Dr. Nikolaus Osterrieder
This proposal is a continuation and extension of our long-term efforts to determine the genomic regions and genes of Equine Herpesvirus Type 1 (EHV-1) that are involved in pathogenesis and determine tissue tropism in vivo . More specifically, our research has been focusing on the differences in membrane (glyco)protein and tegument protein genes between virulent and avirulent strains, because both groups of herpesvirus virus proteins that are constituents of the virus envelope (membrane (glyco)proteins) or the matrix (tegument proteins) have been shown to be crucially involved in cellular tropism and pathogenesis. In addition, we have been concentrating on how the knowledge of EHV-1 virulence factors can be exploited for the generation of efficacious and safe modified live vaccines. The presented proposal aims at two major goals. Firstly, studies are planned on continuing the analysis of the function of individual genes in different genetic virus backgrounds. Secondly, based on the notion that more recently the neuronal form of the disease with devastating sequelae has been observed, the immediate need has arisen to get a molecular handle on recent EHV-1 strains and isolates with putatively increased virulence and/or altered tropism. The severe neurological outbreaks that have occurred in the U.S. and the similar ones reported recently in the United Kingdom , Belgium and Germany have shown the need for extensive research into the pathobiology of EHV-1 infection. In addition, with the continuing threat of EHV-1 outbreaks despite regular vaccinations, the need for improved and novel vaccines that may be preferentially based on these novel EHV-1 isolates is even more pressing.
EHV-1 affects horse populations worldwide. Within the large family of the Herpesviridae it has been classified under the subfamily of the Alphaherpesvirinae . Owing to its biological properties and genomic features, it forms the genus Varicellovirus together with its close relatives Equine Herpesvirus Type 4 (EHV-4), Varicella zoster virus, Bovine Herpesvirus Type 1 and Suid Herpesvirus Type 1 (Pseudorabies Virus).
EHV-1 targets three organ systems in the natural host: the respiratory tract, the reproductive organs, and the central nervous system (CNS). The affection of these three organ systems determines the clinical signs observed after EHV-1 infection, which are respiratory distress including nasal discharge, dyspnoea, and coughing, late-time abortions in pregnant mares, and neurological disease. The latter is characterized by a paralysis of the hind limbs and the organs innervated by the lumbar nerves like the bladder.
More recently, the neurological form of the disease has been predominant and caused considerable losses to the equine industry. In devastating recent outbreaks of EHV-1 in herds, studs and/or on racetracks, unusually high numbers of horses exhibited the neurological form of the disease. This is alarming, because in the past the neurological form of EHV-1 disease used to be sporadic and restricted to individual animals of an affected herd. The recent reports, however, suggest the emergence of an epidemiological picture that resembles the well known “abortion storms” caused by EHV-1, i.e. that large numbers of animals per affected premise are developing severe clinical disease. For example, in the case of EHV-1 infection at the University riding school in Findlay , OH , more than 90% of the 138 horses developed neurological disease and 12 horses died or had to be euthanized. Another striking feature is that the recent neuronal cases of EHV-1 infection in both Virginia and Ohio occurred despite regular vaccinations in very short intervals, indicating the possibility of the circulation of EHV-1 variants that are able to break vaccine protection ( http://www.thehorse.com , article numbers # 3683, 4104, 4127 and 4272).
Generally speaking, the pathogenesis of EHV-1 abortions and neurological disease is still somewhat enigmatic, but it is now generally accepted that the neurological form of the disease is probably a consequence of an infection of the endothelia of small blood vessels, which results in inflammation of the blood vessels (vasculitis) and thrombosis. The thrombosis in turn causes impaired microcirculation in affected areas, including oxygen deprivation (hypoxia) and finally neuronal death . Abortions and stillbirths may also be caused by the same pathogenic mechanism of vasculitis, thrombus formation, and hypoxia, with a complete or partial detachment of the placenta and malnutrition of the fetus. It has been shown previously that EHV-1 membrane (glyco)protein and tegument proteins are crucially involved in the early stages of infection and in virus assembly and spread, mechanisms that are extremely important with regard to tissue tropism and virulence. Besides the homologues of the Herpes simplex virus type 1 (HSV-1) glycoproteins (g), referred to as gB, gC, gD, gE, gH, gI, and gM, which had been analyzed in some detail, our previous studies and those in the current year of funding have concentrated on the large EHV-1 specific glycoprotein gp2 and the role of gp2 mutations in attenuation of the vaccine strain KyA, as well as the dependence of these mutations on other mutations present in the unique-short segment of the EHV-1 genome.
Objectives and Experimental approach
The objectives of this proposal are twofold. We plan to logically continue our studies on the role of membrane (glyco)protein and tegument proteins in EHV-1 assembly and pathogenesis by generating and analyzing virus mutants based on virulent and avirulent strains. In addition, we plan to include more recent U.S. EHV-1 isolates into our studies, for which the first step, i.e. cloning of recently isolated EHV-1, will be taken. Finally, the efficacy testing of vaccine candidates based on these novel strains is planned. We shall test in horses candidate modified live vaccines that have shown promise in a murine model of EHV-1 immunization and infection. New means to prevent EHV-1-caused disease are urgently needed because currently available vaccines have reached the end of their useful lives.
The specific aims in the first year of the proposal are to clone as a bacterial artificial chromosome (BAC) and preliminary characterize recent EHV-1 isolates obtained from the outbreaks of neurological disease in Virginia (summer 2002) and Ohio (winter 2003). In addition, a 2003 New York State isolate of EHV-1 that induced an abortion storm in a stud shall be similarly cloned as a BAC. The cloning of the different EHV-1 isolates shall be performed exactly as previously described and the mini-F plasmid sequences will be inserted into the gene 71 (gp2-encoding) locus. In addition, selected mutant viruses derived from these novel BAC-cloned viruses shall be generated and tested. Based on preliminary studies, gM, gE-gI and gp2 deletion mutants have good potential as vaccine candidates, therefore we aim at deleting either the gM open reading frame (ORF) or gene 71 from the newly generated BACs and shall test for their growth properties in cultured cells and in the murine immunization and infection model. The viruses' phenotypes will be compared to the parental viruses reconstituted from the BACs and to already available mutants that are based on the virulent RacL11 and avirulent KyA strains of EHV-1. In addition, we plan to continue work on the interplay of the glycoproteins encoded in the unique short region of the genome.
In the second year of the proposal, we plan to finalize the animal experiments in mice and to extend the studies to the behavior of selected mutants in horses. Groups of horses shall be inoculated with glycoprotein-negative viruses generated in the first year of the study. Both the residual virulence of the generated mutants as well as the vaccine potential shall be investigated. It is planned to immunize groups of 5 horses by the intranasal route with the three most promising vaccine candidates emanating from the preliminary studies performed in cultured cells and in mice. We shall examine the induction of clinical disease after vaccination and challenge as well as the quantity and quality of the immune response. The local (nasal secretion) and systemic serological response shall be determined by examining the magnitude of secretory IgA levels, as well as that of the circulating immunoglobulin isotypes (IgGa, IgGb, IgGc, IgG(T), IgA or IgM). The determination of the immunoglobulin isotypes may allow the identification of a specific immune response that has a predictive value with regard to protection against EHV-1 caused disease by vaccination.