Although viruses are simple in structure and composition, their interactions with host cells are complex. Merely to gain entry, animal viruses make use of a repertoire of cellular processes that involve hundreds of cellular proteins. Although some viruses have the capacity to penetrate into the cytosol directly through the plasma membrane, most depend on endocytic uptake, vesicular transport through the cytoplasm, and delivery to endosomes and other intracellular organelles. The internalization may involve clathrin-mediated endocytosis (CME), macropinocytosis, caveolar/lipid raft-mediated endocytosis, or a variety of other still poorly characterized mechanisms. This review focuses on the cell biology of virus entry and the different strategies and endocytic mechanisms used by animal viruses.

Virus entry by endocytosis.

Porcine circovirus type 2 (PCV2)-associated disease (PCVAD) continues to be an important differential diagnosis on pig farms in the United States and worldwide. Case trend analyses indicate that the incidence of PCVAD is on the rise in the United States. Accurate diagnosis is important in order to implement appropriate intervention strategies. PCVAD can manifest as a systemic disease, as part of the respiratory disease complex, as an enteric disease, as porcine dermatitis and nephropathy syndrome, or as reproductive problems. PCVAD may be only a sporadic individual animal diagnosis; however, PCVAD may also manifest as a severe herd problem accelerated and enhanced by concurrent virus or bacterial infections. This article is intended to discuss the most common disease manifestations, pathogenesis, diagnostic approaches, and intervention strategies associated with PCVAD in North America.

https://journals.sagepub.com/doi/pdf/10.1177/104063870701900601

Porcine circovirus type 2-associated disease: Update on current terminology, clinical manifestations, pathogenesis, diagnosis, and intervention strategies

Porcine circovirus type 2 (PCV2) is a member of the family Circoviridae, a recently established virus family composed of small, non-enveloped viruses, with a circular, single-stranded DNA genome. PCV2, which is found all over the world in the domestic pig and probably the wild boar, has been recently associated with a number of disease syndromes, which have been collectively named porcine circovirus diseases (PCVD). Postweaning multisystemic wasting syndrome (PMWS), porcine dermatitis and nephropathy syndrome (PDNS) and reproductive disorders are the most relevant ones. Among them, only PMWS is considered to have a severe impact on domestic swine production. PMWS mainly affects nursery and/or fattening pigs; wasting is considered the most representative clinical sign in this disease. Diagnosis of this disease is confirmed by histopathological examination of lymphoid tissues and detection of a moderate to high amount of PCV2 in damaged tissues. Since PMWS is considered a multifactorial disease in which other factors in addition to PCV2 are needed in most cases to trigger the clinical disease, effective control measures have focused on the understanding of the co-factors involved in individual farms and the control or elimination of these triggers. PDNS, an immuno-complex disease characterized by fibrino-necrotizing glomerulonephritis and systemic necrotizing vasculitis, has been linked to PCV2, but a definitive proof of this association is still lacking. PCV2-associated reproductive disease seems to occur very sporadically under field conditions, but it has been characterized by late-term abortions and stillbirths, extensive fibrosing and/or necrotizing myocarditis in fetuses and the presence of moderate to high amounts of PCV2 in these lesions. Taking into account that scientific information on PCV2 and its associated diseases has been markedly expanded in the last 8 years, the objective of this review is to summarize the current state of knowledge of the most relevant aspects of PCV2 biology and PCVD.

https://www.cambridge.org/core/services/aop-cambridge-core/content/view/S1466252305000083

Porcine circovirus diseases.

Abstract Porcine circovirus type 2 (PCV2) is a member of the family Circoviridae, a recently established virus family composed of small, non-enveloped viruses, with a circular, single-stranded DNA genome. PCV2, which is found all over the world in the domestic pig and probably the wild boar, has been recently associated with a number of disease syndromes, which have been collectively named porcine circovirus diseases (PCVD). Postweaning multisystemic wasting syndrome (PMWS), porcine dermatitis and nephropathy syndrome (PDNS) and reproductive disorders are the most relevant ones. Among them, only PMWS is considered to have a severe impact on domestic swine production. PMWS mainly affects nursery and/or fattening pigs; wasting is considered the most representative clinical sign in this disease. Diagnosis of this disease is confirmed by histopathological examination of lymphoid tissues and detection of a moderate to high amount of PCV2 in damaged tissues. Since PMWS is considered a multifactorial disease in which other factors in addition to PCV2 are needed in most cases to trigger the clinical disease, effective control measures have focused on the understanding of the co-factors involved in individual farms and the control or elimination of these triggers. PDNS, an immuno-complex disease characterized by fibrino-necrotizing glomerulonephritis and systemic necrotizing vasculitis, has been linked to PCV2, but a definitive proof of this association is still lacking. PCV2-associated reproductive disease seems to occur very sporadically under field conditions, but it has been characterized by late-term abortions and stillbirths, extensive fibrosing and/or necrotizing myocarditis in fetuses and the presence of moderate to high amounts of PCV2 in these lesions. Taking into account that scientific information on PCV2 and its associated diseases has been markedly expanded in the last 8 years, the objective of this review is to summarize the current state of knowledge of the most relevant aspects of PCV2 biology and PCVD.

