The Harry M. Zweig Memorial Fund for Equine Research


Detection of WNV Antibodies and Discrimination
Between Vaccinated and Infected Horses

Dr. Amy Glaser

During the late summer and fall of 1999, several cases of encephalitis of unknown cause occurred in humans in New York City. Initial investigations by public health laboratories and the Centers for Disease Control (CDC) implicated St. Louis encephalitis virus as the cause. This virus is present in the US, but had not recently been identified in the NYC area. Within the same period, Ward Stone at the New York State Department of Environmental Conservation began receiving reports of crows dying in and near the New York City area. At the same time, Dr. Tracy McNamara, Director of Pathology at the Wildlife Conservation Society headquartered at Bronx zoo, was puzzled by the patterns of deaths she was seeing in zoo and wild birds, the majority of which had lesions of viral encephalitis. Samples from the wild and zoo birds were sent to the National Veterinary Services Laboratories (NVSL) in Ames, Iowa and the National Wildlife Health Laboratories in Madison, Wisconsin where virus isolation was performed. Virus was isolated at both laboratories that could not be readily identified and these viruses were forwarded to the CDC for further identification. They were eventually determined to be West Nile virus (WNV), a member of the Flavivirus genus of the family Flaviviridae. Further diagnostic tests on human samples revealed that the same virus was responsible for the human illness. West Nile virus is one of a number of related viruses whose other known members include Japanese encephalitis, Kunjin, Murray Valley encephalitis, St. Louis encephalitis and several others. These viruses are all transmitted by mosquito or tick vectors and have a natural host range that includes many species of birds and mosquitoes. In areas where WNV was previously known to occur, infection cycles alternate between a wild life cycle in a rural environment and an urban cycle. Wildlife cycles involve birds (usually in wetland settings) and mosquitoes that feed exclusively on birds, while urban cycles involve domestic and wild birds and mosquitoes feeding on both birds and mammals

WNV had not previously been identified in North or South America and the source of the virus is unknown. Sequence analysis indicates that this virus was almost identical to a virus isolated from a goose in Israel in 1998. While its presence was discovered in 1999, it is unlikely that we will be able to determine the precise time WNV was introduced to this continent, however it reappeared in 2000 and is making an early appearance in 2001 with a wider geographic distribution. It is likely that we will find it again in the future because it appears to have become well established in wildlife and mosquito populations.

Humans and horses are the mammalian species most likely to have clinical signs of illness following WNV infection. Historically, WNV causes disease only in a small percentage of horses infected and an even smaller percentage of people. Both humans and horses are considered dead end hosts because virus levels in blood during infection are very low. As a consequence, virus is not transmitted from infected people or horses to mosquitoes or other animals.

Clinical disease in horses in 1999 attributable to WNV infection occurred in 25 animals, all in Suffolk or Nassau Counties, New York. The year 2000 saw an increase in the number of equine cases identified, with 61 cases in a much broader geographic distribution; 23 of the confirmed cases died or were euthanized. Clinical signs in horses included acute onset of ataxia, depression, weakness, and muscle tremors. Many cases were quite mild, with horses only slightly ataxic and weak. Clinical signs are not specific for WNV infection and other equine illnesses that cause neurologic signs should also be considered in the differential diagnosis. These include rabies, equine herpes myelitis, equine protozoal myelitis, eastern equine encephalitis, spinal chord compression due to malformation

During the late summer and fall of 1999, several cases of encephalitis of unknown cause occurred in humans in NY City. Initial investigations by public health laboratories and the Centers for Disease Control (CDC) implicated St. Louis encephalitis virus as the cause. This virus is present in the US, but had not recently been identified in the NYC area. Within the same period, Ward Stone at the New York State Department of Environmental Conservation began receiving reports of crows dying in and near the New York City area. At the same time, Dr. Tracy McNamara, Director of Pathology at the Wildlife Conservation Society headquartered at Bronx zoo, was puzzled by the patterns of deaths she was seeing in zoo and wild birds, the majority of which had lesions of viral encephalitis. Samples from the wild and zoo birds were sent to the National Veterinary Services Laboratories (NVSL) in Ames, IA and the National Wildlife Health Laboratories in Madison, Wisconsin where virus isolation was performed. Virus was isolated at both laboratories that could not be readily identified and these viruses were forwarded to the CDC for further identification. They were eventually determined to be West Nile virus (WNV), a member of the Flavivirus genus of the family Flaviviridae. Further diagnostic tests on human samples revealed that the same virus was responsible for the human illness. West Nile virus is one of a number of related viruses whose other known members include Japanese encephalitis, Kunjin, Murray Valley encephalitis, St. Louis encephalitis and several others. These viruses are all transmitted by mosquito or tick vectors and have a natural host range that includes many species of birds and mosquitoes. In areas where WNV was previously known to occur, infection cycles alternate between a wild life cycle in a rural environment and an urban cycle. Wildlife cycles involve birds (usually in wetland settings) and mosquitoes that feed exclusively on birds, while urban cycles involve domestic and wild birds and mosquitoes feeding on both birds and mammals.

