Thesis (Ph. D.)--University of Sydney, 2004. / Title from title screen (viewed 5 May 2008). Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the Faculty of Medicine. Includes list of published articles and presentations. Includes bibliographical references. Also available in print form.

Dissertation (Ph.D.)--Auburn University, 2007. / Abstract. Vita. Includes bibliographic references (p.281-340).

Thesis (MMedVet. (Wildlife Diseases))--University of Pretoria, 2007. / Includes bibliographical references.

Thesis (Ph.D.)--University of California, San Francisco, 2004. / Includes bibliographical references. Also available online.

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

California sea lions (CSLs) have an unusually high occurrence of urogenital cancer (UGC), with studies revealing metastatic carcinoma in 26 % of CSLs admitted to a rehabilitation centre between 1998 and 2012. It is likely that the aetiology of this disease is multi-factorial as genetics, viral infection and exposure to contaminants have been associated with this cancer to date. The goal of this study was to investigate the association of a number of factors using a case-control study design on animals admitted to a rehabilitation centre. The study additionally concentrates on two main areas; (i) genetic factors and (ii) the presence of herpesvirus. Previous investigations identified cancer to be more likely in animals with specific microsatellite alleles. In the present study genotyping of CSLs at three microsatellite loci revealed that homozygosity at one marker (Pv11) was significantly associated with the presence of the disease. Pv11 was found to be located within a gene called heparanase 2 (HPSE2) and investigations into the expression of its protein revealed differences according to Pv11 genotype. The presence of herpesvirus was investigated by two PCR methods and identified the gammaherpesvirus OtHV-1. The results of the two methods were contradictory with one method identifying a highly significant relationship between the presence of OtHV-1 and UGC whereas the other did not. Complicating factors such as potential differences in sensitivity of the tests along with the possible presence of closely related viruses or variants of OtHV-1 may explain this. The availability of necropsy data for the CSLs in the study allowed the inclusion of body condition data in the statistical analysis to evaluate other potential risk factors. Final analysis revealed the presence of three risk factors; Pv11 genotype, OtHV-1 presence and thinner blubber. This study is the largest study undertaken so far in order to investigate the involvement of risk factors associated with UGC in the CSL and supports a multi-factorial aetiology of this disease.

MSc (Microbiology) / Department of Microbiology / Background: Human Herpes Virus-8 (HHV-8) and Human Immunodeficiency Virus (HIV) are endemic in sub-Saharan Africa. However, the prevalence of HHV-8 in HIV-infected individuals in sub-Saharan Africa has not been fully described and characterized. Objectives: The objective of this study was to determine the seroprevalence and genetic subtypes of HHV-8 in an HIV-infected population in Cameroon. Methodology: KSHV/HHV-8 Enzyme-linked Immunosorbent Assay (ELISA) kit (Advanced Biotechnologies Inc., USA) was used to detect IgG antibodies in the plasma of 406 HIV-infected outpatients of the Mutengene Baptist Health Centre, Cameroon. To detect the viral presence, a 233 bp fragment of the ORF 26 gene of HHV-8 was targeted by polymerase chain reaction (PCR) in total DNA purified from patients’ whole blood. A 453 bp of the K1 gene was amplified by nested PCR, sequenced and phylogenetically analysed to infer subtypes. The online tool, Synonymous Non-synonymous Analysis Program (SNAP), was used to determine the rate of synonymous and nonsynonymous mutations in the K1 gene. The genetic variability among the derived K1 nucleotide sequences was determined by mean genetic distance analysis. Results: Of the 406 participants, an HHV-8 seroprevalence of 79.1% was obtained. There was a statistically significant association of seroprevalence with age (p= 0.00), CD4+ cell count (p= 0.02), marital status (p= 0.02) and ownership of a transistor radio set (p= 0.00). Seventy samples (23.3%) were successfully amplified for ORF 26 gene confirming the presence of replicating virus. K1 sequences were obtained for 14 of the 20 (70%) K1 amplified DNAs. The mean genetic diversity of K1 sequences ranges from 0.0%-22.3%. Phylogenetic analysis revealed two infecting viral subtypes in the study cohort: subtype A5 (57.1%), and subtype B (35.7%). Greater positive selection and genetic diversity were observed in A5 subtype compared to B subtype of K1. Interestingly, one sample (BM 547) clustered with an unclassifiable sequence from South Africa. Conclusions and recommendation: This study revealed the endemicity of HHV-8 infection in the studied population, with subtypes A5 and B as the most important epidemiological genetic variants. In addition, targeting the ORF 26 region by PCR could be an approach to detect replicating virus in individuals. Further studies should investigate the association between HHV-8 infection and KS development in the study area which is endemic for HIV. This study contributes data to the HIV/HHV-8 co-infection landscape in the study area and in Africa at large.

