Host immune responses to porcine circovirus type 2d infection in C57BL/6 mice: lymphocyte activation, thymic T-cell dynamics, and Treg expansion

Abstract

Porcine circovirus type 2 (PCV2), a major immunosuppressive pathogen, causes substantial economic losses to the global swine industry. PCV2d is the dominant genotype in multiple regions, but its immunopathological mechanisms, particularly virus‒host immune crosstalk, remain unclear. Here, we evaluated immune responses to a rescued replication-competent PCV2d in C57BL/6 mice, focusing on lymphocyte activation, thymic T-cell apoptosis and dynamics, T-cell immunophenotype shifts, and splenic regulatory T (Treg) cell dynamics. CD69-based assessment of lymphocyte activation revealed time-specific patterns: CD8⁺ T cells were activated throughout infection, with stronger activation at 7–14 days post-infection (dpi); γδ T cells were activated only early; CD4⁺ T cells and natural killer (NK) cells were activated late (14–28 dpi); and B cells were persistently activated. Thymic analysis revealed subset-specific apoptosis; for example, CD8⁺ T cells underwent early apoptosis across all stages, immature CD4⁻CD8⁻ T cells were vulnerable late, and altered dynamics were detected: CD4⁺ and CD8⁺ T cells first increased and then decreased from early to late infection, while CD4⁺CD8⁺ T cells showed the opposite trend. T-cell immunophenotyping with CD44 and CD62L revealed reduced numbers of naive CD4⁺/CD8⁺ T cells, increased numbers of memory T cells, and upregulated the expression of PD-1, a marker of T-cell exhaustion. Splenic Treg cells (CD4⁺CD25⁺Foxp3⁺) expanded, with a peak at 21 dpi. These findings demonstrate the multifaceted immunomodulatory effects of PCV2d, including innate activation, adaptive immune suppression, thymic dysfunction, T-cell exhaustion, and Treg-mediated immunosuppression. This work improves our understanding of PCV2d pathogenesis, aiding the development of targeted control strategies and vaccines for swine PCV2.

Keywords

PCV2d / Lymphocyte activation / Thymic T-cell apoptosis / T-cell exhaustion / Regulatory T (Treg) cells / C57BL/6 mouse model

References

[1] Agata Y, Kawasaki A, Nishimura H, Ishida Y, Tsubata T, Yagita H, Honjo T. Expression of the PD-1 antigen on the surface of stimulated mouse T and B lymphocytes. International Immunology, 1996, 8(5): 765-772

[2] Akondy RS, Fitch M, Edupuganti S, Yang S, Kissick HT, Li KW, Youngblood BA, Abdelsamed HA, McGuire DJ, Cohen KW, et al. . Origin and differentiation of human memory CD8 T cells after vaccination. Nature, 2017, 552(7685): 362-367

[3] Barber DL, Wherry EJ, Masopust D, Zhu B, Allison JP, Sharpe AH, Freeman GJ, Ahmed R. Restoring function in exhausted CD8 T cells during chronic viral infection. Nature, 2006, 439(7077): 682-687

[4] Belkaid Y, Rouse BT. Natural regulatory T cells in infectious disease. Nature Immunology, 2005, 6(4): 353-360

[5] Bertoletti A, Gehring AJ. The immune response during hepatitis B virus infection. Journal of General Virology, 2006, 87(Pt 6): 1439-1449

[6] Boettler T, Spangenberg HC, Neumann-Haefelin C, Panther E, Urbani S, Ferrari C, Blum HE, von Weizsäcker F, Thimme R. T cells with a CD4+CD25+ regulatory phenotype suppress in vitro proliferation of virus-specific CD8+ T cells during chronic hepatitis C virus infection. Journal of Virology, 2005, 79(12): 7860-7867

[7] Brandt L, Benfield T, Mens H, Clausen LN, Katzenstein TL, Fomsgaard A, Karlsson I. Low level of regulatory T cells and maintenance of balance between regulatory T cells and TH17 cells in HIV-1-infected elite controllers. Journal of Acquired Immune Deficiency Syndromes, 2011, 57(2): 101-108

[8] Chen Y, Fan G, Yang B, Fan X, Chen H, Ma Z, Lou J, Xu J, Wang Y, Zhang S. The single-cell transcriptional landscape of lung cells from PCV2d-infected mice. Frontiers in Microbiology, 2025, 16: 1554961

[9] Cho H, Kang H, Kim CW, Kim HY, Jang JW, Yoon SK, Lee CD. Phenotypic Characteristics of PD-1 and CTLA-4 Expression in Symptomatic Acute Hepatitis A. Gut Liver, 2016, 10(2): 288-294

