Strain-dependent porcine circovirus type 2 (PCV2) entry and replication in T-lymphoblasts

Porcine circovirus type 2 (PCV2) is the etiological agent of PCV2-associated diseases (PCVAD). PCV2 targets lymphoblasts, and pigs suffering from PCVAD display lymphocyte depletion in lymphoid tissues. PCV2 infection of lymphoblasts has not been studied. Here, the replication cycle of PCV2 (abortion strain 1121 and PMWS strain Stoon1010) in T-lymphoblasts was examined. The expression of Rep and Cap were found for both viral strains, while progeny virus was detected for Stoon1010 but not for 1121. PCV2 attached to 11-26% (1121-Stoon1010) of the T-lymphoblasts while 2.6-12.7% of cells showed virus internalization. Chondroitin sulfate (CS) was present on 25% of T-lymphoblasts, and colocalized with PCV2 on 31-32% of the PCV2+ cells. Enzymatic removal of CS reduced PCV2 infection. PCV2 infection was decreased by chlorpromazine, cytochalasin D and Clostridium difficile toxin B for both viral strains and by amiloride for 1121 but not for Stoon1010. Inhibiting either endosome acidification or serine proteases strongly reduced PCV2 infection. Three-dimensional analysis of Cap structure demonstrated a better Cap-nucleic acid affinity for Stoon1010 than for 1121. Taken together, PCV2 binds to T-lymphoblasts partially via CS, enters via clathrin-mediated endocytosis, and disassembles under functions of a pH-drop and serine proteases. Strain Stoon1010 displayed an enhanced viral binding, a specific receptor-mediated endocytosis, an increased Cap-nucleic acid affinity, and a more productive infection in T-lymphoblasts than 1121 did, indicating an evolution from 1121 to Stoon1010

Changes on the viral capsid surface during the evolution of porcine circovirus type 2 (PCV2) from 2009 till 2018 may lead to a better receptor binding

Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated diseases (PCVAD). Three major PCV2 genotypes (PCV2a, PCV2b, and PCV2d) have been identified globally. Despite their worldwide distribution, the prevalence and genetic evolution of PCV2 in Belgium has not previously been determined. In this study, 319 samples from animals suffering from diseases likely to be associated with PCV2 were collected from 2009 to 2018 and analysed by virus titration. The overall prevalence of PCV2 in PCVAD-suspected cases was 15.7 per cent (50/319). The phylogenetic analysis demonstrated that at least three genotypes (PCV2a, PCV2b, and PCV2d) circulated in Belgium from 2009 till 2018, and that PCV2 evolved from PCV2a to PCV2b and from PCV2d-1 to PCV2d-2. Sequence comparison among the forty-three PCV2 isolates showed that they had 89.7-100 per cent nucleotide-sequence and 88.5-100 per cent amino-acid-sequence identities. Three amino acid sites were under positive selection. Three-dimensional analysis of genotype-specific amino acids revealed that most of the mutations were on the outside of the cap protein with a few conserved mutations present on the inner side. Mutations toward more basic amino acids were found on the upper and tail parts of two connecting capsid proteins which form one big contact region, most probably involved in receptor binding. The lower part was relatively conserved. This polarity change together with the formation of an extruding part drive the virus to a more efficient GAG receptor binding. Taken together, these results showed a genotype shift from PCV2a to PCV2b and later on from PCV2d-1 to PCV2d-2, and a PCV2 evolution toward a better receptor binding capacity

Dissecting clinical outcome of porcine circovirus type 2 with in vivo derived transcriptomic signatures of host tissue responses

Background: Porcine Circovirus Type 2 (PCV2) is a pathogen that has the ability to cause often devastating disease manifestations in pig populations with major economic implications. How PCV2 establishes subclinical persistence and why certain individuals progress to lethal lymphoid depletion remain to be elucidated. Results: Here we present PorSignDB, a gene signature database describing in vivo porcine tissue physiology that we generated from a large compendium of in vivo transcriptional profiles and that we subsequently leveraged for deciphering the distinct physiological states underlying PCV2-affected lymph nodes. This systems genomics approach indicated that subclinical PCV2 infections suppress a myeloid leukocyte mediated immune response. However, in contrast an inflammatory myeloid cell activation is promoted in PCV2 patients with clinical manifestations. Functional genomics further uncovered STAT3 as a druggable PCV2 host factor candidate. Moreover, IL-2 supplementation of primary lymphocytes enabled ex vivo study of PCV2 replication in its target cell, the lymphoblast. Conclusion: Our systematic dissection of the mechanistic basis of PCV2 reveals that subclinical and clinical PCV2 display two diametrically opposed immunotranscriptomic recalibrations that represent distinct physiological states in vivo, which suggests a paradigm shift in this field. Finally, our PorSignDB signature database is publicly available as a community resource (http://www.vetvirology.ugent.be/PorSignDB/, included in Gene Sets from Community Contributors http://software.broadinstitute.org/gsea/msigdb/contributed_genesets.jsp) and provides systems biologists with a valuable tool for catalyzing studies of human and veterinary disease. Finally, a primary porcine lymphoblast cell culture system paves the way for unraveling the impact of host genetics on PCV2 replication

