Comparative study on the in vitro and in vivo properties of two bovine herpesvirus-5 reference strains

<p>Abstract</p> <p>Background</p> <p>Bovine herpesvirus 5 (BoHV-5) is an alphaherpesvirus responsible for meningoencephalitis in young cattle and it is antigenically and genetically related to bovine herpesvirus 1. BoHV-5 outbreaks are sporadic and restricted in their geographical distribution, being mostly detected in the Southern hemisphere. The N569 and A663 strains are prototypes of the "a" and "b" subtypes of BoHV-5, however, scarce information about their <it>in vitro </it>and <it>in vivo </it>properties is currently available.</p> <p>Methods</p> <p>For the <it>in vitro </it>comparison between BoHV-5 A663 and N569 strains, viral growth kinetics, lysis and infection plaque size assays were performed. Additionally, an experimental infection of cattle with BoHV-5 A663 and N569 strains was carried out. Viral excretion, development of neurological signs, presence of specific antibodies in serum and nasal swabs and presence of latent BoHV-5 DNA in trigeminal ganglion, were analyzed. Histopathological examination of samples belonging to inoculated animals was also performed.</p> <p>Results</p> <p>The lytic capacity and the cell-to-cell spread was lower for the A663 strain compared to the N569 strain, however, the production of total infectious viral particles was similar between both strains. Concerning the <it>in vivo </it>properties, the A663 and N569 strains are able to induce similar degrees of pathogenicity in cattle.</p> <p>Conclusions</p> <p>Our results show that the A663 strain used in this study is less adapted to <it>in vitro </it>replication in MDBK cells than the N569 strain and, although slight differences were observed, both strains are able to induce a similar degree of virulence in the natural host.</p

Characterization of BoHV-5 field strains circulation and report of transient specific subtype of bovine herpesvirus 5 in Argentina

<p>Abstract</p> <p>Background</p> <p>Bovine herpesvirus 5 (BoHV-5) is a member of the subfamily <it>Alphaherpesvirinae </it>responsible for meningo-encephalitis in young cattle. The first case of bovine meningo-encephalitis associated with a herpesvirus infection was reported in Australia. The current geographical distribution of BoHV-5 infection is mainly restricted to South America, especially Brazil and Argentina. Outbreaks of BoHV-5 are regularly observed in Argentina suggesting the circulation of the virus in the bovine population.</p> <p>Results</p> <p>Seventeen field strains of BoHV-5 isolated from 1984 to now were confirmed by differential PCR and subjected to restriction endonuclease analysis (REA). Viral DNA was cleaved with BstEII which allows the differentiation among subtypes a, b and non a, non b. According to the REA with BstEII, only one field strain showed a pattern similar to the Argentinean A663 strain (prototype of BoHV-5b). All other isolates showed a clear pattern similar to the Australian N569 strain (prototype of BoHV-5a) consistent with the subtypes observed in Brazil, the other South-American country where BoHV-5 is known to be prevalent. The genomic region of subtype b responsible for the distinct pattern was determined and amplified by PCR; specifically a point mutation was identified in glycoprotein B gene, on the BstEII restriction site, which generates the profile specific of BoHV-5b.</p> <p>Conclusions</p> <p>This is the first report of circulation of BoHV-5a in Argentina as the prevailing subtype. Therefore the circulation of BoHV-5b was restricted to a few years in Argentina, speculating that this subtype was not able to be maintained in the bovine population. The mutation in the gB gene is associated with the difference in the restriction patterns between subtypes "a" and "b".</p

MageA6 expression increases MageA11 protein levels.

<p>(A)Western blot showing Flag-MageA11 when co-expressed with increasing quantities of HA-MageA6 (0, 250, 500, 1000 and 1500 ng). GFP expression is the internal control. Membrane was probed with the indicated antibodies. (B) Western blot showing Flag-MageA11 when co-expressed with increasing quantities of HA-MHD-MageA6 (0, 250, 500, 1000 and 1500 ng). GFP expression is the internal control. Membrane was probed with the indicated antibodies. (C) Western blot showing Flag-MageA11 when co-expressed with increasing quantities of HA-MageA2 (0, 250, 500, 1000 and 1500 ng). GFP expression is the internal control. Membrane was probed with the indicated antibodies. (D) Western blot of LNCaP cells silenced (siA6/2) or not (siC) for MageA6 expression. Anti-pan MAGE-A antibody (6C1, Santa Cruz) was used to detect Mage-A proteins. 65KDa band corresponds to MageA11 while 45KDa band could correspond to different Mage-A proteins. GAPDH was used as loading control. (E)Left panel: Western Blot showing the endogenous levels of MageA11 in LNCaP stably expressing MageA6 (A6) or empty vector (EV). Extracts of HEK293T cells transfected with Flag-MageA11 (F-A11), HA-MageA6 (HA-A6) or empty vector (EV) were used as controls. MAGE-A detection was performed with anti-pan MAGE antibody (6C1, Santa Cruz). 65KDa band corresponds to MageA11 while 45KDa band could correspond to different Mage-A proteins. The observed increment in 45KDa band in LNCaP-A6 is caused by MageA6 stable expression. Right panel: quantification of MAGE-A11 vs β-tubulin band intensity corresponding to Fig 4E, lanes 4 and 5. (F) RT-qPCR for the determination of MageA11 mRNA levels in LNCaP-A6 (A6) and LNCaP-EV (EV). MageA11 mRNA was normalized to GAPDH mRNA levels. (G) RT-qPCR for the determination of PSA mRNA levels in LNCaP-A6 (A6) and LNCaP-EV (EV). PSA mRNA was normalized to GAPDH mRNA levels. Error bars indicate mean S.D. Student’s t test was used for statistical analysis. ** p < 0.001. * unspecific band. Triangles show the corresponding protein band and dashes mark the MW.</p

MageA6 and MageA11 co-expression in prostate cancer.

<p>Analysis of cBioPortal Cancer Genomics data sets form Prostate Adenocarcinoma and Testicular Germ Cell Cancer (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178370#sec002" target="_blank">Material and Methods</a>) as indicated in the two main columns. Gene overexpression was calculated by Z-score, defined as the relative expression of an individual gene to the gene’s expression distribution in a reference population. The indicated percent of over-expression refers to the number of samples over-expressing a given gene over the total of samples. Dot-plot graphics shows the correlation between MageA6 and MageA11 gene expression. Insets indicate Pearson and Spearman correlation scores.</p

MageA6 enhances MageA11-dependent AR transcriptional activity.

<p>(A) Reporter gene assay for GR, MR and AR activity using specific gene-reporter in the presence or absence of MageA6 or MageA11 expression. Cells were treated with dexamethasone (Dx), Aldosterone (Aldo) or dihidrotestosterone (DHT) for 24 h prior to harvesting. The assay was performed in HEK293T cells. ev, empty vector. (B) Similar to A but combining MageA11 and MageA6 expression as indicated. (C) Determination of PSA mRNA levels through RT-qPCR. LNCaP cells were transfected with a siRNA control (siC) or a siRNA to silence MageA6 expression (siA6/2). DHT was added to cells as indicated. PSA mRNA levels were normalized to GAPDH mRNA levels. Error bars indicate mean S.D. Student’s t test was used for statistical analysis. * p < 0.05. ** p < 0.001.</p