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FGPV Open Reading Frames. Location and function of FGPV open reading frames and comparison to their closest orthologues (PDF 756 kb)

Evidence is presented to show that variola and monkeypox viruses evolved independently from a common ancestor. An open reading frame (ORF), potentially coding for a protein of 341 amino acid residues, was found to be conserved in two strains of variola virus (Harvey and Somalia), but degenerate in the Denmark strain of monkeypox virus. Monkeypox virus had a deletion of 391 bp, two 24bp deletions and a single base pair deletion within the coding region of this single copy ORF. The ORF corresponds to the E5R ORF in the published sequence of the Copenhagen strain of vaccinia virus, and the DNA sequence was determined for an additional strain of vaccinia virus, Dairen. A number of other Orthopoxviruses were found to contain this ORF, strengthening confidence in its presence in an ancestral Orthopoxvirus. The equivalent DNA sequence was determined for a number of monkeypox virus strains from West and Central Africa. The Denmark strain was identical to one from Liberia, indicating that this virus probably originated from West Africa. A third virus from West Africa, Benin, was found to have >99% base similarity and the same pattern of deletions as the other two monkeypox viruses. The Zaire strains were identical to one another and different from the West African strains. Like the West African strains, they contained the two 24bp deletions and single base pair deletion. In place of the large deletion they had three smaller deletions of 5-, 9- and 127-bp as well as a single base pair insertion. They also had additional deletions of land 2-bp and an insertion of 3bp. The West African strains have the potential to code for a truncated gene product of 107 amino acid residues, whereas the Zaire strains have no significant ORF. This clearly shows that monkeypox virus has diverged into two geographically isolated groups (Zaire and West Africa). There was >99% base similarity between the two groups, suggesting that the divergence occurred recently. Phylogenetic analysis, by the neighbour-joining method, was undertaken on the corresponding DNA sequences from variola (2 strains), monkeypox (6 strains), vaccinia (1 strain + 2 published sequences), cowpox (2 strains), taterapox, camel pox and ectromelia viruses. For every species gerbilpox virus was the nearest neighbour, suggesting that taterapox virus may be the species most closely related to the common ancestral Orthopoxvirus. Within the variola and cowpox virus species there was >99% DNA sequence conservation. Between species, camelpox virus was the most closely related species to gerbilpox virus, with variola virus, and, more distantly, vaccinia virus, falling into the same group. Cowpox virus was the most diverged species examined. Ectromelia virus shared a branch with cowpox virus. A comparison was made of the intergenic DNA sequence between this ORF and the adjacent downstream ORF. Variation was found, both within and between species, in the form of insertions and deletions. The interrelationships between the different Orthopoxvirus species more or less parallels that of the E5R-equivalent comparison. Some of the viruses had clusters of direct repeats. A pentameric repeated unit was found in 2, 10 and 17 copies in camelpox, gerbilpox and ectromelia viruses respectively. Raccoon poxvirus had a 7bp unit in 13 adjacent copies. The two cowpox viruses had a more complex arrangement of repeated sequences. It was thought that the ESR ORF may prove to be nonessential for virus replication. This was tested by interruption of the E5R gene in vaccinia virus; this did not affect the ability of the virus to form plaques in cell culture, but appeared to reduce the pathogenicity of the 'virus for rabbits. The deduced amino acid sequences were analysed for conserved and variable regions within the gene, to which no specific function has yet been assigned.