Effects of PRRSV Infection on the Porcine Thymus

Please cite this article in press as: Wang et al., Effects of PRRSV Infection on the Porcine Thymus, Trends in Microbiology (2019), https://doi.org/ 10.1016/j.tim.2019.10.009

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Review

Effects of PRRSV Infection on the Porcine Thymus Gang Wang,1,2,5,* Ying Yu,1,4,5 Xuehui Cai,1 En-Min Zhou,3 and Jeffrey J. Zimmerman2,* Porcine reproductive and respiratory syndrome virus (PRRSV) dramatically affects the thymus and its ability to carry out its normal functions. In particular, infection incapacitates PRRSV-susceptible CD14pos antigen-presenting cells (APCs) in the thymus and throughout the body. PRRSV-induced autophagy in thymic epithelial cells modulates the development of T cells, and PRRSV-induced apoptosis in CD4posCD8pos thymocytes modulates cellular immunity against PRRSV and other pathogens. Pigs are less able to resist and/or eliminate secondary infectious agents due the effect of PRRSV on the thymus, and this susceptibility phenomenon is long recognized as a primary characteristic of PRRSV infection.

PRRSV Adversely Affects Immune Responses Porcine reproductive and respiratory syndrome virus (PRRSV) (see Glossary) exists as two distinct virus species, that is, PRRSV-1 and PRRSV-2 [1,2], with both species subject to frequent mutation and viral recombination events. The immunological response by pigs to PRRSV infection is vigorous, but inefficient in terms of establishing sterilizing immunity, as evidenced by prolonged viremia, transmission of virus to comingled animals 99 days post-inoculation (DPI) [3], the detection of infectious PRRSV in lymphoid tissues from 10 of 11 pigs at 105 DPI [4], and the recovery of virus from buccal swab samples collected at 157 DPI [5]. Research has shown that PRRSV infection affects both innate and adaptive immune responses, that is, suppresses type I interferon induction [6–8], delays the development of neutralizing antibody [9,10], and deregulates cytokine expression [11–13]. Relevant to our understanding of the total impact of PRRSV infection on the porcine immune system, recent studies have shown that PRRSV can directly affect the process of thymocyte differentiation or the thymic microenvironment itself. Herein we review the current information on the pathogenesis of this process, the mechanism of PRRSV-induced immunomodulation, and the consequences for the immune system.

The Thymus and Its Role in Immunity The ontogeny of the thymus varies among species, but the developing thymus can be identified in the fetal pig by 22 days of gestation (DG), is completely formed by 36 DG, and undergoes a period of allometric growth from 36 to 114 DG [14,15]. At birth, the thymus consists of two lobes that partially cover the anterior portion of the heart and extend along each side of the trachea to the larynx (Figure 1A). The gland is covered by a capsule of connective tissue that extends into the gland forming ’trabeculae’ that define the borders of individual thymic lobules (Figure 1D). Each lobule is organized as an outer cortex composed of immature thymocytes along with epithelial cells, macrophages, and dendritic cells, and an inner core (medulla) consisting of mature thymocytes, epithelial cells, and dendritic cells [16]. Although the process is not clearly understood, the thymus gradually decreases in size as pigs age (thymic involution) [17]. The thymus serves as the primary site of T cell proliferation, differentiation, and selection, thereby playing a central role in cell-mediated immunity. In the fetal pig, prothymocytes derived from stem cells in primary hemopoietic centers of the fetal liver can be detected in the thymus at about 40 DG, although fully maturated T cells are not detected until after birth [15,18]. After birth, prothymocytes are generated in red bone marrow. These T cell precursors translocate to the thymic cortex via the circulatory system, therein to develop and become fully functional T cells or die via apoptosis [16]. In brief, thymocytes undergo four stages of development, beginning with the translocation of prothymocytes into the thymic cortex [19,20]. This process requires that thymocytes receive the appropriate signals for migration, proliferation, and differentiation from thymic APCs (Box 1) positioned in specific thymic microenvironments, that is, cortical and medullary epithelial cells, macrophages, dendritic

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Highlights PRRSV induces thymic atrophy and thymocyte apoptosis, with the virus isolate an important factor in determining the degree of the effect. Virus-susceptible thymic CD14pos APCs induce CD4posCD8pos thymocyte apoptosis and thymic epithelial cell autophagy. Extrathymic consequences of PRRSV infection include a reduction in the production of prothymocytes derived from bone marrow. PRRSV may affect thymus functions by reduction of bone marrowderived prothymocytes and the production of proinflammatory mediators and glucocorticoids. The impact of PRRSV on thymus functions contributes to anomalous immune responses, that is, suppression of type I interferon, deregulated cytokine expression, and delayed development of neutralizing antibodies.

