Browsing by Author "Vukea, Phillia Rixongile."

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Characterisation of infectious bursal disease virus (IBDV) polyprotein processing.
(2011) Vukea, Phillia Rixongile.; Coetzer, Theresa Helen Taillefer.
Infectious bursal disease virus (IBDV) is a birnavirus that infects the B-cells in the bursa of Fabricius of young chickens, causing Gumboro disease. The IBDV 114 kDa polyprotein (NH2-pVP2-VP4-VP3-COOH) is thought to be processed at 512Ala-Ala513 and 755Ala-Ala756 through the proteolytic activity of VP4, a serine protease which uses a Ser/Lys catalytic dyad, to release pVP2, VP4 and VP3. Precursor VP2 (pVP2) is further processed at its C-terminus to generate VP2 and structural peptides through the cleavage of the 441Ala-Phe442, 487Ala-Ala488, 494Ala-Ala495 and 501Ala-Ala502 peptide bonds to release VP2 and four structural peptides, pep46, pep7a, pep7b and pep11. While the processing at the 441Ala-Phe442 site was shown to be mediated by the endopeptidase activity of VP2, the processing at the other two sites is not well understood. The products resulting from the processing of the IBDV polyprotein were previously identified by anti-VP2 and anti-VP3 antibodies. The present study used anti-VP4 peptide antibodies to identify products resulting from the IBDV polyprotein processing. It was hypothesised that VP4 exists in two forms, the embedded form which exists as an integral part of the polyprotein and a mature form which is released after the processing. In order to characterise the two forms of VP4, six different fragments i.e. full-length polyprotein (Met1-Glu1012), truncated polyprotein (Ile227-Trp891), VP4-RA (Arg453-Ala755), VP4-RK (Arg453-Lys722), VP4-ΔVP3 (Ala513-Trp891, called VP4-AW for the sake of simplicity) and VP4-AA (Ala513-Ala755) were amplified from the IBDV dsRNA, cloned into a T-vector and sub-cloned into several expression vectors. The constructs were sequenced prior to expression. The sequence of the polyprotein coding region was used to determine the pathotype of the isolate used for viral dsRNA isolation. This isolate was from IBDV-infected bursae harvested from commercial chickens during an IBD outbreak in KwaZulu-Natal, South Africa in 1995, thus naming the isolate SA-KZN95. The comparison of the deduced amino acid sequence of SA-KZN95 polyprotein with 52 sequences of other IBDV strains highlighted 21 residues which could be molecular markers of different IBDV pathotypes. The residues of SA-KZN95 were identical to those of the Malaysian very virulent UPM94/273 strain. The constructs representing the embedded and mature forms of VP4 were recombinantly expressed. Processing was observed from the expression of the full-length polyprotein, truncated polyprotein, VP4-RA, VP4-RK and VP4-AW, but not from VP4-AA expression. The mutation of the Ser/Lys catalytic dyad in the full-length polyprotein, truncated polyprotein, VP4-RA, VP4-RK and VP4-AW, prevented processing thus verifying that the proteolytic activity was due to VP4. Anti-VP4 peptide antibodies were raised in chickens for the identification of the polyprotein cleavage products. The anti-VP4 peptide antibodies detected more cleavage products than expected from the polyprotein, suggesting that additional or different cleavage sites may be used. The characterisation of the cleavage products suggested that the processing for the release of VP4 occurs either at the 487Ala-Ala488 or the 512Ala-Ala513 site in a single polyprotein molecule. Ultimately, an IBDV polyprotein processing strategy that would explain the release of the additional products was proposed in the present study. The present study also illustrated the importance of Pro377 in the processing of the polyprotein where its replacement with Leu induced a prominent change in polyprotein processing. The mutation seemed to induce structural changes that may possibly affect the cleavage sites. Although no autocatalytic activity was observed during the expression of VP4-AA (mature form), it cleaved mutant VP4-RK in trans. It seemed to be active as a dimer on a gelatine gel but no activity was observed against a dialanyl fluorogenic peptide substrate. It also appeared to form peptidase-inhibitor complexes with anti-thrombin III. The present study also describes attempts to detect native VP4 in IBDV-infected bursa homogenates by anti-VP4 peptide antibodies on a western blot and by proteolytic activity determination on gelatine-containing SDS-PAGE gels. The findings of the study provide new information that may contribute to the development of anti-viral agents. These anti-viral agents may target polyprotein processing, capsid assembly and thus prevent virus replication during IBDV infection.
