This dissertation, "Molecular Epidemiology of and Vaccine Development Against Foot-and-mouth Disease Virus in Hong Kong" by Kin-hi, Raymond, Hui, 許建熙, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled MOLECULAR EPIDEMIOLOGY OF AND VACCINE DEVELOPMENT AGAINST FOOT-AND-MOUTH DISEASE VIRUS IN HONG KONG Submitted by Raymond Kin-hi Hui for the degree of Doctor of Philosophy at The University of Hong Kong in December 2004 Foot-and-mouth disease (FMD) is an endemic viral infectious disease in the Hong Kong porcine industry. This disease is caused by the Foot-and-mouth disease virus (FMDV) and disease outbreak causes a great impact on the local agricultural economy. Vaccination is a prompt and an effective measure to prevent the disease. To develop a specific vaccine against local strains of virus, epidemiology of the disease was first studied by nucleotide sequence analysis of the G-H loop in the VP1 region of the viral genome. Among 1056 samples collected from 42 local pig farms during the past four years, 109 were positive for the disease. Sequence data reveal that viruses isolated in Hong Kong belong to the Cathay topotype showing that the virus is introduced from neighboring Asian countries. The non-synonymous and the total substitution rates of -2 -3 FMDV circulating in Hong Kong were 1.26 10 and 4.83 10 substitutions/nucleotide/year respectively. Linear regression analysis estimates that the virus was introduced to Hong Kong between 1962 and 1968. Sequence data also indicate that geographical factors affect the spread of the virus, but the major causative event is most likely caused by human activities. In order to quantify FMDV precisely during vaccine development, a Sybr green real-time RT-PCR assay was developed. Sensitivity of the assay was 2.9 log units higher than that of conventional plaque assay and the dynamic detection range of the assay was at least eight orders of magnitude, and it was also capable of detecting a single copy of viral genome molecule. The assay also reflects the level of neutralizing antibody in serum samples by monitoring the virus proliferation in virus neutralization assay. Melting curve analysis of amplicons reveals that a single nucleotide change in the amplicon contributed to alternation in melting temperature by 0.5 C. This characteristic can be applied as a tool in quality control during inactivated vaccine production. The effectiveness of various vaccine candidates was investigated. In this study, vaccine candidates designed according to the local virus strain were used to immunize BALB/c mice. FMDV-specific antibodies and their neutralizing activity were evaluated with ELISA and real-time RT-PCR mediated neutralizing assay respectively. Among these candidates, Escherichia coli expressing the VP1 protein elicited the antibody titer and neutralizing activity most similar as compared to those immunized with a commercial inactivated vaccine. The same viral gene fragment immunized in form of pcDNA3.1-SP DNA plasmid and recombinant adenovirus also induced humoral responses; nevertheless, the specific antibody titer was lower than those vaccinated with bacterial expressed protein and conferred only partial neutralization in cell culture. In summary, strains of FMDV circulating in Hong Kong pig farms are localized, and therefore a specific vaccine is needed to protect the animals. Bacterial expressed VP1 protein designed according to local virus strains is a potential cost-effective vaccine candidate. DOI: 10.5353/th_b3154854 Subjects: