Edward P. Rybicki

RDP: detection of recombination amongst aligned sequences

SUMMARY Recombination Detection Program (RDP) is a program that applies a pairwise scanning approach to the detection of recombination amongst a group of aligned DNA sequences. The software runs under Windows95 and combines highly automated screening of large numbers of sequences with a highly interactive interface for examining the results of the analyses.

Revision of taxonomic criteria for species demarcation in the family Geminiviridae, and an updated list of begomovirus species.

Members of the family Geminiviridae characteristically have circular single-stranded DNAgenomes packaged within twinned (so-called geminate) particles. Geminiviruses are currentlydivided into four genera on the basis of their genome organizations and biological properties[2,20].Thosethathaveamonopartitegenomeandaretransmittedbyleafhoppervectors,primarilyto monocotyledonous plants, are included in the genus Mastrevirus, of which Maize streak virus isthe type species. Viruses that have monopartite genomes distinct from those of the mastrevirusesand that are transmitted by leafhopper vectors to dicotyledonous plants are included in thegenus Curtovirus, with Beet curly top virus as the type species. The genus Topocuvirus, recentlyrecognized by the International Committee on Taxonomy of Viruses (ICTV) [18], has only onemember (also the type species), Tomato pseudo-curly top virus, which has a monopartite genomeandistransmittedbyatreehoppervectortodicotyledonousplants.ThegenusBegomoviruscontainsviruses that are transmitted by the whitefly Bemisia tabaci (Gennadius) to dicotyledonous plants,with Bean golden yellow mosaic virus (originally Bean golden mosaic virus – Puerto Rico)asthetype species. Many begomoviruses have bipartite genomes (DNA A and DNA B components),although numerous begomoviruses with a monopartite genome occur in the Old World, and thereare some for which a single component is not infectious yet no DNA B component has been found.Geminiviruses cause significant yield losses to many crop plants throughout the world [5, 7].Because of their economic importance and the relative ease with which their DNA genomescan be cloned, many geminiviruses have been isolated and characterized. Guidelines for naming

Plant-made vaccines for humans and animals.

Summary 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 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.

A phylogenetic and evolutionary justification for three genera of Geminiviridae

SummaryGene-by-gene phylogenetic analyses of all of the viruses for which sequences are known, as well as analysis of the coding capacities, clearly demonstrated that there are two major groups of viruses in the taxonomic familyGeminiviridae. These are of the Subgroup I type, with one genomic component, which mainly infect monocots and are leafhopper-transmitted; and of the Subgroup III type, with one or two genomic components, which infect dicots and are whitefly-transmitted. The existence of “New World” and “Old World” clusters of Subgroup III viruses was confirmed, as well as the possession by the latter of an AV1 ORF not present in New World viruses. A third minor generic group is defined by viruses of the Subgroup II type, which have a single genomic component, infect dicots, and are leafhopper-transmitted. The latter group appear to be the result of an ancient recombination event between a Subgroup III-like and a Subgroup I-like virus. The question of whether one- and two-component Subgroup III viruses should be in the same taxon appears hard to resolve: the only distinguishing feature of the one-component Subgroup III viruses is that they apparently have no second component, as gene-for-gene comparisons of the “A” components of the viruses with other Subgroup III viruses place them within a larger Old World group of viruses, most of which are two component. The possibility exists that these viruses may either have independently lost their B components, or possess a B component that has simply not yet been found. Possible nomenclatural changes to accommodate viruses with the same name which are not closely related to one another, and possible evolutionary scenarios to account for the observed familial, generic and specific diversity of geminiviruses, are discussed.

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 wide 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.

Oral Immunogenicity of Human Papillomavirus-Like Particles Expressed in Potato

ABSTRACT Human papillomavirus-like particles (HPV VLPs) have shown considerable promise as a parenteral vaccine for the prevention of cervical cancer and its precursor lesions. Parenteral vaccines are expensive to produce and deliver, however, and therefore are not optimal for use in resource-poor settings, where most cervical HPV disease occurs. Transgenic plants expressing recombinant vaccine immunogens offer an attractive and potentially inexpensive alternative to vaccination by injection. For example, edible plants can be grown locally and can be distributed easily without special training or equipment. To assess the feasibility of an HPV VLP-based edible vaccine, in this study we synthesized a plant codon-optimized version of the HPV type 11 (HPV11) L1 major capsid protein coding sequence and introduced it into tobacco and potato. We show that full-length L1 protein is expressed and localized in plant cell nuclei and that expression of L1 in plants is enhanced by removal of the carboxy-terminal nuclear localization signal sequence. We also show that plant-expressed L1 self-assembles into VLPs with immunological properties comparable to those of native HPV virions. Importantly, ingestion of transgenic L1 potato was associated with activation of an anti-VLP immune response in mice that was qualitatively similar to that induced by VLP parenteral administration, and this response was enhanced significantly by subsequent oral boosting with purified insect cell-derived VLPs. Thus, papillomavirus L1 protein can be expressed in transgenic plants to form immunologically functional VLPs, and ingestion of such material can activate potentially protective humoral immune responses.