[10] Darwich L, Pié S, Rovira A, Segalés J, Domingo M, Oswald IP, Mateu E. Cytokine mRNA expression profiles in lymphoid tissues of pigs naturally affected by postweaning multisystemic wasting syndrome. Journal of General Virology, 2003, 84(Pt 8): 2117-2125

[11] Du Q, Zhang H, He M, Zhao X, He J, Cui B, Yang X, Tong D, Huang Y. Interleukin-10 promotes porcine circovirus type 2 persistent infection in mice and aggravates the tissue lesions by suppression of T-cell infiltration. Frontiers in Microbiology, 2019, 10 2050

[12] Eclercy J, Larcher T, Andraud M, Renson P, Bernard C, Bigault L, Ledevin M, Paboeuf F, Grasland B, Rose N, et al. . PCV2 coinfection does not impact PRRSV MLV1 safety but enhances virulence of a PRRSV MLV1-like strain in infected SPF pigs. Veterinary Microbiology, 2020, 244 108656

[13] Franzo G, Segalés J. Porcine circovirus 2 genotypes, immunity and vaccines: Multiple genotypes but one single serotype. Pathogens, 2020

[14] Fu Q, Hou L, Xiao P, Guo C, Chen Y, Liu X. CD44 deficiency leads to decreased proinflammatory cytokine production in lung induced by PCV2 in mice. Research in Veterinary Science, 2014, 97(3): 498-504

[15] Fuller MJ, Callendret B, Zhu B, Freeman GJ, Hasselschwert DL, Satterfield W, Sharpe AH, Dustin LB, Rice CM, Grakoui A, et al. . Immunotherapy of chronic hepatitis C virus infection with antibodies against programmed cell death-1 (PD-1). Proc Natl Acad Sci U S A, 2013, 110(37): 15001-15006

[16] Ghosh C, Luong G, Sun Y. A snapshot of the PD-1/PD-L1 pathway. Journal of Cancer, 2021, 12(9): 2735-2746

[17] Gupta S, Su H, Agrawal S. Immune response to SARS-CoV-2 vaccine in 2 men. International Archives of Allergy and Immunology, 2022, 183(3): 350-359

[18] Hamel AL, Lin LL, Nayar GP. Nucleotide sequence of porcine circovirus associated with postweaning multisystemic wasting syndrome in pigs. Journal of Virology, 1998, 72(6): 5262-5267

[19] Harding JC, Ellis JA, McIntosh KA, Krakowka S. Dual heterologous porcine circovirus genogroup 2a/2b infection induces severe disease in germ-free pigs. Veterinary Microbiology, 2010, 145(3-4): 209-219

[20] Jeurissen SH, Wagenaar F, Pol JM, van der Eb AJ, Noteborn MH. Chicken anemia virus causes apoptosis of thymocytes after in vivo infection and of cell lines after in vitro infection. Journal of Virology, 1992, 66(12): 7383-7388

[21] Jiang CG, Wang G, Tu YB, Liu YG, Wang SJ, Cai XH, An TQ. Genetic analysis of porcine circovirus type 2 in China. Archives of Virology, 2017, 162(9): 2715-2726

[22] Jiang H, Wang D, Wang J, Zhu S, She R, Ren X, Tian J, Quan R, Hou L, Li Z, et al. . Induction of porcine dermatitis and nephropathy syndrome in piglets by infection with porcine circovirus type 3. Journal of Virology, 2019, 93(4 e02045-18

[23] Josefowicz SZ, Lu LF, Rudensky AY. Regulatory T cells: Mechanisms of differentiation and function. Annual Review of Immunology, 2012, 30: 531-564

[24] Joshi NS, Kaech SM. Effector CD8 T-cell development: A balancing act between memory cell potential and terminal differentiation. Journal of Immunology, 2008, 1803): 1309-1315

[25] Kaech SM, Cui W. Transcriptional control of effector and memory CD8+ T-cell differentiation. Nature Reviews. Immunology, 2012, 12(11): 749-761

[26] Karuppannan AK, Opriessnig T. Porcine circovirus type 2 (PCV2) vaccines in the context of current molecular epidemiology. Viruses, 2017

[27] Klenerman P, Oxenius A. T-cell responses to cytomegalovirus. Nature Reviews Immunology, 2016, 16(6): 367-377

[28] Lai GH, Lien YY, Lin MK, Cheng JH, Tzen JT, Sun FC, Lee MS, Chen HJ, Lee MS. VP2 of chicken anemia virus interacts with apoptin for downregulation of apoptosis through dephosphorylated threonine 108 on apoptin. Scientific Reports, 2017, 7(1): 14799

[29] Lanzavecchia A, Sallusto F. Dynamics of T lymphocyte responses: Intermediates, effectors, and memory cells. Science, 2000, 290(5489): 92-97

