Analysis of bovine trigeminal ganglia following infection with bovine herpesvirus 1.

Following primary infection of the eye, oral cavity, and/or nasal cavity, bovine herpesvirus 1 (BHV-1) establishes latency in trigeminal ganglionic (TG) neurons. Virus reactivation and spread to other susceptible animals occur after natural or corticosteroid-induced stress. Infection of calves with BHV-1 leads to infiltration of lymphocytes in TG and expression of IFN-gamma (interferon-gamma), even in latently infected calves. During latency, virus antigen and nucleic acid positive non-neural cells were occasionally detected in TG suggesting there is a low level of spontaneous reactivation. Since we could not detect virus in ocular or nasal swabs, these rare cells do not support high levels of productive infection and virus release or they do not support virus production at all. Dexamethasone (DEX) was used to initiate reactivation in latently infected calves. Foci of mononuclear or satellite cells undergoing apoptosis were detected 6h after DEX treatment, as judged by the appearance of TUNEL+ cells (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling). BHV-1 antigen expression was initially detected in lymphocytes and other non-neural cells in latently infected calves following DEX treatment. At 24h after DEX treatment, viral antigen expression and nucleic acid were readily detected in neurons. Our data suggest that persistent lymphocyte infiltration and cytokine expression occur during latency because a low number of cells in TG express BHV-1 proteins. Induction of apoptosis and changes in cytokine expression following DEX treatment correlates with reactivation from latency. We hypothesize that inflammatory infiltration of lymphoid cells in TG plays a role in regulating latency.

Persistence and reactivation of bovine herpesvirus 1 in the tonsils of latently infected calves.

Bovine herpesvirus 1 (BHV-1), like other members of the Alphaherpesvirinae subfamily, establishes latent infection in sensory neurons. Reactivation from latency can occur after natural or corticosteroid-induced stress culminating in recurrent disease and/or virus transmission to uninfected animals. Our previous results concluded that CD4(+) T cells in the tonsil and other adjacent lymph nodes are infected and undergo apoptosis during acute infection (M. T. Winkler, A. Doster, and C. Jones, J. Virol. 73:8657-8668, 1999). To test whether BHV-1 persisted in lymphoreticular tissue, we analyzed tonsils of latently infected calves for the presence of viral DNA and gene expression. BHV-1 DNA was consistently detected in the tonsils of latently infected calves. Detection of the latency-related transcript (LRT) in tonsils of latently infected calves required nested reverse transcription-PCR (RT-PCR) suggesting that only a few cells contained viral DNA or that LRT is not an abundant transcript. bICP0 (immediate-early and early transcripts), ribonucleotide reductase (early transcript), and glycoprotein C (late transcript) were not detected by RT-PCR in latently infected calves. When reactivation was initiated by dexamethasone, bICP0 and ribonucleotide reductase transcripts were detected. Following dexamethasone treatment, viral nucleic acid was detected simultaneously in trigeminal ganglionic neurons and lymphoid follicles of tonsil. LRT was detected at 6 and 24 h after dexamethasone treatment but not at 48 h. Dexamethasone-induced reactivation led to apoptosis that was localized to tonsillar lymphoid follicles. Taken together, these findings suggest that the tonsil is a site for persistence or latency from which virus can be reactivated by dexamethasone. We further hypothesize that the shedding of virus from the tonsil during reactivation plays a role in virus transmission.

Bovine herpesvirus 1 can infect CD4(+) T lymphocytes and induce programmed cell death during acute infection of cattle.

Acute infection of cattle with bovine herpesvirus 1 (BHV-1) represses cell-mediated immunity, which can lead to secondary bacterial infections. Since BHV-1 can induce apoptosis of cultured lymphocytes, we hypothesized that these virus-host interactions occur in cattle. To test this hypothesis, we analyzed lymph nodes and peripheral blood mononuclear cells (PBMC) after calves were infected with BHV-1. In situ terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) staining of lymphoid tissues (pharyngeal tonsil, cervical, retropharyngeal, and inguinal) was used to detect apoptotic cells. Calves infected with BHV-1 for 7 days revealed increased apoptotic cells near the corticomedullary junction in lymphoid follicles and in the subcapsular region. Increased frequency of apoptotic cells was also observed in the mucosa-associated lymphoid tissue lining the trachea and turbinate. Immunohistochemistry of consecutive sections from pharyngeal tonsil revealed that CD2(+) T lymphocytes were positive for the BHV-1 envelope glycoprotein gD. The location of these CD2(+) T lymphocytes in the germinal center suggested that they were CD4(+) T cells. Electron microscopy and TUNEL also revealed apoptotic and herpesvirus-infected lymphocytes from this area. Fluorescence-activated cell sorting analyses demonstrated that CD4(+) and CD8(+) T cells decreased in lymph nodes and PBMC after infection. The decrease in CD4(+) T cells correlated with an increase in apoptosis. CD4(+) but not CD8(+) lymphocytes were infected by BHV-1 as judged by in situ hybridization and PCR, respectively. Immediate-early (bovine ICP0) and early (ribonucleotide reductase) transcripts were detected in PBMC and CD4(+) lymphocytes prepared from infected calves. In contrast, a late transcript (glycoprotein C) was not consistently detected suggesting productive infection was not efficient. Taken together, these results indicate that BHV-1 can infect CD4(+) T cells in cattle, leading to apoptosis and suppression of cell-mediated immunity.

