Dr. Nikolaus Osterrieder
Equine herpesvirus type 1 (EHV-1) is widely known as a major cause of abortion, respiratory disease and neurological disorders in horse populations world-wide. Of particular importance is the repeated occurrence of so-called abortion storms, which result in severe economical losses and may eliminate valuable offspring and threaten the breeding potential of affected studs. In addition, performance of EHV-1 and/or EHV-4-infected horses on race tracks is significantly reduced, and herpesvirus infection is often a disease upon which secondary bacterial infection can ensue (O'Callaghan & Osterrieder, 1999).
EHV-1 is a member of the Alphaherpesvirinae subfamily of the Herpesviridae and belongs to the Genus Varicellovirus. During the course of EHV-1 infection of a horse, several tissues and organs are utilized to establish the infection, i.e. the virus must enter, replicate and egress various cell types of the host organism. EHV-1 enters the body through mucosal surfaces and then replicates predominantly in peripheral blood mononuclear cells (PBMC), where it can enter the latent state and lead to the establishment of live-long persistence in affected animals. Starting from infected PBMC, EHV-1 infects endothelial cells and can cause neurological symptoms or so-called ‘sterile’ abortions by secondary obstruction of small blood vessels. Alternatively the virus leads to abortions by infecting fetal tissues, especially the liver, spleen, and kidney of the fetus. The infection of the fetus is thought to occur by migration of infected PBMC through the placenta, which then reach the target tissues via the umbilical cord.
The fact that EHV-1 needs to infect and replicate in various cell types is true not only for virulent wild-type strains but also for avirulent modified live vaccine strains, which have been shown to induce some level of protection against EHV-1-induced abortions and respiratory disease. Modified live vaccines are in wide use in the U.S. (Rhinomune, Pneumabort K), however, their efficacy sometimes is questionable. For a rational design of novel and more potent modified live vaccines, which confer complete protection and yet are fully apathogenic, as well as for the judgment of virulence of individual EHV-1 strains, it is important to understand the mechanisms by which EHV-1 is able to egress from an infected cell in order to be transported over long distances, or to infect a neighboring cell and spread locally within an infected tissue or organ.
Figure 1. Electron microscopic picture of an EHV-1 virion. Given are the structural components; the bar represents 100nm.
Main players involved in the processes of virus entry into cells, direct transmission of infectivity from one cell to a neighboring uninfected cell as well as virus egress are EHV-1 glycoproteins and the proteins of the so-called herpesvirus tegument. Mature herpesvirus particles consist of a nucleocapsid that contains the linear double-stranded viral DNA genome and associated proteins (the core), a proteinaceous layer around the nucleocapsid (tegument), and a viral envelope which is derived from host cell membranes and in which the virus-encoded membrane (glyco)proteins are incorporated (Fig. 1). Glycoproteins of the Alphaherpesvirinae are named according to the nomenclature established for the prototype member of the subfamily, Herpes simplex virus type 1 (HSV-1). Homologues of glycoproteins (g) gB, gC, gD, gE, gG, gH, gI, gK, gL, gM have been identified in the EHV-1 genome. The glycoprotein genes are found throughout the 150 kbp EHV-1 genome, which was sequenced in its entirety (Telford et al., 1992) , however, a cluster of membrane (glyco)protein genes is found in the so-called unique-short (US) region of the genome, which is only 14 kbp in length and harbors 9 genes in the wild-type Ab4 strain (Telford et al., 1992); Fig. 2). The functions of some of the EHV-1 glycoproteins, i.e. gB, gC, gD, gE, gI and gM were determined, but the interplay between individual glycoproteins or between membrane (glyco) proteins and the tegument proteins in especially virus egress and virus cell-to-cell spread are only rudimentarily understood (Osterrieder et al., 1998).
From the 9 EHV-1 US genes, as many as 6 are membrane (glyco)protein genes. These genes are believed to have evolved from a common ancestor by gene duplication (Fig. 2). The gG gene, the unique gp2 gene, which is only present in the genomes of EHV-1 and its close relative EHV-4, the gD gene as well as the gE and gI genes are concentrated in this small portion of the genome. In addition, the non-glycosylated type II membrane protein US9 is located in this genomic region (Telford et al., 1992) . Mutations in the US region leading to an attenuation of viruses and reduced replication capacity has been described for closely related Alphaherpesvirinae, like pseudorabies virus (PRV) infecting pigs, bovine herpesvirus type 1 (BHV-1) and human HSV-1. Continuous passage of these viruses in cultured cells leads to abrogation of gE and/or gI and/or US9 expression, which is associated with an impaired ability of the viruses to efficiently migrate from cell-to-cell and invariantly results in avirulence (Balan et al., 1994; Card et al., 1992; Mulder et al., 1996; van Engelenburg et al., 1995). In contrast, EHV-1 vaccine strain KyA, which lacks the gE, gI and US9 genes, efficiently grows in cultured cells. However, a number of additional mutations when compared to other EHV-1 strains has occurred in this small region of the KyA genome. These mutations may be compensatory mutations by which strain KyA has overcome its growth restrictions in cultured cells.
In this proposal, we aim at identifying the impact of deletions and mutations in the US (glyco)protein genes on virus egress and cell-to-cell spread in vitro and in vivo and to analyze the possible interplay between these important viral structures. The studies will be performed by exchanging portions of the genomes using infectious clones of EHV-1 strains of different origin and virulence (virulent RacL11 and avirulent KyA). Our studies should lead to a better understanding of growth properties of individual EHV-1 strains both in cultured cells and in animals, which can eventually be exploited in the design of novel efficacious vaccines to combat one of the most important viral diseases of the horse.