Porcine Parvovirus Replication Research Articles

Proteomics analysis of PK-15 cells infected with porcine parvovirus and the effect of PCBP1 on PPV replication.

Porcine parvovirus (PPV) is a cause of reproductive failure in the swine industry. Our knowledge of PPV remains limited, and there is no effective treatment for PPV infection. Proteomics of PPV-infected PK-15 cells was conducted to identify differentially expressed proteins at 6 hours post-infection (hpi) and 36 hpi. Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed that various pathways participate in PPV infection. Poly (rC) binding protein 1 was confirmed to inhibit PPV replication, which provided potential targets for anti-PPV infection. Our findings improve the understanding of PPV infection and pave the way for future research in this area.

Chaperonin CCT5 binding with porcine parvovirus NS1 promotes the interaction of NS1 and COPƐ to facilitate viral replication

Porcine parvovirus (PPV) is an important pathogen causing reproductive disorders in first pregnant sows. The non-structure protein NS1 of PPV is a multifunctional protein playing a key role in viral replication. Chaperonin-containing T-complex polypeptide complex (CCT), containing CCT1-CCT8 subunits, belongs to the type II chaperones that interact with proteins to help in folding and maintaining. In this study, CCT5, for the first time, was found to be one of the host interacting proteins of PPV NS1, and CCT5 was directly bound with NS1. Interference of CCT5 expression by specific siRNA and knockout of CCT5 expression by CRISPR/Cas9 suppressed PPV replication, while overexpression of CCT5 promoted PPV replication in PK-15 cells. The interaction of CCT5 and PPV NS1 was dependent on the 36–42 aa motif at the N-terminal end of NS1. More importantly, CCT5 was also found interacting with COPƐ, which has previously been demonstrated to promote PPV replication by regulating type I interferon. Interference and knockout of CCT5 expression significantly reduced the interaction of PPV NS1 and host protein COPƐ, and promoted the IFN-β expression. These results show that CCT5 mediates the interaction of PPV NS1 and COPƐ to regulate viral replication, providing new insight into the mechanism of PPV replication.

Coatomer protein COPƐ, a novel NS1-interacting protein, promotes the replication of Porcine Parvovirus via attenuation of the production of type I interferon

Porcine Parvovirus (PPV) is a pathogen causing porcine reproductive disorders. Non-structural protein NS1 appears diverse functions acting as a predominant regulator in promoting PPV replication. In this study, we identified a PPV NS1 binding protein coatomer subunit epsilon (COPƐ), and found that COPƐ is a critical regulator during PPV replication. In NS1 transfected or PPV infected cells, COPƐ was interacted with NS1 and translocated into nucleus together with NS1. Knockout of COPƐ could inhibit PPV production by increasing the expression levels of IFN-β, while overexpression of COPƐ enhanced PPV production by reducing the expression levels of IFN-β. Furthermore, the domain mapping assay showed that the N-terminal amino acids domain of NS1 (25-EAFSYVF-31) were required for the interaction of COPƐ with NS1. Sequence alignment result displays that parvovirus NS1 (EAFSYVF) amino acids domain is highly conservative among PPV, CPV, FPV and MEV, and down-regulation of COPƐ could also significantly reduce the replication of these viruses. Notably, we found that the interaction of COPƐ with NS1 play an important role in promoting the production of type I interferon during PPV or CPV infection, which affect the replication of these viruses. Taken together, the results presented here show a novel function of NS1 interaction with COPƐ that regulates the parvovirus replication through modulating the type I interferons signaling pathway, provided a potential target for the control of parvovirus-associated diseases.

Porcine circovirus type 2 infection attenuates the K63-linked ubiquitination of STING to inhibit IFN-β induction via p38-MAPK pathway

