Introduction

The focus of our research is on equine herpesvirus type 1 (EHV-1), Marek's disease virus (MDV) and varicella zoster virus (VZV). Working on viruses that are closely related allows us to address the overall biological theme of the laboratory's research, elucidation of alphaherpesvirus replication and pathogenesis. Special emphasis is put on virus assembly, egress and cell-to-cell spread as well as the interaction with the immune system. More specifically, we seek to identify proteins, which are required for the specific interaction of the viruses with their respective hosts and/or represent virulence factors. A related program focuses on the development of EHV-1 into a universal vector for immunization and gene therapy.

Equine herpesvirus 1

Generation and mutagenesis of BACs of recent EHV-1 strains from the US and Europe

Previously, our laboratory has generated BACs of EHV-1 strains RacH, RacL11 and KyA. In order to extend the repertoire of strains that are accessible to Escherichia coli mutagenesis, we have begun and will be continuing to construct BACs from recently isolated strains of EHV-1 that either caused the abortigenic or the recently more prevalent neurological disease. The behavior of these strains in vitro and in vivo (in mice and horses) will then be analyzed and mutant viruses are constructed with the aim to identify regions in the genome that are responsible for the putative differences in tropism. These studies are done in close collaboration with Dr. Nick Davis-Poynter at the Animal Health Trust in Newmarket, UK and Dr. George Allen at the University of Kentucky. One of the targets is the recently identified polymorphism in the polymerase gene. We have developed fast and simple mutagenesis methods that will allow exchange of sequences in one step and should lead to rapid identification of mutations that are responsible for different pathotypes of the disease.

Determination of the role of EHV-1 proteins involved in virus egress and cell-to-cell spread

Although EHV-1 and MDV are related viruses, the biological properties with regard to replication in cell culture and egress from infected cells are quite different. EHV-1 releases infectivity into the supernatant and expresses unique glycoproteins, such as gp2 located in the US region of the genome. We have shown that the requirement for gp2 in cell-to-cell spread is influenced by the presence or absence of gE-gI and a mutation in gD as observed in the KyA strain. By constructing and analyzing a number of gE-gI negative mutants that harbor different forms of gp2 and/or gD we shall analyze the suspected co-evolution of unique-short glycoprotein genes in strains of different virulence. We will also analyze the contributions of the US9 protein and gC in EHV-1 (neuro)virulence in horses.

Immunomodulatory EHV-1 proteins

In addition to their structural role, some EHV-1 glycoproteins have been shown to have or may have immunomodulatory properties. For example, glycoprotein G (gG) binds C, CC, and CXC chemokines from various species with high affinity. In addition, the major glycoprotein gp2 is expressed both as a membrane-bound and secreted form and may act as an immune mediator by virtue of sequestration of soluble host factors. Only recently, the gM complex partner, the UL49.5 product, has been shown to possess TAP inhibitory activity, i.e. it prevents transport of proteasome-generated peptides into the ER and thereby their presentation by MHC class molecules (Danijela Lalic et al., Herpesvirus Workshop 2004, Reno, NV).
All our EHV-1 mutants generated from a virulent strain were based on RacL11, which was isolated in the 1950's in Poland. We have now cloned as bacterial artificial chromosomes two EHV-1 strains that were isolated recently in the US. One of the isolates is an "abortion only" (NY2003), one is an isolate from a horse that was euthanized following severe neurological symptoms (VA2002). Based on these novel isolates, we shall construct mutants that are either unable to express gG or unable to produce the secreted form of gp2. The gG mutants will be constructed in analogy to the RacL11 mutants by complete deletion of the open reading frame, the construction of viruses that are unable to express secreted gp2 will be done by targeted mutagenesis of the proteolytic cleavage site that was determined by Millar Whalley, and co-workers from Maquarie University in Sydney. After preliminary testing of these mutant viruses in mice, we also plan to inoculate horses to determine the growth properties and virulence in the natural host. Finally, to evaluate the role of immunomodulatory proteins in EHV-1 pathogenesis, we will test the growth properties of a UL49.5-negative virus in horses. Besides these in vivo studes, we plan to elucidate on a molecular level the mechanism underlying TAP inhibition by the UL49.5 product and its possible functional interaction with its complex partner gM in this process and the molecules that are possibly targeted by gp2. The studies on the immunomodulatory proteins of EHV-1 are done in close collaboration with Dr. Dennis O'Callaghan, LSU, Shreveport, LA, and Dr. Emmanuel Wiertz's group at the University of Leiden, The Netherlands.

Further development of EHV-1 as an immunization and/or gene therapy vector

We have started a program to investigate the possibilities of using EHV-1 as a vector to deliver foreign genes - to both equines and other mammals, including humans. We have generated mutants expressing antigens from the equine pathogens West Nile virus, equine influenza virus and Venezuelan equine encephalitis virus, the bovine pathogen bovine viral diarrhea virus, as well as the human pathogen hepatitis C virus. These constructs will be tested first in mice and then either in the target species (animals) or primates for the induction of insert-specific and sustained immune responses.

Marek's Disease Virus

MHC class I down-regulation and production of mutant MDV random mutagenesis

A collaboration with Dr. Henry Hunt, USDA-ADOL, East Lansing, MI, on MDV-induced MHC class I down regulation has been established. We have identified a possible candidate, the US3 orthologous protein that may play an important role in this process. However, based on results with other herpesviruses, we expect more proteins to be involved in this targeted interference with the host's immune response to infection. By generating and testing more MDV mutants that will be generated by transposon-based mutagenesis of MDV BAC clones, more viral proteins involved in MHC class I down regulation will be identified.

Role of gC in tumor formation

It has been shown previously that loss of tumorigenicity of MDV is concomitant with a significant reduction or absence of gC expression in cultured cells. The hypothesis to be tested is that reconstitution of gC expression is responsible for MDV tumor formation because efficient lytic replication in the animal can take place in the presence of gC only. We have restored gC expression in the avirulent MDV BAC20 clone available and have deleted the gC ORF in the virulent pRB-1B clone. In addition, a number of mutant viruses carrying point mutations in the start codon or viruses over-expressing gC have been generated. In the very near future, we will conduct an in-depth study to assess the effect of these manipulations on the viruses' behavior in vivo.

Varicella zoster virus

Generation of a BACs and preliminary testing of selected mutant viruses

The understanding of VZV pathogenesis and the development of novel well-characterized vaccines have been complicated by the difficulties in mutant virus generation and the lack of relevant cell culture systems. We plan to introduce BAC technology to VZV research, and to begin an analysis of the structural function of the VZV ORF9 product, a putative tegument protein. Firstly, we shall establish a virulent VZV strain, the sequenced P-Oka strain, as an infectious BAC clone in Escherichia coli. Secondly, the P-Oka BAC clone (pP-Oka) shall be mutagenized in E. coli, and mutants that lack open reading frame 9 (ORF9) encoding the major tegument protein VP22, will be generated. We hypothesize that the VZV ORF9 protein plays a major structural role in virus cell-to-cell spread and, but may be less important for virus egress via apical surfaces in a skin organ culture system that allows for the production of free infectious VZV. This novel cell culture system was developed by our collaborator in these studies, Dr. Jennifer Moffat at SUNY Upstate Medical Center in Syracuse, NY. The goal of these studies is to generate infectious clones of VZV to get a better molecular handle on the virus and to rationally generate and test more potent and efficacious vaccine candidates.