Thesis (Ph.D.)--Murdoch University, 2008. / Thesis submitted to the Faculty of Health Sciences. Includes bibliographical references (leaves 197-242).

Thesis (M.S.)--Virginia Polytechnic Institute and State University, 1994. / Vita. Abstract. Includes bibliographical references (leaves 70-96). Also available via the Internet.

Thesis (Ph.D.)--Murdoch University, 2003. / Thesis submitted to the Division of Health Sciences. Bibliography: leaves 164-198.

No description available.

The objectives of this study were to: 1) develop a technique to analyze the <i>in vitro</i> cytotoxic activity of lymphocytes from adult horses against equine herpes virus-1 (EHV-1) infected allogenic equine dermal fibroblasts (EDF); 2) evaluate the ability of a 72 hour in vitro incubation with interleukin-2 (I L-2) to enhance the lymphocytic cytolytic activity against EHV-1 infected EDF; 3) compare the cytotoxic activity among lymphocytes isolated from pregnant mares and non-pregnant mares against EHV-1 infected EDF; 4) ascertain if any correlations existed between the percent cytotoxicity and percentage of lymphocytes phenotypically identified by five different mouse-anti-equine monoclonal antibodies; and 5) determine if any correlation existed between virus-neutralizing antibody titers and the percent cytotoxicity. Results of the study indicate that <i>in vitro</i> cytotoxic activity of equine lymphocytes against EHV-1 infected allogenic fibroblasts can be measured with a standard 4 hour 51Cr release assay. This activity was enhanced by an <i>in vitro</i> incubation with IL-2. The cytolytic activity of freshly isolated lymphocytes was greater for non-pregnant than pregnant mares. However, after IL-2 stimulation the cytolytic activity was greater for lymphocytes from pregnant mares. A positive correlation was not detected between the percentage of phenotypically identified cells and the percent cytoxicity, although several negative correlations were present. This suggests that the cytotoxic activity was either not mediated by any of the phenotypically identified cell populations or that the activity was mediated by several different cell populations. No correlation was detected between virus neutralizing antibody titers and the percent cytotoxicity. / Master of Science

Formalized epidemiological field investigations were conducted to compare and contrast the transmission dynamics of EHV-1 neurological disease among horses stabled at Churchill Downs Racetrack, Louisville, Kentucky and of EHV-1 respiratory illness among horses stabled in the student barn at Murray State University. Differences were assessed by means of statistical and mathematical modeling techniques applied to survey and biological data collected over the course of the respective disease events. Regression methods applied to survey data enabled the construction of a statistical model to predict a date of onset of illness for horses within each equine cohort. Comparisons of the epidemic curves revealed that the Murray State University outbreak was 4.5 times longer (9 weeks versus 14 days) than the Churchill Downs Racetrack event. Survival analysis was used to explore the relationship between time to infection for each equine cohort. Horses stabled in the affected barn at Churchill Downs racetrack had a 3.02 times greater daily risk (p < 0.001) for contracting EHV-1 infection relative to horses stabled in the student barn at Murray State University. Estimates of the basic R0 number, calculated using mathematical formulae that incorporated the duration of the infectious period for neuropathogenic and nonneuropathogenic strains of EHV-1, were 10.25 and 2.94 for the Churchill Downs racetrack and Murray State University outbreaks, respectively. The generation time for the Churchill Downs outbreak was 6.1 times shorter (0.39 days versus 2.38 days) than for the Murray State University event. An assessment of the temporal occurrence of symptomatic infection is similar for each event and suggests that the appearance of clinical illness is constant over the course of an outbreak. A Reed-Frost model was constructed for each EHV-1 event where values of the transmission parameters (q, p and k) were estimated by fitting a model that most closely matched the observed profile of EHV-1 cases. The value of prophylactic vaccination on the spread of EHV-1 was assessed by making adjustments to these fitted models for varying levels of herd immunity. The results indicate that the prevention of EHV-1 neurological illness requires a higher level of herd immunity than EHV-1 respiratory illness.

