Veterinary Quarterly

2023, VOL. 43, NO. 1, 1–18

https://doi.org/10.1080/01652176.2023.2234430

Mechanisms of circovirus immunosuppression and pathogenesis with a

focus on porcine circovirus 2: a review
Enikő Fehéra,b , Ferenc Jakabb and Krisztián Bányaia,c
Veterinary Medical Research Institute, Budapest, Hungary; bNational Laboratory of Virology, Szentágothai Research Centre,
a

University of Pécs, Pécs, Hungary; cDepartment of Pharmacology and Toxicology, University of Veterinary Medicine, Budapest,
Hungary

ABSTRACT ARTICLE HISTORY

Certain pathogens, due to their adverse effects on the immune reaction, aggravate the course Received 21 September
of concomitant heterologous infections. Here we summarize mechanisms by which circoviruses, 2022
including the most studied porcine circovirus 2, and other mammalian and avian circoviruses, Accepted 3 July 2023
trigger their own replication and confound the hosts’ immune response. At different stages of KEYWORDS
infection, from latent state to disease induction, these viruses markedly influence the cellular Pig; porcine; circovirus;
signaling pathways. Circoviruses have been found to interfere with interferon and PCV2;
proinflammatory cytokine producing and responsive pathways. Apoptotic processes, altered immunosuppression;
cellular transport and constraint of the mitotic phase all support the viral replication. The pathogenesis; review
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.

1. Introduction IFN molecules as well as the proinflammatory

The formation of an antiviral state in host cells is the
result of sequential steps of variable cascade mecha-
nisms 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 para-
crine signaling that ultimately contributes to expres-
sion 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; macro-
phages) and components of the adaptive immunity
(Schneider et al. 2014; Rojas et al. 2021). Another piv-
otal 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).
pathways act in concert and ensure feedback to each
to restrict viral propagation in and between suscep-
tible cells. To by-pass this fine-tuned network, viruses
evolved countermeasures at variable points by tar-
geting 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-associ-
ated Toll-like receptors (TLRs), the RIG-I-like receptors

CONTACT Enikő Fehér feher.eniko@vmri.hu Veterinary Medical Research Institute, Hungária krt. 21, H-1143 Budapest, Hungary.
© 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/),
which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article
has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.
2 E. FEHÉR ET AL.
(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 reticu-
lum (ER) and peroxisome (Ablasser and Hur 2020;
Rojas et al. 2021). MAVS activates inhibitor of kap-
pa-B kinase epsilon (IKKε) and TANK binding kinase 1
(TBK1), two enzymes that phosphorylate either inter-
feron regulatory factor (IRF) 3 or IRF7. The homodi-
mers 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 stim-
ulate 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 macro-
phage activation, and as an immunomodulatory and
proinflammatory protein it contributes to the activa-
tion 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 het-
erodimer is associated with the IκB (NF-κB inhibitor)
inhibitory complex. Phosphorylation of IκB by IκB
kinase (IKK), followed by polyubiquitination and deg-
radation, 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 appara-
tus and recruit TBK1 for downstream signaling. At
this point, the processes overlap with pathways con-
nected 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 recep-
tor 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 tran-
scription (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) pro-
moter sequences of ISGs (Lee and Ashkar 2018).
After binding to its receptor, IFN-γ activates Jak1/
Jak2, and controls the expression of further regula-
tory elements (i.e. transcription factors) by phosphor-
ylated 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 num-
ber 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, replica-
tive 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 sup-
port 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 impor-
tance, which comes from their capacity to induce
marked immunosuppression. Beside the direct patho-
genic 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 dis-
ease virus (BFDV), goose circovirus (GoCV), duck cir-
covirus (DuCV), pigeon circovirus (PiCV), finch
circovirus and canary circoviruses, may generate leth-
argy, weight loss, as well as beak deformation, feath-
ering 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 infiltra-
tion, 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-occur-
rence can worsen the course of infection and makes
the etiological role of circoviruses in disease develop-
ment 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 reproduc-
tive 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 depend-
ing on the condition of the host. The PCV2-asociated
immunosuppression is characterized by lymphope-
nia, lymphoid cell (B- and T-cell) depletion and
altered cytokine production (Darwich et al. 2003b;
Saikumar and Das 2019). PCV2 is most often diag-
nosed 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 interfer-
ence with the host immune system, PCV2 and con-
current 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 dis-
eases. PCV3 is commonly detected in swine with
acute porcine dermatitis and nephropathy syn-
drome-like signs, reproductive failures, cardiac dis-
eases and multisystemic inflammation (Phan et al.
2016; Palinski et al. 2017). Histological findings
include dermatitis and epidermitis, necrotizing vas-
culitis, pneumonia, glomerulonephritis, granuloma-
tous 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
Veterinary Quarterly 3
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 sug-
gest that PCV4 might be pathogenic alone; the virus
recovered from a cloned genome induced inflamma-
tion 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 cir-
coviruses, co-infections contribute probably to the
development or exacerbation of CaCV related dis-
eases (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 interac-
tions (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 identi-
fied in monocytic cells and other blood cells, in lym-
phoid 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 plas-
macytoid DCs (pDC). These cells are not the main
virus factories but promote dissemination of the
virus into variable tissues (Figure 1) (Gilpin et al.
4 E. FEHÉR ET AL.

