Influence of equine herpesvirus type 2 infection on monocyte chemoattractant protein 1 gene transcription in equine blood mononuclear cells M. DUNOWSKA* y , J. MEERS y , R. D. JOHNSON z , C. R. WILKS y y Institute of Veterinary, Animal and Biomedical Sciences, Massey University, New Zealand, z Division of Plant and Soil Sciences, West Virginia University, 401 Brooks Hall, PO Box 6057, Morgantown WV, 26506-6057, USA SUMMARY Representational difference analysis (RDA) was used to compare gene expression in equine mononuclear cells either infected with equine herpesvirus-2 (EHV-2) or adsorbed with inactivated EHV-2. Seven clones identified in non-infected cells after three rounds of selective subtraction and enrichment for differentially expressed genes contained sequences homologous to equine monocyte chemoattractant protein 1 (MCP-1). This suggested that EHV-2 may down-regulate MCP-1 transcription in infected cells. These findings correlate well with similar findings described for human cytomegalovirus and support the view that EHV-2 may have the ability to modify the chemokine environment of infected cells. This may constitute an important feature of EHV-2 biology, because such an ability has the potential to compromise host defence mechanisms and predispose to infection with other pathogens. # 2001 Harcourt Publishers Ltd SEVERAL studies have indicated that equine herpes- virus type 2 (EHV-2) may be involved in equine respiratory disease and poor performance (Fu et al 1986, Schlocker et al 1995, Murray et al 1996, Borchers et al 1997). However, little is known about the patho- genicity of EHV-2 infection and the exact role it may play in equine respiratory disease remains to be estab- lished. An indirect proof that EHV-2 may interact with the host immune response and influence the suscept- ibility of infected foals to secondary infections was provided by Nordengrahn et al (1996), who showed that EHV-2 vaccination prevented the occurrence of Rhodococcus equi pneumonia in foals. EHV-2 has been shown to be latent in B lymphocytes (Drummer et al 1996). Additionally, examination of the genome of EHV-2 revealed that it codes for an interleukin-10-like protein and two, or possibly three, proteins that have a structure characteristic of G- protein-coupled receptors (GPCR) (Telford et al 1995). These findings indicate that EHV-2 has the potential to modulate host immune responses. Herpesviruses are well adapted to their hosts (Banks and Rouse 1992). They rarely cause serious diseases in adult, immunocompetent individuals, and can establish latent or persistent infection that may last for a life- time. Clinical signs observed in a field situation fol- lowing infection with EHV-2 may be due to secondary infections, rather than be a direct consequence of a primary herpesviral infection. Thus, the importance of EHV-2 infection may be difficult to assess based on the observation of clinical signs following experimental challenge if the secondary pathogens are not also pre- sent. In contrast, interactions that occur at a molecular level between a virus and the infected cell may provide more reliable information about the relationships between the microorganism and its host. In this study, representational difference analysis (RDA) was used to investigate differences, at a mole- cular level, between equine peripheral blood leucocytes (PBL) infected with EHV-2 and those adsorbed with inactivated EHV-2. An EHV-2 isolate was cultured from the PBL of a foal showing clinical signs of upper respiratory disease. The isolate was confirmed to be EHV-2 by PCR and Southern hybridisation with an EHV-2 specific probe (Dunowska 1999). The virus was passaged seven times in equine foetal kidney (EFK) cells and purified by ultracentrifugation at 100,000 g for 90 minutes through a 25 per cent sucrose cushion, re-suspended in phosphate buffered saline, pH 70(PBS), and stored at 70  C until use. Virus inactiva- tion was achieved by incubation at 56  C for 30 minutes, followed by UV treatment for 15 minutes. Inactivation was successful, as no cytopathic effect (CPE) was observed in EFK cells adsorbed with inactivated EHV-2 after two 1-week passages. Approximately 120 ml of blood were collected in heparin from a healthy, adult thoroughbred horse, that had been negative for EHV-2 infection on several occasions prior to the experiment, as tested by PCR, Research in Veterinary Science 2001, 71, 111±113 doi:10.1053/rvsc.2001.0493, available online at http://www.idealibrary.com on 0034-5288/01/020111  03 $35.00/0 # 2001 Harcourt Publishers Ltd *Corresponding author: Magdalena Dunowska, Department of Pathology, Colorado State University, Fort Collins, CO 80523-1671, USA. Tel.: (970) 491 6144; Fax: (970) 491 0603; E-mail: mdunowsk@lamar.colostate.edu