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2, SARS-CoV-2. Given the ongoing COVID-19 pandemic and the continued evolution of the virus to escape host immunity, new vaccines and refinement of first-generation vaccines to improve protection against SARS-CoV-2 variants of concern is vital. In Africa, the cost of vaccine manufacturing as well as the scarcity in resources for storage and distribution have all contributed to the inequitable access to vaccines and heavy reliance on donations. Modified Vaccinia Virus Ankara (MVA) is a low-cost production vector platform which is suitable in this context. This project falls into a bigger study where our group compared different vector platforms, including MVA. The project serves as a proof of concept that this platform can be used to produce vaccines encompassing different variants of SARS-CoV-2 as they emerge. The most recent variant, Omicron, has proven to be highly immune evasive and demonstrates this need well. As the virus mutates, the variants of concern each present with differing characteristics and subsequently differ in immunogenicity and pathogenicity. Sub-Saharan Africa has been ransacked by the pandemic, resulting in loss of lives and livelihoods; the effects of which will undoubtedly be felt for decades to come. This study had two aims: 1. The development of a candidate vaccine, MVA-SARS-CoV2-S∆TM by using the widely used MVA platform and poxvirus recombinant vaccine strategies used in our research group, 2. The testing of this vaccine's immunogenicity in mice. The MVA-based vaccine was constructed by infection of BHK21 cells with wildtype MVA, and transfection with transfer vector pMVA-FNK2. The transfer vector contains a truncated form of the SARS-CoV-2 spike glycoprotein gene, the vaccinia virus host-range gene K1L and reporter gene eGFP, flanked by gene sequences to allow homologous recombination into MVA. The presence of the K1L gene in the recombinant virus allowed for selection by passaging in RK13 cells, which were not permissive for the parent MVA. Following the potentially successful isolation, the recombinant MVA-SARS-CoV2-S∆TM was validated and characterized by PCR, Sanger sequencing, Western blot analysis and immunofluorescence, which all confirmed the presence and expression of the spike protein. Large scale propagation of the vaccine was done in RK13 cells, and the stock was titrated to yield a titre of 3.9 x 106 ffu/ml. Balb/c mice were inoculated three times with MVA-SARS-CoV2-S∆TM at a dose of 3 x 105 to assess immunogenicity of the vaccine. Results from the immunogenicity assessments demonstrated that the vaccine induced T-cell responses shown by an enzyme-linked immunosorbent spot (ELISpot) assay. An enzyme-linked immunosorbent assay (ELISA) confirmed the ability of MVA-SARSCoV2-S∆TM to induce increasing titres of binding antibodies in mice over a period of 56 days. However, the vaccine did not induce neutralizing antibodies against a matched SARS-CoV-2 pseudovirus, highlighting the need for further refinement of the vaccine. In conclusion, recombinant MVA expressing a truncated SARS-CoV-2 spike protein was successfully constructed and tested for immunogenicity in mice. The candidate vaccine induced good T-cell responses and binding antibodies, but not neutralizing antibodies. This study provides additional evidence that MVA can be used as a platform for a SARS-CoV-2 vaccine, as has been previously demonstrated by others, and this could potentially be adapted for emerging variants of concern. This work also allows for the direct comparison of the MVA platform to other platforms employed by our group (DNA and plant-based subunit) as SARS-CoV-2 vaccines.