1State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China 2Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA, USA 3Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China 4College of Veterinary Medicine, Qingdao Agricultural University, Qingdao, China 5These authors contributed equally to this work.

*Correspondence: [email protected], [email protected], [email protected]

https://doi.org/10.1016/j.tim.2019.10.009 ª 2019 Elsevier Ltd. All rights reserved.

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cells, and fibroblasts and extracellular matrix components [16,21]. Early developing thymocytes, 5% of the total thymic lymphocyte population, express neither T cell receptor (TCR) nor cluster of differentiation (CD) markers (CD3, CD4, and CD8). Second stage thymocytes, 80% of the thymocytes at the outer cortex of the thymus, express both CD4 and CD8 molecules (CD4posCD8pos). At the third stage, interactions with MHC I/II molecules (positive selection) trigger CD4posCD8pos thymocytes to become T helper cells (CD4pos), T cytotoxic cells (CD8pos), or initiate apoptosis (negative selection). The developing thymocytes then migrate to the thymic medulla where non-self-tolerant cells are eliminated via apoptosis. The remaining cells complete their development and exit the thymus as mature CD4pos and CD8pos cells (naı¨ve T cells) with the capacity for T cell-mediated immunity [19,20,22,23].

PRRSV and the Thymus The presence of PRRSV in the thymus was described shortly after the discovery of the virus [24]. In the initial report, virus was isolated from 3-week-old pigs inoculated with isolate Canada/LHVA-92-1, and virus-positive cells were observed in thymic tissues using immunohistochemical techniques [24]. Subsequently, viral antigen was detected in the thymus of 3-week-old pigs inoculated with isolate PRRSV-2 ATCC VR-2386 by using immunohistochemistry (IHC). Virus-antigen-positive cells were frequently identified as macrophages and were located near necrotic areas in the medulla and, less often, in the cortex [25]. Differences in virus concentration were observed in 5-week-old piglets inoculated with PRRSV-2 isolates of varying virulence (HuN4 and CH-1a) using TaqMan fluorescent quantitative reverse transcription PCR (RT-PCR), that is, the thymus of pigs inoculated with a highly pathogenic isolate (HuN4) showed higher virus concentration than pigs inoculated with a nonpathogenic isolate (CH-1a) (P <0.01, Student’s t-test) [26]. Similar differences in virus concentration were also observed in 5-week-old pigs inoculated with PRRSV-1 isolates of varying virulence (LV, SU1bel, and 215-06). By IHC, the number of immunostained cells in pigs inoculated with the more pathogenic isolate (SU1-bel) was significantly higher than in pigs inoculated with isolates LV or 205-06, and the lowest number of immunostained cells was observed in the group inoculated with the least pathogenic isolate (215-06) (P = 0.003, Pearson and Spearman tests) [27]. Other investigators also confirmed the presence of PRRSV in the thymus using various PRRSV isolates [13,28–35].

