Molecular basis for the different PCV2 susceptibility of T-lymphoblasts in Landrace and Piétrain pigs.

Clinically, Landrace pigs are more susceptible to porcine circovirus-associated diseases (PCVADs) than Piétrain pigs. We previously found that porcine circovirus type 2 (PCV2) can infect T-lymphoblasts. The present study examined the replication kinetics of six PCV2 strains in the lymphoblasts of Landrace and Piétrain pigs. The results showed that T-lymphoblasts from Landrace pigs are much more susceptible to PCV2 infection than those from Piétrain pigs. In addition, PCV2 replication was strain-dependent. PCV2 binding to T-lymphoblasts was partially mediated by chondroitin sulfate (CS) and dermatan sulfate (DS). Phosphacan, an effective internalization mediator in monocytes that contains several CS chains, was also demonstrated to be involved in PCV2 internalization. Viral binding and internalization were not different between the two breeds, however, the subsequent step, the disassembly was. Although inhibition of serine proteases blocked PCV2 replication in both Landrace and Piétrain pigs, this only occurred at a neutral pH in Piétrain pigs, whereas this occurred also at a low pH in Landrace. This suggested that more proteases can cleave PCV2 in Landrace lymphoblasts than in Piétrain lymphoblasts, explaining the better replication. Through co-localization studies of viral particles with endo-lysosomal markers, and quantitative analysis of organelle sizes during viral internalization, it was observed that PCV2 may exhibit a higher propensity for viral escape from late endosomes in Landrace pigs (smaller) compared to Piétrain pigs. These results provide new understandings of the different PCV2 susceptibility in Landrace and Piétrain pigs.

PCV2 Uptake by Porcine Monocytes Is Strain-Dependent and Is Associated with Amino Acid Characteristics on the Capsid Surface.

Porcine circovirus type 2 (PCV2) is associated with several economically important diseases that are described as PCV2-associated diseases (PCVADs). PCV2 is replicating in lymphoblasts, and PCV2 particles are taken up by monocytes without effective replication or complete degradation. Glycosaminoglycans (GAGs) have been demonstrated to be important receptors for PCV2 binding and entry in T-lymphocytes and continuous cell lines. The objective of this study was to determine whether differences exist in viral uptake and outcome among six PCV2 strains from different disease outbreaks in primary porcine monocytes: Stoon-1010 (PCV2a; PMWS), 1121 (PCV2a; abortion), 1147 (PCV2b; PDNS), 09V448 (PCV2d-1; PCVAD with high viral load in lymphoid tissues [PCVADhigh]), DE222-13 (PCV2d-2; PCVADhigh), and 19V245 (PCV2d-2; PCVADhigh). The uptake of PCV2 in peripheral blood monocytes was different among the PCV2 strains. A large number of PCV2 particles were found in the monocytes for Stoon-1010, DE222-13, and 19V245, while a low number was found for 1121, 1147, and 09V448. Competition with, and removal of GAGs on the cell surface, demonstrated an important role of chondroitin sulfate (CS) and dermatan sulfate (DS) in PCV2 entry into monocytes. The mapping of positively/negatively charged amino acids exposed on the surface of PCV2 capsids revealed that their number and distribution could have an impact on the binding of the capsids to GAGs, and the internalization into monocytes. Based on the distribution of positively charged amino acids on PCV2 capsids, phosphacan was hypothesized, and further demonstrated, as an effective candidate to mediate virus attachment to, and internalization in, monocytes. IMPORTANCE PCV2 is present on almost every pig farm in the world and is associated with a high number of diseases (PCV2-associated diseases [PCVADs]). It causes severe economic losses. Although vaccination is successfully applied in the field, there are still a lot of unanswered questions on the pathogenesis of PCV2 infections. This article reports on the uptake difference of various PCV2 strains by peripheral blood monocytes, and reveals the mechanism of the strong viral uptake ability of monocytes of Piétrain pigs. We further demonstrated that: (i) GAGs mediate the uptake of PCV2 particles by monocytes, (ii) positively charged three-wings-windmill-like amino acid patterns on the capsid outer surface are activating PCV2 uptake, and (iii) phosphacan is one of the potential candidates for PCV2 internalization. These results provide new insights into the mechanisms involved in PCVAD and contribute to a better understanding of PCV2 evolution. This may lead to the development of resistant pigs.

Porcine circovirus type 2 ORF5 protein induces endoplasmic reticulum stress and unfolded protein response in porcine alveolar macrophages.

Porcine circovirus type 2 (PCV2) is the essential infectious agent causing porcine circovirus-associated disease (PCVD) in pigs and one of the important viruses that severely jeopardize the swine husbandry industry. PCV2 elicits the unfolded protein response (UPR) via activation of the PERK pathway, and its capsid protein (Cap) has also been found to induce UPR with subsequent activation of apoptosis. The open reading frame 5 (ORF5) protein is a recently discovered non-structural protein, and its function in PCV2 pathogenesis remains unknown. The aim of this study was to determine whether the PCV2 ORF5 protein could induce endoplasmic reticulum stress (ERS) and UPR in porcine alveolar macrophages (PAMs). pEGFP-tagged ORF5 protein was transiently overexpressed in PAMs. Transmission electron microscopy (TEM) was employed to examine changes in ER morphology, and quantitative real-time PCR and western blotting analysis were used to measure UPR-related cell signaling alterations. We found that the ORF5 protein triggers swelling and degranulation of the ER and upregulates the expression of ERS markers. Further experiments demonstrated that the PCV2 ORF5 protein induces ERS and UPR via the PERK (RNA-activated protein kinase-like endoplasmic reticulum kinase), ATF6 (activating transcription factor 6) and IRE1 (inositol requiring enzyme 1) signaling pathways. Together with previous studies, we provide new information on the ERS-UPR induced by the PCV2 ORF5 protein.