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 repli-
cation 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.

2003; Vincent et al. 2003; 2005; Chang et al. 2006;
Darwich and Mateu 2012). PCV2 binds heparan sul-
fate, chondroitin sulfate B and dermatan sulfate
receptors and enters the monocytic cells via clath-
rin-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 replica-
tion 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).
In cultured DCs, PCV2 alone does not influence
cell differentiation, antigen processing and presenta-
tion, 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 expres-
sion 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 deteri-
oration 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) phos-
phorylation (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 sug-
gested 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 lym-
phoid 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 sec-
ondary and opportunistic infections as a likely conse-
quence of suppressed immune functions (Abadie
et al. 2001).
As the above-mentioned data imply, the main tar-
gets of circoviruses are monocytic cells. After dissem-
ination and invasion of permissive cells, circoviruses
obtain excessive replication and immune impairment
that predisposes the host to be invaded by patho-
gens, advocating disease development.
5. Immunomodulatory elements of circoviruses
The genomic DNA of circoviruses has immunomodu-
latory 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 oli-
godeoxynucleotides), that are often used to study
DNA motifs, act on the IFN expression via TLR depen-
dent (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 second-
ary 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 cyto-
skeletal rearrangements via the impairment of actin
polymerization, a mechanism that inhibits endocyto-
sis, secretion of cytokines as well as antigen presen-
tation in DCs (Balmelli et al. 2011).
Veterinary Quarterly 5
In addition to the viral DNA, virus-encoded pro-
teins 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 sig-
naling 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 indepen-
dent manner, probably at the step of virus attach-
ment/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 expres-
sion via cGAS activation, a process that promotes
virus replication (Wang et al. 2020).
The viral Cap modulates the cellular transport pro-
cesses that trigger virus replication. PCV2 Cap aug-
ments 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 infec-
tion, 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 down-
regulation 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 nucleophos-
min-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 activa-
tion 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 tran-
scription 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
6 E. FEHÉR ET AL.

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.