Effects Vary among PRRSV Isolates PRRSV-induced lesions described in early reports included the depletion of lymphocytes in the thymic cortex of 6-day-old piglets inoculated with Lelystad virus [36] and necrotic foci with pyknosis and karyorrhexis in the thymic medulla of 3-week-old pigs inoculated with PRRSV-2 isolate ATCC VR-2386 [25]. However, no lesions were observed in the thymus of 1-, 4-, and 10-week-old pigs inoculated with PRRSV-2 isolate ATCC VR-2332 [33]. These contradictory observations were resolved when research showed that the virus isolate was an important factor in determining the effect of PRRSV infection on the thymus. For example, severe thymic lesions (Figure 1C), characterized by numerous apoptotic cells and atrophy of the cortex, smaller thymic lobules, and a breakdown of the boundaries between the cortex and medulla (Figure 1F), were observed in 5-week-old pigs inoculated with PRRSV-2 isolate HuN4 [13,26]. In contrast, PRRSV-2 isolate CH-1a-inoculated pigs showed mild thymic atrophy, minor pathological changes (Figure 1G), and fewer apoptotic cells (P <0.01, Student’s t-test) [26]. Using TUNEL labeling to detect cell death, it was found that the number of TUNELpos cells per mm2 in the thymus of HuN4-inoculated pigs was significantly higher than that of CH-1a-inoculated pigs (P <0.01, Student’s t-test). Differences in the degree of pathology were also observed in 5-week-old pigs inoculated with PRRSV-1 isolates of varying virulence (LV, SU1-bel, and 215-06). Pigs inoculated with the most virulent isolate (SU1-bel) showed the most severe thymic pathology, primarily in the cortex at 7 DPI (Figure 1H), in association with a significant increase in apoptotic thymocytes at 3 and 7 DPI [27]. Using a variety of PRRSV isolates, other investigators [37–41] confirmed that thymic lesions were related to virus virulence and showed that attenuation of PRRSV by sequential passage on Marc-145 cells resulted in isolates that produced fewer thymic lesions than the parent isolate [42]. Likewise, the degree of alteration of thymus T cell populations is affected by virus isolate. In 5-weekold pigs, flow cytometric analyses at 0, 3, 7, and 14 DPI found a significant increase in the proportions

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Glossary CD3: cluster of differentiation 3 is a multimeric protein complex initially expressed in the cytoplasm of T cell precursors, that is, the stem cells from which T cells arise in the thymus. CD3 migrates to the cell membrane at the beginning of T cell precursor differentiation and covalently links with TCR. CD3 helps to activate both the CD8pos T cells and CD4pos T cells [101,102]. CD4: cluster of differentiation 4 is a glycoprotein found on the surface of various immune cells, for example, T helper cells, monocytes, and dendritic cells. CD4 is a coreceptor of the TCR and binds to an MHC molecule; specifically, the class II MHC protein [103]. CD8: cluster of differentiation 8 is a transmembrane glycoprotein that serves as a coreceptor for the TCR. Like TCR, CD8 binds to an MHC molecule – specifically, the class I MHC protein [104]. CD14: cluster of differentiation 14 is a glycoprotein, found mainly on the surface of monocytes and macrophages, that serves as a receptor for complexes of lipopolysaccharide (LPS) and lipopolysaccharide-binding protein (LBP). MHC: major histocompatibility complex is a set of cell-surface proteins essential for the acquired immune system to recognize foreign molecules in vertebrates. PRRSVs: porcine reproductive and respiratory syndrome viruses are small, enveloped, positive-sense, single-stranded RNA viruses in the genus Porartervirus, family Arteriviridae, order Nidovirales. Two species are recognized: PRRSV-1 and PRRSV-2. Regulatory T (Treg) cells: a population of CD4pos T cells characterized by the expression of transcription factor FOXP3 which plays important roles in preventing autoimmunity, maintaining tolerance, and modulating ongoing immune responses. TCR: T cell receptor (TCR) is a molecule on the surface of T cells responsible for recognizing fragments of antigen as peptides bound to major histocompatibility complex (MHC) molecules. Binding between TCR and antigen peptides is of relatively low affinity and degenerate, that is, many TCRs recognize the same antigen

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of CD4pos and CD8pos thymocytes and a reduction in CD4posCD8pos thymocytes when compared with CH-1a-infected pigs (P <0.01, Student’s t-test) and control pigs (P < 0.01, Student’s t-test) at each time point [26]. Differences in the degree of change in thymocyte populations changes were also observed in 5-week-old pigs inoculated with PRRSV-1 isolates of varying virulence (LV, SU1-bel, and 215-06), with pigs inoculated with the most virulent isolate (SU1-bel) showing the lowest proportion of CD3pos thymocytes [27].

