| dc.description.abstract | Rift Valley fever (RVF) is a mosquito-borne zoonotic disease endemic to Africa that presents a significant health threat to both humans and animals. Outbreaks have serious economical implications. Two facts
cause concern. Firstly, recent outbreaks in the Arabian Peninsula and effects of global warming have the implication that the virus might spread further into non-endemic RVF regions since it utilizes a wide range of mosquito vetors. Secondly, RVFV is a potential bio-terrorism agent. Therefore, quick, safe, robust and cost-effective methods of detection and appropriate diagnostic reagents are needed for early diagnosis, disease surveillance and bio-defence field monitoring. The development of several effective ELlSAs for the detection of antibodies to RVFV have been reported (Paweska et al., 2003a, 2003b and 2005a;
2005b). A serious drawback of these ELlSAs is that they use an inactivated virus as antigen. The availability of a recombinant antigen would lower the safety risks of antigen preparation and the costs of diagnosis. The purpose of this study was to clone and express a recombinant RVFV nucleoprotein (N) from a southern African RVFV strain (Zim 688/78), and to evaluate its suitability as a diagnostic antigen in an ELISA for the detection of antibodies to RVFV in humans and animals. The gene encoding the N protein was amplified by RT-PCR using primers specifically designed to contain
restriction enzyme sites for cloning. The nucleic acid sequence of the gene was determined and compared to that of the 7 other published N gene sequences of different strains. The N gene of the Zim688178 strain had 5 unique nucleic acid differences, all in wobble positions which did not cause amino acid changes. The nucleic acid sequence of the Zim688I78 strain was the closest related to the Clone1 3 strain (98.6%), and its amino acid sequence was identical to Clone 13. A fragment of the gene encoding the glycoprotein G2 was also amplified, its nucleic acid sequence was determined and compared to the published G2 sequences of other strains. The G2 fragment was closest related to the BEgy93 (98.7%), HEgy93 (98.7%) and ZH548 (98.7%) strains. The Zim688/78 N gene was cloned into two bacterial expression system vectors, pGEX4T-1 and pET32(a)+ and three baculovirus expression vectors, pFastBac-1, pFastBacHT-B and pBACgus-1. Low amounts of completely insoluble recombinant N protein (rN) were expressed by baculovirus recombinants generated using pFastBacHT-B. The expression level of rN using pFastBac-I was higher and rN was more soluble. These constructs, including the pBACgus-1 construct, was sent to Biovac (Pinelands, South Africa) for further optimization of baculovirus expression. Bacterial expression using the pGEX4T-1
vector resulted in a high yield of rN, but it was mostly insoluble. However, IPTG induced bacterial expression with the pET32(a)+ construct resulted in a high yield of soluble rN. The recombinant nucleoprotein was purified with Ni-affinity column purification, using the pET32(a)+ fusion tag with 6x His
residues. The purified rN was of high quality and therefore it could be used for direct coating of immunoplates in indirect ELISA (I-ELI%). Various coating procedures were tested. Overnight coating in carbonatelbicarbonate buffer (pH 9.6) on a MaxiSorp (Nunc) plate gave the best results. The recombinant 5 nucleoprotein antigen based I-ELISA was used to measure IgG and IgM antibodies in titrated sera from vaccinated personnel and naturally infected humans respectively. The test was able to distinguish between sera with high and low concentrations of antibodies. The rN-based I-ELISA was also used to measure IgG and IgM antibody responses in vaccinated and experimentally infected sheep. The test was
able to show seroconversion in both vaccinated and infected sheep, track the rise and decline of IgM antibodies and detect the expected lower levels of the immune response in sheep vaccinated with live attenuated virus compared to that of sheep infected with wild type virus. The rN also proved to be suitable as an antigen in IgG sandwich and IgM capture ELlSA formats.
The production of the RVFV rN under experimental procedures used was quick, easy, relatively inexpensive and safe. The findings of this project demonstrated that the recombinant is a high quality diagnostic antigen for use in I-ELISAs for the detection of antibodies in humans and animals. | |
