Equine Herpesvirus-1 : An Elusive Target

  Equine herpesvirus -1; an elusive target    Infectious diseases are not uncommon in racehorses in training, breeding stock, and pleasure horses. Some of the more serious diseases can be financially devastating to the animal’s owners and to the equine industry on the whole. Viruses belonging to the herpesvirus family cause some of the most well characterized equine infectious diseases, and the most problematic of these is equine herpesvirus 1 (EHV-1; species Equid alphaherpesvirus 1). EHV-1 is ubiquitous in most horse populations in the world. It is responsible for major economic and welfare problems causing respiratory disease, neurological disease (mainly seen in adult horses), and abortion and neonatal foal death in pregnant mares. This was most notably highlighted by the multiple abortion outbreak recorded in Hertfordshire, England, between February and April 2016 in fully vaccinated animals ( http://www.aht.org.uk/cms-display/interim-report16-april2.html ). Studies have determined that EHV-1 is a common cause of abortion. Occasional cases have also been linked to EHV-4 infection, but this is much rarer and doesn’t account for episodes of multiple abortion, as is seen occasionally with EHV-1.     The virus    EHV-1 was first isolated from an equine abortion in the U.S. in the 1930s. At the time of first isolation the vets weren’t sure what it was, but they knew it was infectious. Subsequent genetic analysis much later led to the classification of the virus in the genus Varicellovirus (family Herpesviridae), together with its close relatives equine herpesvirus 4 (EHV-4; species Equid alphaherpesvirus 4) and equine herpesvirus 8 (EHV-8; species Equid alphaherpesvirus 8). Interestingly it is grouped with, and is therefore genetically similar to, the human herpesvirus responsible for chickenpox, the Varicella Zoster virus. Initial infection of horses was thought to occur around weaning, when virus-neutralizing antibodies transferred to the foal from the mare’s colostrum had declined enough to make them susceptible to infection. However, virus has been isolated from foals as young as seven days old with high antibody levels but without any significant clinical signs. Immunity to re-infection after primary infection is relatively short-lived, lasting between three-six months, but it is rare for naturally infected mares to abort in consecutive pregnancies.     Disease processes    The virus first enters the horses’ body via the respiratory tract, usually by direct contact with infected animals, contaminated surfaces, or equipment such as tack or veterinary instruments. Direct contact with infected aborted fetuses or placental tissues is also a major source of virus, which experience indicates can cause serious problems if they occur in open barns or large groups of horses. Once the cells in the respiratory tract are infected, the virus spreads cell-to-cell until it finds its way to the regional lymph nodes, where it can infect white blood cells called lymphocytes. These lymphocytes circulate through the body carrying the virus with them, which is known as a “cell associated viraemia.” The infected lymphocytes can come into contact with and infect numerous cell types, including cells known as “endothelial cells,” which line the inside of blood vessels of the central nervous system and the pregnant uterus. With EHV-1 infection, these endothelial cells undergo an inflammatory response which can lead to bleeding, cell death, and blood clot formation, which in narrow veins disrupts blood supply. This process results in subsequent tissue damage and serious complications such as placental separation (occasionally with delivery of a virus-negative fetus) and/or leakage of virus across the separating placenta (most frequently with delivery of a virus-positive fetus). Similar mechanisms play a role in neurological disease, a condition called equine herpesvirus myeloencephalopathy or EHM. This condition is also sometimes referred to as an equine stroke, as it is caused by the cellular inflammatory response rather than direct virus infection of nerve cells, which occurs with some other herpesviruses. Less serious clinical signs of infection can include fever, lethargy, inappetence, enlarged lymph nodes, and profuse clear nasal discharge, although not all infected animals will display clinical signs. Recently published work from the Irish Equine Centre has identified EHV-8 as also being occasionally responsible for abortions in mares.  Cases of EHV-8 abortion have also been detected retrospectively by the Animal Health Trust (AHT) among its pathology caseload, as this virus, which is genetically almost identical to EHV-1, triggers positive results in the EHV-1 tests. The frequency and clinical relevance of EHV-8 at this stage is unclear. Of 100 viruses presumed to be EHV-1 and whose genetic material were recently analyzed by the AHT, three were actually confirmed as EHV-8.    Latency    EHV-1 and its ancestor viruses have evolved alongside horses for millions of years. This has allowed them to become very well adapted to their host species. The majority of EHV-1 infections in horses are relatively benign. Published studies have shown that between 50-80% of horses have antibodies in their blood suggestive of exposure to EHV-1 at some point in their lives. This makes evolutionary sense, as EHV-1 relies on the horse cells for replication and subsequent dissemination, and ultimately it is not in the virus’ interest to kill off its host. Like all herpesviruses, EHV-1 is able to establish a state of dormant infection (termed “viral latency”) after the primary infection cycle, and this is the major immune evasion mechanism employed by the virus. During latency there is no viral replication and viral transmission does not occur; this may potentially last years. When in the latent state, the virus is protected from the host immune response.  Up to 70% of horses in some studies have been shown to carry latent virus in their lymph nodes. However, virus in a latent state can periodically become active again, in a process known as reactivation. The dynamics of EHV-1 latency and reactivation of the virus are not well understood. Latent virus has been identified in lymph nodes and nervous tissue, including trigeminal ganglia in the base of the brain, and specific white blood cells (leukocytes) in the blood. After reactivation, the virus can cause subclinical disease in the horse but still be shed by what are sometimes referred to as silent shedders, and these horses infect other animals with which they come into contact. The virus infection can develop and cause the serious clinical signs of abortion and/or neurological disease and is thought to be responsible for the sporadic and sometimes apparently completely spontaneous abortions that are reported internationally every year in geographically isolated populations/animals.      