Effect of PRRSV Infection on Specific Cell Types Early reports described thymic lesions, but did not identify the affected cell types. Virus-infected cells in 4-week-old pigs inoculated with PRRSV VR-2385 were generally located in the medulla, were round or triangular in shape, and resembled macrophages (Figure 2A, small arrow), or were stellate with at least one long cytoplasmic process more characteristic of interdigitating cells (Figure 2A, large arrow), or were unidentified multinucleated cells (Figure 2A, arrow head) [29]. Similar virus-infected cells were observed in aborted fetuses, stillborn piglets, or 7-day-old live-born piglets infected with PRRSV-2 SNUVR980606 in utero (Figure 2B) [43,44] or 12-day-old piglets infected with PRRSV-2 SD-23983 in utero (Figure 2C) [45].

peptide, and many antigen peptides are recognized by the same TCR [105,106]. T follicular helper cells: a CD4pos T cell subset essential for germinal center formation and maintenance and the development of longlived humoral immunity during infection and vaccination. T helper 17 (Th17) cells: a CD4pos T cell subset involved in the development of autoimmune diseases and host defense against fungi and extracellular bacteria. TUNEL: terminal dUTP nick endlabeling is used to detect DNA fragmentation, a key feature of late-stage apoptosis.

Figure 1. Thymus of Neonatal Pig and Different Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Isolates Induced Thymic Atrophy. (A) Thymus of a neonatal specific pathogen free (SPF) pig. (B,C) Thymus of an age-matched negative control piglet and the thymus of a PRRSV HuN4-infected piglet showing serious atrophy at 28 days post-infection (DPI), respectively [13] (with the permission of the publisher – 4598060710104). (D) Thymic histological sections stained with hematoxylin and eosin (H&E) from a neonatal SPF pig. (E–G) Thymic histological sections (H&E) from an age-matched negative control pig, HuN4-infected pig, and a CH-1a-infected pig at 10 DPI, respectively [26] (with the permission of the publisher – 4598061155158). (H–K) Thymic histological sections (H&E) from age-matched negative control, SU1-bel-infected, LV-infected, and 215-06-infected pigs at 7 DPI, respectively [27] (with the permission of the publisher – 4687540122202).

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Box 1. Positive and Negative Selection of Thymocytes Positive and negative selection during thymocyte development guarantees the production of functional, healthy T cells. Positive selection occurs in the thymic cortex when CD4posCD8pos cells expressing low levels of TCR are triggered to differentiate into CD4pos or CD8pos cells by weak interactions with MHC I/II molecules expressed on the surface of cortical epithelial cells. Negative selection occurs in both the cortex and medulla. The mechanism of negative selection in the cortex is not clearly understood, but occurs when CD4posCD8pos cells strongly interact with MHC ligands, which thereby induces these cells to undergo apoptosis [22]. In the thymic medulla, epithelial cells express tissue-restricted antigens that interact or are transferred to dendritic cells and, in turn, interact with developing CD4pos and CD8pos T cells. Autoreactive T cells are induced to undergo apoptosis, thereby assuring the production of self-tolerant mature T cells [19–23].

Advances in methodology, for example, the availability of anti-pig CD14 antibody and monoclonal anti-N protein antibody (SDOW17), made it possible to establish that PRRSV-infected cells in the thymus were CD14pos APC (Figure 2D) [46]. This is an immunologically important cell population, and the consequences of infection are discussed below. Double-labeling using IHC to detect virusinfected cells, and TUNEL to identify cell death, revealed that the majority of labeled cells were either PRRSV-infected or TUNELpos, but not both. In fact, TUNELpos cells were more numerous than PRRSVpos cells in live-born piglets inoculated with PRRSV-1 isolate SNUVR100058 or PRRSV-2 isolate SNUVR100059 [47]. Similar results were observed in 5-week-old piglets inoculated with PRRSV-2 isolate HuN4 [46]. Subsequent flow cytometric analyses showed a significant reduction in CD4posCD8pos cells in the thymus of HuN4-infected piglets (Figure 2E) [26] and led to the conclusion that the TUNELpos cells colocalized with CD3pos cells (Figure 2F) [46] were CD4posCD8pos cells. In addition to PRRSV infection in CD14pos APCs, and bystander apoptosis in CD4posCD8pos cells, PRRSV-induced autophagy has also been identified in the thymus of 4-week-old pigs inoculated with PRRSV-2 isolate HuN4 [48]. Using antikeratin antibody to identify thymic epithelial cells [49], and microtubule-associated protein 1 light chain 3 (LC3) as a marker of autophagy [50], it was shown that activated LC3pos cells were thymic epithelial cells (Figure 2G) [48]. This is significant because thymic epithelial cells are important APCs and likewise play a role in positive and negative selection of thymocytes via their cell surface MHC antigens (Box 1) [21,23].

