...funding research, sharing discoveries.
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BMC biotechnology 10
Use of the piggyBac transposon to create HIV-1 gag transgenic insect cell lines for continuous VLP production.
BACKGROUND: Insect baculovirus-produced Human immunodeficiency virus type 1 (HIV-1) Gag virus-like-particles (VLPs) stimulate good humoral and cell-mediated immune responses in animals and are thought to be suitable as a vaccine candidate. Drawbacks ... expand abstractto this production system include contamination of VLP preparations with baculovirus and the necessity for routine maintenance of infectious baculovirus stock. We used piggyBac transposition as a novel method to create transgenic insect cell lines for continuous VLP production as an alternative to the baculovirus system. RESULTS: Transgenic cell lines maintained stable gag transgene integration and expression up to 100 cell passages, and although the level of VLPs produced was low compared to baculovirus-produced VLPs, they appeared similar in size and morphology to baculovirus-expressed VLPs. In a murine immunogenicity study, whereas baculovirus-produced VLPs elicited good CD4 immune responses in mice when used to boost a prime with a DNA vaccine, no boost response was elicited by transgenically produced VLPs. CONCLUSION: Transgenic insect cells are stable and can produce HIV Pr55 Gag VLPs for over 100 passages: this novel result may simplify strategies aimed at making protein subunit vaccines for HIV. Immunogenicity of the Gag VLPs in mice was less than that of baculovirus-produced VLPs, which may be due to lack of baculovirus glycoprotein incorporation in the transgenic cell VLPs. Improved yield and immunogenicity of transgenic cell-produced VLPs may be achieved with the addition of further genetic elements into the piggyBac integron. collapse abstract
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Plant biotechnology journal 2010 May; 8(5)
Plant-made vaccines for humans and animals.
The concept of using plants to produce high-value pharmaceuticals such as vaccines is 20 years old this year and is only now on the brink of realisation as an established technology. The original reliance on transgenic plants has largely given way to... expand abstract transient expression; proofs of concept for human and animal vaccines and of efficacy for animal vaccines have been established; several plant-produced vaccines have been through Phase I clinical trials in humans and more are scheduled; regulatory requirements are more clear than ever, and more facilities exist for manufacture of clinic-grade materials. The original concept of cheap edible vaccines has given way to a realisation that formulated products are required, which may well be injectable. The technology has proven its worth as a means of cheap, easily scalable production of materials: it now needs to find its niche in competition with established technologies. The realised achievements in the field as well as promising new developments will be reviewed, such as rapid-response vaccines for emerging viruses with pandemic potential and bioterror agents. collapse abstract
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Archives of virology 2010 Feb; 155(3)
A unique isolate of beak and feather disease virus isolated from budgerigars (Melopsittacus undulatus) in South Africa.
Beak and feather disease virus (BFDV), the causative agent of psittacine beak and feather disease (PBFD) infects psittaciformes worldwide. We provide an annotated sequence record of three full-length unique genomes of BFDV isolates from budgerigars (... expand abstractMelopsittacus undulatus) from a breeding farm in South Africa. The isolates share >99% nucleotide sequence identity with each other and ~96% nucleotide sequence identity to two recent isolates (Melopsittacus undulatus) from Thailand but only between 91.6 and 86.6% identity with all other full-length BFDV sequences. Maximum-likelihood analysis and recombination analysis suggest that the South African budgerigar BFDV isolates are unique to budgerigars, are non-recombinant in origin, and represent a new genotype of BFDV. collapse abstract
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Molecular plant pathology 2009 Dec; 11(1)
Maize streak virus: an old and complex 'emerging' pathogen.