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 non-
essential factor in respect to PCV2 replication, it sup-
ports the virus propagation by induction of
prolonged S-phase and arresting of cell proliferation
in alveolar macrophage cells with the downregula-
tion 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, local-
ized in the ER, enhances the replication of PCV2 by
inducing ER associated stress that leads to autoph-
agy, 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 pro-
cesses 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 proinflam-
matory 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 acti-
vate 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 monophos-
phates-activated protein kinase and the activation of
ERK1/2 that downregulates the cell survival support-
ing 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 sup-
ports autophagosome formation and induces auto-
phagy 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 genera-
tion 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 accu-
mulated 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-tris-
phosphate 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 pro-
teins, release of progeny viruses, whilst triggering
apoptosis (Figure 2) (Wei et al. 2009). Similarly to
ORF5, ORF3 is nonessential with regard to virus rep-
lication, 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 sta-
bilization 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 repli-
cation 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
Veterinary Quarterly 7
(Xiang et al. 2012; Lv et al. 2014; 2015; Rosario et al.
2017). Apoptosis was linked to viral protein expres-
sion 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-infec-
tion with other pathogens or vaccination. A huge
variety of experimental design has been imple-
mented 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 immu-
nized pigs, PCV2 and most of the CpG-ODNs did not
stimulate the production of IFN-γ and IL-2, while sig-
nificant 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
8 E. FEHÉR ET AL.
influence significantly the expression of these cyto-
kines 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 measure-
ment 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 varia-
tion seen in cytokine production of blood cells seems
to depend on composition of the tested cell popula-
tion, on the stage of infection, on the vaccination
status or the presence of co-infective agents. In gen-
eral, 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 gno-
tobiotic 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 with-
out detectable PCV2, while lower neutralizing anti-
body 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 cyto-
kines are typically downregulated by PCV2, but
increased levels have been detected as well depend-
ing 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 com-
monly 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 lev-
els were observed in PCV2 infected alveolar macro-
phages 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 con-
trols (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 mea-
sured 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 cyto-
kine 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 degrada-
tion 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 activa-
tion (Zhu et al. 2016). It was concluded that anti-in-
flammatory 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 cyto-
kine response could be measured in PCV1 inoculated
animals and cell cultures, these changes are not sig-
nificant 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 cir-
covirus 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 com-
pared 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 immunomodula-
tory sequence motifs or proteins, PCV2 generates
insignificant levels of IFNs and decreases IFN expres-
sion 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 initia-
tion in early stages of infection.
An increased internalization of PCV2 VLPs is seen
in the presence of externally added IFN-γ that prob-
ably 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 replica-
tion 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 pro-
moter 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-act-
ing) 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 rep-
lication, 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
Veterinary Quarterly 9
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 expres-
sion 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 repli-
cation, 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 ubiquiti-
nated 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 pro-
duction 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
10 E. FEHÉR ET AL.

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 expres-
sion 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-in-
fected PK-15 cells (Li et al. 2022). Along with Cap,
one has to highlight the role of ORF5 in IFN antago-
nism and support of PCV2 replication. As revealed by
mRNA analysis, the ORF5 protein of PCV2 antago-
nizes 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 upregu-
lated 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-in-
flammatory properties, produced primarily by mono-
cytic cells and B-cells. IL-10 hampers excessive
inflammation, allergy or autoimmune responses of
the host. Some pathogens exploit its anti-inflamma-
tory effect to evade the immune response (Driessler
et al. 2004; Cao et al. 2006). The often seen downreg-
ulation of proinflammatory cytokines has been found

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 posttransla-
tional decrease of the IL-12 level.

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).
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 infec-
tion 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 gly-
cosylase induces p38 phosphorylation, and the acti-
vated 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 overex-
pressed 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 mono-
cytic 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 antago-
nizing 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 propor-
tion 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
Veterinary Quarterly 11
infected animals. Moreover, PCV2 promoted replica-
tion 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 posttransla-
tional 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 downregula-
tion in macrophages and this prevents the binding
of the transcription factor to the il12B (IL-12p40) pro-
moter (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 mea-
sured 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 mono-
cytic 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 condi-
tions. At present, the largest data set is available for
PCV2, a readily culturable circovirus with well-estab-
lished animal model, thus presentation of the circo-
virus-associated immunosuppression could be
formulated for it.
Monocytic cells, including macrophages/mono-
cytes and DCs, have been identified as primary tar-
gets 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 modifica-
tions is the interaction of this protein with the cellu-
lar gC1qR molecule (Choi et al. 2015; Du et al. 2016;
2018; Wang et al. 2019; 2021b; 2022c). PCV2 not only
12 E. FEHÉR ET AL.
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 infec-
tive 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 pro-
voke 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 cel-
lular 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 pro-
longation of the S-phase and progression of the
infection are supported by both structural and
non-structural viral proteins. Furthermore, the repli-
cative, intermediate dsDNA inhibits transport pro-
cesses, 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 initia-
tion 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 concur-
rent infections, it is crucial to gather more informa-
tion 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 treat-
ment strategies.
Disclosure statement
The authors report there are no competing interests to
declare.
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 fund-
ing scheme.
ORCID
Enikő Fehér http://orcid.org/0000-0001-7778-9116
Krisztián Bányai http://orcid.org/0000-0002-6270-1772
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