Downloaded from www.microbiologyresearch.org by IP: 54.204.244.33 On: Mon, 17 Oct 2016 08:53:40 Journal of General Virology (1997), 78, 2147–2151. Printed in Great Britain ............................................................................................................................................................................................................... SHORT COMMUNICATION Deletion of glycoprotein gM of pseudorabies virus results in attenuation for the natural host Johannes M. Dijkstra, Volker Gerdts, Barbara G. Klupp and Thomas C. Mettenleiter Institute of Molecular and Cellular Virology, Friedrich-Loeffler-Institutes, Federal Research Centre for Virus Diseases of Animals, D-17498 Insel Riems, Germany Glycoprotein M (gM) is one of the very few non- essential glycoproteins conserved throughout the herpesvirus family. Despite this conservation little is known about its function in virus replication. To test for the importance of gM in vivo in a natural virus–host system, 6-week-old piglets were intranasally infected with a gM N mutant of the alphaherpesvirus pseudorabies virus (PrV). Follow- ing infection virus excretion from the nasal mucosa was decreased ca. 100-fold compared to wild-type or revertant virus. Clinical signs were limited to transiently elevated temperature. In contrast, animals infected by wild-type or revertant virus exhibited high fever, severe respiratory symptoms and affliction of the central nervous system. Prior infection with gM N PrV conferred protection against challenge infection and animals mounted an anti- body response against gM after wild-type virus infection. Thus, gM is important for efficient virus replication in vivo and deletion of gM may con- tribute to development of live attenuated, geneti- cally marked vaccines. The alphaherpesvirus pseudorabies virus (PrV) causes Aujeszky’s disease (AD) in pigs, characterized by respiratory symptoms and central nervous disorders. Manifestation of disease depends on the virulence and infectious dose of the virus strain, age of the animal and route of infection. Naturally, infection occurs oronasally resulting in primary replication in the respiratory tract leading to respiratory distress. In addition, the virus enters nerve endings in trigeminal and olfactory pathways and ascends into the central nervous system. Whereas younger animals may exhibit a high mortality due to severe central nervous disorders, older animals usually survive with only moderate respiratory symptoms (Pensaert & Kluge, 1989). To prevent economic losses, vaccination with live attenuated and inactivated vaccines is performed. Although Author for correspondence : Thomas C. Mettenleiter. Fax 49 38351 7151. e-mail mettenleiterrie.bfav.de PrV is able to infect most mammals except higher primates, including humans, pigs are the only animal species capable of surviving a productive PrV infection. They are, therefore, considered the natural host of PrV. The observation that immunogenic glycoproteins are absent in several attenuated live vaccine strains prompted intensive research into the structure and function of PrV glycoproteins. Until now, eleven PrV glycoproteins, gB, gC, gD, gE, gG, gH, gI, gK, gL, gM and gN, have been identified (reviewed in Mettenleiter, 1996). Whereas homologues to most of them are present in other alphaherpesviruses, only gB, gH, gL, gM and gN are conserved throughout the herpesvirus family. gB, gH and gL are necessary for productive replication in all viruses analysed in this respect. In contrast, gM is dispensable for replication of herpes simplex virus 1 (HSV-1 ; Baines & Roizman, 1991, 1993 ; MacLean et al., 1991, 1993), equine herpesvirus 1 (Osterrieder et al., 1996) and PrV (Dijkstra et al., 1996). In these viruses, lack of gM resulted in a decrease of replicative ability in cell culture represented by 10- to 50- fold lower virus titres, a delay in entry into target cells, and slightly reduced plaque size. For gM of the betaherpesvirus human cytomegalovirus a role in attachment was inferred from its heparin-binding properties (Kari et al., 1994). These limited phenotypic alterations in the absence of gM contrast the high conservation of gM structure. All deduced gM homologues share a charged carboxy terminus, eight putative trans- membrane domains and an N-glycosylation motif between the first and second hydrophobic domain (Dijkstra et al., 1996). So far, only one investigation has dealt with growth properties of a herpesvirus gM - mutant in animals (MacLean et al., 1993). gM - HSV-1, after footpad inoculation into mice, was impaired in its ability to grow at the periphery, and spread to the nervous system. After intracranial infection, the LD  of the gM - mutant proved to be 35-fold higher than that of the isogenic revertant. Mice, however, are not natural hosts for HSV-1. To analyse a gM - mutant in a natural virus–host system, we constructed a gM deletionlacZ insertion mutant and isogenic rescuant from PrV strain Ka (Kaplan & Vatter, 1959). Briefly, in PrV-ΔgMβ approximately 60 % of the UL10 gene, which encodes gM, is deleted and replaced by a gG–β- galactosidase expression cassette (Mettenleiter & Rauh, 1990 ; 0001-4847  1997 SGM CBEH