Maize streak virus (MSV; Genus Mastrevirus, Family Geminiviridae) occurs throughout Africa, where it causes what is probably the most serious viral crop disease on the continent. It is obligately transmitted by as many as six leafhopper species in th... expand abstracte Genus Cicadulina, but mainly by C. mbila Naudé and C. storeyi. In addition to maize, it can infect over 80 other species in the Family Poaceae. Whereas 11 strains of MSV are currently known, only the MSV-A strain is known to cause economically significant streak disease in maize. Severe maize streak disease (MSD) manifests as pronounced, continuous parallel chlorotic streaks on leaves, with severe stunting of the affected plant and, usuallly, a failure to produce complete cobs or seed. Natural resistance to MSV in maize, and/or maize infections caused by non-maize-adapted MSV strains, can result in narrow, interrupted streaks and no obvious yield losses. MSV epidemiology is primarily governed by environmental influences on its vector species, resulting in erratic epidemics every 3-10 years. Even in epidemic years, disease incidences can vary from a few infected plants per field, with little associated yield loss, to 100% infection rates and complete yield loss. TAXONOMY: The only virus species known to cause MSD is MSV, the type member of the Genus Mastrevirus in the Family Geminiviridae. In addition to the MSV-A strain, which causes the most severe form of streak disease in maize, 10 other MSV strains (MSV-B to MSV-K) are known to infect barley, wheat, oats, rye, sugarcane, millet and many wild, mostly annual, grass species. Seven other mastrevirus species, many with host and geographical ranges partially overlapping those of MSV, appear to infect primarily perennial grasses. PHYSICAL PROPERTIES: MSV and all related grass mastreviruses have single-component, circular, single-stranded DNA genomes of approximately 2700 bases, encapsidated in 22 x 38-nm geminate particles comprising two incomplete T = 1 icosahedra, with 22 pentameric capsomers composed of a single 32-kDa capsid protein. Particles are generally stable in buffers of pH 4-8. DISEASE SYMPTOMS: In infected maize plants, streak disease initially manifests as minute, pale, circular spots on the lowest exposed portion of the youngest leaves. The only leaves that develop symptoms are those formed after infection, with older leaves remaining healthy. As the disease progresses, newer leaves emerge containing streaks up to several millimetres in length along the leaf veins, with primary veins being less affected than secondary or tertiary veins. The streaks are often fused laterally, appearing as narrow, broken, chlorotic stripes, which may extend over the entire length of severely affected leaves. Lesion colour generally varies from white to yellow, with some virus strains causing red pigmentation on maize leaves and abnormal shoot and flower bunching in grasses. Reduced photosynthesis and increased respiration usually lead to a reduction in leaf length and plant height; thus, maize plants infected at an early stage become severely stunted, producing undersized, misshapen cobs or giving no yield at all. Yield loss in susceptible maize is directly related to the time of infection: infected seedlings produce no yield or are killed, whereas plants infected at later times are proportionately less affected. DISEASE CONTROL: Disease avoidance can be practised by only planting maize during the early season when viral inoculum loads are lowest. Leafhopper vectors can also be controlled with insecticides such as carbofuran. However, the development and use of streak-resistant cultivars is probably the most effective and economically viable means of preventing streak epidemics. Naturally occurring tolerance to MSV (meaning that, although plants become systemically infected, they do not suffer serious yield losses) has been found, which has primarily been attributed to a single gene, msv-1. However, other MSV resistance genes also exist and improved resistance has been achieved by concentrating these within individual maize genotypes. Whereas true MSV immunity (meaning that plants cannot be symptomatically infected by the virus) has been achieved in lines that include multiple small-effect resistance genes together with msv-1, it has proven difficult to transfer this immunity into commercial maize genotypes. An alternative resistance strategy using genetic engineering is currently being investigated in South Africa. Useful websites: http://www.mcb.uct.ac.za/MSV/mastrevirus.htm; http://www.danforthcenter.org/iltab/geminiviridae/geminiaccess/mastrevirus/Mastrevirus.htm. collapse abstract
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The Journal of general virology 2010 Mar; 91(Pt 4)
Replicative intermediates of maize streak virus found during leaf development.
Geminiviruses of the genera Begomovirus and Curtovirus utilize three replication modes: complementary-strand replication (CSR), rolling-circle replication (RCR) and recombination-dependent replication (RDR). Using two-dimensional gel electrophoresis,... expand abstract we now show for the first time that maize streak virus (MSV), the type member of the most divergent geminivirus genus, Mastrevirus, does the same. Although mastreviruses have fewer regulatory genes than other geminiviruses and uniquely express their replication-associated protein (Rep) from a spliced transcript, the replicative intermediates of CSR, RCR and RDR could be detected unequivocally within infected maize tissues. All replicative intermediates accumulated early and, to varying degrees, were already present in the shoot apex and leaves at different maturation stages. Relative to other replicative intermediates, those associated with RCR increased in prevalence during leaf maturation. Interestingly, in addition to RCR-associated DNA forms seen in other geminiviruses, MSV also apparently uses dimeric open circular DNA as a template for RCR. collapse abstract
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Biotechnology progress 25(4)
Optimization of chimeric HIV-1 virus-like particle production in a baculovirus-insect cell expression system.