[30] Larrubia JR, Benito-Martínez S, Miquel J, Calvino M, Sanz-de-Villalobos E, Parra-Cid T. Costimulatory molecule programmed death-1 in the cytotoxic response during chronic hepatitis C. World Journal of Gastroenterology, 2009, 15(41): 5129-5140

[31] Li J, Yu T, Zhang F, Wang X, Zhou J, Gao X, Gao S, Liu X. Inactivated chimeric porcine circovirus (PCV) 1-2 vaccines based on genotypes 2b and 2d exhibit similar immunological effectiveness in protecting pigs against challenge with PCV2b strain 0233. Archives of Virology, 2017, 162(1): 235-246

[32] Li S, Liu J, Meng L, Yin S, Wu H, Zou J, Yuan D, He H, Yin G, Jia X, et al. . Cellular immune signatures and differences of four porcine circovirus type 2 vaccines to heterologous PCV2d infection. NPJ Vaccines, 2025, 10(1): 92

[33] Liu J, Chen I, Kwang J. Characterization of a previously unidentified viral protein in porcine circovirus type 2-infected cells and its role in virus-induced apoptosis. Journal of Virology, 2005, 79(13): 8262-8274

[34] Mankertz A, Mankertz J, Wolf K, Buhk HJ. Identification of a protein essential for replication of porcine circovirus. Journal of General Virology, 1998, 79(2): 381-384

[35] Mankertz A, Caliskan R, Hattermann K, Hillenbrand B, Kurzendoerfer P, Mueller B, Schmitt C, Steinfeldt T, Finsterbusch T. Molecular biology of porcine circovirus: Analyses of gene expression and viral replication. Veterinary Microbiology, 2004, 98(2): 81-88

[36] Mazimpaka E, Ringo RS, Hirooka T, Okabayashi T. Efficacy of PCV2 vaccination under natural conditions: A longitudinal study using PCR and virus isolation. Veterinary Sciences, 2025

[37] Nilsson J, Boasso A, Velilla PA, Zhang R, Vaccari M, Franchini G, Shearer GM, Andersson J, Chougnet C. HIV-1-driven regulatory T-cell accumulation in lymphoid tissues is associated with disease progression in HIV/AIDS. Blood, 2006, 108(12): 3808-3817

[38] Noteborn MH, de Boer GF, van Roozelaar DJ, Karreman C, Kranenburg O, Vos JG, Jeurissen SH, Hoeben RC, Zantema A, Koch G. Characterization of cloned chicken anemia virus DNA that contains all elements for the infectious replication cycle. Journal of Virology, 1991, 65(6): 3131-3139

[39] Pinchuk LM, Filipov NM. Differential effects of age on circulating and splenic leukocyte populations in C57BL/6 and BALB/c male mice. Immunity & Ageing, 2008, 5 1

[40] Qin Y, Xu Z, Wang Y, Li X, Cao H, Zheng SJ. VP2 of infectious bursal disease virus induces apoptosis by triggering oral cancer overexpressed 1 (ORAOV1) protein degradation. Frontiers in Microbiology, 2017, 8: 1351

[41] Raidal SR, Firth GA, Cross GM. Vaccination and challenge studies with psittacine beak and feather disease virus. Australian Veterinary Journal, 1993, 70(12): 437-441

[42] Rovira A, Balasch M, Segalés J, García L, Plana-Durán J, Rosell C, Ellerbrok H, Mankertz A, Domingo M. Experimental inoculation of conventional pigs with porcine reproductive and respiratory syndrome virus and porcine circovirus 2. Journal of Virology, 2002, 76(7): 3232-3239

[43] Rutebemberwa A, Ray SC, Astemborski J, Levine J, Liu L, Dowd KA, Clute S, Wang C, Korman A, Sette A, et al. . High-programmed death-1 levels on hepatitis C virus-specific T cells during acute infection are associated with viral persistence and require preservation of cognate antigen during chronic infection. The Journal of Immunology, 2008, 181(12): 8215-8225

[44] Sakaguchi S, Yamaguchi T, Nomura T, Ono M. Regulatory T cells and immune tolerance. Cell, 2008, 133(5): 775-787

[45] Sallusto F, Lenig D, Förster R, Lipp M, Lanzavecchia A. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature, 1999, 401(6754): 708-712

[46] Sann S, Heng B, Vo HTM, Arroyo Hornero R, Lay S, Sorn S, Ken S, Ou TP, Laurent D, Yay C, et al. . Increased frequencies of highly activated regulatory T cells skewed to a T helper 1-like phenotype with reduced suppressive capacity in dengue patients. Mbio, 2024, 15(6 e0006324

