PubMed Health⌕ Search

Biomedical subjects

E P Rybicki

Publications and source records attributed to E P Rybicki.

At least 19 recordsLinked to original sources
PubMed2006

Plant-produced cottontail rabbit papillomavirus L1 protein protects against tumor challenge: a proof-of-concept study.

The native cottontail rabbit papillomavirus (CRPV) L1 capsid protein gene was expressed transgenically via Agrobacterium tumefaciens transformation and transiently via a tobacco mosaic virus (TMV) vector in Nicotiana spp. L1 protein was detected in concentrated plant extracts at concentrations up to 1.0 mg/kg in transgenic plants and up to 0.4 mg/kg in TMV-infected plants. The protein did not detectably assemble into viruslike particles; however, immunoelectron microscopy showed presumptive pentamer aggregates, and extracted protein reacted with conformation-specific and neutralizing monoclonal antibodies. Rabbits were injected with concentrated protein extract with Freund's incomplete adjuvant. All sera reacted with baculovirus-produced CRPV L1; however, they did not detectably neutralize infectivity in an in vitro assay. Vaccinated rabbits were, however, protected against wart development on subsequent challenge with live virus. This is the first evidence that a plant-derived papillomavirus vaccine is protective in an animal model and is a proof of concept for human papillomavirus vaccines produced in plants.

Agrobacterium tumefaciens↗
PubMed2005

Strategies for the prevention of cervical cancer by human papillomavirus vaccination.

As cervical cancer is causally associated with 14 high-risk types of human papillomavirus (HPV), a successful HPV vaccine will have a major impact on this disease. Although some persistent HPV infections progress to cervical cancer, host immunity is generally able to clear most HPV infections. Both cell-mediated and antibody responses have been implicated in influencing the susceptibility, persistence or clearance of genital HPV infection. There have been two clinical trials that show that vaccines based on virus-like particles (VLPs) made from the major capsid protein, L1, are able to type specifically protect against cervical intra-epithelial neoplasia and infection. However, there is no evidence that even a mixed VLP vaccine will protect against types not included in the vaccine, and a major challenge that remains is how to engineer protection across a broader spectrum of viruses. Strategies for production of HPV vaccines using different vaccine vectors and different production systems are also reviewed.

Antibodies, Viral↗
PubMed2003

Expression of Human papillomavirus type 16 major capsid protein in transgenic Nicotiana tabacum cv. Xanthi.

The production of vaccine antigens in plants is a safe and potentially very cost-effective alternative to traditional expression systems. We investigated the possibility of transgenic plant expression of the Human papillomavirus (HPV) type 16 L1 major capsid protein, with and without nuclear localisation signals, in Nicotiana tabacum cv. Xanthi plants. The genes were stably integrated into the N. tabacum genome, and both the expressed proteins were capable of assembling into capsomers and virus-like particles. The proteins in concentrated leaf extracts (L1(Tr)) were tested for antigenicity using a panel of characterised monoclonal antibodies (Mabs). Neutralising and conformation-specific Mabs (H16:V5 and H16:E70) were shown to bind to both types of the plant-produced particles. We estimated the L1(Tr) product yield to be 2-4 microg per kg of fresh leaf material. Rabbits immunised with small doses of plant-produced particles elicited a weak anti-HPV-16 L1 immune response. Our results support the feasibility of using transgenic plants for the production of HPV vaccines.

Animals↗
PubMed2002

Investigation of Maize streak virus pathogenicity determinants using chimaeric genomes.