A baculovirus-insect cell expression system potentially provides the means to produce prophylactic HIV-1 virus-like particle (VLP) vaccines inexpensively and in large quantities. However, the system must be optimized to maximize yields and increase p... expand abstractrocess efficiency. In this study, we optimized the production of two novel, chimeric HIV-1 VLP vaccine candidates (GagRT and GagTN) in insect cells. This was done by monitoring the effects of four specific factors on VLP expression: these were insect cell line, cell density, multiplicity of infection (MOI), and infection time. The use of western blots, Gag p24 ELISA, and four-factorial ANOVA allowed the determination of the most favorable conditions for chimeric VLP production, as well as which factors affected VLP expression most significantly. Both VLP vaccine candidates favored similar optimal conditions, demonstrating higher yields of VLPs when produced in the Trichoplusia ni Pro insect cell line, at a cell density of 1 x 10(6) cells/mL, and an infection time of 96 h post infection. It was found that cell density and infection time were major influencing factors, but that MOI did not affect VLP expression significantly. This work provides a potentially valuable guideline for HIV-1 protein vaccine optimization, as well as for general optimization of a baculovirus-based expression system to produce complex recombinant proteins. collapse abstract
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Plant biotechnology journal 2009 Dec; 8(1)
High level protein expression in plants through the use of a novel autonomously replicating geminivirus shuttle vector.
We constructed a novel autonomously replicating gene expression shuttle vector, with the aim of developing a system for transiently expressing proteins at levels useful for commercial production of vaccines and other proteins in plants. The vector, p... expand abstractRIC, is based on the mild strain of the geminivirus Bean yellow dwarf virus (BeYDV-m) and is replicationally released into plant cells from a recombinant Agrobacterium tumefaciens Ti plasmid. pRIC differs from most other geminivirus-based vectors in that the BeYDV replication-associated elements were included in cis rather than from a co-transfected plasmid, while the BeYDV capsid protein (CP) and movement protein (MP) genes were replaced by an antigen encoding transgene expression cassette derived from the non-replicating A. tumefaciens vector, pTRAc. We tested vector efficacy in Nicotiana benthamiana by comparing transient cytoplasmic expression between pRIC and pTRAc constructs encoding either enhanced green fluorescent protein (EGFP) or the subunit vaccine antigens, human papillomavirus subtype 16 (HPV-16) major CP L1 and human immunodeficiency virus subtype C p24 antigen. The pRIC constructs were amplified in planta by up to two orders of magnitude by replication, while 50% more HPV-16 L1 and three- to seven-fold more EGFP and HIV-1 p24 were expressed from pRIC than from pTRAc. Vector replication was shown to be correlated with increased protein expression. We anticipate that this new high-yielding plant expression vector will contribute towards the development of a viable plant production platform for vaccine candidates and other pharmaceuticals. collapse abstract
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Virology journal 6
Comparative analysis of Panicum streak virus and Maize streak virus diversity, recombination patterns and phylogeography.
BACKGROUND: Panicum streak virus (PanSV; Family Geminiviridae; Genus Mastrevirus) is a close relative of Maize streak virus (MSV), the most serious viral threat to maize production in Africa. PanSV and MSV have the same leafhopper vector species, lar... expand abstractgely overlapping natural host ranges and similar geographical distributions across Africa and its associated Indian Ocean Islands. Unlike MSV, however, PanSV has no known economic relevance. RESULTS: Here we report on 16 new PanSV full genome sequences sampled throughout Africa and use these together with others in public databases to reveal that PanSV and MSV populations in general share very similar patterns of genetic exchange and geographically structured diversity. A potentially important difference between the species, however, is that the movement of MSV strains throughout Africa is apparently less constrained than that of PanSV strains. Interestingly the MSV-A strain which causes maize streak disease is apparently the most mobile of all the PanSV and MSV strains investigated. CONCLUSION: We therefore hypothesize that the generally increased mobility of MSV relative to other closely related species such as PanSV, may have been an important evolutionary step in the eventual emergence of MSV-A as a serious agricultural pathogen.The GenBank accession numbers for the sequences reported in this paper are GQ415386-GQ415401. collapse abstract
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Expert review of vaccines 2009 Aug; 8(9)
Third International Conference on Plant-Based Vaccines and Antibodies.
