Dr. Gillian Perkins and Dr. Nikolaus Osterrieder
Dr. Gillian Perkins
Dr. Nikolaus Osterrieder
This proposal is focused on an important, yet poorly researched area with respect to the control of Equine Herpesvirus Type 1 (EHV-1), namely the metaphylaxis and therapy of EHV-1 infection. Historically, and rightly so, attention has focused on improving the immune prophylaxis of the disease by developing novel, more efficacious vaccines and to define parameters that are surrogate markers for protection. These studies are ongoing in many university laboratories, including ours, and in pharmaceutical companies, and it is important that they be continued. However, recent outbreaks at race-tracks and boarding facilities involving large numbers of animals have raised the well-justified question of whether vaccination by itself will be sufficient to protect contact animals when high virus loads are shed from infected and sometimes poorly immunized horses, e.g. in an outbreak scenario. In addition, recent studies assessing the efficacy of standard anti-herpesvirus drugs, such as acyclovir or even the more readily bioavailable valacyclovir, in horses have been sobering in that therapeutically efficacious levels could not be reached in the serum, let alone in the target tissues such as the central nervous system or the reproductive organs, even after continuous administration at high doses.
Here we will examine whether a recently discovered approach to reduce virus replication and shedding, RNA interference (RNAi) mediated by small inhibitory RNA (siRNA) would present an alternative therapeutic approach. Only recently has siRNA-mediated repression of a related virus, Herpes simplex Virus 1 (HSV-1), in a murine model system been demonstrated . The salient findings of this study were that siRNA that was topically administered by cationic liposomes could repress virus replication at mucosal sites and clinical signs (vulvovaginitis) quite effectively. It could be used both therapeutically, i.e. shortly before and for up to 6 h after challenge infection, but also metaphylactically as evidenced by the fact that siRNA antiviral responses were still detectable 9 days after application. Importantly, the authors of this report were unable to detect viruses that could mutate to circumvent siRNA-mediated protection from disease [9,18].
Based on our preliminary data that show that EHV-1 replication can be reduced to almost undetectable levels by certain siRNA’s in vitro, we believe that siRNA treatment of horses could be a valuable therapeutic alternative in outbreak situations or as a metaphylactic measure. This is especially important at stud farms, racetracks, sales, and other places with dense horse populations and where health status of other animals may be unknown. It is important to note that siRNA seems to be an ideal complement to vaccination, which is a critical quality when we discuss novel regimens to contain local and population-wide spread of the virus. Background. 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 organization, it has been grouped into the genus Varicellovirus together with its close relative Equine Herpesvirus Type 4 (EHV-4) [16,17,33]. 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 systems determines the clinical signs observed after EHV-1 infection, which are respiratory disease including nasal discharge, dyspnoea, and coughing, late-time abortions in pregnant mares, and neurological disease. The latter is usually characterized by a paralysis of the hind limbs and the organs that are innervated by the lumbar nerves (e.g., the bladder) [17,33]. More recently, the neurological form of the disease has been predominant and caused considerable losses to the equine industry. Large-scale epidemics of EHV-1 paralytic disease have been documented in the past years, and more than 25 outbreaks have been recorded all over the United States since 2001, 11 of which occurred in 2006. This latest surge has resulted in the classification of paralytic equine herpesvirus infection or equine herpesvirus-induced myelo-encephalopathy (EHM) as a potentially emerging disease by the United States Department of Agriculture . These recent cases also 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 became clinically sick, 35% developed neurological disease and 9% (12 horses) died or had to be euthanized. Another striking feature is that recent neurological cases of EHV-1 infection occurred despite regular vaccinations in very short intervals, indicating the possibility of the circulation of EHV-1 variants that can be maintained in the equine population despite vaccinal protection. The pathogenesis of EHV-1 abortions and neurological disease is probably a consequence of an infection and damage of the endothelia of small blood vessels, which results in inflammation (vasculitis)and thrombosis. The thrombosis in turn causes impaired microcirculation in affected areas, including oxygen deprivation of predominantly the white matter (hypoxia), axonal swelling, and ultimately neuronal death. Abortions and stillbirths can 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 [24-26]. A matter of considerable controversy and an active area of research is the identification of correlates of virulence of individual strains and to identify cellular and tissue tropisms of various strains. The recent discovery of a polymorphism in one of the important virus enzymes, the DNA polymerase , and the recent proof that this mutation is at least necessary for the neurologic potential of certain EHV-1 strains, which our laboratory provided in collaboration with the Animal Health Trust in Newmarket (see Appendix 1, Goodman et al, PLoS Pathogens, in revision), seems to support the notion of two different EHV-1 pathotypes circulating in the equine population. Expected advantages and possible limitations of siRNA therapy and metaphylaxis of EHV-1 infection.
Since horses acquire EHV-1 infections through contact with infectious secretions or via inhalation of infectious aerosols, and the primary site of replication is the epithelium of the upper respiratory tract , blockade of virus at the port of entry at mucosal surfaces seems a reasonable target for intervention. After the virus has penetrated the epithelial barrier, reaches deeper tissues of the respiratory tract and finally draining lymph nodes and the circulation via infected B and T cells [10,11,23], intervention to avoid infection is no longer possible and much less likely to be successful . During an EHV-1 infection, substantial viral shedding occurs via the nose and can be observed from day 1 up until approximately day 7, but low levels of virus – as determined by quantitative real-time PCR – are continually shed to a lesser extent at least until day 21 after infection [5,20].
As mentioned above, there currently is no efficacious antiviral treatment available for EHV-1 infections, and management of an outbreak is mainly by biosecurity measures and supporting therapy of diseased animals . Over the last few years, interest in using RNA interference to prevent and treat viral infections has grown. Double-stranded siRNAs are designed such that they are complementary to a specific mRNA, shuttled to a so-called silencing complex, which is present in virtually all cell types. This complex cleaves double-stranded siRNA resulting in a small, single-stranded RNA which can then bind to its target mRNA. The binding results in degradation of target mRNA and translation of protein can no longer occur. Several siRNAs have been used successfully to inhibit viral infections from HIV to many respiratory viruses [8,22]. We here intend to investigate the ability of siRNA to inhibit the growth and replication of EHV-1 at the primary site of infection, namely the upper respiratory tract. As such, not only viral replication in the affected animals can be controlled, but also, by diminishing viral shedding from the nose, the overall viral load in the population or herd can be reduced. In an outbreak scenario it is conceivable that infected and non-infected contact animals are treated with siRNA in order to prevent clinical manifestation and spread of the disease. Generally, horses can be treated with siRNA before attending races, performances, shows, or any other place where a large number of potential shedders gather, at a reasonable cost of about $5 to $20 per treatment. The versatility of siRNA applications, the lack of interference with vaccination, and the low frequency of viral escape mechanisms to the treatment make this approach an attractive candidate for therapy and metaphylaxis.