls (Kekarainen et al. 2008b; Du et al. 2016; 2018; Wang et al. 2022c). Similarly to PCV2, PCV3 infection can markedly alter the cytokine response, but few data are currently available. Elevated IL-8 and undetectable IL-1β, IL-4, IL-10 levels were reported in serum of pigs showing subclinical PCV3 infection, a finding that is consistent with that of seen in early stages of PCV2 infection (Temeeyasen et al. 2020). The downregulation of IFN-β and upregulation of IL-6 and TNF-α expression were observed in PCV3 inoculated HEK-293T cells (Zhang et al. 2020c). Cytokine response to avian circovirus infection is also poorly studied and results come mainly from vaccine experiments. Recombinant PiCV Cap VLPs are promising vaccine candidates inducing T-cell proliferation and increased IFN-γ level, but decreasing TGF-β expression in lymphocytes from spleen of PiCV uninfected pigeons, when compared to mock control (Huang et al. 2021). The recombinant Cap elevated the CD4, CD8 and IFN-γ expression levels in splenic mononuclear cells both in PiCV infected and uninfected pigeons, but the humoral immunity was delayed in subclinical PiCV cases (Stenzel et al. 2019). In DuCV infected Cherry Valley ducklings, increased IL-10, decreased IL-12 and IFN-γ levels, and decreased soluble CD4 and CD8 amounts could be measured that may indicate an impaired T-cell function. In the same time, DuCV infection was shown to enhance the pathogenicity of avian pathogenic Escherichia coli (APEC) (Wang et al. 2022b).

7. Exploiting of IFNs

As it has been exemplified, via its immunomodulatory sequence motifs or proteins, PCV2 generates insignificant levels of IFNs and decreases IFN expression induced by other microbes (Hasslung et al. 2003; Vincent et al. 2007; Wikstrom et al. 2007; Kekarainen et al. 2008a). Whilst inhibition of the IFN response supports the immune evasion and disease progression, IFNs up to a certain amount has been found to support PCV2 uptake and replication initiation in early stages of infection.

An increased internalization of PCV2 VLPs is seen in the presence of externally added IFN-γ that probably enhances endocytic activity of 3D4/31 cells (Meerts et al. 2005a). IFN-α and IFN-γ increase the number of infected cells and enhance PCV2 replication in PK-15 and 3D4/31 (porcine monocytic) cells, except that pretreatment with IFN-α inhibits viral replication in pK-15 cells (Meerts et al. 2005a; Ramamoorthy et al. 2009; Mutthi et al. 2018). An ISRE-like sequence has been identified in the rep promoter of PCV2 that promotes the propagation of PCV2 in PK-15 and 3D4/31 cells in cooperation with the IFNs (Ramamoorthy et al. 2009). Concerted actions of the ISRE, TATA box and other (e.g. cis-acting) elements, together with variable interacting transcription factors, determine the response of PCV2 to these cytokines (Ramamoorthy et al. 2009).

PCV2-induced IFN production supports PCV2 replication, too. PCV2 upregulates IFN-β production in PK-15 cells via RIG-I/MDA-5/MAVS/IRF3 signaling, as well as through cGAS signaling with enhancement of cGAS expression, STING dimerization and perinuclear translocation (Huang et al. 2017a; 2018; Wang et al. 2020). The cGAS-STING pathway reacts for the viral DNA, while the RIG-I/MDA5/MAVS/IRF3/IRF7 signaling is activated by RNAs connected to viral gene expression processes (Huang et al. 2017a; 2018). In PAMs, PCV2 enhances the expression level of IKKα, IRF3 and IRF7 via MyD88 signaling that leads to IFN-α and IFN-β production (Chen et al. 2016).

The beneficial effect of IFNs on PCV2 replication initiation could explain why PCV2 remains in a latent state until IFN triggering stimuli (including non-circovirus agents) have an impact on the course of infection (Allan et al. 2000; Rovira et al. 2002). PCV2 may promote IFN production under certain conditions, but the underlying factors are not understood.

8. Molecular background of IFN interference

Although IFNs contribute to the induction of PCV2 replication, PCV2 counteracts IFN-I producing and signaling pathways from cGAS receptor signaling to STAT molecules, which supports the excessive replication, and invasion of PCV2 and other pathogens (Wu et al. 2021). Beside silencing of the catalytic activity in early phase of infection, phosphorylation of porcine cGAS at S278 via PI3K/AKT signaling (but not via p38-MAPK, JNK, ERK) facilitates the K48-linked poly-ubiquitination and p62-mediated autophagic degradation of cGAS protein in PK-15 cells (Wang et al. 2021b). The PCV2 Cap-gC1qR connection helps recruit p-PKCδ and promotes histone deacetylase 6 activation that mediates transport of the ubiquitinated cGAS from the cytosol to autolysosomes (Wang et al. 2021b).