Equine Herpesvirus type 1 (EHV-1) is a worldwide threat to the health of horses. It can cause mild respiratory disease, abortions and deaths of newborn foals as well as a potentially fatal neurologic disorder known as Equine Herpesvirus Myeloencephalopathy (EHM). The virus is maintained in populations by stress-induced periodic reactivation of virus in long-term latently infected horses and transmission of the reactivated virus to susceptible individuals. In horses, peripheral blood leukocytes (PBLs) are thought to be an important site for EHV-1 latent genomes. Since the Unfolded Protein Response (UPR) is a cellular response to a variety of stressors that has been linked to reactivation of herpes simplex virus in humans, a virus closely related to EHV-1, I tested the hypothesis that latent EHV-1 relies on the UPR as a pluripotent stress sensor and uses it to reactivate lytic gene expression. Since little work has been done in defining the UPR in horses, I first successfully developed a quantitative real-time polymerase chain reaction (RT-qPCR) assay to detect and quantitate transcripts for selected UPR genes in equine dermal (E.Derm) cells and PBLs. Activation of the UPR was achieved in both cell types using thapsigargin and a difference in gene expression after activation of the UPR in two equine cell types was found. A nested PCR assay to detect and distinguish latent EHV-1 and EHV-4 was evaluated and the sensitivity of the technique to detect EHV-1 was determined. I discovered that the nested PCR technique was not sensitive enough to detect the estimated one latent viral genome in 50,000 PBLs. Lytic EHV-1 infection was characterized by single step growth curve in E.Derm cells and consistent detection of temporal EHV-1 gene expression by RT-qPCR was achieved. The relationship between EHV-1 gene expression and UPR gene expression during lytic infection was investigated. While EHV-1 infection had no effect on UPR gene expression, activation of the UPR appeared to decrease the expression of EHV-1 genes temporarily and reversibly during the first 4 h after infection. Finally, detection of EHV-1 in PBLs from horses presumed to be latently infected by co-cultivation with E. Derm cells permissive to EHV-1 infection was attempted. To detect viral DNA, PBLs were stimulated with thapsigargin or interleukin 2 (IL-2) which was previously reported to induce reactivation of latent EHV-1. I was not able to reproduce previously published experiments of reactivation in vitro of latent EHV-1 by stimulation with IL-2, and virus reactivation did not occur after stimulation of PBLs with thapsigargin. In summary, a RT-qPCR assay to measure the expression of equine UPR genes was developed and activation of the UPR by treatment of E.Derm cells and PBLs with thapsigargin was successfully achieved. A difference in gene expression after activation of the UPR in two equine cell types was found. In contrast to what has been reported for other alphaherpesviruses, there appears to be no, or only little, interaction between the UPR and EHV-1 during viral infection. Detection of latent EHV-1 genomes in PBLs was not achieved by using a nested PCR, as this technique was not sensitive enough to detect the estimated one latent viral genome in 50,000 PBLs. Finally, latent EHV-1 was not detected in presumed latently infected PBLs or reactivated by triggering the UPR in equine PBLs.

Commingling of horses from various populations, together with physiological stress associated with transport and confinement at a sales complex, may be associated with detection and transmission of equine herpesvirus type-1 (EHV-1) and -4 (EHV-4). This prospective cohort study aimed to investigate the currently undefined prevalence of EHV-1 and -4 in young Thoroughbreds at an auction sale in South Africa, and associations between clinical signs, physiological stress and viral detection. Ninety, two-year old Thoroughbreds (51 colts, 39 fillies) were consigned from eight farms and sampled at a South African auction sale. The horses were monitored for pyrexia and nasal discharge. Nasal swabs were collected for quantitative polymerase chain reaction (qPCR) assay to detect EHV-1 and -4 and faecal samples were collected for enzyme immunoassay (EIA) to determine faecal glucocorticoid metabolite (FGM) concentrations. EHV-4 nucleic acid was detected in some and EHV-1 nucleic acid in none of the population. Pyrexia and nasal discharge were poor indicators of EHV-4 status. Variation in FGM concentrations was best explained by transportation and preparation for auction. Peaks in EHV-4 detection and increases in FGM concentrations were identified shortly post-arrival and on the first day of auction. Temporal changes in FGM concentrations of horses from individual farms showed two distinct patterns: Pattern A (biphasic peaks) and Pattern B (single peak). It was concluded that sales consignment was associated with some EHV-4 nucleic acid detection and distinctive physiological stress patterns in this population of young Thoroughbreds. / Dissertation (MSc)--University of Pretoria, 2014. / tm2015 / Companion Animal Clinical Studies / MSc / Unrestricted

