Cellular response markers and cytokine gene expression in the central nervous system of cattle naturally infected with bovine herpesvirus 5

Abstract

The present study reports an investigation on the phenotype of inflammatory and immune cells, cytokine and viral gene expression in the brains of cattle naturally infected with bovine herpesvirus 5 (BHV5). Brain sections of 38 affected animals were analysed for the nature and extent of perivascular cuffs in the Virchow-Robin space and parenchyma. Histopathological changes were severe in the olfactory bulbs (Obs), hippocampus, piriform, frontal, temporal and parietal cortices/lobes and were characterized by inflammatory infiltrates in Virchow-Robin spaces. The histopathological changes correlated positively with the distribution of BHV5 antigens (r = 0.947; P < 0.005). Cells of CD3+ phenotype were predominant in areas with severe perivascular cuffs. Viral antigens and genomic viral DNA were detected in the Obs and piriform lobe, simultaneously (r = 0.987; P < 0.005). Similarly, pro-inflammatory cytokine genes INFG, IL2, TNF and LTBR were expressed in the same brain areas (P < 0.005). These results provide important information on the inflammatory and immunological events accompanying BHV5 neurological infections. Our findings provide the first evidence for increased immune activation followed by inflammatory cytokine expression, positively correlated with viral replication in the cranial areas of the brain. Taken together, these results suggest that the host immune response and inflammation play a crucial role in the pathogenesis of acute encephalitis by BHV5 in cattle.

Expression of miR-155 associated with Toll-like receptors 3, 7, and 9 transcription in the olfactory bulbs of cattle naturally infected with BHV5

“…Brains of 16 cattle with positive diagnosis of acute BHV5 neurological disease (Cardoso et al 2016b) and from eight healthy cattle were obtained from routine pathological diagnostics at the Laboratory of Animal Virology of University of São Paulo state, Brazil. The brain samples were selected from 38 cattle reported previously (Cardoso et al 2016b) based on OB anatomical preservation. All applicable institutional guidelines for the care and use of animals were followed (CEEA 2015/09765).…”

“…Briefly, OB histological slides (infected and controls) prepared as described previously (Cardoso et al 2016b) were hydrated for 15 min followed by incubation overnight at 4°C with 1:50 diluted monoclonal antibody to BHV5 (BHV52F9) (Goldoni et al 2004) and 1:100 diluted N200 monoclonal antibody (Sigma-Aldrich®) produced against neurofilament. After three washes, slides were incubated with the respective goat secondary antibody (1:100) anti-mouse FITC (Sigma-Aldrich®) and nuclear staining was performed with 1 mg/ml of DAPI (4′-6-diamino-2-phenylindole; Sigma-Aldrich®) was diluted in Fluormount™ aqueous medium.…”

“…Briefly, total DNA from each OB region and respective negative control was extracted using DNAzol™ according to manufacturer's instructions (Invitrogen®). An average of 100 ng of genomic DNA were used for quantitative real-time polymerase chain reaction (qPCR) and BHV5 primers and respective probes developed for glycol-C gene were used as described previously (Cardoso et al 2016b). The data obtained from three replicates were carried out by qPCR reaction and analyzed by the software on a StepOnePlus™ real time instrument (Applied Biosystems®).…”

“…The expression of the housekeeping Histone 2a gene was also quantified in a similar way as viral DNA. The qPCR reaction was conducted as described previously (Cardoso et al 2016b).The same amount of cDNA applied on TLR3, 7, and 9 transcription was used to detect sequence mature of bta-miR-155 (cat # 25576) and amplification was performed using Advanced miRNA Assay™ (Applied Biosystems®). All reactions were carried out and analyzed as described for BHV5 DNA detection.…”

“…Cattle experimentally infected with BHV5 showed an overexpression of TLRs 3, 7, and 9 in the frontal cerebral cortex, including the OBs, during acute infection and/or following virus reactivation (Marin et al 2015). We have previously demonstrated that the host immune response and inflammation play a crucial role in the pathogenesis of acute encephalitis by BHV5 in cattle (Cardoso et al 2016b). In many aspects, there is a consensus that BHV5 acute infection induces activation of proinflammatory cytokines and chemokines that eventually lead to brain injury, especially in frontal cortices (Marin et al 2015).…”