Porcine circovirus 2 (PCV2) has been proved to increase the risk of other pathogens infection via immunosuppression. Although the co-infection of PCV2 and porcine parvovirus (PPV) is commonly observed in worldwide, the relative immune mechanisms promoting PPV infection in PCV2-infected piglets are currently unknown. Herein, we found that PCV2 infection suppressed IFN-β expression and promoted PPV infection in the piglets. Consistent with this finding, we confirmed that PCV2 infection significantly inhibited the induction of IFN-β to promote PPV replication in cell level. Furthermore, PCV2 infection attenuated the K63-linked ubiquitination of STING induced by PPV, blocked the formation of complex of STING, TBK1 and IRF3, and further prevented the phosphorylation of TBK1 and IRF3, resulting in a decreased IFN-β transcription response to PPV infection. Consistently, using cGAMP to direct stimulate STING also appeared a reduced STING-K63 ubiquitination and IFN-β induction in PCV2-infected cells. However, we noted that knockdown of p38-MAPK signaling could markedly attenuate the inhibitory effect of PCV2 on STING-K63 ubiquitination, and improve the induction of IFN-β in PCV2-infected whenever theses cells were challenged with PPV infection or cGAMP stimulation. Meanwhile, we found that PCV2 infection promoted the phosphorylation of USP21 to inhibit the K63 ubiquitination of STING and the transcription of IFN-β via activation of p38-MAPK signaling. Taken together, our results demonstrate that PCV2 infection activates the p38-MAPK signaling pathway-mediated USP21 phosphorylation to inhibit the K63 ubiquitination of STING, which prevents the phosphorylation and transportation to the nucleus of IRF3, leading to an increase risk for PPV infection.

Ferulic acid isolated from propolis inhibits porcine parvovirus replication potentially through Bid-mediate apoptosis

Propolis from honeybee hives, which is a traditional Chinese medicine, is widely used in veterinary clinics. Many compounds have been identified and isolated from propolis. Ferulic acid (FA), one of the propolis components, previous studies have proven that it has antiviral effects. To study the mechanism of FA antiviral effects, experiments such as immunofluorescence, quantitative real-time PCR and immunoblotting were introduced. In porcine kidney (PK-15) cells, PPV infection induced the expression of the proapoptotic genes Bid, Bad, Bim and Bak, disrupted mitochondrial membrane potential, promoted mitochondria-mediated, caspase-dependent apoptotic signaling and induced apoptosis. Furthermore, the infected PK-15 cells had increased intracellular reactive oxygen species (ROS) generation. FA treatment, however, reversed these effects and increased cell viability. FA treatment also significantly decreased the PPV-induced expression of Bid, Cyt-c and Apaf-1, suggesting that ROS were involved in the activation of the mitochondria-mediated apoptosis pathway. This in vitro study showed that the antiviral activity of FA was probably associated with inhibiting the replication of PPV by blocking proapoptotic factors such as Bid, Bcl-2 and Mcl-1, and attenuating the mitochondria-mediated response by inhibiting the activation of the Bid-related signaling pathway. Pharmacological inhibitors inhibited PPV-induced apoptosis by blocking Bid, and also suppressed the expression of Caspase family proteins in ppv-induced apoptosis. Taken together, our results suggested that PPV induced PK-15 cell apoptosis via activation of Bid and Bid-related signaling pathways and that the mitochondria act as the mediators of these pathways. FA effectively and extensively attenuated this PPV action, and thus is a potential antiviral agent against PPV.

Autophagy Promotes Porcine Parvovirus Replication and Induces Non-Apoptotic Cell Death in Porcine Placental Trophoblasts.

Autophagy plays important roles in the infection and pathogenesis of many viruses, yet the regulatory roles of autophagy in the process of porcine parvovirus (PPV) infection remain unclear. Herein, we show that PPV infection induces autophagy in porcine placental trophoblasts (PTCs). Induction of autophagy by rapamycin (RAPA) inhibited the occurrence of apoptotic cell death, yet promoted viral replication in PPV-infected cells; inhibition of autophagy by 3-MA or ATG5 knockdown increased cellular apoptosis and reduced PPV replication. Interestingly, we found that in the presence of caspase-inhibitor zVAD-fmk, PPV induces non-apoptotic cell death that was characterized by lysosomal damage and associated with autophagy. Induction of complete autophagy flux by RAPA markedly promoted PPV replication compared with incomplete autophagy induced by RAPA plus bafilomycin (RAPA/BAF) in the early phase of PPV infection (24 h.p.i.). Meanwhile, induction of complete autophagy with RAPA increased lysosomal damage and non-apoptotic cell death in the later phase of PPV infection. Therefore, our data suggest that autophagy can enhance PPV replication and promote the occurrence of lysosomal-damage-associated non-apoptotic cell death in PPV-infected porcine placental trophoblasts.