Genes and intergenic regions were reciprocally exchanged between a highly pathogenic Maize streak virus (MSV) isolate (MSV-MatA) and three less pathogenic isolates (MSV-Kom, MSV-R2, and MSV-VW) to determine the contribution of individual genome constituents to MSV pathogenicity in maize. Comparison of disease symptoms produced by the 54 resulting chimaeras and parental viruses enabled identification of genome constituents that are primarily responsible for the heightened pathogenicity of MSV-MatA in maize. Whereas pathogenicity determinants were detected in all of the MSV genomic regions examined, generally only chimaeras containing the MSV-MatA long intergenic region, coat protein gene, and/or movement protein gene were more pathogenic than the milder MSV isolates from which most of their genomes were derived. The pathogenicity of chimeras was strongly influenced by the relatedness of their parental viruses and evidence was found of nucleotide sequence-dependent interactions between both coding and intergenic regions.

Chimera↗
PubMed2001

Sequence diversity and virulence in Zea mays of Maize streak virus isolates.

Full genomic sequences were determined for 12 Maize streak virus (MSV) isolates obtained from Zea mays and wild grass species. These and 10 other publicly available full-length sequences were used to classify a total of 66 additional MSV isolates that had been characterized by PCR-restriction fragment length polymorphism and/or partial nucleotide sequence analysis. A description is given of the host and geographical distribution of the MSV strain and subtype groupings identified. The relationship between the genotypes of 21 fully sequenced virus isolates and their virulence in differentially MSV-resistant Z. mays genotypes was examined. Within the only MSV strain grouping that produced severe symptoms in maize, highly virulent and widely distributed genotypes were identified that are likely to pose the most serious threat to maize production in Africa. Evidence is presented that certain of the isolates investigated may be the products of either intra- or interspecific recombination.

Geminiviridae↗
PubMed2001

The relative infectivities and genomic characterisation of three distinct mastreviruses from South Africa.

The genomic nucleotide sequences of the cloned agroinfectious genomes of three South African mastreviruses obtained from Zea mays, a Setaria sp., and Panicum maximum (designated MSV-Kom, MSV-Set, and PanSV-Kar respectively), were determined. Additionally, their relative infectivities and virulence were analysed in a range of differentially susceptible wheat, maize, and barley genotypes. MSV-Kom produced moderate to severe streak symptoms in all maize genotypes tested, but only moderate to very mild symptoms in the wheat and barley genotypes. MSV-Set infected only the susceptible to tolerant maize genotypes, but was generally more severe in the barley and wheat genotypes than MSV-Kom. PanSV-Kar was incapable of infecting any of the wheat and barley genotypes and only produced very mild symptoms on the three most sensitive maize genotypes. Genomic characteristics in common with related mastreviruses were identified. Phylogenetic analysis indicated that while MSV-Kom was closely related to previously sequenced MSV isolates, MSV-Set and PanSV-Kar represented distinctly novel strains of MSV and PanSV respectively. In the case of MSV-Set, this is the most distantly related MSV strain yet characterised.

Animals↗
PubMed2001

Investigation of the potential of maize streak virus to act as an infectious gene vector in maize plants.

There has been a great deal of interest in the possibility that geminiviruses might be used as infectious gene vectors for expression of foreign proteins in plants. However, generic mastreviruses such as Maize streak virus (MSV) have no sequences which are dispensable for systemic infection of plants, and there is a strict limitation on the size of viral DNA which can be moved systemically. We attempted to complement the movement functions deleted from a wild-type-sized, replication-proficient gene replacement vector, by co-infecting plants with it and either wild type MSV, or a replication-deficient but putatively movement-proficient viral construct. While ssDNA formation by the gene replacement vector could be complemented in trans by co-transfected wild type virus, true systemic movement of either the vector, or of co-complementing constructs, did not occur. However, recombination between the two complementing viral constructs frequently occurred to generate wild-type virus genomes. The results therefore suggest that formation of ssDNA and size of the viral replicon are not the sole determinants of whether the MSV movement proteins can mobilise viral sequences and move them systemically in plants.

Blotting, Southern↗
PubMed2001

Analysis of the diversity of African streak mastreviruses using PCR-generated RFLPs and partial sequence data.

