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The first objective of this study is to develop multiple primer RT-PCR assay that could detect specific RNA products only present during EHV-1/EHV-4 acute or latent infection in horses. One of the unique feature of EHV-1 latency is that the virus becomes latent not only in TG, but also in some circulating peripheral blood leukocytes. Therefore, we could use blood sample to detect the EHV-1 latency by examining the viral genome in PBL using RT-PCR. During acute and reactivation phase, the virus replicates inside the infected cell, therefore, genes essential for virus replication during the productive phase will be present and thus could be used as markers, such as immediate early genes (ICP0, ICP4), or structural genes (gB, gD, gC). Detection of those essential gene transcripts will be an indication of acute infection or latency reactivation of EHV-1 or EHV-4. LAT is the only RNA produced during the latent infection. If only LAT is detected, the animal is latently infected only. If the gene transcripts present during productive infection are also readily detectable, it indicates the animal has latency reactivation or acute infection to a naive animal. <P>
The second of objective of this study is to develop ELISA assay that diagnose the EHV-1, WNV and WEE virus infection. The acute infection often occurs in young foals from first exposure to the virus, when they have not been vaccinated. When a naive animal is found to have both LAT expression and gene transcription products present from the productive infection, such animals will generally have lower IgG titers than those suffering from recent EHV latency reactivation. For the WEE and WNV recent infected animal, IgM antibodies to WEE or WNV are almost always detectable before the second week of illness and IgM capture enzyme ELISA could be used for the diagnosis. However, for EHV-1 reactivation induced neurological disease, production of IgG antibodies against EHV-1 antigen is the major response to restimulation. In addition, EHV-1 is a routine immunization in the horse, resulting in an enhanced IgG response should latent EHV-1 undergo reactivation. Therefore, we plan to develop ELISA assay that could differentiate reactivated animals from non-reactivated animals even if the horses have been vaccinated with EHV-1/EHV-4. One advantage of this assay is it can be completed in one day instead of 3 to 5 days for the SN test. It will also be specific for IgG response to EHV-1 during reactivation.
NON-TECHNICAL SUMMARY: Equine herpesvirus type 1 (EHV-1) infection and West Nile virus infection can lead to neurological disease in equine. No quick diagnosis method is available to distinguish WNV associated- or EHV-1 acute infection associated-encephalitidies from EHV-1 latent infection reactivation associated encephalitidies The purpose of this study is to develop diagnosis test that could differentiate WNV infection or the EHV-1 acute infection from EHV-1 latent infection reactivation
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APPROACH: 1) Developing a multiple primer RT-PCR assay that could detect specific RNA products only present during EHV-1/EHV-4 acute or latent infection in horses. PCR primer sets will be designed specific for EHV-1 LAT, EHV-4 LAT, EHV-1/EHV-4 essential gene transcript (immediate early gene (ICP0) or late gene (gD) RNA). EHV-1 and EHV-4 RNA will be generated in tissue culture in the Lab of principal investigator. Blood samples from the Oregon State University Veterinary Diagnostic Lab and Veterinary Teaching Hospital will be used to verify the assay. 2) Developing ELISA assays that diagnose the EHV-1, WNV and WEE virus infection. If EHV-1 productive gene products are found in multi-virus RT-PCR assays, and both WNV and WEE serology tests are negative, we will check the EHV-1 IgG titer. To do this, serum from the tested animal will be applied to an EHV-1 coated plate, and the amount of bound IgG will be detected using standard ELISA techniques. In addition, a threshold of the IgG titer will be established to define the titer for reactivation from vaccinated animal and reactivation from non-vaccinated animal.
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PROGRESS: 2005/03 TO 2006/09 <BR>
RT-PCR methods have been developed by using multiple primers specific for RNA products during EHV-1/EHV-4 acute or latent infection in horses. The selected primers are specific for EHV structural gene gB and latency associated transcript (LAT), respectively. The former RNA expression can only been seen during acute infection, the later can be found during the latent infection and productive infection. If we only see LAT but not gB RNA expression, it indicates that the horse only has EHV-1 latent infection. If we see both gB RNA and LAT expression, we have acute EHV-1 infection, which may either come from other horse or from EHV-1 reactivation. Horse with neurological symptoms can be caused by EHV-1 reactivation with mutation in the virulence gene. We are able to use this RT-PCR test to examine the bloods from suspicious horses and to quickly diagnose the EHV infection from WNV and EPM. WE have validate the sensitivity of this assay, are able to identify an EHV-1 productive infection or reactivation from EHV-1 latent infection. It has improved our diagnosis in the EHV-1 related neurological diseases. Samples come from clinical pathology lab for routine blood works have been selected to validate out test. About 60% of the tested samples were found to be positive for EHV-1 LAT. About 10% of the samples collected from the hospital and clinic with neurological symptom were found to have productive infection or reactivation of EHV-1. This RT-PCR test has been used as a confirmative test in the diagnostic lab for horse with neurological disease. Serological test results have been evaluated with our RT-PCR test. The serum neutralization titer before the onset of disease and following the disease were expected to be different depending on status of the EHV-1 infection. We were unable to do following up work in serology test, since many of them were euthanized once they were found to have neurological disease. We found our RT-PCR test is the most effective way in differentiate the EHV-1 neurological disease from WNV and EPM.
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IMPACT: 2005/03 TO 2006/09 <br>
This test has made it possible for us to differentiate EHV-1 associated neurological disease from the WNV and EPM associated diseases. It also reduced the our screening tests for neurological disease diagnosis in horses.