This relatively new biennial meeting - the first was in Prague in 2005 - was chaired by Julian Ma (Guy's Hospital, London, UK), with Mario Pezzotti (University of Verona, Italy) as local organizer, and attracted approximately 180 delegates from 25 co... expand abstractuntries. The theme was 'Plant Expression Systems for Recombinant Pharmacologics': there were 46 talks gathered into two plenaries, 12 themed sessions and 72 posters. Topics covered included publicly funded and commercial developments, innovation, regulation and commercialization, competition with conventional technology, manufacture and new products. collapse abstract
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The Journal of general virology 2009 Nov; 90(Pt 12)
Dating the origins of the maize-adapted strain of maize streak virus, MSV-A.
Maize streak virus (MSV), which causes maize streak disease (MSD), is one of the most serious biotic threats to African food security. Here, we use whole MSV genomes sampled over 30 years to estimate the dates of key evolutionary events in the 500 ye... expand abstractar association of MSV and maize. The substitution rates implied by our analyses agree closely with those estimated previously in controlled MSV evolution experiments, and we use them to infer the date when the maize-adapted strain, MSV-A, was generated by recombination between two grass-adapted MSV strains. Our results indicate that this recombination event occurred in the mid-1800 s, approximately 20 years before the first credible reports of MSD in South Africa and centuries after the introduction of maize to the continent in the early 1500 s. This suggests a causal link between MSV recombination and the emergence of MSV-A as a serious pathogen of maize. collapse abstract
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BMC cancer 9
Human papillomavirus prevalence, viral load and pre-cancerous lesions of the cervix in women initiating highly active antiretroviral therapy in South Africa: a cross-sectional study.
BACKGROUND: Cervical cancer and infection with human immunodeficiency virus (HIV) are both important public health problems in South Africa (SA). The aim of this study was to determine the prevalence of cervical squamous intraepithelial lesions (SILs... expand abstract), high-risk human papillomavirus (HR-HPV), HPV viral load and HPV genotypes in HIV positive women initiating anti-retroviral (ARV) therapy. METHODS: A cross-sectional survey was conducted at an anti-retroviral (ARV) treatment clinic in Cape Town, SA in 2007. Cervical specimens were taken for cytological analysis and HPV testing. The Digene Hybrid Capture 2 (HC2) test was used to detect HR-HPV. Relative light units (RLU) were used as a measure of HPV viral load. HPV types were determined using the Roche Linear Array HPV Genotyping test. Crude associations with abnormal cytology were tested and multiple logistic regression was used to determine independent risk factors for abnormal cytology. RESULTS: The median age of the 109 participants was 31 years, the median CD4 count was 125/mm3, 66.3% had an abnormal Pap smear, the HR-HPV prevalence was 78.9% (Digene), the median HPV viral load was 181.1 RLU (HC2 positive samples only) and 78.4% had multiple genotypes. Among women with abnormal smears the most prevalent HR-HPV types were HPV types 16, 58 and 51, all with a prevalence of 28.5%. On univariate analysis HR-HPV, multiple HPV types and HPV viral load were significantly associated with the presence of low and high-grade SILs (LSIL/HSIL). The multivariate logistic regression showed that HPV viral load was associated with an increased odds of LSIL/HSIL, odds ratio of 10.7 (95% CI 2.0 - 57.7) for those that were HC2 positive and had a viral load of
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Virology journal 6
Experimental evidence indicating that mastreviruses probably did not co-diverge with their hosts.
BACKGROUND: Despite the demonstration that geminiviruses, like many other single stranded DNA viruses, are evolving at rates similar to those of RNA viruses, a recent study has suggested that grass-infecting species in the genus Mastrevirus may have ... expand abstractco-diverged with their hosts over millions of years. This "co-divergence hypothesis" requires that long-term mastrevirus substitution rates be at least 100,000-fold lower than their basal mutation rates and 10,000-fold lower than their observable short-term substitution rates. The credibility of this hypothesis, therefore, hinges on the testable claim that negative selection during mastrevirus evolution is so potent that it effectively purges 99.999% of all mutations that occur. RESULTS: We have conducted long-term evolution experiments lasting between 6 and 32 years, where we have determined substitution rates of between 2 and 3 x 10(-4) substitutions/site/year for the mastreviruses Maize streak virus (MSV) and Sugarcane streak Réunion virus (SSRV). We further show that mutation biases are similar for different geminivirus genera, suggesting that mutational processes that drive high basal mutation rates are conserved across the family. Rather than displaying signs of extremely severe negative selection as implied by the co-divergence hypothesis, our evolution experiments indicate that MSV and SSRV are predominantly evolving under neutral genetic drift. CONCLUSION: The absence of strong negative selection signals within our evolution experiments and the uniformly high geminivirus substitution rates that we and others have reported suggest that mastreviruses cannot have co-diverged with their hosts. collapse abstract
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Vaccine 2009 Oct; 27(46)
Immunogenicity of an HPV-16 L2 DNA vaccine.