Maize streak virus (MSV) is the most economically significant member of a diverse group of African grass-infecting Mastrevirus species in the family Geminiviridae. We designed a single set of degenerate primers which enables the PCR amplification of an approximately 1300 bp DNA fragment spanning both conserved (the RepA gene) and variable (the long intergenic region and MP gene) portions of these viruses' genomes. Using restriction fragment length polymorphism (RFLP) analysis of PCR products obtained from 39 MSV, one SSV, and two PanSV isolates, it was possible to both identify the different virus species, which differ in nucleotide sequence by up to 40%, and to differentiate between MSV isolates sharing up to 99% sequence identity. The reliability of the RFLP data for typing the MSV isolates was verified by the phylogenetic analysis of the partial genomic nucleotide sequences of a representative subset of the MSV isolates. Based on both the RFLP and sequence data, the MSV isolates could be clearly differentiated into the four groups: these were a group of predominantly maize-infecting isolates, and three groups containing grass/wheat-infecting isolates. RFLP analysis also revealed a number of mixed virus infections in which, in certain instances, it was possible to identify individual population members.

Africa↗
PubMed2001

Complete nucleotide sequence and host range of South African cassava mosaic virus: further evidence for recombination amongst begomoviruses.

Complete nucleotide sequences of the DNA-A (2800 nt) and DNA-B (2760 nt) components of a novel cassava-infecting begomovirus, South African cassava mosaic virus (SACMV), were determined and compared with various New World and Old World begomoviruses. SACMV is most closely related to East African cassava mosaic virus (EACMV) in both its DNA-A (85% with EACMV-MH and -MK) and -B (90% with EACMV-UG2-Mld and EACMV-UG3-Svr) components; however, percentage sequence similarities of less than 90% in the DNA-A component allowed SACMV to be considered a distinct virus. One significant recombination event spanning the entire AC4 open reading frame was identified; however, there was no evidence of recombination in the DNA-B component. Infectivity of the cloned SACMV genome was demonstrated by successful agroinoculation of cassava and three other plant species (Phaseolus vulgaris, Malva parviflora and Nicotiana benthamiana). This is the first description of successful infection of cassava with a geminivirus using Agrobacterium tumefaciens.

Agrobacterium tumefaciens↗
PubMed2001

Forced recombination between distinct strains of Maize streak virus.

Recombination between divergent virus genomes is believed to be a major mechanism for generation of novel virus genotypes. We have examined the recombination process in geminiviruses by forcing recombination between two distinct isolates of Maize streak virus (MSV), MSV-Kom and MSV-Set. Heterodimeric agroinfectious constructs containing tandemly cloned mixtures of complete or partial MSV-Set and MSV-Kom genomes were used to simulate a circular dimeric form similar to that which would be expected to occur following a single intermolecular crossing-over event between MSV-Set and MSV-Kom replicative form DNAs at the long intergenic region (LIR)-movement protein gene (MP) interface. We isolated, analysed and biologically characterized many of the recombinant MSV genomes that were generated from the constructs in planta. Apart from having the same simulated breakpoint at the LIR-MP interface, all the genomes examined had a second breakpoint that had been generated through either intramolecular homologous recombination or a replicational release mechanism. The pathogenicities of six predominantly MSV-Kom-like recombinants were tested in maize. While all were capable of producing a symptomatic infection in this host, none was more virulent than MSV-Kom and only two were more virulent than MSV-Set. The two most virulent recombinants were leafhopper transmitted to a range of differentially MSV-resistant maize, wheat and barley genotypes and both were found to have unique biological properties.

Base Sequence↗
PubMed1999

Generation of maize cell lines containing autonomously replicating maize streak virus-based gene vectors.

