Once a system had been developed for generating and selecting homogeneous LSDV recombinants expressing a reporter gene, it was possible to generate and select LSDV recombinants expressing genes of veterinary importance for use as pox-vectored recombinant vaccines.
However, before commencing further, two areas were identified where the current system could be improved upon, viz. the LSDV transfer vector, and, the strategy for selecting homogeneous recombinants. The LSDV transfer vector was streamlined by removal of non-essential sequences, and, a number of unique restriction enzyme (R.E.) sites were restored in the MCS after their uniqueness was lost due to introduction of the gpt positive selectable marker gene (along with a number of R.E. sites).
Although it was possible to select homogeneous LSDV recombinants using the modified MPAselection strategy outlined in chapter 2, it needs to be mentioned that th is strategy is not deemed to be perfect and early attempts to select for homogeneous LSDV recombinants expressing immunogenic genes of veterinary importance (in place of the lacZ reporter gene) were highly frustrated by the presence of contaminating wild-type (parental) LSDV. The lacZ reporter gene provided a useful visual basis for assisting with the selection process, however it was found that attempting to retain this gene in the transfer vector with the addition of immunogenic genes from viruses of veterinary importance led to logistical problems such as size (insertion plasmids would have been greater than – 14 kbp in size).
In order to overcome this problem the gene of the enhanced green fluorescent protein (EGFP) (originally isolated from the jellyfish, Aequorea victoria) (Cheng et al., 1996) was substituted for the lacZ gene and was incorporated into the improved LSDV transfer vector. Not only is this gene much smaller in size than the lacZ gene (1 kbp, as opposed to 3.7 kbp), but it also allows for the in vivo selection of recombinants. The enhanced form of the green fluorescent protein produces a stable continuous emission of visible green light at a wavelength of 511 nm when excited with UV light in the 490 nm range (which coincides with the range used in most laboratory fluorescence microscopes and flow cytometers). The EGFP gene was placed under control of the W late P11 K promoter. Recombinant LSDVs were found to express the protein within 12 hours post~infection making it easier to select for viral foci homogeneous for recombinant virus over foci containing a mixture of both wild-type and recombinant viruses when the cells were viewed under UV light. Contaminating wtlSDV still proved to be a problem though, and other modifications were introduced to the selection process to attempt to overcome this problem (described in the Materials and Methods).
FBT cells were propagated on sterile microscope glass coverslips in 6-well cell culture dishes (Nunclon, Denmark) until approximately 90 % confluent. Some wells were then inoculated with recombinant virus, while others were inoculated with wt virus at an MOl =0.1. Cells were then returned to the CO2 incubator. After approximately four days, or when cpe were visible, the cells were fixed in ice-cold acetone for 20 minutes. The acetone was removed and the cells were washed once in PBSA (PBS without magnesium or calcium). Cells were then flooded in preabsorbed polyclonal antibody for BEFV (obtained from a naturally infected bovid) or polyclonal antibody for RVFV (sheep anti-RVFV polyclonal serum supplied by Mara Schoeman, Virology Department, OVI) diluted in blocking reagent (2% low fat milk powder [Elite, South Africa] diluted in PBSA) (the optimal dilution factor having been determined previously) and incubated for 30 minutes at RT. Incubation was then followed by 4 washes in PBSA (each wash lasting 10 minutes) with gentle rocking on a STR6 Platform Shaker (Stuart Scientific, UK). Rabbit antibovine immunoglobulin type-G (lgG) conjugated to fluorescein isothiocyanate (FITC) (Sigma, USA) was diluted 1 :80 in blocking reagent (containing 0.01 % Evans Blue counterstain [Merck, Germany]) for BEFV detection (and donkey anti-sheep IgG-FITC conjugate (Sigma, USA) diluted 1: 1 00 for RVFV detection), and was added to the cells for a period of 30 minutes at RT.
The four PBSA washes were repeated, followed by a final 10 minute wash in distilled water. The samples were then air dried, mounted on glass microscope slides in mounting fluid (1 : 1 glycerol:PBS, pH = 7.4) and viewed under UV light using a Leica OM IL inverted fluorescence microscope (Germany).
CHAPTER 1: LITERATURE REVIEW
1.1 INTRODUCTION TO POXVIRUSES
1.2 AIMS OF THIS INVESTIGATION
1.3 BIOLOGY OF POXVIRUSES
1.4 LUMPY SKIN DISEASE VIRUS
1.5 POXVIRUSES AS VECTORS FOR RECOMBINANT VACCINES
CHAPTER 2: GENERATION AND SELECTION OF TK-DISRUPTED LSDV RECOMBINANTS
2.2 MATERIALS AND METHODS
CHAPTER 3: GENERATION AND SELECTION OF LSDV RECOMBINANTS EXPRESSING GENES OF VETERINARY IMPORTANCE
3.2 IVIATERIALS AND METHODS
CHAPTER 4 IMMUNE RESPONSES AND PROTECTION STUDIES IN ANIMALS
4.2 MATERIALS AND METHODS
CHAPTER 5 CONCLUDING REMARKS
ARTICLES PUBLISHED AND PRESENTATIONS AT SCIENTIFIC MEETINGS