The ability to elicit cross-neutralizing antibodies makes human papillomavirus (HPV) L2 capsid protein a possible HPV vaccine. We examined and compared the humoral response of mice immunized with a HPV-16 L2 DNA vaccine or with HPV-16 L2 protein. The... expand abstract L2 DNA vaccine elicited a non-neutralizing antibody response unlike the L2 protein. L2 DNA vaccination suppressed the growth of L2-expressing C3 tumor cells, which is a T cell mediated effect, demonstrating that the lack of non-neutralizing antibody induction by L2 DNA was not caused by lack of T cell immunogenicity of the construct. collapse abstract
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Vaccine 2009 Jul; 27(35)
A prime-boost immunisation regimen using recombinant BCG and Pr55(gag) virus-like particle vaccines based on HIV type 1 subtype C successfully elicits Gag-specific responses in baboons.
Mycobacterium bovis BCG is considered an attractive live bacterial vaccine vector. In this study, we investigated the immune response of baboons to a primary vaccination with recombinant BCG (rBCG) constructs expressing the gag gene from a South Afri... expand abstractcan HIV-1 subtype C isolate, and a boost with HIV-1 subtype C Pr55(gag) virus-like particles (Gag VLPs). Using an interferon enzyme-linked immunospot assay, we show that although these rBCG induced only a weak or an undetectable HIV-1 Gag-specific response on their own, they efficiently primed for a Gag VLP boost, which strengthened and broadened the immune responses. These responses were predominantly CD8+ T cell-mediated and recognised similar epitopes as those targeted by humans with early HIV-1 subtype C infection. In addition, a Gag-specific humoral response was elicited. These data support the development of HIV-1 vaccines based on rBCG and Pr55(gag) VLPs. collapse abstract
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Virology journal 6
A highly divergent South African geminivirus species illuminates the ancient evolutionary history of this family.
BACKGROUND: We have characterised a new highly divergent geminivirus species, Eragrostis curvula streak virus (ECSV), found infecting a hardy perennial South African wild grass. ECSV represents a new genus-level geminivirus lineage, and has a mixture... expand abstract of features normally associated with other specific geminivirus genera. RESULTS: Whereas the ECSV genome is predicted to express a replication associated protein (Rep) from an unspliced complementary strand transcript that is most similar to those of begomoviruses, curtoviruses and topocuviruses, its Rep also contains what is apparently a canonical retinoblastoma related protein interaction motif such as that found in mastreviruses. Similarly, while ECSV has the same unusual TAAGATTCC virion strand replication origin nonanucleotide found in another recently described divergent geminivirus, Beet curly top Iran virus (BCTIV), the rest of the transcription and replication origin is structurally more similar to those found in begomoviruses and curtoviruses than it is to those found in BCTIV and mastreviruses. ECSV also has what might be a homologue of the begomovirus transcription activator protein gene found in begomoviruses, a mastrevirus-like coat protein gene and two intergenic regions. CONCLUSION: Although it superficially resembles a chimaera of geminiviruses from different genera, the ECSV genome is not obviously recombinant, implying that the features it shares with other geminiviruses are those that were probably present within the last common ancestor of these viruses. In addition to inferring how the ancestral geminivirus genome may have looked, we use the discovery of ECSV to refine various hypotheses regarding the recombinant origins of the major geminivirus lineages. collapse abstract
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The Journal of general virology 2009 Feb; 90(Pt 3)
Rapid host adaptation by extensive recombination.