Equine herpesvirus type 1 (EHV-1) is a common pathogen of horses that typically causes upper respiratory disease, however is also associated with late-term abortion, neonatal foal death and neurologic disease. Once a horse is infected, the virus concentrates to local lymphoid tissue, where it becomes latent. The virus can recrudesce during times of stress, which can lead to the initiation of devastating outbreaks. Some variants of EHV-1 have been associated with more severe disease outcomes. Appropriate outbreak management focuses on minimizing the movement of potentially exposed horses. This approach lacks a strategy for prevention at the level of latency largely due to a knowledge paucity in regards to carriage rate of latent EHV-1. Biosecurity decisions are also dependent on awaiting currently-available diagnostic testing that often take several days for results. Thus, our work has been focused on understanding the carriage rate of the latent virus in different geographic regions as well as improving diagnostic efficiency, both of which are essential for improving the management of EHV-1 disease. Loop mediated isothermal amplification (LAMP) is a method that amplifies nucleic acid rapidly at a constant temperature and is minimally affected by inhibitors that are often found in clinical samples. This procedure can be followed by multiple detection methods. A new, efficient sequencing method, called nanopore sequencing, has been developed in a handheld device, called MinION, that provides thorough output in a timely manner. When combined with LAMP, it has been referred to as LAMPore. The first objective of our work was to estimate the prevalence of latent EHV-1 and compare the frequency of each variant in the submandibular lymph nodes from horses in Virginia. Our second objective was to perform direct DNA sequencing of EHV-1 using the mobile MinION sequencer in combination with LAMP viral enrichment. Our findings demonstrated a low apparent prevalence of latent EHV-1 DNA in submandibular lymph nodes in this population of horses in Virginia as well as successful detection and identification of EHV-1 in equine nasal swab samples using LAMPore sequencing. / Doctor of Philosophy / Horses can develop disease from a virus called equine herpesvirus type 1 (EHV-1). Symptoms can vary from mild respiratory signs to the inability to rise leading to death or euthanasia. Horses transmit this virus to other nearby horses; however, the virus also becomes dormant once a horse is infected, meaning the virus is not infectious but is present within the animal. When the horse undergoes stress, such as during travel or competition, the virus can become active again, leading to the spread to other horses. This results in outbreaks, many of which are devastating to the equine industry. In order to minimize the risks of this virus spreading and causing disease, management is currently focused on minimizing the movement of horses that may have been exposed to the virus. There is little information regarding the number of horses that harbor the dormant virus and the current methods to detect the infectious virus can take multiple days for results. These limit decision-making during the management of an outbreak. Our work seeks to determine the number of horses in a region that harbor EHV-1 and also to test a new, efficient diagnostic method to identify the virus in samples from horses. Our findings showed a low number of horses in Virginia harbor dormant EHV-1 in the lymph nodes under their mandible, a common site of dormancy. Further, we found that our new method of detection was effective in identifying the virus in samples from nasal secretions from horse.

Equine herpesvirus-1 (EHV-1) can cause acute upper respiratory tract disease, abortion, neonatal death and neurological disease in horses. Rapid, accurate and timely diagnosis of EHV-1 infection in horses is important to curtail the spread of this pathogen. It has been reported that the neuropathogenic phenotype of EHV-1 can result from a single non-synonymous nucleotide substitution at position 2254 (A→G2254) in open reading frame 30 (ORF30). This was the basis for the development of an allelic discrimination, real-time PCR assay to distinguish between potential neuropathogenic and non-neuropathogenic EHV-1 strains. However, PCR analysis of a panel of EHV-1 abortion isolates revealed that other point mutations within ORF30 could produce false negative results with this previously described assay. Therefore, one of the objectives of this dissertation project was to develop a more sensitive and specific allelic discrimination real-time PCR assay for the detection of EHV-1. This was achieved by redesigning the primers and probes targeting ORF30. The new assay was ten times more sensitive than the original assay, with a lower detection limit of 10 infectious virus particles. While mutations within EHV-1’s genome can hinder diagnosis, they can also impact the virulence of the virus. Objective two, therefore, was to determine if sequential cell passage of T953 would induce sufficient attenuation of the EHV-1 genome to produce a low virulence phenotype. Two separate groups of 28 BALB/c mice were inoculated with either the parental strain or passage 135 (T953 P135) of EHV-1 strain T953. The animals were observed for fourteen days, euthanized and their tissues analyzed for the presence of EHV-1. At the conclusion of the fourteen day observation period, all of the mice infected with T953 P135 survived and regained their pre-inoculation body condition. Furthermore, there were significant differences in virus titer and viral DNA concentrations between T953 P135 and the parental strain, further confirming the attenuated phenotype of the virus. Taken together, data from this study clearly demonstrates that sequential cell culture passage of the neuropathogenic T953 strain of EHV-1 results in attenuation for young adult BALB/C mice.