[47] Schmitz I, Schneider C, Fröhlich A, Frebel H, Christ D, Leonard WJ, Sparwasser T, Oxenius A, Freigang S, Kopf M. IL-21 restricts virus-driven Treg cell expansion in chronic LCMV infection. PLoS Pathogens, 2013, 95 e1003362

[48] Shen T, Zheng J, Xu C, Liu J, Zhang W, Lu F, Zhuang H. PD-1 expression on peripheral CD8+ TEM/TEMRA subsets closely correlated with HCV viral load in chronic hepatitis C patients. Virol J, 2010, 7: 310

[49] Terme M, Ullrich E, Aymeric L, Meinhardt K, Desbois M, Delahaye N, Viaud S, Ryffel B, Yagita H, Kaplanski G, et al. . IL-18 induces PD-1-dependent immunosuppression in cancer. Cancer Research, 2011, 7116): 5393-5399

[50] Tischer I, Gelderblom H, Vettermann W, Koch MA. A very small porcine virus with circular single-stranded DNA. Nature, 1982, 295(5844): 64-66

[51] Trautmann L, Janbazian L, Chomont N, Said EA, Gimmig S, Bessette B, Boulassel MR, Delwart E, Sepulveda H, Balderas RS, et al. . Upregulation of PD-1 expression on HIV-specific CD8+ T cells leads to reversible immune dysfunction. Nature Medicine, 2006, 12(10): 1198-1202

[52] Vaziry A, Silim A, Bleau C, Frenette D, Lamontagne L. Chicken infectious anemia vaccinal strain persists in the spleen and thymus of young chicks and induces thymic lymphoid cell disorders. Avian Pathology, 2011, 40(4): 377-385

[53] Wan X, Yang R, Liu G, Zhu M, Zhang T, Liu L, Wu R. Downregulation of cellular prion protein inhibited the proliferation and invasion and induced apoptosis of Marek's disease virus-transformed avian T cells. Journal of Veterinary Science, 2016, 17(2): 171-178

[54] Wang H, Zhang N, Xu R, Ji C, Wei Y, Mi Q. The PD-1/PD-L1 pathway and Epstein-Barr virus. European Journal of Medical Research, 2025, 30(1): 486

[55] Wherry EJ, Teichgräber V, Becker TC, Masopust D, Kaech SM, Antia R, von Andrian UH, Ahmed R. Lineage relationship and protective immunity of memory CD8 T-cell subsets. Nature Immunology, 2003, 4(3): 225-234

[56] Xiao CT, Halbur PG, Opriessnig T. Complete genome sequence of a novel porcine circovirus type 2b variant present in cases of vaccine failures in the United States. Journal of Virology, 2012, 86(22): 12469

[57] Xu D, Fu J, Jin L, Zhang H, Zhou C, Zou Z, Zhao JM, Zhang B, Shi M, Ding X, et al. . Circulating and liver resident CD4+CD25+ regulatory T cells actively influence the antiviral immune response and disease progression in patients with hepatitis B. The Journal of Immunology, 2006, 177(1): 739-747

[58] Xu Y, Chaudhury A, Zhang M, Savoldo B, Metelitsa LS, Rodgers J, Yustein JT, Neilson JR, Dotti G. Glycolysis determines dichotomous regulation of T-cell subsets in hypoxia. The Journal of Clinical Investigation, 2016, 126(7): 2678-2688

[59] Yu S, Shang P. A review of bioeffects of static magnetic field on rodent models. Progress in Biophysics and Molecular Biology, 2014, 114(1): 14-24

[60] Yu C, Cao M, Wei Y, Liu J, Zhang H, Liu C, Feng L, Huang L. Evaluation of cross-immunity among major porcine circovirus type 2 genotypes by infection with PCV2b and PCV2d circulating strains. Veterinary Microbiology, 2023, 283 109796

[61] Zhang Z, Zhang JY, Wherry EJ, Jin B, Xu B, Zou ZS, Zhang SY, Li BS, Wang HF, Wu H, et al. . Dynamic programmed death 1 expression by virus-specific CD8 T cells correlates with the outcome of acute hepatitis B. Gastroenterology, 2008, 134(7): 1938-1949, 1949.e1931-1933

[62] Zhou J, Zhu N, Dai Q, Sun H, Zhao J, Qiu Y, Zhou B, Wang D, Cui Y, Guo J, et al. . DEAD-box RNA helicase 10 inhibits porcine circovirus type 3 replication by interacting with the viral capsid protein and activating interferon responses. Journal of Virology, 2025, 996 e00576-25

Funding

National Key Research and Development Program of China(2022YFD1800300)

RIGHTS & PERMISSIONS

The Author(s)