Experimental investigations into virus recombination can provide valuable insights into the biochemical mechanisms and the evolutionary value of this fundamental biological process. Here, we describe an experimental scheme for studying recombination ... expand abstractthat should be applicable to any recombinogenic viruses amenable to the production of synthetic infectious genomes. Our approach is based on differences in fitness that generally exist between synthetic chimaeric genomes and the wild-type viruses from which they are constructed. In mixed infections of defective reciprocal chimaeras, selection strongly favours recombinant progeny genomes that recover a portion of wild-type fitness. Characterizing these evolved progeny viruses can highlight both important genetic fitness determinants and the contribution that recombination makes to the evolution of their natural relatives. Moreover, these experiments supply precise information about the frequency and distribution of recombination breakpoints, which can shed light on the mechanistic processes underlying recombination. We demonstrate the value of this approach using the small single-stranded DNA geminivirus, maize streak virus (MSV). Our results show that adaptive recombination in this virus is extremely efficient and can yield complex progeny genomes comprising up to 18 recombination breakpoints. The patterns of recombination that we observe strongly imply that the mechanistic processes underlying rolling circle replication are the prime determinants of recombination breakpoint distributions found in MSV genomes sampled from nature. collapse abstract
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Archives of virology 154(2)
Insights into the role and function of L2, the minor capsid protein of papillomaviruses.
Human papillomaviruses (HPV) are responsible for the most common human sexually transmitted viral infections, and high-risk types are responsible for causing cervical and other cancers. The minor capsid protein L2 of HPV plays important roles in viru... expand abstracts entry into cells, localisation of viral components to the nucleus, in DNA binding, capsid formation and stability. It also elicits antibodies that are more cross-reactive between HPV types than does the major capsid protein L1, making it an attractive potential target for new-generation, more broadly protective subunit vaccines against HPV infections. However, its low abundance in natural capsids--12-72 molecules per 360 copies of L1--limits its immunogenicity. This review will explore the biological roles of the protein, and prospects for its use in new vaccines. collapse abstract
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Drug discovery today 2008 Dec; 14(1-2)
Plant-produced vaccines: promise and reality.
Plant-produced vaccines are a much-hyped development of the past two decades, whose time to embrace reality may have finally come. Vaccines have been developed against viral, bacterial, parasite and allergenic antigens, for humans and for animals; a ... expand abstractwide variety of plants have been used for stable transgenic expression as well as for transient expression via Agrobacterium tumefaciens and plant viral vectors. A great many products have shown significant immunogenicity; several have shown efficacy in target animals or in animal models. The realised potential of plant-produced vaccines is discussed, together with future prospects for production and registration. collapse abstract
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Virology journal 5
Experimental observations of rapid Maize streak virus evolution reveal a strand-specific nucleotide substitution bias.
BACKGROUND: Recent reports have indicated that single-stranded DNA (ssDNA) viruses in the taxonomic families Geminiviridae, Parvoviridae and Anellovirus may be evolving at rates of approximately 10(-4) substitutions per site per year (subs/site/year)... expand abstract. These evolution rates are similar to those of RNA viruses and are surprisingly high given that ssDNA virus replication involves host DNA polymerases with fidelities approximately 10,000 times greater than those of error-prone viral RNA polymerases. Although high ssDNA virus evolution rates were first suggested in evolution experiments involving the geminivirus maize streak virus (MSV), the evolution rate of this virus has never been accurately measured. Also, questions regarding both the mechanistic basis and adaptive value of high geminivirus mutation rates remain unanswered. RESULTS: We determined the short-term evolution rate of MSV using full genome analysis of virus populations initiated from cloned genomes. Three wild type viruses and three defective artificial chimaeric viruses were maintained in planta for up to five years and displayed evolution rates of between 7.4 x 10(-4) and 7.9 x 10-4 subs/site/year. CONCLUSION: These MSV evolution rates are within the ranges observed for other ssDNA viruses and RNA viruses. Although no obvious evidence of positive selection was detected, the uneven distribution of mutations within the defective virus genomes suggests that some of the changes may have been adaptive. We also observed inter-strand nucleotide substitution imbalances that are consistent with a recent proposal that high mutation rates in geminiviruses (and possibly ssDNA viruses in general) may be due to mutagenic processes acting specifically on ssDNA molecules. collapse abstract
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The Journal of general virology 2008 Aug; 89(Pt 9)
Recombination, decreased host specificity and increased mobility may have driven the emergence of maize streak virus as an agricultural pathogen.