The expanding aquaculture industry continues to encounter major challenges in the form of highly contagious aquatic viruses. Control and eradication measures targeting the most lethal and economically damaging virus-induced diseases, some of which are notifiable, currently involve ‘stamping out’ policies and surveillance strategies. These approaches to disease control are performed through mass-culling followed by restriction in the movement of fish and fish products, resulting in considerable impacts on trade. Although effective, these expensive, ethically complex measures threaten the sustainability and reputation of the aquatic food sector, and could possibly be reduced by emulating innovative vaccination strategies that have proved pivotal in maintaining the success of the terrestrial livestock industry. DIVA ‘differentiating infected from vaccinated animal’ strategies provide a basis to vaccinate and contain disease outbreaks without compromising ‘disease-free’ status, as antibodies induced specifically to infection can be distinguished from those induced in vaccinated animals. Various approaches were carried out in this study to assess the feasibility of marker/DIVA vaccination for two of the most important disease threats to the global Atlantic salmon and common carp/koi industries, i.e. infectious salmon anaemia (ISA) and koi herpesvirus disease (KHVD), respectively. Antibody responses of Atlantic salmon (Salmo salar L.), following immunisation with an ISA vaccine, administered with foreign immunogenic marker antigens (tetanus toxoid (TT), fluorescein isothiocyanate (FITC) and keyhole limpet hemocyanin (KLH)) were assessed by antigen-specific enzyme linked immunosorbent assay (ELISA). Although antibodies were induced to some markers, these were unreliable and may have been affected by temperature and smoltification. Detectable antibodies to ISAV antigen were also largely inconsistent despite low serum dilutions of 1/20 being employed for serological analysis. The poor antibody responses of salmon to the inactivated ISA vaccine suggested that DIVA vaccination is not feasible for ISA. A similar approach for KHV, utilising green fluorescent protein (GFP) as the marker, similarly failed to induce sufficiently detectable antibody responses in vaccinated carp (Cyprinus carpio L.). However, as high anti-KHV antibody titres were obtained with an inactivated KHV vaccine (≥1/3200), alternative approaches were carried out to assess the feasibility of DIVA vaccination for carp. Investigations of early KHV pathogenesis in vivo and antigen expression kinetics in vitro (0-10 days post infection (dpi)) provided valuable data for the diagnostics necessary for DIVA surveillance strategies. Following viral infection, molecular methods were shown to be the most effective approach for early detection of KHV infected fish prior to sero-conversion, during which time antibodies are not detectable. An experimental immersion challenge with KHV, however, revealed complications in molecular detection during early infection. The KHV DNA was detected in external biopsies of skin and gills, but also internally in gut and peripheral blood leukocytes ≤ 6 hours post infection (hpi), suggesting rapid virus uptake by the host. The gills and gut appeared to be possible portals of entry, supported by detection of DNA in cells by in situ hybridisation (ISH). However, many false negative results using organ biopsies occurred during the first 4 dpi. The gills were the most reliable lethal biopsy for KHV detection by various polymerase chain reaction (PCR) assays, with a PCR targeting a glycoprotein-gene (ORF56) and a real-time PCR assay being the most sensitive of the 7 methods investigated. Importantly, non-lethal mucus samples reduced the number of false negative results obtained by all KHV PCR assays during the earliest infection stages with large levels of viral DNA being detected in mucus (up to 80,000 KHV DNA genomic equivalents 200 μL-1). KHV DNA was consistently detected in the mucus as a consequence of virus being shed from the skin. Determining the expression kinetics of different viral structural proteins can be useful for DIVA serological tests. Analysis of KHV antigen expression in tissues by immunohistochemistry and indirect fluorescent antibody test was inconclusive, therefore 2 novel semi-quantitative immunofluorescence techniques were developed for determining KHV antigen expression kinetics in susceptible cell lines. During the course of KHV infection in vitro, a greater abundance of capsid antigen was produced in infected cells compared to a glycoprotein antigen (ORF56), as determined by detection with antigen-specific monoclonal antibodies (MAbs). The capsid antigen was characterised as a ~100 kDa protein by SDS-PAGE and identified as a product of KHV ORF84 by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF/TOF MS). This antigen was subsequently detected in the serum of >25% of KHV infected/exposed carp (6/17), as well as in carp vaccinated with a live attenuated vaccine (3/4), but not with an inactivated vaccine (0/7), by Western blot making it a potential DIVA target for an inactivated vaccine. Attempts were made to improve the sensitivity of KHV serological testing by taking advantage of recombinant proteins specific for KHV (CyHV-3), rORF62 and rORF68 and eliminating any interference by cross-reacting antibodies to carp pox (CyHV-1). These proteins successfully reacted with anti-KHV antibodies. The feasibility of DIVA strategies for KHVD was determined using these recombinant antigens to coat ELISA plates. Differential antibody responses were detected from carp sera to an internal virus tegument protein (rORF62) and external region of a transmembrane protein (rORF68). Fish vaccinated with an inactivated vaccine produced significantly lower antibody responses to rORF62 than to rORF68, whereas infected, exposed and live attenuated vaccinated fish recognised both proteins allowing differentiation between vaccinated and infected carp. However, the sensitivity of the assay was limited, possibly by high levels of natural antibodies detected at the relatively low serum dilutions (1/200) used. As the capsid antigen (ORF84) and tegument protein (ORF62) are derived from internal KHV structural proteins, they induce non-neutralising antibodies, which may be useful for DIVA strategies. Such antibodies are longer lasting than neutralising antibodies and often comprise the majority of fish anti-viral antibodies. This was noted in a fish surviving experimental challenge, which had an antibody titre of 1/10,000, but neutralising titre of 1/45. Such antigens may therefore hold potential for developing effective serological diagnostic tests for KHV and provide the potential for DIVA strategies against KHVD. Natural antibodies will, however, continue to present a challenge to the development of sensitive and reliable KHV serological tests, and hence the application of DIVA strategies.