Virion sense gene replacement derivatives of maize streak virus (MSV) were constructed with selectable marker expression cassettes based on the bialaphos resistance gene (bar) and the CaMV 35S promoter. The effect on replication of increasing the genomic size was tested by including: (1) the 550-bp maize adhI intron and 68-bp TMV omega RNA leader sequences upstream of the bar genes; and (2) a fusion between the bar and E. coli glutathione reductase (gor) genes. Three recombinant viral vectors ranging in size from 2.7 kb to 4.8 kb replicated efficiently in biolistically transfected cells of suspension cultured Black Mexican sweetcorn (BMS) cells. Deletions greater than 39 bp 3' of the stemloop sequence in the LIR adversely affected replicon release. Transformed bialaphos-resistant BMS cell lines were generated with all three vectors containing the bar gene: between 38 and 60% of cell lines contained replicating viral episomes. The replicons were structurally stable, replicated to copy numbers of over 500 per haploid genome, and were detected for more than one year after introduction. We noted significant enhancement of bar gene expression, both at the protein and RNA levels, associated with the presence of episomal vector DNA. The maize adhI intron and TMV omega RNA leader sequences did not seem to have a significant effect on bar gene expression from replicating constructs, although expression from controls was enhanced. The results suggest that MSV-based constructs would provide a useful system for long-term gene amplification in cereal cell culture systems.

Acetyltransferases↗
PubMed1998

Molecular characterisation of a distinct South African cassava infecting geminivirus.

African cassava mosaic virus (ACMV) and East African cassava mosaic virus (EACMV) are whitefly-transmitted geminiviruses (WTGs) which are widespread in cassava in Africa and cause serious yield losses. Recently, a new geminivirus affecting cassava in South Africa (SACMV) has been reported. In this work SACMV was found to have DNA-A and DNA-B components. Comparisons of amino acid sequences of the putative coat protein, and nucleotide sequences of the common region and a 687-bp DNA B fragment of SACMV with other WTGs, showed that SACMV clustered with the Old World subgroup of the Begomovirus genus of geminiviruses. Despite its bipartite nature, SACMV was most closely related to monopartite TYLCVs, but was sufficiently different to justify designating it as a distinct virus. In serological studies, SACMV grouped biologically with EACMV isolates.

Cloning, Molecular↗
PubMed1997

An association between HIV-1 subtypes and mode of transmission in Cape Town, South Africa.

OBJECTIVES: To determine HIV-1 env and gag subtypes in male homosexual and heterosexual populations in Cape Town, South Africa. DESIGN: DNA was isolated from blood originating from 61 patients attending local clinics. Samples were divided according to presumed mode of transmission: male homosexual (n = 26), heterosexual/vertical (n = 32), blood transfusion (n = 1) and unknown (n = 2). METHODS: Proviral HIV-1 DNA was subtyped by heteroduplex mobility assay (HMA) based on the 799 base-pair V3-V5 region of the env gene (n = 47) or by sequence analysis of the p17 region of the gag gene (n = 33), or both. For HMA, reference plasmids were constructed containing the V1-V5 env region sequences (1.2-kb) representative of local subtypes. Subtype designation of reference subtypes was confirmed by sequence analysis of the V3-loop region. RESULTS: Analysis of the partial gag sequences and HMA of the V3-V5 env region identified three subtypes: B, C and D. A fourth env subtype, subtype E, was also identified by HMA. Subtypes were found to segregate according to mode of transmission, with subtype B viruses found in 96% (25 out of 26) of the male homosexual group and subtype C viruses found in 81% (26 out of 32) of the heterosexual/vertical transmission group. Subtype B viruses were also found in four heterosexual patients, one patient infected by blood transfusion and in two patients with unknown mode of transmission. Subtype D viruses were found in one male homosexual patient and one heterosexual patient. A subtype E virus was identified in a heterosexual patient. No discrepancy was found in subtype designation in samples analysed in both between the gag and env regions (n = 19). CONCLUSIONS: Subtype B viruses were associated with male homosexual transmission and subtype C viruses with heterosexual transmission, suggesting two independent epidemics. This data may have implications in the selection of appropriate vaccines for different risk groups in the country.

Adult↗