Apoptosis in the late replication phase of Bovine alphaherpesvirus 1 in experimentally infected calves

Abstract

Bovine alphaherpesvirus 1 (BoHV-1) is a pathogen causing respiratory and reproductive clinical signs in cattle. Infected animals may develop rhinotracheitis, vulvovaginitis, balanoposthitis, and abortion. Viral latency is generally established in neuronal ganglia simultaneously to a decrease in both genes or genome expression and viral replication. Under stressful conditions, infection is reactivated leading to viral replication and the manifestation of clinical signs. In this study, we evaluated both viral reactivation and apoptosis in trigeminal ganglia cells as BoHV-1 progressed from the latent to the acute phase of infection after dexamethasone administration in experimentally infected calves. To test ganglia cell death as a consequence of BoHV-1 infection, we stained the BoHV-1 samples with TUNEL after the viral shedding by the calves. RT-qPCR of apoptotic genes was also performed, showing the upregulation of the caspase 8 gene in the trigeminal ganglia from cattle experimentally infected with BoHV-1. These results showed the occurrence of apoptosis in ganglion cells of calves infected by BoHV-1.

Keywords: Alphaherpesvirus; Apoptosis; Latency; Neuronal ganglia.

Fig. 1

BoHV-1 titration from experimentally infected cattle in MDBK cells. A Evolution of BoHV-1 titers in nasal secretions from animals from G3 (group resentative of productive infection) during the experimental phase. B Evolution of BoHV-1 titers in nasal secretions of G4 animals (group representative of latent infection) during the experimental phase. The values are expressed in log₂ TCID₅₀/ml; dpi, days post-infection

Fig. 2

Antibody response against BoHV-1 from sera of BoHV-1 experimentally infected cattle. Production of neutralizing antibodies response in cattle sera against BoHV-1 infection by virus neutralization assay. G1: mock-inoculated and treated with dexamethasone group; G2: mock-inoculated but not treated with dexamethasone group; G3: BoHV-1-infected and treated with dexamethasone group (productive infection); and G4: BoHV-1-infected but not treated with dexamethasone group (latent infection). dpi: days post-infection. BLD: below limit of detection

Fig. 3

Identification of +TUNEL cells (nuclei, green) in histological sections of trigeminal ganglia of experimentally infected cattle at day 51 post-infection during a productive infection that was reactivated by dexamethasone (G3) or latent infection (G4). G1: negative control group for G3. G2: negative control group for G4. Arrows denote +TUNEL cells. PC: positive control. Magnification: 200 ×. +TUNEL cells score: [+] low intensity; [+++] high intensity

Fig. 4

Gene profile in trigeminal ganglia collected on day 51. Relative differences in gene expression due to treatment based on the 2^−ΔΔCt analysis of the genes studied. DEX effect: gene expression difference between G1 and G2 due to treatment with dexamethasone (G1). Productive infection: differences between the gene expression in the G1 and G3 groups due to a productive viral infection (G3) (viral reactivation after dexamethasone administration). Latent infection: difference in the gene expression between the G2 and G4 groups due to latent viral infection (G4). A Pro-apoptotic genes with detectable expression. B Anti-apoptotic genes with detectable expression. AIF, apoptosis-inducing factor; BAX, BCL2-associated X protein; BID, BH3 interacting domain death agonist; CAS3, caspase 3; CAS8, caspase 8; p53, tumor suppressor gene; AKT, V-akt murine thymoma viral oncogene homolog 1; BCL2, B-cell lymphoma 2; PI3K, phosphatidylinositol-3-kinase; RAS, Harvey rat sarcoma viral oncogene homolog