Postulated Mechanisms by Which PRRSV May Affect Thymic Function Research into the mechanisms involved in PRRSV-induced thymic atrophy and thymocyte apoptosis is ongoing. Efforts to date have focused on understanding the direct and indirect effects of PRRSV on the thymus (Figure 3, Key Figure), as well as the extrathymic consequences of infection, for example, impact on prothymocytes derived from bone marrow, proinflammatory mediators, and glucocorticoids.

Reduced Prothymocyte Production in Bone Marrow PRRSV induces extensive cell apoptosis in bone marrow, both directly via infection and indirectly via a bystander effect. This results in acute bone marrow failure, that is, hypoplasia characterized by the absence of normal myeloid and erythroid precursors [51–53]. As the primary source of T cell precursors, it is reasonable to hypothesize that the bone marrow of PRRSV-infected pigs could fail to produce sufficient numbers of prothymocytes for populating the thymic cortex. Insufficient numbers of prothymocytes migrating from the bone marrow to the thymus and thymocytes undergoing apoptosis in the thymic cortex could contribute to thymic atrophy during PRRSV infection.

Deleterious Effect on Positive Selection Positive and negative selection during thymocyte development guarantees the production of mature, fully functional T cells (Box 1). This requires that thymocytes receive the appropriate signals for migration, proliferation, and differentiation from thymic APCs (cortical and medullary epithelial cells, macrophages, dendritic cells, etc.) positioned in specific thymic microenvironments [16,21,54]. It has

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Figure 2. Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Infects Thymic CD14pos AntigenPresenting Cells (APCs), Induces Apoptosis in Thymocytes and Autophagy in Thymic Epithelial Cells. (A) PRRSV in macrophages (small arrow), cells that resemble interdigitating cells (large arrow), and multinucleated cells (arrowhead) following inoculation with PRRSV VR2385. Immunoperoxidase, and hematoxylin counterstain. Bar = 21 mm [29] (with the permission of the publisher – 4687550433409). (B) In a stillborn pig inoculated in utero with PRRSV-2 SNUVR980606, PRRSV-positive (red reaction) macrophages in the medulla at 9 days post-infection (DPI). Fast red, hematoxylin counterstain. Magnification, 3193 [43] (with the permission of the publisher – 4687551300997). (C) Following inoculation with PRRSV-2 SD 23983, thymus cells were positive for virus antigen by immunohistochemistry using SDOW 17 mAb. Arrows indicate PRRSV-positive cells. Magnification, 3300 [45] (with the permission of the publisher – 4687560489344). (D) Following inoculation with PRRSV-2 HuN4, CD14pos APCs stain green, PRRSVpos cells are red, and virus-infected CD14pos APC cells (double-stained cells) are yellow [46] (with the permission of the publisher – 4598070263077). (E) Flow cytometric comparison of CD4posCD8pos thymocytes in HuN4-infected, CH-1a-infected, and control pigs (*P < 0.05, **P < 0.01) [26] (with the permission of the publisher – 4598061155158). (F) Inoculation with PRRSV-2 HuN4, TUNELpos cells are red, CD3pos cells are green, and virus-induced apoptotic CD3pos cells (double-stained cells) are yellow [46] (with the permission of the publisher – 4598070263077). (G) In a pig inoculated with PRRSV-2 HuN4, activated LC3pos cells are red, keratinpos cells are green, and virus induced autophagic thymic epithelial cells (double-stained cells) are yellow [48].

