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

ABSTRACT

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.