Porcine circovirus diseases

Porcine circovirus type 2 (PCV2) infections: clinical signs, pathology and laboratory diagnosis.

In a previous study, it was demonstrated that high replication of Porcine circovirus 2 (PCV2) in a gnotobiotic pig was correlated with the absence of PCV2-neutralizing antibodies. The aim of the present study was to investigate if this correlation could also be found in SPF pigs in which PMWS was experimentally reproduced and in naturally PMWS-affected pigs. When looking at the total anti-PCV2 antibody titres, PMWS-affected and healthy animals seroconverted at the same time point, and titres in PMWS-affected animals were only slightly lower compared to those in healthy animals. In healthy animals, the evolution of PCV2-neutralizing antibodies coincided with that of total antibodies. In PMWS-affected animals, neutralizing antibodies could either not be found (sera from field studies) or were detected in low titres between 7 and 14 DPI only (sera from experimentally inoculated SPF pigs). Differences were also found in the evolution of specific antibody isotypes titres against PCV2. In healthy pigs, IgM antibodies persisted until the end of the study, whereas in PMWS-affected pigs they quickly decreased or remained present at low titres. The mean titres of other antibody isotypes (IgG1, IgG2 and IgA), were slightly lower in PMWS-affected pigs compared to their healthy group mates at the end of each study. This study describes important differences in the development of the humoral immune response between pigs that get subclinically infected with PCV2 and pigs that experience a high level of PCV2-replication which in 3 of 4 experiments led to the development of PMWS. These observations may contribute to a better understanding of the pathogenesis of a PCV2-infection.

/pdf/correlation-between-the-presence-of-neutralizing-antibodies-17pe3kttyo.pdf

Correlation between the presence of neutralizing antibodies against porcine circovirus 2 (PCV2) and protection against replication of the virus and development of PCV2-associated disease

Monocyte/macrophage lineage cells are target cells in vivo for porcine circovirus 2 (PCV2) replication. The porcine monocytic cell line 3D4/31 supports PCV2 replication in vitro, and attachment and internalization kinetics of PCV2 have been established in these cells. However, PCV2 receptors remain unknown. Glycosaminoglycans (GAG) are used by several viruses as receptors. The present study examined the role of GAG in attachment and infection of PCV2. Heparin, heparan sulfate (HS), chondroitin sulfate B (CS-B), but not CS-A, and keratan sulfate reduced PCV2 infection when these GAG were incubated with PCV2 prior to and during inoculation of 3D4/31 cells. Enzymatic removal of HS and CS-B prior to PCV2 inoculation of 3D4/31 cells significantly reduced PCV2 infection. Similarly, when PCV2 virus-like particles (VLP) were allowed to bind onto 3D4/31 cells in the presence of heparin and CS-B, attachment was strongly reduced. Titration of field isolates and low- and high-passage laboratory strains of PCV2 in the presence of heparin significantly reduced PCV2 titers, showing that the capacity of PCV2 to bind GAG was not acquired during in vitro cultivation but is an intrinsic feature of wild-type virus. When Chinese hamster ovary (CHO) cells were inoculated with PCV2, relative percentages of PCV2-infected cells were 27% ± 8% for HS-deficient and 12% ± 10% for GAG-deficient cells compared to wild-type cells (100%). Furthermore, it was shown using heparin-Sepharose chromatography that both PCV2 and PCV2 VLP directly interacted with heparin. Together, these results show that HS and CS-B are attachment receptors for PCV2.

Porcine Circovirus 2 Uses Heparan Sulfate and Chondroitin Sulfate B Glycosaminoglycans as Receptors for Its Attachment to Host Cells

Porcine circovirus 2 (PCV2) replication is characterized by high variation among infected pigs. This study investigated the role of immunologic responses in causing this variation. Twelve gnotobiotic pigs were inoculated with PCV2. Four of these pigs were treated with cyclosporin A (CysA) to monitor the effect of the adaptive immunity on the development of the PCV2 infection. Through lymph node biopsies at 10, 15, and 21 days postinoculation (DPI), PCV2 replication in lymphoid tissues was monitored. The production of total PCV2-specific and PCV2-neutralizing antibodies was followed, together with interferon-gamma (IFN-gamma) mRNA expression levels in peripheral blood monocytes as a marker for cellular immunity. In general, the CysA-treated pigs showed the highest PCV2 titers, indicating that the adaptive immunity is necessary to restrain PCV2 replication. Three different PCV2 replication patterns were observed in non-CysA-treated pigs. Pattern 1: In two pigs, PCV2 was not detected. They had the highest neutralizing antibody titers, appearing from 15 DPI. In these pigs a good cellular response was indicated by a peak in IFN-gamma mRNA at 15 DPI. Pattern 2: Five pigs contained low to moderate PCV2 titers at 15 DPI, remaining constant or decreasing towards 21 DPI. Lower neutralizing antibody titers were observed and no rise in IFN-gamma was detected. Pattern 3: In one pig, a low PCV2 titer at 15 DPI dramatically increased toward 21 DPI. Although an antibody response against PCV2 was mounted, no PCV2-neutralizing antibodies were detected. This pig also showed no rise in IFN-gamma. The study findings indicate that variation in the onset of the adaptive immunity may account for variation in PCV2 replication among pigs. Absence of PCV2-neutralizing antibodies may be an important factor in the development of an increased virus replication.