Maize streak virus (MSV; family Geminiviridae, genus Mastrevirus), the causal agent of maize streak disease, ranks amongst the most serious biological threats to food security in subSaharan Africa. Although five distinct MSV strains have been current... expand abstractly described, only one of these - MSV-A - causes severe disease in maize. Due primarily to their not being an obvious threat to agriculture, very little is known about the 'grass-adapted' MSV strains, MSV-B, -C, -D and -E. Since comparing the genetic diversities, geographical distributions and natural host ranges of MSV-A with the other MSV strains could provide valuable information on the epidemiology, evolution and emergence of MSV-A, we carried out a phylogeographical analysis of MSVs found in uncultivated indigenous African grasses. Amongst the 83 new MSV genomes presented here, we report the discovery of six new MSV strains (MSV-F to -K). The non-random recombination breakpoint distributions detectable with these and other available mastrevirus sequences partially mirror those seen in begomoviruses, implying that the forces shaping these breakpoint patterns have been largely conserved since the earliest geminivirus ancestors. We present evidence that the ancestor of all MSV-A variants was the recombinant progeny of ancestral MSV-B and MSV-G/-F variants. While it remains unknown whether recombination influenced the emergence of MSV-A in maize, our discovery that MSV-A variants may both move between and become established in different regions of Africa with greater ease, and infect more grass species than other MSV strains, goes some way towards explaining why MSV-A is such a successful maize pathogen. collapse abstract
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The Journal of general virology 2008 Aug; 89(Pt 9)
HIV-1 subtype C Pr55gag virus-like particle vaccine efficiently boosts baboons primed with a matched DNA vaccine.
A DNA vaccine expressing human immunodeficiency virus type 1 (HIV-1) southern African subtype C Gag (pTHGag) and a recombinant baculovirus Pr55gag virus-like particle prepared using a subtype C Pr55gag protein (Gag VLP) was tested in a prime-boost in... expand abstractoculation regimen in Chacma baboons. The response of five baboons to Gag peptides in a gamma interferon (IFN-gamma) enzyme-linked immunospot (ELISPOT) assay after three pTHGag immunizations ranged from 100 to 515 spot-forming units (s.f.u.) per 10(6) peripheral blood mononuclear cells (PBMCs), whilst the response of two baboons to the Gag VLP vaccine ranged from 415 to 465 s.f.u. per 10(6) PBMCs. An increase in the Gag-specific response to a range of 775-3583 s.f.u. per 10(6) PBMCs was achieved by boosting with Gag VLPs the five baboons that were primed with pTHGag. No improvement in Gag responses was achieved in this prime-boost inoculation regimen by increasing the number of pTHGag inoculations to six. IFN-gamma responses were mapped to several peptides, some of which have been reported to be targeted by PBMCs from HIV-1 subtype C-infected individuals. Gag VLPs, given as a single-modality regimen, induced a predominantly CD8+ T-cell IFN-gamma response and interleukin-2 was a major cytokine within a mix of predominantly Th1 cytokines produced by a DNA-VLP prime-boost modality. The prime-boost inoculation regimen induced high serum p24 antibody titres in all baboons, which were several fold above that induced by the individual vaccines. Overall, this study demonstrated that these DNA prime/VLP boost vaccine regimens are highly immunogenic in baboons, inducing high-magnitude and broad multifunctional responses, providing support for the development of these products for clinical trials. collapse abstract
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BMC biotechnology 8
Expression of HIV-1 antigens in plants as potential subunit vaccines.
BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) has infected more than 40 million people worldwide, mainly in sub-Saharan Africa. The high prevalence of HIV-1 subtype C in southern Africa necessitates the development of cheap, effective vacci... expand abstractnes. One means of production is the use of plants, for which a number of different techniques have been successfully developed. HIV-1 Pr55Gag is a promising HIV-1 vaccine candidate: we compared the expression of this and a truncated Gag (p17/p24) and the p24 capsid subunit in Nicotiana spp. using transgenic plants and transient expression via Agrobacterium tumefaciens and recombinant tobamovirus vectors. We also investigated the influence of subcellular localisation of recombinant protein to the chloroplast and the endoplasmic reticulum (ER) on protein yield. We partially purified a selected vaccine candidate and tested its stimulation of a humoral and cellular immune response in mice. RESULTS: Both transient and transgenic expression of the HIV antigens were successful, although expression of Pr55Gag was low in all systems; however, the Agrobacterium-mediated transient expression of p24 and p17/p24 yielded best, to more than 1 mg p24/kg fresh weight. Chloroplast targeted protein levels were highest in transient and transgenic expression of p24 and p17/p24. The transiently-expressed p17/p24 was not immunogenic in mice as a homologous vaccine, but it significantly boosted a humoral and T cell immune response primed by a gag DNA vaccine, pTHGagC. CONCLUSION: Transient agroinfiltration was best for expression of all of the recombinant proteins tested, and p24 and p17/p24 were expressed at much higher levels than Pr55Gag. Our results highlight the usefulness of plastid signal peptides in enhancing the production of recombinant proteins meant for use as vaccines. The p17/p24 protein effectively boosted T cell and humoral responses in mice primed by the DNA vaccine pTHGagC, showing that this plant-produced protein has potential for use as a vaccine. collapse abstract
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Archives of virology 153(8)
Two dicot-infecting mastreviruses (family Geminiviridae) occur in Pakistan.
