GENETIC TRANSFORMATION OF CERATOTHECA TRILOBA FOR THE PRODUCTION OF ANTHRAQUINONES FROM HAIRY ROOT CULTURES

Background: Ceratotheca triloba was found to contain three anthraquinones (9, 10-anthracenedione, 1-hydroxy-4-methylanthraquinone and 5, 8-dimethoxy-2, 3, 10, 10a-tetrahydro-1H, 4aH-phenanthrene-4, 9-dione [DTP]) in its roots. Inhibition of the human topoisomerase II enzyme is the basis of some currently used cancer drugs such as doxorubicin which is shown to be cardio-toxic. For this reason we decided to investigate anthraquinones from C. triloba as a possible anticancer drug, however the main limitation was the large quantities of roots that are required to obtain a good yield of the active compound. Therefore the aim of this research was to obtain a higher yield of anthraquinones in hairy roots cultures than the parent plant as well as to compare yields of hairy root, cell suspension and shoot cultures. Materials and Methods: Protocols for seed sterilization, seed germination, shoot cultivation, callus induction, A. rhizogenes mediatedtransformation and hormone supplementations of hairy roots were developed. Results:The results revealed that stem explants was susceptible to transformation by Agrobacterium rhizogenes at a low optical density of 0.2. Induced hairy roots were decontaminated by exposure to cefotaxime at 500mg.l-1 for five days and then 200mg.l-1 for eight days. Visualization of culture extract profiles by TLC revealed anthraquinones were present in all cultures. Analysis of the culture extracts by HPLC showed the highest yield of anthraquinones was produced in hairy root cultures supplemented with 1-Naphthaleneacetic acid [NAA] (8 mg). This was a 17 fold increase compared to field roots (0.47 mg). Conclusion:Therefore C. triloba hairy root cultures are the preferable biological system for anthraquinones production over shoot (0.13 mg) and cell suspension cultures (0.70 mg)

Protection of cattle elicited using a bivalent lumpy skin disease virus-vectored recombinant Rift Valley fever vaccine

Lumpy skin disease and Rift Valley fever are two high-priority livestock diseases which have the potential to spread into previously free regions through animalmovement and/or vectors, as well as intentional release by bioterrorists. Since the distribution range of both diseases is similar in Africa, it makes sense to use a bivalent vaccine to control them. This may lead to the more consistent and sustainable use of vaccination against Rift Valley fever through a more cost-effective vaccine. In this study, a recombinant lumpy skin disease virus was constructed in which the thymidine kinase gene was used as the insertion site for the Gn and Gc protective glycoprotein genes of Rift Valley fever virus using homologous recombination. Selection markers, the enhanced green fluorescent protein and Escherichia coli guanidine phosphoribosyl transferase (gpt), were used for selection of recombinant virus and in amanner enabling a second recombination event to occur upon removal of the gpt selection-pressure allowing the removal of both marker genes in the final product. This recombinant virus, LSD-RVF.mf, was selected to homogeneity, characterized and evaluated in cattle as a vaccine to show protection against both lumpy skin disease and Rift Valley fever in cattle. The results demonstrate that the LSD-RVF.mf is safe, immunogenic and can protect cattle against both diseases.This work was generously supported by the GALVmed organization (recombinant virus construction, selection, and characterization) (under grant no. 710/CAP/07/003) and the Canadian International Development Research Centre in collaboration with Global Affairs Canada, within the Canadian International Food Security Research Fund (CIFSRF) Grant no. 107848-002 (cattle trial). IDRC have also kindly agreed to pay the APF.https://www.frontiersin.org/journals/veterinary-science#am2020Veterinary Tropical Disease

Production of recombinant lumpy skin disease virus A27L and L1R proteins for application in diagnostics and vaccine development

Vaccination using live attenuated vaccines (LAVs) is considered the most effective method for control of lumpy skin disease (LSD). However, this method is limited by safety concerns, with reports of adverse reactions following vaccination. This study evaluates A27L and L1R which are essential proteins for virus attachment and membrane fusion as recombinant sub-unit vaccines against LSD. These proteins were recombinantly expressed in Escherichia coli and purified using affinity chromatography. Purified proteins were formulated individually (A27L or L1R) and in combination (A27L and L1R) with 10% (w/w) Montanide™ Gel 01 PR adjuvant at a final antigen dose of 20 µg per protein. The safety and immunogenicity of these formulations were evaluated in rabbits in a 42-day clinical trial. Animals were vaccinated on day 0 and boost injection administered 21 days later. No reduced morbidity, increased temperature and any other clinical signs were recorded in vaccinated animals for all three vaccine formulations. The highest neutralizing antibody response was detected on day 42 post-primary vaccination for all formulations when using serum neutralising assay. The neutralisation data correlates with antibody titres quantified using a whole cell ELISA. Evaluating the combination of A27L and L1R as potential diagnostic reagents showed highest sensitivity for detection of antibodies against LSD when compared to individual proteins. This study reports the immunogenicity of recombinant A27L and L1R combination for successful application in LSD vaccine development. Furthermore, these proteins demonstrated the potential use in LSD diagnostics