been shown that autophagy in epithelial cells compromises MHC II-restricted thymic T cell selection in vivo [50,55]. Therefore, the presence of large numbers of apoptotic cells in the PRRSV-infected thymus suggests the possibility that virus-infected CD14pos APCs and autophagic thymic epithelial cells are unable to interact correctly with the developing thymocytes, thereby leading to a breakdown in the process of positive selection and a reduction in the number of developmentally mature T cells

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Key Figure

Postulated Mechanisms of Thymocyte Apoptosis and Immunomodulation Induced by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)

Figure 3. Postulated mechanisms by which PRRSV may affect thymic functions include reduced prothymocyte production in bone marrow; alterations in the normal process of positive selection; delivery of proapoptotic signals to thymocytes by thymic macrophages; and glucocorticoids. Consequences include T cell lymphopenia and abnormal peripheral blood mononuclear cell (PBMC) populations; disordered cytokine responses [interferon (IFN)-g, interleukin (IL)1b, IL-4, IL-10, tumor necrosis factor (TNF)-a, etc.] as a result of altered innate and cellular-mediated immune responses; regulatory T cell (Treg) and compromised antigen processing for PRRSV and other pathogens; and delayed development of neutralizing antibodies against PRRSV. Thus, the net effect on the thymus is chronic, persistent PRRSV infection and compromised immune functions resulting in polymicrobial infections.

available to the pig’s immune system [46,48,56]. If this hypothesis were substantiated, then exploration of a therapeutic approach to stimulate the production of positively selected thymocytes in PRRSV-infected pigs would be justified. Exogenous strategies for achieving the recovery of thymic immune function have not been defined, but therapies currently in clinical trials include exogenous administration of recombinant keratinocyte growth factor (KGF), interleukin (IL)-7, and IL-22, hormonal modulation, and the use of precursor T cells and thymus bioengineering (see review in [57]).

Thymic Macrophages May Deliver Proapoptotic Signals to Thymocytes Tumor necrosis factor-a (TNF-a) is a proapoptotic cytokine and, in HIV infection, CD8pos apoptosis is mediated by the interaction between TNF-a bound to the membrane of macrophages and TNF-receptor II on CD8pos T cells [58]. In the PRRSV-1 infected thymus, immunostaining detected TNF-a primarily in the cytoplasm of thymic macrophages and, to a lesser extent, in the cytoplasm of neutrophils and lymphocytes in the thymic medulla [27]. Whether TNF-a in thymic macrophages contributes to thymocyte apoptosis is undetermined, but warrants further investigation.

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Glucocorticoids Induced by PRRSV Contribute to Thymocyte Apoptosis CD4posCD8pos thymocytes are particularly sensitive to glucocorticoids via the activation of caspase-3, -8, and -9 [59–61]. PRRSV-2 infection was shown to upregulate serum glucocorticoid levels in infected pigs from 3 DPI to 10 DPI [62] and induced intrathymic caspase-3, -8, and -9 activation at 10 DPI [63]. On the other hand, inhibition of glucocorticoid production prior to rabies virus infection prevented virus-induced thymocyte depletion in mice, providing further evidence that thymocyte apoptosis is related to increased serum glucocorticoid levels [64,65]. To date, attempts to moderate the thymocyte apoptotic response by reducing the level of serum TNF-a levels, stimulated by PRRSV-2 infection [66,67], have not met with success. That is, intramuscular administration of dexamethasone (DEX) during HP-PRRSV-2 (HuN4) infection inhibited the upregulation of serum TNF-a, but accelerated thymic atrophy and clinical signs (J. Tong, MS Thesis, Chinese Academy of Agricultural Sciences, 2015) [62]. Likewise, intraperitoneal administration of Mifepristone (RU486), a competitive glucocorticoid receptor antagonist [68], during PRRSV-2 HuN4 infection enhanced the expression of clinical disease and induced more severe thymic atrophy (J. Tong, MS Thesis, Chinese Academy of Agricultural Sciences, 2015) [62]. Possible explanations for these results include several possibilities, for example, PRRSV infection-induced thymic atrophy might occur independently of increased systemic glucocorticoids levels; or glucocorticoids might synergize with other mediators within the thymus to induce thymocyte apoptosis; perhaps the route of administration affects the outcome at the cellular level.

