Replication kinetics of neurovirulent versus non-neurovirulent equine herpesvirus type 1 strains in equine nasal mucosal explants Annelies P. Vandekerckhove, 1 S. Glorieux, 1 A. C. Gryspeerdt, 1 L. Steukers, 1 L. Duchateau, 2 N. Osterrieder, 3 G. R. Van de Walle 1,2 3 and H. J. Nauwynck 1 3 Correspondence G. R. Van de Walle gerlinde.vandewalle@UGent.be Received 18 December 2009 Accepted 25 April 2010 1 Department of Virology, Parasitology and Immunology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium 2 Department of Physiology and Biometrics, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium 3 Institut fu ¨ r Virologie, Freie Universitaet Berlin, Philippstrasse 13, 10115 Berlin, Germany Equine herpesvirus type 1 (EHV-1) is the causative agent of equine herpes myeloencephalopathy, of which outbreaks are reported with increasing frequency throughout North America and Europe. This has resulted in its classification as a potentially emerging disease by the US Department of Agriculture. Recently, it was found that a single nucleotide polymorphism (SNP) in the viral DNA polymerase gene (ORF30) at aa 752 (NAD) is associated with the neurovirulent potential of EHV-1. In the present study, equine respiratory mucosal explants were inoculated with several Belgian isolates typed in their ORF30 as D 752 or N 752 , to evaluate a possible difference in replication in the upper respiratory tract. In addition, to evaluate whether any observed differences could be attributed to the SNP associated with neurovirulence, the experiments were repeated with parental Ab4 (reference neurovirulent strain), parental NY03 (reference non-neurovirulent strain) and their N/D revertant recombinant viruses. The salient findings were that EHV-1 spreads plaquewise in the epithelium, but plaques never cross the basement membrane (BM). However, single EHV-1-infected cells could be observed below the BM at 36 h post-inoculation (p.i.) for all N 752 isolates and at 24 h p.i. for all D 752 isolates, and were identified as monocytic cells and T lymphocytes. Interestingly, the number of infected cells was two to five times higher for D 752 isolates compared with N 752 isolates at every time point analysed. Finally, this study showed that equine respiratory explants are a valuable and reproducible model to study EHV-1 neurovirulence in vitro, thereby reducing the need for horses as experimental animals. INTRODUCTION Equine herpesvirus 1 (EHV-1) is a ubiquitous respiratory viral pathogen that causes serious economic losses in the horse industry worldwide (Allen & Bryans, 1986; Bryans & Allen, 1989; Brosnahan & Osterrieder, 2009). EHV-1 exerts its impact by causing respiratory tract disease, but it can also cause abortion, neonatal foal death and nervous system disorders (Patel & Heldens, 2005; Lunn et al., 2009). The mucosa of the upper airway tract is the first line of defence against respiratory diseases (Timoney, 2004). It is also the primary replication site of EHV-1, as it is for most alphaherpesviruses (Kydd et al., 1994a; Van Maanen, 2002; Van Maanen & Cullinane, 2002; Gryspeerdt et al., 2010). Subsequently, the virus disseminates via a leukocyte-associated viraemia, which enables EHV-1 to reach end-vessel endothelia in the uterus and central nervous system (Allen & Bryans, 1986; Kydd et al., 1994b). In these organ systems, virus replication can result in vasculitis and perivasculitis, ultimately resulting in abor- tion and myeloencephalopathy, respectively. Devastating outbreaks of equine herpes myeloencephalopathy are reported with increasing frequency throughout North America and Europe (Kohn et al., 2006; Perkins et al., 2009; Pusterla et al., 2009), resulting in its classification as a potentially emerging disease by the US Department of Agriculture’s Animal and Plant Health Inspection Service (USDA APHIS, 2007). Recently, it was shown by Nugent et al. (2006) that a single nucleotide polymorphism (SNP) in the catalytic subunit of the viral DNA polymerase, encoded by open reading frame 3These authors contributed equally to this work. Journal of General Virology (2010), 91, 2019–2028 DOI 10.1099/vir.0.019257-0 019257 G 2010 SGM Printed in Great Britain 2019