The identification of novel marine bacteria, and the construction of single chain fragment variable antibodies for the control of a viral pathogen

Isolation, identification, and characterization of members of the yet uncultured bacterioplankton is fundamental in understanding the roles they play in the marine environment, and their relationships with other members of the marine environment, such as viruses, phytoplankton, and zooplankton etc. In the first part of the thesis, I have used modified culture media in the traditional cultivation approach and isolated three novel bacterial strains from the marine environment in Hong Kong and China. Using the powerful polyphasic taxonomic approach, I determined that the two isolates from Hong Kong belonged to two novel genera in the family Cryomorphaceae. Both strains were characterized and published as Owenweeksia hongkongensis gen. sp. nov. and Lishizhenia caseinilytica gen. sp. nov. in the "International Journal of Systematic and Evolution Microbiology (IJSEM)", the leading journal in the field. The new strain isolated from a pearl-oyster culture pond, in Hainan Province, China, has been identified as a new species belonging to the genus Marinomonas, and was named and published as Marinomonas ostrestagni sp. nov. in IJSEM.; With the increased demand of shrimp for food, shrimp aquaculture has expanded considerably in many countries. In the last decade, the production of shrimp from aquaculture was drastically reduced due to the infection of novel viruses like the White Spot Syndrome Virus (WSSV) and Yellow Head Virus. Traditional treatments such as the sterilization of culture ponds and/or usage of antibiotics did not stop the problem. The repeated infection incidences suggested that the native immune system of shrimp is not sufficient to defend against the viral infection. In the second part of the thesis, I have constructed murine single chain fragment variable antibodies (ScFvs) phage display libraries against two major structural proteins of WSSV, VP26 and VP28. The ScFvs consisted of variable light chains of immunoglobulin joined to variable heavy chains by a peptide linker of 7 amino acids (short linker) or 16 amino acids (long linker). The size of the short linker libraries and long linker libraries were 3 x 107 and 1.7 x 108 clones, respectively. BstO I fingerprinting patterns indicated that the independent clones in each phage library were diverse. Screening of these libraries led to the identification of three groups of ScFvs that bind to independent epitopes on r-VP28. Two VP28 binding ScFvs delayed the onset of mortality in an in vivo neutralization assay in the crayfish Procambarus clarkii, confirming the role of VP28 in WSSV infection. These ScFvs have potentials to be developed into prophylactic reagents against WSSV. The new Cryomorphaceae bacteria that I isolated and identified could also be evaluated for its potential probiotics due to their high carotenoids content that may relieve the oxidative stress caused by WSSV infection.