WNV had not previously been identified in North or South America and the source of the virus is unknown. Sequence analysis indicates that this virus was almost identical to a virus isolated from a goose in Israel in 1998. While its presence was discovered in 1999, it is unlikely that we will be able to determine the precise time WNV was introduced to this continent, however it reappeared in 2000 and is making an early appearance in 2001 with a wider geographic distribution. It is likely that we will find it again in the future because it appears to have become well established in wildlife and mosquito populations.

Humans and horses are the mammalian species most likely to have clinical signs of illness following WNV infection. Historically, WNV causes disease only in a small percentage of horses infected and an even smaller percentage of people. Both humans and horses are considered dead end hosts because virus levels in blood during infection are very low. As a consequence, virus is not transmitted from infected people or horses to mosquitoes or other animals.

Clinical disease in horses in 1999 attributable to WNV infection occurred in 25 animals, all in Suffolk or Nassau Counties, NY. The year 2000 saw an increase in the number of equine cases identified, with 61 cases in a much broader geographic distribution; 23 of the confirmed cases died or were euthanized. Clinical signs in horses included acute onset of ataxia, depression, weakness, and muscle tremors. Many cases were quite mild, with horses only slightly ataxic and weak. Clinical signs are not specific for WNV infection and other equine illnesses that cause neurologic signs should also be considered in the differential diagnosis. These include rabies, equine herpes myelitis, equine protozoal myelitis, eastern equine encephalitis, spinal chord compression due to malformation or trauma, and lead poisoning. WNV encephalitis cases in horses generally occur well after the virus has been identified in bird, mosquito pools and humans in a geographic area. However, illness in horses was the first indication of the presence of WNV in an area in some cases. The first cases in the northeast appeared the third to fourth week of August with most cases in September and October. Equine cases in Florida have already occurred in June of 2001 and coincided with the detection of virus in birds, suggesting that the WNV transmission season in the south may be significantly extended.

Since the clinical signs are nonspecific, laboratory testing is required to determine the cause of illness. The only specific test for detection of WNV antibodies is a serum neutralization test. Use of the virus in this test requires laboratory facilities which are not commonly available. The currently available ELISA detects antibodies to the viral envelope protein. The envelope protein is one of the most antigenic proteins in the virus and is the protein against which neutralizing antibodies are directed. Other proteins expressed by the virus in an infected cell are antigenic and are recognized by antibodies after infection with virus. NS1 is one of these other proteins, which is not in the virus particle, but is expressed by virus in cells and is secreted from the cell. Diagnosis of WNV infection in horses currently depends on detection of antibody to the envelope protein. Test interpretation could be complicated in populations of vaccinated horses if envelope is the only protein used by testing strategies for the detection of WNV antibodies.

At least two vaccines for WNV are in development. Vaccination of people with whole inactivated virus induces antibody to the envelope glycoprotein, but not to NS1, suggesting that detection of NS1 antibodies may be useful to indicate infection. There is no information available regarding the proteins recognized by a humoral immune response to WNV infection in horses. It is likely that several viral proteins may generate an antibody response, including NS1, NS5 (viral polymerase, and NS3). We propose to determine if viral proteins other than envelope are recognized by antibodies produced in horses after natural infection with WNV. We will focus on NS1 initially because it has been shown to be a good antigen in humans, but may investigate the antibody response to other proteins if necessary. Once we have defined the proteins which are recognized by antibodies in equine serum samples after natural infection, we will test the suitability of this/these proteins for use in an ELISA test that can differentiate between vaccinated and naturally infected horses.