Porcine parvovirus replication is suppressed by activation of the PERK signaling pathway and endoplasmic reticulum stress-mediated apoptosis

Endoplasmic reticulum (ER) stress is associated with numerous mammalian diseases, especially viral diseases. Porcine parvovirus (PPV) is the causative agent of reproductive failure in swine. Here, we observed that the PPV infection of porcine kidney 15 and porcine testis cells resulted in the activation of ER stress sensors mediated by protein kinase R-like ER kinase (PERK), but not inositol-requiring enzyme 1 and activating transcription factor 6 (ATF6). ER stress activation obviously blocked PPV replication. Depletion of proteins, such as PERK, eukaryotic initiation factor 2, and ATF4, by small interfering RNA significantly enhanced PPV replication. Moreover, the pro-apoptotic factor C/EBP homologous protein was identified a key factor in the inhibition of PPV replication. These data demonstrate that PPV infection activates ER stress through the PERK signaling pathway and that ER stress inhibits further PPV replication by promoting apoptosis.

In vitro antiviral activity of germacrone against porcine parvovirus.

Porcine parvovirus (PPV) infections can lead to significant losses to the swine industry by causing reproductive failure in pigs. Germacrone has been reported to efficiently suppress the replication of influenza virus. In this report, the antiviral activity of germacrone on PPV in swine testis (ST) cells was investigated. Here, we show for the first time that germacrone protects cells from PPV infection and suppresses the synthesis of viral mRNA and protein. Furthermore, we show that germacrone inhibits PPV replication at an early stage in a dose-dependent manner. These findings suggest that germacrone is a potential candidate for anti-PPV therapy.

Antiviral effect of lithium chloride on infection of cells by porcine parvovirus.

Porcine parvovirus (PPV) causes reproductive failure in pigs, which leads to economic losses to the industry. As reported previously, LiCl efficiently impairs the replication of a variety of viruses, including the coronavirus infectious bronchitis virus (IBV), transmissible gastroenteritis virus (TGEV), and pseudorabies herpesvirus. We demonstrate for the first time that inhibition of PPV replication in swine testis (ST) cells by LiCl is dose-dependent, and that the antiviral effect of LiCl occurred in the early phase of PPV replication. These results indicate that LiCl might be an effective anti-PPV drug to control PPV disease. Further studies are required to explore the mechanism of the antiviral effect of LiCl on PPV infection in vivo.

Nitric oxide inhibits the replication cycle of porcine parvovirus in vitro

This study investigated the inhibitory effect and mechanism of nitric oxide (NO) on porcine parvovirus (PPV) replication in PK-15 cells. The results showed that two NO-generating compounds, S-nitroso-l-acetylpenicillamine (SNAP) and l-arginine (LA), at a noncytotoxic concentration could reduce PPV replication in a dose-dependent manner and that this anti-PPV effect could be reversed by the NO synthase (NOS) inhibitor N-nitro-l-arginine methyl ester (l-NAME). By assaying the steps of the PPV life cycle, we also show that NO inhibits viral DNA and protein synthesis. This experiment provides a frame of reference for the study of the anti-viral mechanism of NO.

A monoclonal antibody which recognizes cell surface antigen and inhibits porcine parvovirus replication.

Monoclonal antibody technologies were applied to the study of early events in porcine parvovirus (PPV) infections in vitro. Balb/c mice were immunized with whole swine testicle cells and hybridomas were produced following fusion with myeloma cells. Resultant clones were screened firstly in an ELISA system, to detect monoclonal antibody recognition of swine testicle cells, and secondly, in a fluorescent antibody test to detect monoclonal antibody which inhibited production of PPV antigen. One clone, 1H11, which satisfied these screening requirements, recognized proteins present in cell lines both permissive and non-permissive for porcine parvovirus replication and inhibited the production of virus progeny of several PPV isolates. A linear staining pattern of cross-linked plasma membranes, indicative of monoclonal antibody binding at the cell membrane, was demonstrated by indirect immunofluorescence assays. In immunoblotting experiments, 1H11 recognized a polypeptide of approximately 40 kDa in size, present in both permissive and non-permissive cell lines.

Inhibition of porcine parvovirus replication by empty virus particles.