Lumpy skin disease (LSD) is a notifiable viral infection due both to its morbidity in cattle and its severe economic burden. The disease was confined to Sub-Saharan Africa but has in recent years spread to the Middle East and Europe. Vaccination is the only way of preventing LSD. Live attenuated lumpy skin disease virus (LSDV) has been used as a vaccine against LSD. The most successful LSD vaccine is the Neethling vaccine strain (nLSDV) from South Africa. There are however, reports of nLSDV being too attenuated or too virulent in different breeds of cattle. A South African produced vaccine strain of LSDV, Herbivac, was said to be more immunogenic than nLSDV (personal communication, Deltamune). Whole genomic sequence comparison of Herbivac with nLSDV revealed a single potentially significant change in open reading frame (ORF) 131. This ORF encodes a superoxide dismutase (SOD) homologue. The mutation identified in Herbivac is a 2bp deletion which causes a frameshift mutation that restores the SOD homologue to resemble the full-length SOD homolog encoded by the virulent field strain. This SOD homologue gene is truncated in nLSDV. Protein structural alignment of SOD homologues from LSDV and other characterised SOD homologues was done. Both the truncated and full-length SOD homologues lacked the catalytic arginine at position 142 which is involved in SOD activity. Some similarities with known SOD homologues which act as SOD decoys, such as the Leporipoxviruses myxoma and Shope Fibroma virus, were observed. Like Leporipoxvirus’ SOD homologues, the full-length Herbivac SOD homologue contained regions of homology with the copper chaperone for SOD (CCS). The putative SOD protein from Herbivac, and not nLSDV, contained 6 out of 8 metal binding residues. Unlike the SOD decoy from myxoma virus, the full-length SOD homolog from Herbivac did not include a cysteine molecule at position 56 that stabilizes the SOD-CCS heterodimer. The alignment suggests that all SOD homologs from LSDV are inactive as enzymes. Transcriptome analysis of messenger RNA from spleens of mice infected with nLSDV or Herbivac for 24hrs was carried out to determine the effect of Herbivac or nLSDV infection on host gene expression. Compared to the PBS control, nLSDV and Herbivac induced the differential expression of 98 genes in common, largely related to response to viral infection. nLSDV differed in the unique expression of 6 genes and Herbivac differed in the unique expression of 36 genes, including granzyme A and Poly (ADP-ribose) polymerase (Parp 9). Herbivac upregulated genes associated with pathogen pattern recognition, interferon response, immune response and cell death. More Gene ontology (GO) processes were enriched after Herbivac infection than nLSDV infection. Amongst these processes were immune response processes, the interferon and cell death related responses. To characterise the SOD homolog with respect to SOD activity, cell death and whether it plays a role in growth of viruses expressing it, recombinant viruses were constructed. The first set of recombinants included a SOD knock-out virus where the SOD gene from nLSDV was replaced with reporter gene GFP. A SOD knock-in virus was constructed with the SOD homologue gene altered to improve the stability of the gene. A reporter gene mCherry was also inserted. Histological examination of CAMs infected with nLSDVdSOD-M showed vacuolation and oedema to a greater degree than nLSDV, Herbivac and nLSDVSODis-M. Herbivac showed greater immune cell infiltration. SOD knock-in, nLSDVSODis-M showed increased epithelial hyperplasia and fibroplasia. Another set of recombinants without marker genes was made, nLSDVdSOD-UCT (SOD knock-out), and nLSDVSODis-UCT (SOD knock-in). Deleting the SOD homolog reduced virus yield by approximately ten-fold in MDBK cells. Histologically, the presence of the SOD homolog in nLSDVSODis-UCT caused greater inflammatory changes in the mesoderm after 5 days of infection compared to nLSDVdSOD-UCT. In vitro functional studies were done in MDBK cells which are permissible to LSDV infection. No difference in SOD activity could be detected amongst the different viruses. Differences could, however, be detected in induction and inhibition of apoptosis. There was increased induction of apoptosis following infection by all viruses containing full-length SOD compared to infection with truncated or SOD knock-out virus. Viruses expressing a full-length SOD showed greater inhibition of camptothecin induced apoptosis than nLSDV or the SOD knockout. Similarly, full-length SOD induced cell death by necrosis to a greater extent than the SOD knock-out or nLSDV; and all viruses inhibited camptothecin induced necrosis. These results suggest that the presence of a SOD homolog in a vaccine could be advantageous with respect to growth of the vaccine to higher titres and to improved immunogenicity of the vaccine. Further work is required to test this hypothesis in a bovine animal model.