Diagnostics    Identification of EHV-1 infection in the laboratory relies on either direct virus detection or indirect detection by looking for antibodies against the virus in blood, which is suggestive of recent virus exposure and response by the horse’s immune system. As with any diagnostic test, the selection of the most appropriate sample and timing of sample collection are critical in obtaining accurate results. The Horserace Betting Levy Board Code of Practice for EHV-1 (HBLB CoP) recommends prompt examination of aborted fetuses and placentae or dead foals by an experienced pathologist and laboratory that are used to confirming EHV-1 infection. Their investigations will look for characteristic pathological changes and demonstrate presence of virus in the tissues of cases using sensitive and specific assays such as immunohistochemistry and PCR. These same methods may be used to examine fatal cases of EHV-1 neurological disease in adult horses, confirming infection in the central nervous system. Infection may also be confirmed by detecting EHV-1 being shed from the respiratory tract on a nasopharyngeal swab, which samples the nose and throat of the horse. Virus shedding from the respiratory tract lasts between five to ten days and tends to wane with time, so swabs collected at a later date have less virus for detection and thus infections can be missed. There are also occasions when it is recommended to test blood samples for seroconversion, which is the rise in antibodies after the horse is exposed to the virus. Detection by seroconversion can be carried out using the complement fixation test (CFT). Seroconversion can be demonstrated by a fourfold or greater rise in antibody level between paired blood samples taken when signs are first noted and then again two-to-four weeks later. The CFT is particularly useful for detecting recent exposure to EHV-1 or vaccination because antibody levels rise rapidly after exposure and then drop relatively quickly over two-to-three months to a low baseline level, when they are able to detect further infections.     Control strategies and vaccination    If EHV-1 is confirmed on a premises, the HBLB CoP recommends isolation of the infected animals, movement restrictions, and hygiene measures for at least 28 days from the date of the last EHV abortion, foal death, or case of neurological disease.  On an individual horse level, control of EHV-1 infection is currently conducted by bi-annual vaccination of non-pregnant animals or in pregnant mares at five, seven, and nine months of gestation in an attempt to maintain protective immunity. Commercially available vaccines against EHV-1 have been shown to protect against respiratory disease and reduce the amount and duration of virus shedding from the infected animal. However, they do not protect against neurological disease and only provide partial protection against abortion. There are a variety of vaccines available for EHV-1, and published data has shown that these can reduce the number of abortions in an experimental model of EHV-1 infection. However, abortions still occur in the horse population and there is a need for better vaccines to protect against the serious clinical signs of EHV-1 infection. Numerous experimental EHV-1 vaccines have been developed using different techniques over the last 50 years. Some of these have provided no protection, others have only induced partial protection, and no vaccine against EHV-1, experimental or commercial, has been able to completely protect against virus shedding or viraemia. There is a school of thought that the best way to induce a protective immune response against EHV-1 is to stimulate two arms of the host immune system: the humoral response (due to production of antibodies) and the cell mediated response (due to production of a type of white blood cell known as a “cytotoxic t-lymphocyte”). One way to do this is to use live attenuated vaccines. This technique uses a partially disabled EHV-1 virus that does not cause clinical signs yet retains the ability to induce protective immune responses in the same way that the fully virulent natural virus does after infection. This view is reinforced by work showing that repeated infections in younger animals results in protection from respiratory disease as animals get older and that vaccination is substantially more effective in horses that have experienced prior infection.     Historically, there has been a distrust of using modified live EHV-1 vaccines in equines, mainly because of some well-publicized problems with some early examples back in the 1960s. There are currently two commercially produced live attenuated vaccines in use in some parts of the world; Rhinomune in the U.S. (Boehringer Ingelheim Vetmedica) and Prevaccinol in Germany (MSD) are licensed for use for the prevention of respiratory disease.  However, experimental studies in pregnant animals showed that both Rhinomune and Prevaccinol did not protect horses from viraemia and abortion after viral challenge.     Modern molecular biology techniques allow scientists to accurately target specific parts of the virus to attenuate it and consequently restrict the cells in which they grow, which results in much safer vaccines. Live attenuated vaccines have been developed to protect against herpesviruses in other species, including herpes zoster in humans caused by Varicella Zoster virus, Feline Herpesvirus type 1 in cats, Bovine Herpesvirus 1 in cattle, and Pseudorabies virus in pigs, and there is a lot of ongoing research into vaccines to protect humans against Herpes Simplex virus type 2.      Future work    Research groups are continuing to work towards better vaccines to protect against the serious clinical signs of EHV-1 infection. Over the last 50 or so years we have learned that making an effective vaccine to protect horses from both abortion and neurological disease caused by EHV-1 is and will continue to be technically difficult. However, its importance to both pleasure horses and the equine racing and breeding industry means that we must continue to work towards this aim.  At the Animal Health Trust we have begun work on a new industry-funded program involving both national and international collaborators to determine the amount of protection that can be achieved by using newly designed live attenuated vaccines. Candidate vaccines are being designed using specific scientific rationales based on work from world renowned experts, which will then be characterized in the laboratory and tested for their ability to provide protection. We are looking to involve commercial partners at an early stage to allow us to fully develop any effective vaccines for the benefit of the horse.     