Primary replication and invasion of the bovine gammaherpesvirus BoHV-4 in the genital mucosae

Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus that is widespread in cattle. Ex vivo models with bovine genital tract mucosa explants were set up to study molecular/cellular BoHV-4-host interactions. Bovine posterior vagina, cervix and uterus body were collected from cows at two stages of the reproductive cycle for making mucosa explants. The BoHV-4 replication kinetics and characteristics within the three different mucosae of animals in the follicular and luteal phase were assessed by virus titration. The number of plaques, plaque latitude and number of infected cells were determined by immunofluorescence. BoHV-4 replicated in a productive way in all genital mucosal tissues. It infected single individual cells in both epithelium and lamina propria of the genital mucosae at 24 hours post-inoculation (hpi). Later, small BoHV-4 epithelial plaques were formed that did not spread through the basement membrane. 50% of the number of BoHV-4 infected cells were identified as cytokeratin(+) and CD172a(+) cells in the three parts of the genital tract at 24 hpi. Upon a direct injection of genital explants with BoHV-4, fibrocytes became infected, indicating that the unidentified 50% of the infected cells are most probably fibrocytes. In this study, in vivo-related in vitro genital tract models were successfully established and the early stage of the pathogenesis of a genital infection was clarified: BoHV-4 starts with a productive infection of epithelial cells in the reproductive tract, forming small foci followed by a non-productive infection of surveilling monocytic cells which help BoHV-4 to invade into deeper tissues

Breed differences in PCV2 uptake and disintegration in porcine monocytes

Porcine circovirus type 2 (PCV2) is associated with various diseases which are designated as PCV2-associated diseases (PCVADs). Their severity varies among breeds. In the diseased pigs, virus is present in monocytes, without replication or full degradation. PCV2 entry and viral outcome in primary porcine monocytes and the role of monocytes in PCV2 genetic susceptibility have not been studied. Here, virus uptake and trafficking were analyzed and compared among purebreds Pietrain, Landrace and Large White and hybrid Pietrain x Topigs20. Viral capsids were rapidly internalized into monocytes, followed by a slow disintegration to a residual level. PCV2 uptake was decreased by chlorpromazine, cytochalasin D and dynasore. The internalized capsids followed the endosomal trafficking pathway, ending up in lysosomes. PCV2 genome was nicked by lysosomal DNase II in vitro, but persisted in monocytes in vivo. Monocytes from purebred Pietrain and the hybrid showed a higher level of PCV2 uptake and disintegration, compared to those from Landrace and Large White. In conclusion, PCV2 entry occurs via clathrin-mediated endocytosis. After entry, viral capsids are partially disintegrated, while viral genomes largely escape from the pathway to avoid degradation. The degree of PCV2 uptake and disintegration differ among pig breeds

Gammaherpesvirus BoHV-4 infects bovine respiratory epithelial cells mainly at the basolateral side

Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus that is widespread in cattle. However, only a few studies about the pathogenesis of BoHV-4 primary infection have been reported. In the present study, ex vivo models with bovine nasal and tracheal mucosa explants were used to study the cellular BoHV-4-host interactions. Infection was observed in nasal but not in tracheal epithelial cells. To find a possible correlation between the integrity and restricted infection of the respiratory epithelium, both nasal mucosal and tracheal explants were treated with EGTA, a drug that disrupts the intercellular junctions, before inoculation. The infection was analyzed based on the number of plaques, plaque latitude and number of infected single cells, as determined by immunofluorescence. BoHV-4 infection in nasal mucosal explants was enhanced upon opening the tight junctions with EGTA. Infection in tracheal explants was only found after treatment with EGTA. In addition, primary bovine respiratory epithelial cells (BREC) were isolated, grown at the air-liquid interface and infected either at the apical or basolateral side by BoHV-4. The results showed that BoHV-4 preferentially bound to and entered BREC at the basolateral surfaces of both nasal and tracheal epithelial cells. The percentage of BoHV-4 infection was significantly increased both from nasal and tracheal epithelial cells after treatment with EGTA, which indicates that the BoHV-4 receptor is mainly located at the basolateral surface of these cells. Thus, our findings demonstrate that integrity of the respiratory epithelium is crucial in the host's innate defense against primary BoHV-4 infections

CD44ICD promotes breast cancer stemness via PFKFB4-mediated glucose metabolism.