Viral Genetic Determinants The degree of PRRSV-induced thymic atrophy is associated with the virulence of the infecting isolate, as discussed above in the section ’Effects Vary among PRRSV Isolates’. Genetic studies initially led to the hypothesis that nonstructural protein (Nsp) 9 played a central role in the virulence of Chinese PRRSV-2 isolates [69–71]. Specifically, alignment of full-length sequences from 204 PRRSV-2 isolates revealed two amino acid mutations (positions 519 and 544 in Nsp 9) that differed consistently between highly pathogenic and classical PRRSV-2 isolates. HuN4 and CH-1a infectious cDNA clones (rHuN4-T519S-A544T, and rCH-1a-S519T-T544A) were constructed by the replacement of double amino acids from CH-1a to HuN4 or from HuN4 to CH-1a, respectively. Comparison of thymus-tobody weight ratios of 5-week-old piglets inoculated with rHuN4-T519S-A544T, rCH-1a-S519TT544A, rCH-1a, or rHuN4 showed that the two consistent amino acid mutations (positions 519 and 544 in Nsp 9) played a role, but were not the only factors that contributed to HuN4-induced thymic atrophy [71]. Thus, the other virus-related factors that contribute to thymic atrophy and thymocyte apoptosis remain to be identified.

The PRRSV-Compromised Thymus PRRSV induces abnormal innate and adaptive immune responses, that is, it suppresses type I interferon (IFN) induction [6–8], deregulates cytokine expression [11–13], and delays the development of neutralizing antibody [9,10]. Achieving a full understanding of the mechanisms involved is a work-in-progress, but thymic atrophy may be part of an overall PRRSV strategy to subvert immune responses and achieve chronic persistent infection in the host.

Consequences for the Porcine Fetus PRRSV can be detected in the thymus of piglets infected in utero, including aborted fetuses, stillborn, and live-born piglets [72]. The concentration of PRRSV in fetal thymus is higher than it is in other fetal organs and, on that basis, the thymus is considered a primary site for PRRSV replication [47,73–77]. Neonatal piglets that survive in utero PRRSV infection show effects similar to those previously discussed, that is, thymic atrophy and apoptosis, altered thymocyte populations, and alterations to innate and adaptive immune responses [45,47,78,79].

Consequences for the Innate Immune Response PRRSV-susceptible thymic CD14pos APCs and other monocyte-macrophage lineage cells [46,53,80–82], including immature or mature dendritic cells, play a variety of important immunological roles, for example, antigen recognition, processing, and presentation to T and B cells, as well as production of

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innate immune response-related cytokines [83]. The ability of PRRSV infection to suppress type I IFN induction has been extensively documented [84–86]. An additional complexity in understanding the mechanisms involved in this process is the fact that changes in cytokine responses are variable among PRRSV isolates, that is, infection by PRRSV-2 (HuN4) or PRRSV-1 (Lena) elevate levels of IFN type I and other cytokines associated with innate immunity [66,67,86,87]. As the first line of defense against microbial infection, PRRSV-susceptible thymic CD14pos APCs and other related monocyte-macrophage lineage cells’ contribution and role in the PRRSV-modulated innate immune response requires further investigation.

Consequences for Cell-Mediated Immunity Thymic activity is essential for maintaining or reconstituting a pool of functional peripheral T cells [88]. PRRSV infection affects normal thymic functions, which then leads to a decline and alteration of naı¨ve T cells among newly differentiated T cells. This manifests as PRRSV-related lymphopenia in peripheral blood mononuclear cells (PBMCs), that is, a decrease in CD4pos T cells and an increase in CD8pos and CD4posCD8pos T cells, from 3 to 14 DPI [26,79,89–91]. The consequence is a weak and delayed adaptive immune response, as indicated by changes in T cell differentiation and function, for example, as manifested by disordered cytokine responses (IFN-g, IL-1b, IL-4, IL-10, TNF-a, etc.) [84–86,92,93]. Ongoing research on the mechanisms by which PRRSV alters cell-mediated immunity, including its effect on thymic functions, will eventually provide a clearer understanding of the process.