Identification of infectious bursal disease virus (IBDV) receptors through the use of recombinant capsid protein, VP2.
(2014) Brien, Kayleen Fransiena.; Vukea, Phillia Rixongile.; Coetzer, Theresa Helen Taillefer.
Infectious bursal disease virus (IBDV) is a non-enveloped Birnavirus which infects the immature antibody producing B-cells of the bursa of Fabricius in young chickens. The virus causes infectious bursal disease (IBD) which is highly contagious and immunosuppressive. A compromised immune system in infected chickens leaves them susceptible to other opportunistic pathogens and as a result increases their mortality rate. Major economic losses in the commercial poultry industry are subsequently experienced in affected regions. Currently vaccines are used to control IBDV infection, however, their efficacy is affected by factors such as the presence of maternally derived antibodies in young chickens which reduces vaccine load, the continuous emergence of new virulent IBDV strains and bursal atrophy caused by some vaccines. It is therefore important to consider new ways of controlling the virus such as targeting specific stages in the virus life cycle. Since virus attachment to host cell receptor(s) is the most crucial step in the virus life cycle, developing novel antiviral agents which prevent viral entry represents a good alternative strategy for IBDV control. Identification of receptor binding proteins and receptors of host cell membranes is required for antiviral development. The receptor binding protein and outer capsid of IBDV is VP2, however, the receptor(s) utilised by IBDV to gain entry into host cells have not been conclusively identified. Recombinant VP2 was used to identify possible IBDV receptor(s) on bursal plasma membranes using a virus overlay protein binding assay (VOPBA) and affinity chromatography. Therefore, VP2 was heterologously expressed in an Escherichia coli and a Pichia pastoris expression system as a 64 kDa fusion protein and a 47 kDa protein respectively. In addition, both systems expressed VP2 as high molecular mass proteins which were confirmed by electro-elution and western blotting. Although purification of VP2 expressed in the E. coli system was a challenge because VP2 expressed as inclusion bodies, polyclonal chicken anti-VP2 antibodies were produced using VP2 expressed in this system. Purification of VP2 expressed in P. pastoris was easier and produced a greater yield of VP2 which was used to produce a VP2-coupled affinity matrix for the purification of chicken anti-VP2 antibodies and for the purification of VP2-binding proteins of the bursal plasma membrane. Moreover, peptides were selected from the VP2 amino acid sequence and use to raise polyclonal chicken anti-VP2 peptide antibodies for comparative identification against chicken anti-VP2 antibodies of possible IBDV receptor(s). Two IBDV VP2-binding proteins with molecular masses of 70 and 32 kDa of the bursal plasma membrane were identified in a VOPBA using recombinant VP2 or IBDV and chicken anti-VP2 antibodies. In addition to the VOPBA, four IBDV VP2-binding proteins with molecular masses of 70, 60, 45 and 32 kDa were affinity purified on a VP2-coupled affinity matrix. Analysis of the affinity purified proteins by mass spectrometry identified five proteins which share common peptides which include, the Ig-gamma chain and Ig-lambda chain of Gallus gallus, outer major protein of Serratia marcescens, the 60 kDa chaperonin of Pseudomonas fluorescens and elongation factor-Tu of Yersinia pestis. The results strongly suggest that an Ig-receptor like protein may form part of the IBDV receptor, however, much further work is required in order to establish whether the chicken homologues of the identified bacterial sequences are part of the putative bursal receptor. It is believed that the bacterial proteins contain common peptides with chicken proteins of the chicken genome which has not been fully annotated as yet. Taken together, this study successfully used VP2 to identify possible IBDV receptor(s) on bursal plasma membranes which could ultimately lead to the development of antiviral agents targeted at IBDV entry.
Recombinant expression of, and characterisation of antibodies against variable surface glycoproteins : LiTat 1.3 and LiTat 1.5 of Trypanosoma brucei gambiense.