CD44 is a single-pass cell surface glycoprotein that is distinguished as the first molecule used to identify cancer stem cells in solid tumors based on its expression. In this regard, the CD44high cell population demonstrates not only the ability to regenerate a heterogeneous tumor, but also the ability to self-regenerate when transplanted into immune-deficient mice. However, the exact role of CD44 in cancer stem cells remains unclear in part because CD44 exists in various isoforms due to alternative splicing. Methods: Gain- and loss-of-function methods in different models were used to investigate the effects of CD44 on breast cancer stemness. Cancer stemness was analyzed by detecting SOX2, OCT4 and NANOG expression, ALDH activity, side population (SP) and sphere formation. Glucose consumption, lactate secretion and reactive oxygen species (ROS) levels were detected to assess glycolysis. Western blot, immunohistochemical staining, ELISA and TCGA dataset analysis were performed to determine the association of CD44ICD and PFKFB4 with clinical cases. A PFKFB4 inhibitor, 5MPN, was used in a xenograft model to inhibit breast cancer development. Results: In this report, we found that the shortest CD44 isoform (CD44s) inhibits breast cancer stemness, whereas the cleaved product of CD44 (CD44ICD) promotes breast cancer stemness. Furthermore, CD44ICD interacts with CREB and binds to the promoter region of PFKFB4, thereby regulating PFKFB4 transcription and expression. The resultant PFKFB4 expression facilitates the glycolysis pathway (vis-à-vis oxidative phosphorylation) and promotes stemness of breast cancer. In addition, we found that CD44ICD and PFKFB4 expressions are generally up-regulated in the tumor portion of breast cancer patient samples. Most importantly, we found that 5MPN (a selective inhibitor of PFKFB4) suppresses CD44ICD-induced tumor development. Conclusion: CD44ICD promotes breast cancer stemness via PFKFB4-mediated glycolysis, and therapies that target PFKFB4 (e.g., 5MPN therapy) may lead to improved outcomes for cancer patients

Preferential use of Siglec-1 or Siglec-10 by type 1 and type 2 PRRSV strains to infect PK15S1-CD163 and PK15S10-CD163 cells

Cellular entry mediators define whether the cell is permissive to PRRSV infection. Porcine sialoadhesin (pSn, Siglec-1) and CD163 are main entry mediators facilitating infection of porcine macrophages by PRRSV. Recently, Siglec-10 was demonstrated to be an alternative receptor for PRRSV. To examine if virulence and pathogenicity of PRRSV strains could be correlated with the use of different Siglecs, a PK15 cell line recombinantly expressing Siglec-1 and CD163 (PK15(S1-CD163)) and a PK15 cell line recombinantly expressing Siglec-10 and CD163 (PK15(S10-CD163)) were used to compare the virus replication of 7 genotype 1 subtype 1 strains (G1s1), 2 genotype 1 subtype 3 (G1s3) strains and 5 genotype 2 (G2) strains. Some strains (08VA (G1s1), 13V117 (G1s1), 17V035 (G1s1), VR2332 (G2)) were poor virus producers (= 10(6) TCID50/mL. PK15(S10-CD163) cells exhibited a higher efficiency in virus production per infected cell than the PK15(S1-CD163) cells. The G1s1 strains LV and 07V063 infected more cells in the PK15(S1-CD163), whereas the 94V360 and 08VA strains preferred PK15(S10-CD163). The highly virulent G1s3 strains Lena and Su1-Bel showed a strong preference for PK15(S1-CD163). The G2 strains MN-184, SDSU-73, Korea17 had a much higher infection rate in PK15(S10-CD163), while the reference strain VR2332 and the NADC30 strain had a slight preference for -PK15(S1-CD163). Differences in receptor use may influence the outcome of a PRRSV infection in pigs and explain in part the virulence/pathogenicity of PRRSV strains

Effect of bifidobacteria on intestinal injury and flora in a mouse model of ulcerative colitis

Purpose: To investigate the effect of bifidobacteria on intestinal injury and flora in a mouse model of ulcerative colitis (UC).Methods: Mouse model of UC was produced using dextran sulphate sodium (DSS). The mice were divided into seven groups, viz, reference group, MRS-L medium-negative control group, mesalaminepositive control group, high dose bifidobacteria (MIMBb75) group, middle dose MIMBb75 group and low dose MIMBb75 group. Normal mice were used as control. All mice were sacrificed at day 7 of treatment, and colon length, hemoglobin concentration and intestinal flora were determined.Results: Bifidobacteria inhibited UC-induced decreases in mice hemoglobin and UC-induced colon shortening. In addition, it augmented the diversity of intestinal flora and increased the number of bacteroides and Clostridium leptum.Conclusion: Bifidobacteria plays a therapeutic role in UC via regulation of intestinal microflora.Keywords: Bifidobacteria, MIMBb75, Ulcerative colitis, Intestinal injury, Intestinal flor