The PCV2 dependent inhibition of IFN-α and IFN-β production enhances PPV infection in piglets compared to those infected solely by PPV (Ouyang et al. 2019; Wu et al. 2021). PCV2 hampers the production of IFN-α and IFN-β that is accompanied by decreased ISG (such as ISG15; ISG56; IFIT2, IFN-induced protein with tetratricopeptide repeats; CXCL10, C-X-C motif chemokine ligand 10) mRNA levels in PBMCs of PCV2/PPV co-infected piglets (Wu et al. 2021). PCV2 infection blocks the cGAMP induced IFN-β production in PPV co-infected PK-15 culture by inhibiting K63 ubiquitination of STING through activation of p38-MAPK pathway. p38-MAPK signaling leads to phosphorylation and activation of the USP21 deubiquitinating enzyme that hydrolyzes polyubiquitin chains of STING. Deubiquitination of STING causes lower phosphorylation levels for IRF3 and TBK1, weaker interaction of these molecules and decreased translocation to the nucleus (Wu et al. 2021). The pIRF3 translocation can be inhibited at other signaling points as well. Nuclear transport of IRF3 is mediated by KPNA3 and KPNA4 (Zhang et al. 2020b). PCV2 has been reported to be able to disrupt the interaction of KPNA3 with p-IRF3 in order to block pIRF3 translocation to the nucleus for the inhibition of IFN-β induction in PK-15 cells (Li et al. 2018; Zhang et al. 2020b).

The Cap of PCV2, in concert with the cellular gC1qR protein, inhibits Type I IFN response as well. Cap decreases the phosphorylation level of STAT1 and STAT2 and their interaction with IRF9 upon IFN-α treatment of PK-15 cells (Wang et al. 2022c). Heterotrimer formation, translocation of ISGF3 to the nucleus and binding to ISRE promoter of ISGs are inhibited by both the Rep and Cap. Thus, PCV2 reduces activation of the ISRE promoter and expression of ISGs (CXCL10; IFIT1; MX1, interferon-induced GTP-binding protein) (Wang et al. 2022c). Downregulation of IFN-β production and JAK/STAT signaling has been also reported in PCV2-PRV co-infected PK-15 cells (Li et al. 2022). Along with Cap, one has to highlight the role of ORF5 in IFN antagonism and support of PCV2 replication. As revealed by mRNA analysis, the ORF5 protein of PCV2 antagonizes the transcription of genes involved in Type I IFN production and IFN response in PK-15 cells, including RIG-I (DDX58), LGP2 (DHX58), MDA5 (IFIH1), IFIT1 and IFIT3, IFITM2 and IFITM3 (IFN induced transmembrane proteins), IRF7 and IRF9, ISG15 and TLR3 (Choi et al. 2018).

G3BP1-cGAS interaction promotes DNA binding of cGAS and downstream signaling that lead to IFN-β expression via TBK1 and IRF3 phosphorylation in PK-15 and HEK-293T cells (Zhang et al. 2020c). The interaction between PCV3 Cap and G3BP1 prevents cGAS from recognizing the viral DNA that inhibits IFN-β production (Zhang et al. 2020c). In the same time, IFN stimulated gene expression (ISG15, OAS1, MX1, MX2, IFIT3) have been shown to be upregulated in lung samples of PCV3 infected piglets, but the mechanisms behind are unexplored (Jiang et al. 2020).

9. Molecular background of IL-10/IL-12 pathway modulations

IL-10 is an immunoregulatory cytokine with anti-inflammatory properties, produced primarily by monocytic cells and B-cells. IL-10 hampers excessive inflammation, allergy or autoimmune responses of the host. Some pathogens exploit its anti-inflammatory effect to evade the immune response (Driessler et al. 2004; Cao et al. 2006). The often seen downregulation of proinflammatory cytokines has been found to associate with elevated IL-10 expression and with disease development in PCV2 infections (Figure 3) (Darwich et al. 2003a; 2003b; Sipos et al. 2004; Stevenson et al. 2006; Darwich et al. 2008; Shi et al. 2010; Borghetti et al. 2013; Gao et al. 2014a; Richmond et al. 2015; Du et al. 2019). IL-10 supports PCV2 replication in the late phase of infection and the viral load correlates with the IL-10 level (Darwich et al. 2008; Du et al. 2019). The IL-10 mRNA level has been shown to increase in the lymphoid tissues and has been related with the degree of lymphocyte depletion in the thymus of pigs with PCVAD (Darwich et al. 2003b). IL-10 causes CD4+ and CD8+ T-cell depletion in the spleen, and inhibits the infiltration of these cells into the lung in mice (Du et al. 2019).

