Short Communication Absence of viral antigens on the surface of equine herpesvirus-1-infected peripheral blood mononuclear cells: a strategy to avoid complement-mediated lysis Karen M. van der Meulen, Hans J. Nauwynck and Maurice B. Pensaert Correspondence Hans J. Nauwynck hans.nauwynck@rug.ac.be Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium Received 27 September 2002 Accepted 3 October 2002 Equine herpesvirus-1 (EHV-1) may cause abortion in vaccination- and infection-immune horses. EHV-1-infected peripheral blood mononuclear cells (PBMCs) play an important role in virus immune evasion. The mechanisms by which infected PBMCs can avoid destruction by EHV-1- specific antibody and equine complement were examined. The majority of EHV-1-infected PBMCs (68?6 %) lacked surface expression of viral antigens and these cells were not susceptible to complement-mediated lysis. In infected PBMCs with surface expression of viral antigens, 63 % showed focal surface expression, whereas 37 % showed general surface expression. General surface expression rendered infected PBMCs susceptible to lysis by antibody and complement (from 5?4 to 31?2 % lysed cells depending on the concentration of antibody and complement). Infected PBMCs with focal surface expression showed significant lysis only in the presence of high concentrations of antibody and complement. Thus, the absence of surface expression protects infected PBMCs against complement-mediated lysis. Equine herpesvirus-1 (EHV-1), a member of the genus Alphaherpesvirus, is an important pathogen of horses. After exposure, EHV-1 replicates in the respiratory tract. Replication is followed by a leukocyte-associated viraemia which enables EHV-1 to reach internal organs where its replication can result in abortion, neonatal death or ner- vous system disorders (Allen & Bryans, 1986). Viraemia may occur in the presence of virus-neutralizing antibodies in infection-immune (Doll & Bryans, 1963; Gleeson & Coggins, 1980; Mumford et al., 1987) and vaccination- immune (Bu ¨rki et al., 1990; Heldens et al., 2001) horses. Apparently, recognition of circulating EHV-1-infected peri- pheral blood mononuclear cells (PBMCs) by the antibody- mediated immune system is inefficient. Following infection of cells with enveloped viruses, viral glycoproteins are incorporated into cellular membranes. Binding of virus-specific antibodies to glycoproteins pre- sent in the plasma membrane makes infected cells recog- nizable for the classical complement pathway, phagocytes and natural killer cells, leading to lysis of the cell (Harper, 1994). Several herpesviruses have developed strategies to avoid antibody-dependent cell lysis. For pseudorabies virus (PRV)-infected monocytes, addition of PRV-specific anti- bodies results in clearance of viral glycoproteins from the plasma membrane by antibody-induced internalization (Favoreel et al., 1999). Clearance of the plasma membrane renders infected monocytes significantly less susceptible towards antibody-dependent, complement-mediated lysis (G. R. Van de Walle, H. W. Favoreel, H. J. Nauwynck and M. B. Pensaert, unpublished results). In human cytomegalo- virus (HCMV)-infected monocyte-derived macrophages, transport of viral glycoproteins to the plasma membrane is prevented due to the destruction of the microtubule network (Fish et al., 1996). The absence of HCMV glycoproteins on the cell surface may be another strategy of avoiding recognition by antibody-dependent immune responses. Finally, several herpesviruses are known to encode proteins that interfere with the activation of the complement cascade (reviewed by Favoreel et al., 2000). The main purpose of the present study was to investigate how EHV-1-infected PBMCs are able to avoid recognition and destruction by antibody-dependent immune responses. PBMCs were isolated from infection-immune horses by density centrifugation on Ficoll–Hypaque. After isolation, PBMCs were incubated for 24 h in medium supplemented with 0?5 mM ionomycin (IONO) and 10 nM phorbol dibu- tyrate (PDB) (Sigma) to favour EHV-1 replication (van der Meulen et al., 2001). Rabbit kidney (RK13) and equine embryonic lung (EEL) cells were maintained in minimal essential medium supplemented with 5 % foetal bovine serum, 100 U penicillin ml 21 ,0?1 mg streptomycin ml 21 , 0?1 mg kanamycin ml 21 and 0?3 mg glutamine ml 21 , until trypsinization and inoculation. All cells were inoculated 0001-8864 G 2003 SGM Printed in Great Britain 93 Journal of General Virology (2003), 84, 93–97 DOI 10.1099/vir.0.18864-0