By Neil Bryant 

Infectious diseases are not uncommon in racehorses in training, breeding stock, and pleasure horses. Some of the more serious diseases can be financially devastating to the animal’s owners and to the equine industry on the whole. Viruses belonging to the herpesvirus family cause some of the most well characterized equine infectious diseases, and the most problematic of these is equine herpesvirus 1 (EHV-1; species Equid alphaherpesvirus 1). EHV-1 is ubiquitous in most horse populations in the world. It is responsible for major economic and welfare problems causing respiratory disease, neurological disease (mainly seen in adult horses), and abortion and neonatal foal death in pregnant mares.

This was most notably highlighted by the multiple abortion outbreak recorded in Hertfordshire, England, between February and April 2016 in fully vaccinated animals (http://www.aht.org.uk/cms-display/interim-report16-april2.html). Studies have determined that EHV-1 is a common cause of abortion. Occasional cases have also been linked to EHV-4 infection, but this is much rarer and doesn’t account for episodes of multiple abortion, as is seen occasionally with EHV-1.

The virus

EHV-1 was first isolated from an equine abortion in the U.S. in the 1930s. At the time of first isolation the vets weren’t sure what it was, but they knew it was infectious. Subsequent genetic analysis much later led to the classification of the virus in the genus Varicellovirus (family Herpesviridae), together with its close relatives equine herpesvirus 4 (EHV-4; species Equid alphaherpesvirus 4) and equine herpesvirus 8 (EHV-8; species Equid alphaherpesvirus 8). Interestingly it is grouped with, and is therefore genetically similar to, the human herpesvirus responsible for chickenpox, the Varicella Zoster virus. Initial infection of horses was thought to occur around weaning, when virus-neutralizing antibodies transferred to the foal from the mare’s colostrum had declined enough to make them susceptible to infection. However, virus has been isolated from foals as young as seven days old with high antibody levels but without any significant clinical signs. Immunity to re-infection after primary infection is relatively short-lived, lasting between three-six months, but it is rare for naturally infected mares to abort in consecutive pregnancies.

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