There are two broad aims to this project. The first aim is to compare and characterise two lumpy skin disease virus (LSDV) vaccines namely the vaccine based on attenuated Neethling LSDV (nLSDV) and Herbivac®LS (Herbivac). The second aim is to construct a recombinant LSDV expressing Rift Valley fever virus (RVFV) genes. An LSDV vaccine is critical for sustainable control of lumpy skin disease (LSD). There are four commercially available live attenuated vaccines for LSDV, nLSDV, Herbivac, Lumpyvax and the Kenyan strain sheeppox virus (KS-1). In this study Herbivac was characterised by comparing it to its parent, nLSDV. Growth curves of the two viral strains were conducted in cell culture as well as in embryonated hens’ eggs. No notable difference in the growth rate of the two strains could be detected when the viruses were grown in cell culture, however a notable difference was detected when the viruses were grown on the chick allantoic membranes (CAMs) of embryonated hens’ eggs. When grown on CAMs a faster growth rate was observed for nLSDV compared to Herbivac. nLSDV also killed the embryos at 4 d.p.i where Herbivac did not. The two strains were then further characterised through histological analysis of CAMs after infection with each of the viruses. Overall, higher levels of hyperplasia and hypertrophy were observed in CAMs infected with either nLSDV or Herbivac compared to uninfected CAMs. Herbivac-infected CAMs resulted in thicker chorionic membranes and larger pocks compared to nLSDV. RVFV and LSDV both contribute to the disease burden among cattle in Africa and the Arabian Peninsula. The main aim of this study was to construct a recombinant Herbivac which expresses immunogenic proteins of Rift Valley fever virus (Herbivac-RVFV). Herbivac-RVFV was designed to express specific RVFV genes selected for their antigenic properties. The genes selected are also representative of the genes from recent viral outbreaks in the horn of Africa. The selection of outbreak relevant RVFV genes involved phylogenetic analysis of all full length M-segment and NC gene sequences available on Genbank. Phylogenetic trees were constructed for M-segments and NC genes and groups identified which were highly representative of sequences from recent outbreaks of the virus. Consensus sequences were derived from these groups and included in the transfer vector. The phylogenetic analysis also revealed that the sequences of current RVFV vaccines are phylogenetically distant from viruses isolated from current outbreaks, although high levels of sequence conservation was maintained across all viral strains. This is the first study in which the RVFV genes coding for proteins that will induce a protective immune response (Gn and Gc, as well as the nucleocapsid (NC) gene) were selected so as to be representative of current outbreak strains of the virus. These genes were inserted between LSDV ORFs 49 and 50, a novel insertion site. The transfer vector also contained an eGFP marker gene and an ECO-GPT selection gene, located outside of the LSDV flanking sequences. This meant a two-step isolation procedure, first to isolate the recombinant containing the entire transfer vector with eGFP and ECO-GPT, and then to isolate a recombinant with only the RVFV genes and not eGFP and ECO-GPT. Transient expression of RVFV proteins in cells infected with Herbivac and then transfected with the transfer vector was confirmed via western blotting and immunofluorescence. Here the proteins Gn, Gc and NC were shown to be expressed. In the present study, a single crossover Herbivac-RVFV recombinant was isolated through multiple passaging of cell lysates, originally obtained from Herbivac-infected FBT cells transfected with the transfer vector, in the presence of mycophenolic-acid selection medium.

Includes bibliographical references (leaves 131-137)., Of the many vaccine trials which have taken place, the most promising results have been obtained from the recent phase 3 clinical trial which tested the ability of a dual protein and Canarypox virus recombinant to protect humans against HIV-1 infections. Because poxviruses are being developed as vaccine vectors against a number of diseases, it is important to continue the search for novel poxvirus vectors, in particular, those that do not cross-neutralize one another. This thesis describes the preliminary work performed on the development of Penguinpox virus (PEPV) as a vaccine vector.

Includes bibliographical references (leaves 96-114)., Cowpox virus has been found to encode the inflammation modulatory protein (IMP) (Miller, C.G., 1997), a homologue vaccinia virus complement control protein (VCP). VCP belongs to regulation of complement activation (RCA) protein superfamily. It has been shown to inhibit both the alternative and classical pathways of complement activation by binding to the proteins C3 and C4, thereby preventing complementmediated opsonisation of virus, antibody-mediated lysis of infected cells and migration of inflammatory cells into the site of infection. VCP also possesses heparin binding sites.