Figure 3. Effect of porcine circovirus 2 (PCV2) infection on IL-10 and IL-12 production in monocytic cells. PCV2 capsid protein (Cap) interacts with gC1qR or other cellular proteins, activating PI3K/akt pathway typically in the early phase of infection, and p38-MAPK and ERK in late phases. The activated transcription factors enhance IL-10 expression, while inhibition of NF-κB p65/p50 and the upregulation of virus-triggered miRNAs result in IL-12 and IFN-γ transcription downregulation, and posttranslational decrease of the IL-12 level.

The primary targets of PCV2 are monocytic cells in which the virus activates cellular pathways leading to IL-10 expression. IL-10 production in PAMs is induced when viral Cap interacts with the ubiquitous cellular protein gC1qR. Both in early and late phase of infection this complex triggers PI3K/Akt signaling that cooperates with the NF-κB pathway, while p38-MAPK signaling is highly active in later phases of infection (Figure 2) (Du et al. 2016). Furthermore, the ERK pathway can be upregulated by the Cap in parallel with p38-MAPK independent of gC1qR. At last, a number of transcription factors, including NF-κB p50, AP1 and CREB (activated via phosphorylated PI3K/Akt), as well as SP1 (activated via phosphorylated p38-MAPK and ERK) bind the IL-10 promoter and induce transcription of IL-10 (Figure 3) (Du et al. 2016). While Cap induces IL-10 expression early after PCV2 enters the cells, Rep promotes IL-10 activation in later phase of infection in PAMs. Interaction of PCV2 Rep with the cellular protein thymine DNA glycosylase induces p38 phosphorylation, and the activated p38-MAPK pathway, but not ERK and PI3K/Akt, induces binding of NF-κB p50 and Sp1 to the IL-10 promoter (Figure 3) (Wu et al. 2019). The overexpressed IL-10, as one of the main targets, blocks IκBα degradation, thus inhibiting the release of NF-κB p65/p50 and the nuclear translocation of p65. Furthermore, IL-10 aids the translocation and DNA binding of the NF-κB1 p50/p50 complex in monocytic cells; p50 transactivates the IL-10 gene and mediates anti-inflammatory effect of IL-10 protein (Figure 3) (Driessler et al. 2004; Cao et al. 2006).

IL-12, a heterodimeric molecule composed of p35 and p40 protein chains, is produced primarily by antigen presenting cells, and plays a pivotal role in cytotoxic T-lymphocyte and NK cell activation, and CD4+ T lymphocyte maturation to IFN-γ producing Th1 cells (Ma et al. 2015). As mentioned above, PCV2 induced IL-10 appears to be crucial in the impairment of proinflammatory processes antagonizing the IL-12 and IFN-γ expression (Kekarainen et al. 2008a; 2008b; Gao et al. 2014a; Du et al. 2019). IL-12p40 and IFN-γ levels, as well as Th1 cell proportion were shown to decrease in PBMCs, and lung and pulmonary lymph nodes in pigs challenged with PRRSV and Haemophilus parasuis following PCV2 infection, compared to PCV1 and mock infected animals. Moreover, PCV2 promoted replication of the PRRSV and H. parasuis (Du et al. 2018).

PCV2 Cap-gC1qR interaction has a key role in IL-12p40 decrease in PAMs with miR-23a, miR-23b and miR-29b upregulation via PI3K/Akt1, and with miR-29a and miR-29b upregulation via the p38 MAPK pathway (Du et al. 2018). miR-23a and miR-23b act at posttranscriptional level, while miR-29a and miR-29b influence both transcription and the posttranslational level of IL-12p40. miR-23a and miR-29b were shown to be the most important suppressors of IL-12p40 expression (Figure 3). The activation of Akt1 and p38 signaling leads to NF-κB p65 downregulation in macrophages and this prevents the binding of the transcription factor to the il12B (IL-12p40) promoter (Figure 3) (Du et al. 2018).

In contrast to results related to downregulation of IL-12 in monocytic cells, the decrease of IL-4 and enhancement of IL-12 production has been measured in lymphocytes. It could be obtained via the NF-κB pathway by TLR2, TLR3, TLR4, TLR9 and MyD88 signal adaptor activation (Duan et al. 2014). It is in accordance with studies revealing that the monocytic cell fraction has a primary role in reduction of IL-12 in the PBMC populations (Kekarainen et al. 2008a; 2008b; Gao et al. 2014a).

10. Summary of the circovirus-associated immunosuppressive mechanisms

The evaluation of published data on cellular and immunological changes caused by animal circovirus infection is challenging due to the complexity of the molecular signaling and regulation of this network, and to the differences of the experimental conditions. At present, the largest data set is available for PCV2, a readily culturable circovirus with well-established animal model, thus presentation of the circovirus-associated immunosuppression could be formulated for it.