Consequences for the Development of Neutralizing Antibodies Thymocytes require the appropriate signals from thymic APCs to correctly carry out the process of migration, proliferation, and differentiation. The correct signaling required for this process is altered in PRRSV-infected APCs and autophagic thymic epithelial cells. Clearly established is the fact that CD4pos T cells (T follicular helper cells) provide signals to B cells for antibody affinity maturation and direct immunoglobulin class switching to the antiviral IgG2a/c subclass during viral infection [94,95]. In PRRSV infection, decreased numbers of peripheral helper T cells, caused by the loss of precursor CD4posCD8pos thymocytes, explains the delayed appearance of high-affinity neutralizing antibodies against the virus [9,10]. Combined with the function of CD4pos T cells in contributing to neutralizing antibody production [94,95], the relationship between the PRRSV-induced decline in CD4pos T cells and the delayed appearance of neutralizing antibodies against PRRSV should be a priority for future investigation. It has been hypothesized that PRRSV infection might also affect the emerging T cell repertoire and their ability to recognize foreign epitopes, thereby resulting in abnormal peripheral regulatory T (Treg) cells, T helper (Th)17 cells, potentially autoreactive cells, and PRRSV-specific T cell tolerance [10,78,96–99]. Both the known immediate effects of PRRSV and the possibility of residual or longterm effects on the immune system represent a serious compromise for pig health and the pigs’ ability to resist other pathogens (Figure 3).

Concluding Remarks Economic studies have shown that PRRSV inflicts major losses on swine health and productivity, for example, in Europe and North America the cost of PRRSV to the swine industry has been estimated at $6.25 to $15.25 USD per pig marketed [100]. The routinely reliable control of PRRSV is presently beyond our reach, but the pathway forward must be built on a well-founded understanding of the mechanisms by which PRRSV exerts its effects. At one time considered a vestigial structure with no significant function, the thymus is now regarded as an essential organ in the immune system [21]. Although few studies have considered whether and how specific pathogens affect thymic functions, recent studies have shown that PRRSV infection has both direct and indirect effects on the thymus, including the disruption of thymic architecture and function and a cascading impact on immune responses against PRRSV and other infections (Figure 3). These observations raise questions that must be resolved (see Outstanding Questions) if we are to develop methods able to defend the thymus from the effects of PRRSV infection and maintain the

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Outstanding Questions Although PRRSV compromises T cell immunity, thymic atrophy might represent a strategy employed by the body to avoid the generation of an immune repertoire tolerant to PRRSV. This raises the question of whether thymic atrophy is caused by PRRSV infection or by the body’s responses to PRRSV. Is atrophy beneficial for the body or does it benefit PRRSV? What are the precise mechanisms of infection-induced thymocyte apoptosis? Could we manipulate the signal pathway to protect the healthy function of the thymus and restore the normal immune response? The limited data currently available preclude drawing general conclusions on the mechanism(s) of thymocyte apoptosis, but research on this question may ultimately lead to new strategies against the disease. Since PRRSV infection results in a decline in the number of CD4pos T cells, T follicular helper cells would likewise be decreased. Does the PRRSV-induced decrease in CD4pos T cells explain the delay in the production of neutralizing antibodies against PRRSV? Does PRRSV infection impose residual or long-term effects on the ability of the thymus to function normally? That is, does PRRSV infection have a long-lasting effect on the immune system? The efficacy of traditional vaccines relies on typical immune responses to specific antigens. However, PRRSV infection affects thymus and bone marrow and, thereby, modulates normal immune responses. Can alternative vaccines or therapeutics be developed to provide better efficacy against PRRSV infection?

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integrity of the immune system. Even more intriguing is the prospect that understanding the details of the interactions between PRRSV and the thymus could lead to the development of new vaccines or drugs able to protect against other pathogens that target the immune system, for example, HIV, classical swine fever virus (CSFV), or African swine fever virus.

Acknowledgments We thank Dr Tongqing An and Fandan Meng (Harbin Veterinary Research Institute) for critical review of the manuscript. This work was supported by grants from the Heilongjiang Province Natural Science Foundation (grant number C2016068), the State Key Laboratory of Veterinary Biotechnology Foundation (grant number SKLVBP2018002), and the China Scholarship Council (grant number No. 201703250019).

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