Graduation date: 1994

published_or_final_version / Microbiology / Master / Master of Medical Sciences

Typescript (photocopy). / Digitized by Kansas Correctional Industries

Background: Respiratory diseases are common worldwide, which are caused by various respiratory viruses. As symptoms caused by these viruses are similar, laboratory diagnosis is essential to distinguish the virus. Conventionally, respiratory viruses are isolated by cell culture with a panel of cell lines. However, handling of several cell lines is labour intensive, and the turnaround time of conventional culture is long. In previous study, the use of human colon adeno-carcinoma (Caco-2) in conventional culture was investigated. The study has proven that Caco-2 is generally susceptible to the eight common respiratory viruses, i.e. Adenovirus, Influenza A and B, Respiratory Syncytial virus, Parainfluenza virus 1, 2,3 and 4. As turnaround time of conventional culture is long; therefore, in this study, rapid shell vial culture using Caco-2 cells were evaluated. Moreover, the application of Caco-2 shell vial culture on recovering human metapneumovirus (hMPV) was also investigated. Materials and methods: This study consisted of four stages. First, recovery of viruses by conventional culture and shell vial culture of Caco-2 were compared. Specimens were added to conventional culture and shell vial simultaneously. For conventional culture, formation of CPE was examined daily and IF staining was performed when CPE was indicated; meanwhile, shell vial culture were incubated for seven days and stained with IF to detect infected cells. In stage two, the effect of incubating shell vial culture in rolling drum was investigated. Shell vials inoculated with the same specimen in duplicate were incubated in rolling drum and without rolling drum simultaneously. IF staining was performed in day 2, and results were obtained. For those which are IF negative in day 2, second shell vial was further incubated to seven days before harvest. In the next stage, a large batch of samples was used to evaluate on the use of Caco-2 shell vial culture in day 2 and day 7. Lastly, Caco-2 shell vial and conventional culture and LLC-MK2 conventional culture were tested for isolation of hMPV. Results: Compared to Caco-2 conventional culture, recovery rate of shell vial culture was elevated slightly. When experimenting on the effect of incubation in rolling drum, results showed that recovery rate was raised in shell vial with rolling drum in day 2, moreover, the percentage of positive cells were increased significantly (p value < 0.05). Furthermore, in the evaluation of Caco-2 shell vial in day 2 and day 7, 75% of samples were isolated in day 2 while 85% were recovered in day 7. Lastly, in the investigation on recovery of hMPV, 53%, 42% and 17% hMPV positive cases were isolated by Caco-2 shell vial, Caco-2 conventional culture and LLC-MK2 conventional culture respectively. Conclusion: First, although recovery rate by shell vial and conventional culture were similar, turnaround time was reduced from a week to a few days by shell vial culture. Therefore, Caco-2 shell vial culture is a more efficient than Caco-2 conventional culture in isolating respiratory viruses. The study also showed that incubation of shell vial in rolling drum able to increase the number of positive cells. Furthermore, in this study, Caco-2 cells were also shown to be more efficient in isolating hMPV when compare to LLC-MK2 cells. / published_or_final_version / Microbiology / Master / Master of Medical Sciences

This thesis describes the development of serum and whole blood immunodiagnostic assays for Maedi-Visna virus (MVV). All previously described recombinant MVV ELISA assays utilised either the core p25 or transmembrane (TM) proteins alone, or combined, but as individual proteins. The p25 and TM genes of MVV were cloned individually into the pGEX-2T expression vector. Both proteins were expressed as a combined fusion protein in frame with glutathione S-transferase (GST). The purified recombinant antigens (GST-TM and GST-TM-p25) were used to develop a MVV ELISA. Sera from 46 positive and 46 negative sheep were tested using the GST-TM and GST-TM-p25 ELISAs and a commercial p25 EIA kit. A two-graph receiver operating characteristic (TG-ROC) analysis program was used to interpret the data. The GST-TM-p25 ELISA was more sensitive than the commercial assay which is based on the p25 antigen alone and more specific than the GST-TM ELISA. The GST-TM-p25 ELISA showed a sensitivity and specificity of 100%. The human AIDS lentivirus transmembrane (TM) glycoprotein portion of the envelope viral protein has been identified as the antigen most consistently recognised by antibodies. There is suggestive evidence that the same applies to MVV as the GST-TM fusion protein, expressed in E. coli, has comparable sensitivity to the GST-TM-p25 fusion protein, but lacks specificity. However, due to the hydrophobic nature of the MVV TM protein, purification of the expressed fusion protein required lengthy purification protocols. This was despite the fact that only a truncated version of the TM protein was expressed. This prompted investigating an alternative expression system that could possibly circumvent the above mentioned problems. The yeast Pichia pastoris is known to be suitable for the high-level expression of heterologous proteins which are secreted into the culture supernatant. These features made P. pastoris an attractive host for the expression of the hydrophobic TM protein of MVV. However, limited success was achieved as only low expression levels were obtained and detection and quantification was only accomplished by means of ELISA. Evaluation of the diagnostic performance of the P. pastoris expressed MVV TM-polypeptide was performed using a panel of 36 confirmed negative and positive sera, and evaluated using a TG-ROC analysis programme, which yielded an equal Se and Sp of 83%. The use of a novel rapid immunoassay system, which allows the detection of circulating antibodies in whole blood, has been investigated for use as a MVV diagnostic assay. The central feature of this immunoassay lies in a monoclonal antibody against a glycophorin epitope present on all sheep erythrocytes. A Fab'-peptide conjugate was constructed by coupling a synthetic peptide, corresponding to a sequence from MVV TM protein, to the hinge region of the Fab' fragment of the antisheep erythrocyte antibody. Within the limited number of 10 seronegative and 10 seropositive samples the autologous red blood cell agglutination assay had a sensitivity of 90% and a specificity of 80%. Despite the limitations and difficulties encountered, the use of such rapid whole blood immunodiagnostic assays for MVV holds promise. / Thesis (Ph.D.)-University of Natal, Durban, 1997.

African horse sickness (AHS) is a viral disease with high mortality rates, vectored by the Culicoides midge and affecting members of the Equidae family. AHS is endemic to South Africa, and, as a result, affects export and international competitiveness in equine trade, and impacts significantly on the South African racehorse and performance horse industries. AHS also has devastating consequences for rural and subsistence equine ownership. The protocol developed in this dissertation has the potential to serotype and confirm the AHS virus within a few hours at significantly less cost than current methods. It will ease the financial and time constraints of studying an outbreak in real time and has the potential to solve many of the unknown factors surrounding AHS, particularly and most importantly, the role that each serotype plays in outbreaks and the form of the disease contracted by horses. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.