Monocytic cells, including macrophages/monocytes and DCs, have been identified as primary targets of PCV2 and PiCV. These cells are not the main location of viral replication but serve as a site for latent infection (Gilpin et al. 2003; Vincent et al. 2003; 2005; Chang et al. 2006; Darwich and Mateu 2012). In early phase of PCV2 infection most functions of monocytic cells are intact, but in case of a ligand stimulus (e.g. concomitant infections) PCV2 impairs the cytokine response and raises an imbalance in the activation of immune cells, hence interferes with the innate and adaptive immunity (Vincent et al. 2005; 2007; Ablasser and Hur 2020). The alterations are achieved in a replication independent manner by structural components of PCV2, such as the genomic DNA and Cap protein (Vincent et al. 2007; Wikstrom et al. 2007; Kekarainen et al. 2008a; Balmelli et al. 2011). A key element of the Cap induced modifications is the interaction of this protein with the cellular gC1qR molecule (Choi et al. 2015; Du et al. 2016; 2018; Wang et al. 2019; 2021b; 2022c). PCV2 not only silences, but at some stages of infection triggers and exploits cytokines to initiate its own replication and protein expression, and to promote invasion of the target cells (Huang et al. 2017a; 2018; Wang et al. 2020). At early stages of infections and subclinical cases upregulation of some cytokines (TNF-α, IL-1β, IL-8) may help the survival and dissemination of monocytic cells that serve as portals of PCV2 to enter various tissues, while elevated IL-10 level results in T-cell imbalances (Gilpin et al. 2003; Vincent et al. 2003; 2005; Chang et al. 2006; Darwich and Mateu 2012; Chen et al. 2016). Meanwhile, the ORF4 protein and Cap support the maintenance of anti-apoptotic state of the cells (He et al. 2013; Liu et al. 2013; Gao et al. 2014b).

PCV2 itself does not typically cause severe disease and in PCVAD the virus is often detected together with co-infective microbes. As recorded, the swine pathogens may generate alone less serious disease than in concert with PCV2 (Kim et al. 2006; Kekarainen et al. 2008b; Shi et al. 2010; Sinha et al. 2011; Gao et al. 2014a; Ouyang et al. 2019; Opriessnig et al. 2020). IFNs, induced by PCV2 or concomitant infective agents, may trigger expression of the viral Rep through the ISRE motifs in the rep promoter, thus advocate the virus replication in permissive cells (Ramamoorthy et al. 2009; Sinha et al. 2011; Huang et al. 2017a). At later phase of infection circoviruses (as seen for PCV2 and PiCV) are detected in different cell types and organs (Rosell et al. 1999; Darwich et al. 2004). On the other hand, with progression of the infection, PCV2 reduces IFN production and provoke severe immunosuppression via more intense production of IL-10 that has detrimental effect on the proinflammatory cytokine expression and on the activation of the adaptive immunity (Kekarainen et al. 2008a; 2008b).

The excessive replication of PCV2 requires the cellular machinery, thus the viral life cycle is connected to the nucleus of mitotic cells. Modification of the cytokine expression, arrest of the cell cycle with prolongation of the S-phase and progression of the infection are supported by both structural and non-structural viral proteins. Furthermore, the replicative, intermediate dsDNA inhibits transport processes, endocytosis, antigen presentation and cytokine secretion (Liu et al. 2005; 2006; Balmelli et al. 2011; Tang et al. 2013; Lv et al. 2015; Guo et al. 2018; Ablasser and Hur 2020; Lv et al. 2020). Lymphocyte depletion, typical for PCVAD, may be the consequence of altered cytokine expression and intense virus propagation that is enhanced by initiation of ER stress, autophagy and apoptotic processes supported by the Cap, Rep, ORF3 and ORF5 proteins (Tang et al. 2013; Lv et al. 2015; Zhou et al. 2016; Guo et al. 2018; Lv et al. 2020). At last, these changes generate multiorgan failures and fatal events in the host.

PCV2 is fairly well controlled by vaccines. However, emerging of novel PCV genotypes may lead to escape from immunity raised by vaccines currently in use (Saikumar and Das 2019). Also, the increasing number of newly identified species highlights the diversity of circoviruses. As circoviruses have direct pathological role and affect the outcome of concurrent infections, it is crucial to gather more information about the genetics, evolution, pathomechanisms and immunomodulatory strategies of these viruses and gain further insight into the interactions in the affected hosts. These data will be essential to identify new targets relevant to disease prevention or treatment strategies.

Disclosure statement

The authors report there are no competing interests to declare.

Additional information

Funding

This work was supported by the (RRF-2.3.1-21-2022-00010) of the University of Pécs, Hungary. Project no. TKP2021-NVA-07 has been implemented with the support provided from the financed under the TKP2021-NVA funding scheme.