A protein encoded by the latency-related gene of bovine herpesvirus 1 is expressed in trigeminal ganglionic neurons of latently infected cattle and interacts with cyclin-dependent kinase 2 during productive infection.

Despite productive viral gene expression in the peripheral nervous system during acute infection, the bovine herpesvirus 1 (BHV-1) infection cycle is blocked in sensory ganglionic neurons and consequently latency is established. The only abundant viral transcript expressed during latency is the latency-related (LR) RNA. LR gene products inhibit S-phase entry, and binding of the LR protein (LRP) to cyclin A was hypothesized to block cell cycle progression. This study demonstrates LRP is a nuclear protein which is expressed in neurons of latently infected cattle. Affinity chromatography indicated that LRP interacts with cyclin-dependent kinase 2 (cdk2)-cyclin complexes or cdc2-cyclin complexes in transfected human cells or infected bovine cells. After partial purification using three different columns (DEAE-Sepharose, Econo S, and heparin-agarose), LRP was primarily associated with cdk2-cyclin E complexes, an enzyme which is necessary for G1-to-S-phase cell cycle progression. During acute infection of trigeminal ganglia or following dexamethasone-induced reactivation, BHV-1 induces expression of cyclin A in neurons (L. M. Schang, A. Hossain, and C. Jones, J. Virol. 70:3807-3814, 1996). Expression of S-phase regulatory proteins (cyclin A, for example) leads to neuronal apoptosis. Consequently, we hypothesize that interactions between LRP and cell cycle regulatory proteins promote survival of postmitotic neurons during acute infection and/or reactivation.

Study of neutralizing monoclonal antibodies to bovine herpes virus type-1 (Cooper strain) by immunoperoxidase and immunoelectron microscopy.

Three neutralizing monoclonal antibodies (mAbs) that are specific against bovine herpes virus Type-1 (BHV-1) were studied as to their viral specificity by immunoperoxidase and immunoelectron microscopy. Microscopic examination of GBK BHV-1 infected cells revealed peroxidase activity represented by red-brown granular deposits in the nucleus and cytoplasm. No immunoperoxidase activity was observed in negative controls. For the ultrastructural observations, two approaches were used. Firstly we tested a pre-embedding technique using GBK infected cells, mAbs and gold conjugated-protein A. Gold particles were observed linked to the viral envelopes and to the host cell membrane. Alternatively, a second technique employed BHV-1 purified by potassium tartrate gradients, mAbs and gold conjugated-protein A. After performing the immune reaction, the samples were adsorbed to formvar-coated grids, stained with phosphotungstic acid and observed in a transmission electron microscope. Gold particles were mainly attached to the virion envelope.

Anticuerpos monoclonales específicos para herpesvirus bovino tipo-1 (cepa Cooper)

Anticuerpos monoclonales fueron preparados contra herpesvirus bovino tipo 1 (BHV-1) purificado y se seleccionaron varios de acuerdo con su habilidad de neutralizar la infectividad viral. El virus fue purificado por dos métodos alternativos de concentración, con polietilenglicol seguido de ultracentrifugación sobre un colchón de sacarosa al 25 por ciento (p/p) o usando un gradiente linear de tartrato de potasio. De un total de 204 líneas celulares que segregaban anticuerpos para el virus, obtenidas en dos fusiones, se seleccionaron y expandieron clonalmente 39 hibridomas. La selección se basó en su reactividad específica por ELISA. De éstos, 11 anticuerpos monoclonales fueron capaces de neutralizar BHV-1 total o parcialmente, con o sin el agregado de complemento. Siete anticuerpos monoclonales fueron producidos (en la forma de fluido ascítico) y purificados por precipitación en sulfato de amonio. De éstos, dos fueron capaces de prevenir la penetración de virión luego de su adherencia a la células. Asimismo, tres anticuerpos monoclonales fueron seleccionados para ser usados en las pruebas de inmunoperoxidasa para la detección del BHV-1 en cultivos celulares infectados.

Monoclonal antibodies (McAbs) were prepared against purified Bovine Herpesvirus Type-(BHV-1) and selected for their ability to neutralize viral infectivity. Viral purification was performed by polyethylene glycol concentration and ultracentrifugation on a 25% (w/w) sucrose cushion, and by potassium tartrate linear gradient. Out of 204 cell lines expressing antobodies to the virus, obtained in two fusions, 39 hybridomas were selected and cloned based on enzyme-linked immunosorbent assay (ELISA) reactivity. Eleven McAbs were able to neutralize BHV-1 partially or totally, with or without complement. Seven McAbs were produced as ascitic fluid, and ammonium sulfate-purified. Of these, two were able to prevent virion penetration into the cell after attachment. Three neutralizing McAbs were selected for use in immunoperoxidase tests for detecting BHV-1 in infected cell cultures.