Correlation between type of adaptive immune response against porcine circovirus type 2 and level of virus replication.

In this in vitro study, the replication kinetics of porcine circovirus type 2 (PCV2) in porcine alveolar macrophages (PAM) and fetal cardiomyocytes (FCM), two target cells in vivo, was compared with that in PK-15 cells. Cultures were inoculated with either the postweaning multisystemic wasting syndrome (PMWS)-associated strain Stoon-1010 or the abortion-associated strain 1121. Viral proteins were visualized and virus production was determined. In PK-15 cells, the capsid protein was expressed between 6 and 12 hours post inoculation (hpi), it relocated to the nucleus between 12 and 24 hpi. At that time, Rep protein was also detected in the nucleus. This sequence of events also occurred in FCM and PAM but nuclear localized antigens appeared later (48 hpi) and in a lower percentage of cells. In PAM, clear differences in susceptibility were seen between pigs. In PAM from two out of five tested pigs, nuclear localized antigens were not detected, whereas in PAM from three other pigs they were seen in up to 20% of the antigen-positive cells. Virus production was observed in PK-15 but not in PAM or FCM cultures. In a second study, the replication kinetics of seven different PCV2 strains were compared in PK-15 cells. It was shown that the two abortion-associated strains had a different replication kinetics in comparison with PMWS or porcine dermatitis and nephropathy syndrome associated strains. With the abortion-associated strains, a higher number of infected cells was observed at 24 hpi and the percentage of infected cells with nuclear localised antigens was lower compared to that of other strains.

/pdf/replication-kinetics-of-different-porcine-circovirus-2-56vs4ylvkp.pdf

Replication kinetics of different porcine circovirus 2 strains in PK-15 cells, fetal cardiomyocytes and macrophages.

Porcine circovirus 2 (PCV2) is associated with post-weaning multisystemic wasting syndrome and reproductive problems in pigs. Cells of the monocyte/macrophage lineage are important target cells in PCV2-infected pigs, but the method of binding and entry of PCV2 into these cells is unknown. Therefore, binding and entry of PCV2 to the porcine monocytic cell line 3D4/31 were studied by visualization of binding and internalization of PCV2 virus-like particles (VLPs) by confocal microscopy and chemical inhibition of endocytic pathways (clathrin- and caveolae-mediated endocytosis and macropinocytosis), followed by evaluation of the level of PCV2 infection. It was shown that PCV2 VLPs bound to all cells, with maximal binding starting from 30 min post-incubation. Bound PCV2 VLPs were internalized in 47±5·0 % of cells. Internalization was continuous, with 70·5±9·7 % of bound PCV2 VLPs internalized at 360 min post-incubation. Internalizing PCV2 VLPs co-localized with clathrin. PCV2 infection was decreased significantly by chemical inhibitors that specifically blocked (i) actin-dependent processes, including cytochalasin D (75·5±7·0 % reduction) and latrunculin B (71·0±3·0 % reduction), and (ii) clathrin-mediated endocytosis, including potassium depletion combined with hypotonic shock (50·2±6·3 % reduction), hypertonic medium (56·4±5·7 % reduction), cytosol acidification (59·1±7·1 % reduction) and amantadine (52·6±6·7 % reduction). Inhibiting macropinocytosis with amiloride and caveolae-dependent endocytosis with nystatin did not decrease PCV2 infection significantly. PCV2 infection was reduced by the lysosomotropic weak bases ammonium chloride (47·0±7·9 % reduction) and chloroquine diphosphate (49·0±5·6 % reduction). Together, these data demonstrate that PCV2 enters 3D4/31 cells predominantly via clathrin-mediated endocytosis and requires an acidic environment for infection.

Peter Meerts

Papers

Correlation between the presence of neutralizing antibodies against porcine circovirus 2 (PCV2) and protection against replication of the virus and development of PCV2-associated disease

Porcine Circovirus 2 Uses Heparan Sulfate and Chondroitin Sulfate B Glycosaminoglycans as Receptors for Its Attachment to Host Cells

Correlation between type of adaptive immune response against porcine circovirus type 2 and level of virus replication.

Replication kinetics of different porcine circovirus 2 strains in PK-15 cells, fetal cardiomyocytes and macrophages.

Binding and entry characteristics of porcine circovirus 2 in cells of the porcine monocytic line 3D4/31.