A Sensitive and Specific PCR/dot Blot Hybridization assay for the Detection of Ovine Herpesvirus 2, a Gamma Herpesvirus

Polymerase chain reaction (PCR) provides a powerful technique for identifying viruses and studying the homology between viral nucleic acids. However, PCR assay has limitations in its susceptibility to contamination or to enzymatic inhibitors. In order to avoid problems related to nucleic acid amplification, efforts have been made to obtain specific hybridization assays, such as dot blot hybridization (DBH). DBH has higher specificity and lower sensitivity than PCR. The aims of the present study were to develop a sensitive and specific assay for the detection of ovine herpesvirus 2 (OvHV-2), a gamma herpesvirus. PCR/DBH assay for detecting OvHV-2 DNA was developed and evaluated for its sensitivity and specificity. OvHV-2 specific primer pairs, 755/556, were used for the amplification of target DNA. When PCR product was visually detected, the limit of detection of the PCR test was 102 viral copies. For DBH, the amplified DNA with OvHV-2 specific primer pairs, 556/555, was labeled by the incorporation of digoxigenin (DIG). This DIGlabeled probe was capable of detecting 104 viral copies of purified OvHV-2 DNA by DBH. On the other hand, PCR/ DBH was more sensitive than either PCR or DBH and also very specific. The results showed that the sensitivity of PCR/DBH was higher and stronger than that of PCR and DBH alone. This PCR/DBH assay can be applied efficiently to confirm the presence of OvHV-2 virus on clinical samples and to differentiate specifically between OvHV-2 infection and other viral infections.

The Gamma Herpesvirus Alcelaphine Herpesvirus 1 Causes Apoptotic Infection in Permissive Cell Lines

Apoptosis is a host defense mechanism that the cell uses to limit production of infectious virus. Although many viruses can induce apoptosis in infected cells, large DNA viruses, such as poxviruses, herpesviruses and adenoviruses, usually exhibit the ability to suppress the induction of apoptosis in the infected cells. Several publications have attested to the ability of herpesviruses to protect cells against apoptosis. We investigated the ability of the virus to protect cells in continuous cultivation from apoptosis induced by the virus itself. The gamma herpesvirus alcelaphine herpesvirus 1 (AlHV-1) has been shown to harbor genes with antiapoptotic potentialities. However, here we have demonstrated that productive infection of adherent, permissive cell lines by AlHV-1 resulted in a cytopathic effect characterized by induction of apoptosis. This phenomenon was confirmed using different techniques to detect apoptosis and using different virus strains and cell lines. Therefore, despite the presence of antiapoptotic genes in its genome, AlHV-1 could complete its cycle of productive infection while inducing apoptosis of infected cells. This finding might have implications for the pathobiology of AlHV-1 and other gamma herpesviruses in vivo.

Application of Laser Microdissection to Identify the Cells Susceptible to Ovine Herpesvirus 2

Laser microdissection (LMD) is an important method for obtaining pure cell samples for genetic and proteomic analysis. In general, immunohistochemistry (IHC) and in situ hybridization (ISH) are useful techniques for targeting virus-specific cell populations. However, until now, there have been no IHC and ISH methods available for detecting ovine herpesvirus (OvHV-2). Previous reports have strongly suggested that lytic replication might occur in the respiratory epithelial cells of OvHV-2 infected animals. The aim of the present study was to confirm respiratory epithelial cells as the susceptible cells for the OvHV-2 by using LMD as an alternative method for localizing viral distribution. The microdissection of target cells by LMD was performed using paraffin-embedded tissues from 5 sheep with high viral copies, which were suspected as the status of reactive lytic replication, and 3 sheep with low viral copies, which were suspected as the status of latent infection. Then, OvHV-2-specific polymerase chain reaction (PCR) and real-time PCR were conducted with the extracted DNAs from the microdissected cells. Our results first demonstrate that OvHV-2 DNAs can be detected in the respiratory epithelial cells of high shedder reactive animals, from which inflammatory cells infected latently by OvHV-2 was excluded. These findings indicate that respiratory epithelial cells are susceptible to OvHV-2 and may be associated with its replication in a natural host. Also, in this study, LMD showed the possibility of wide application for the sensitive localization of low copy viral sequences within specific phenotype cells in the investigation of the role of viruses in a variety of clinical conditions.