Most mastreviruses (family Geminiviridae) infect monocotyledonous hosts and are transmitted by leafhopper vectors. Only two mastrevirus species, Tobacco yellow dwarf virus from Australia and Bean yellow dwarf virus (BeYDV) from South Africa, have bee... expand abstractn identified whose members infect dicotyledonous plants. We have identified two distinct mastreviruses in chickpea stunt disease (CSD)-affected chickpea originating from Pakistan. The first is an isolate of BeYDV, previously only known to occur in South Africa. The second is a member of a new species with the BeYDV isolates as its closest relatives. A PCR-based diagnostic test was developed to differentiate these two virus species. Our results show that BeYDV plays no role in the etiology of CSD in Pakistan, while the second virus occurs widely in chickpea across Pakistan. A genomic clone of the new virus was infectious to chickpea (Cicer arietinum L.) and induced symptoms typical of CSD. We propose the use of the name Chickpea chlorotic dwarf Pakistan virus for the new species. The significance of these findings with respect to our understanding of the evolution, origin and geographic spread of dicot-infecting mastreviruses is discussed. collapse abstract
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Archives of virology 153(7)
A new African streak virus species from Nigeria.
The African streak viruses (AfSVs) are a diverse group of mastrevirus species (family Geminiviridae) that infect a wide variety of annual and perennial grass species across the African continent and its nearby Indian Ocean islands. Six AfSV species (... expand abstractof which maize streak virus is the best known) have been described. Here we report the full genome sequences of eight isolates of a seventh AfSV species: Urochloa streak virus (USV), sampled from various locations in Nigeria. Despite there being good evidence of recombination in many other AfSV species, we found no convincing evidence that any of the USV sequences were either inter- or intra-species recombinants. The USV isolates, all of which appear to be variants of the same strain (their genome sequences are all more than 98% identical), share less than 69% nucleotide sequence identity with other currently described AfSV species. collapse abstract
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Virology journal 5
Viable chimaeric viruses confirm the biological importance of sequence specific maize streak virus movement protein and coat protein interactions.
BACKGROUND: A variety of interactions between up to three different movement proteins (MPs), the coat protein (CP) and genomic DNA mediate the inter- and intra-cellular movement of geminiviruses in the genus Begomovirus. Although movement of viruses ... expand abstractin the genus Mastrevirus is less well characterized, direct interactions between a single MP and the CP of these viruses is also clearly involved in both intra- and intercellular trafficking of virus genomic DNA. However, it is currently unknown how specific these MP-CP interactions are, nor how disruption of these interactions might impact on virus viability. RESULTS: Using chimaeric genomes of two strains of Maize streak virus (MSV) we adopted a genetic approach to investigate the gross biological effects of interfering with interactions between virus MP and CP homologues derived from genetically distinct MSV isolates. MP and CP genes were reciprocally exchanged, individually and in pairs, between maize (MSV-Kom)- and Setaria sp. (MSV-Set)-adapted isolates sharing 78% genome-wide sequence identity. All chimaeras were infectious in Zea mays c.v. Jubilee and were characterized in terms of symptomatology and infection efficiency. Compared with their parental viruses, all the chimaeras were attenuated in symptom severity, infection efficiency, and the rate at which symptoms appeared. The exchange of individual MP and CP genes resulted in lower infection efficiency and reduced symptom severity in comparison with exchanges of matched MP-CP pairs. CONCLUSION: Specific interactions between the mastrevirus MP and CP genes themselves and/or their expression products are important determinants of infection efficiency, rate of symptom development and symptom severity. collapse abstract
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