The influence of empty porcine parvovirus (PPV) particles on viral replication was examined in cell cultures and in swine. Following extensive purification, homogeneous preparations of full and empty PPV preparations were obtained and used for in vitro and in vivo analyses. In the first in vitro experiment, swine testes cells were infected with mixtures of various ratios of empty and full (E/F) particles. The production of both intracellular and extracellular virus was markedly inhibited in the presence of empty particles. This inhibition was dependent upon the concentration of empty particles present in the mixture. In the second in vitro study, various concentrations of empty particles were added prior to full virus infection. Again, marked inhibition of progeny virus production was evident and related to the concentrations of empty particles added. Based on the results of in vitro studies, the influence of empty particles on PPV infection in swine was tested by infecting mid-term and late-term gestation swine fetuses with various E/F particle ratios. Both mid-term and late-term fetuses exposed to 0:1, 1:1 and 5:1 E/F ratios displayed gross pathological evidence of PPV infection whereas fetuses exposed to E/F ratios of 30:1 or greater were grossly normal in appearance. However, fetuses infected with 30:1, 50:1 and 300:1 E/F ratios showed evidence of virus in their tissues by DNA hybridization. Regardless of the E/F ratios, late-term infected fetuses responded with high antibody titers ranging from 1024 to 4096. The results from these studies suggested that empty particles interfered with viral replication in both cell culture and in animals.

Identification and characterization of a porcine parvovirus nonstructural polypeptide.

Sera from porcine parvovirus (PPV)-infected swine fetuses immunoprecipitated and 84- to 86-kilodalton polypeptide in addition to the A and B virion structural proteins. This polypeptide, designated NS-1, was present in PPV-infected cell lysates but not in purified virions. Partial proteolysis mapping revealed that NS-1 was not related to the A and B viral structural proteins. All three proteins in infected cells were phosphorylated at serine residues, and NS-1 also contained phosphothreonine. From pulse-labeling experiments with either 32Pi or [35S]methionine, NS-1 was found to first appear 5 to 7 h postinfection, whereas the viral structural polypeptides were first synthesized 9 to 11 h postinfection. Pulse-chase experiments revealed that NS-1 initially appeared as an 84-kilodalton protein and was subsequently structurally modified to forms of slower electrophoretic mobilities. The time of appearance of NS-1 after virus infection coincided with the initiation of viral DNA synthesis, suggesting that this polypeptide (and the modified forms thereof) may be involved in PPV replication.

Studies on the multiplication of a porcine parvovirus

A porcine parvovirus has been characterized with regard to its replication in foetal porcine kidney cells and certain biophysical properties. Electron microscopy of infected cells at selected times postinfection revealed that porcine parvovirus replication took place within or near a series of granular intranuclear inclusions which may be contiguous with cellular heterochromatin. Developing virions were observed to aggregate into a nuclear-like amorphous mass which gradually disrupted as cellular integrity was lost. Purified virions were found to have a buoyant density in CsCl of 1.38 g/ml, while ‘empty’ particles has a buoyant density of 1.29 g/ml. The particle diameter was calculated to be approximately 22 nm.

Replication of porcine parvovirus in peripheral blood lymphocytes, monocytes, and peritoneal macrophages.

Porcine peripheral blood lymphocytes (PBL), peripheral blood monocytes, and peritoneal macrophages were examined for their ability to support porcine parvovirus (PPV) replication. The cell cultures were infected with the NADL-2 strain of PPV at 0.1 multiplicity of infection. PBL cultures were stimulated with the following phytomitogens: phytohemagglutinin M, concanavalin A, and pokeweed mitogen. Unstimulated PBL cultures infected with PPV and uninfected PBL stimulated with phytomitogens served as controls. All cultures were examined daily for PPV-specific immunofluorescence and hemagglutinin. PPV replicated in PBL cultures stimulated with all phytomitogens. Both viral hemagglutinin in culture fluids and nuclear immunofluorescence in cells were detected. In contrast, unstimulated PBL did not support viral replication; however, PPV antigen was detected in the cytoplasm. PPV persisted in unstimulated PBL for 21 days (duration of the experiment) without replication, but replicated each time with the addition of phytohemagglutinin M at 0, 3, 7, 14, and 21 days after infection. Uninfected PBL stimulated with phytomitogens lacked both viral hemagglutinin and immunofluorescence. Simultaneous detection of lymphocyte surface marker and viral antigens in pokeweed mitogen-stimulated PBL revealed that both T and non-T cells (B and null cells) are able to support PPV replication. Peripheral blood monocytes and peritoneal macrophages phagocytized PPV but did not support virus replication.