(2013) Mnkandla, Sanele Michelle.; Coetzer, Theresa Helen Taillefer.; Vukea, Phillia Rixongile.
Human African Trypanosomiasis (HAT), also known as sleeping sickness is one of the many life threatening tropical diseases posing a serious risk to livelihoods in Africa. The disease is restricted to the rural poor across sub–Saharan Africa, where tsetse flies that transmit the disease, are endemic. Sleeping sickness is known to be caused by protozoan parasites of the genus Trypanosoma brucei, with the two sub-species: T. b. gambiense and T. b. rhodesiense, responsible for causing infection in humans. The disease develops in two stages, firstly, the infection is found in the blood and secondly, when the parasites cross the blood-brain barrier entering the nervous system. To date, no vaccines have been developed, however, there is a range of drugs and treatments available which depend on the type of infection (T. b. gambiense or T. b. rhodesiense) as well as disease stage. The trypanosome parasites have a two-host life cycle i.e. in the mammalian host as well as the tsetse fly vector. Throughout the cycle, the parasite undergoes changes, one of them being the acquisition of a variable surface glycoprotein (VSG) coat prior to entry into the human host bloodstream. Once in the host, the infection progresses and through a phenomenon known as antigenic variation, the parasite expresses a different VSG coat periodically, enabling the parasites to constantly evade the host’s immune response, facilitating their survival. The VSG genes coding for the proteins are activated by an intricate process involving the encoding of a gene which is kept silent, until activated in one of several expression sites. Despite the constant switching of VSG surface coats, there are VSG forms that are predominantly expressed in T. b. gambiense namely VSGs LiTat 1.3, LiTat 1.5 and LiTat 1.6 which are used in diagnostic tests, as antigens to detect antibodies in infected sera of HAT patients. The acquisition of these VSG antigens is, however, of high risk to staff handling the parasites, and so the first part of the study was aimed at cloning, recombinantly expressing and purifying the two VSGs known to be recognised by all gambiense HAT patients: LiTat 1.3 and LiTat 1.5, for possible use as alternative antigens in diagnostic tests. The genes encoding both VSGs, LiTat 1.3 and LiTat 1.5, were first amplified from either genomic or complementary DNA (gDNA or cDNA), cloned into a pTZ57R/T-vector and sub-cloned into pGEX or pET expression vectors prior to recombinant expression in E. coli BL21 DE3 and purification by Ni-affinity chromatography. Amplification and subsequent cloning yielded the expected 1.4 kb and 1.5 kb for the LiTat 1.3 and LiTat 1.5 genes respectively. Recombinant expression in E. coli was only successful with the constructs cloned from cDNA, i.e. the pGEX4T-1-cLiTat 1.3 and pET-28a-cLiTat 1.3 clones. Purification of the 63 kDa cLiTat 1.3His protein following solubilising and refolding did not yield pure protein and there were also signs of protein degradation. For comparison, expression was also carried out in P. pastoris and similar to the bacterial system, expression was only successful with the LiTat 1.3-SUMO construct yielding a 62.7 kDa protein. Purification of LiTat 1.3SUMO also surpassed that of cLiTat 1.3His with no degradation. The diagnostic tests based on VSGs LiTat 1.3 and LiTat 1.5 as antigens operate by binding with antibodies in infected sera, to confirm infection. These antibody detection tests have their limitations, hence an alternative would be antigen detection tests, which use antibodies to detect the respective antigens in infected sera. The second part of the study therefore involved antibody production, where chickens were immunised with the native VSGs LiTat 1.3, LiTat 1.5 as well as recombinant RhoTat 1.2 (a VSG expressed in T. evansi). Antibody production was analysed by ELISA and characterised by western blotting, prior to immunolabelling of T. b. brucei Lister 427 parasites. The chicken IgY showed a response to the immunogens, and were able to detect their respective proteins in the western blot. Interestingly, the anti-nLiTat 1.3, anti-nLiTat 1.5 and anti-rRhoTat 1.2 antibodies were able to detect their respective VSGs on the T. b. brucei trypanosome parasites in the immunofluorescence assay, thus demonstrating cross reactivity. As the antibodies showed specificity, they could potentially detect antigens in infected sera of HAT patients in an antigen detection based test.