Classical swine fever virus non-structural protein 4B binds tank-binding kinase 1.

Classical swine fever (CSF) is a contagious disease with a high mortality rate and is caused by classical swine fever virus (CSFV). CSFV non-structural protein 4B (NS4B) plays a crucial role in CSFV replication and pathogenicity. However, precisely how NS4B exerts these functions remains unknown, especially as there are no reports relating to potential cellular partners of CSFV NS4B. Here, a yeast two-hybrid (Y2H) system was used to screen the cellular proteins interacting with NS4B from a porcine alveolar macrophage (PAM) cDNA library. The protein screen along with alignment using the NCBI database revealed 14 cellular proteins that interact with NS4B: DDX39B, COX7C, FTH1, MAVS, NR2F6, RPLP1, PSMC4, FGL2, MKRN1, RPL15, RPS3, RAB22A, TP53BP2 and TBK1. These proteins mostly relate to oxidoreductase activity, signal transduction, localization, biological regulation, catalytic activity, transport and metabolism by GO categories. Tank-binding kinase 1 (TBK1) was chosen for further confirmation. The NS4B-TBK1 interaction was further confirmed by subcellular co-location, co-immunoprecipitation and glutathione S-transferase pull-down assays. This study offers a theoretical foundation for further understanding of the diversity of NS4B functions in relation to viral infection and subsequent pathogenesis.

Rab5 Enhances Classical Swine Fever Virus Proliferation and Interacts with Viral NS4B Protein to Facilitate Formation of NS4B Related Complex.

Classical swine fever virus (CSFV) is a fatal pig pestivirus and causes serious financial losses to the pig industry. CSFV NS4B protein is one of the most important viral replicase proteins. Rab5, a member of the small Rab GTPase family, is involved in infection and replication of numerous viruses including hepatitis C virus and dengue virus. Until now, the effects of Rab5 on the proliferation of CSFV are poorly defined. In the present study, we showed that Rab5 could enhance CSFV proliferation by utilizing lentivirus-mediated constitutive overexpression and eukaryotic plasmid transient overexpression approaches. On the other hand, lentivirus-mediated short hairpin RNA knockdown of Rab5 dramatically inhibited virus production. Co-immunoprecipitation, glutathione S-transferase pulldown and laser confocal microscopy assays further confirmed the interaction between Rab5 and CSFV NS4B protein. In addition, intracellular distribution of NS4B-Red presented many granular fluorescent signals (GFS) in CSFV infected PK-15 cells. Inhibition of basal Rab5 function with Rab5 dominant negative mutant Rab5S34N resulted in disruption of the GFS. These results indicate that Rab5 plays a critical role in facilitating the formation of the NS4B related complexes. Furthermore, it was observed that NS4B co-localized with viral NS3 and NS5A proteins in the cytoplasm, suggesting that NS3 and NS5A might be components of the NS4B related complex. Taken together, these results demonstrate that Rab5 positively modulates CSFV propagation and interacts with NS4B protein to facilitate the NS4B related complexes formation.

Classical Swine Fever Virus Infection and Its NS4A Protein Expression Induce IL-8 Production through MAVS Signaling Pathway in Swine Umbilical Vein Endothelial Cells.

Classical swine fever virus (CSFV) infection causes a severe disease of pigs, which is characterized by hemorrhage, disseminated intravascular coagulation, and leucopenia. IL-8, a main chemokine and activator of neutrophils, regulates the permeability of endothelium, which may be related to the hemorrhage upon CSFV infection. Until now, the molecular mechanisms of IL-8 regulation during CSFV infection are poorly defined. Here, we showed that CSFV infection induced IL-8 production and the upregulation of IL-8 required virus replication in swine umbilical vein endothelial cells (SUVECs). Additionally, MAVS expression was increased and was required for IL-8 production upon CSFV infection. Moreover, ROS was involved in CSFV-induced IL-8 production. Subsequent studies demonstrated that ROS was involved in MAVS-induced IL-8 production and CSFV induced ROS production through MAVS pathway. These results indicate that CSFV induces IL-8 production through MAVS pathway and production of ROS. The role of NS4A in the pathogenesis of CSFV is not well-understood. In this study, we further demonstrated that CSFV NS4A induced IL-8 production through enhancing MAVS pathway and promoted CSFV replication. In addition, we discovered that CSFV NS4A was localized in the cell nucleus and cytoplasm, including endoplasmic reticulum (ER) and mitochondria. Taken together, these results provide insights into the mechanisms of IL-8 regulation and NS4A functions during CSFV infection.