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 700 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.

Human Papillomavirus Virus-Like Particles Are Efficient Oral Immunogens when Coadministered with Escherichia coli Heat-Labile Enterotoxin Mutant R192G or CpG DNA

ABSTRACT Certain human papillomaviruses (HPVs) cause most cervical cancer, which remains a significant source of morbidity and mortality among women worldwide. HPV recombinant virus-like particles (VLPs) are promising vaccine candidates for controlling anogenital HPV disease and are now being evaluated as a parenteral vaccine modality in human subjects. Vaccines formulated for injection generally are more costly, more difficult to administer, and less acceptable to recipients than are mucosally administered vaccines. Since oral delivery represents an attractive alternative to parenteral injection for large-scale human vaccination, the oral immunogenicity of HPV type 11 (HPV-11) VLPs in mice was previously investigated; it was found that a modest systemic neutralizing antibody response was induced (R. C. Rose, C. Lane, S. Wilson, J. A. Suzich, E. Rybicki, and A. L. Williamson, Vaccine 17:2129–2135, 1999). Here we examine whether VLPs of other genotypes may also be immunogenic when administered orally and whether mucosal adjuvants can be used to enhance VLP oral immunogenicity. We show that HPV-16 and HPV-18 VLPs are immunogenic when administered orally and that oral coadministration of these antigens with Escherichia coli heat-labile enterotoxin (LT) mutant R192G (LT R192G) or CpG DNA can significantly improve anti-VLP humoral responses in peripheral blood and in genital mucosal secretions. Our results also suggest that LT R192G may be superior to CpG DNA in this ability. These findings support the concept of oral immunization against anogenital HPV disease and suggest that clinical studies involving this approach may be warranted.

The Molecular Biology of Mastreviruses

Publisher Summary This chapter discusses the molecular biology of mastreviruses, drawing analogies where necessary from research on curtoviruses and begomoviruses. The genomic organization of mastreviruses, curtoviruses, and begomoviruses is illustrated in the chapter. In comparison with mastreviruses, curto- and begomoviruses seem to have a far more complex set of genes and regulatory mechanisms. This may only reflect the fact that these viruses have been more comprehensively investigated—nevertheless, it is clear that all members of the Geminiveridae have common life strategies, but in some cases these viruses have gained extra genes to help them accomplish similar functions, perhaps in a more efficient manner. Mastreviruses have a simpler genomic organization than other geminiviruses, with only three functional genes compared with up to eight genes in some Old World begomoviruses: these are (1) rep, which may be alternatively spliced to produce two different protein products, Rep and RepA, (2) a movement protein gene, and (3) a coat protein gene.

A protocol for the rapid isolation of full geminivirus genomes from dried plant tissue

A high-throughput method of isolating and cloning geminivirus genomes from dried plant material, by combining an Extract-n-Amp-based DNA isolation technique with rolling circle amplification (RCA) of viral DNA, is presented. Using this method an attempt was made to isolate and clone full geminivirus genomes/genome components from 102 plant samples, including dried leaves stored at room temperature for between 6 months and 10 years, with an average hands-on-time to RCA-ready DNA of 15 min per 20 samples. While storage of dried leaves for up to 6 months did not appreciably decrease cloning success rates relative to those achieved with fresh samples, efficiency of the method decreased with increasing storage time. However, it was still possible to clone virus genomes from 47% of 10-year-old samples. To illustrate the utility of this simple method for high-throughput geminivirus diversity studies, six Maize streak virus genomes, an Abutilon mosaic virus DNA-B component and the DNA-A component of a previously unidentified New Word begomovirus species were fully sequenced. Genomic clones of the 69 other viruses were verified as such by end sequencing. This method should be extremely useful for the study of any circular DNA plant viruses with genome component lengths smaller than the maximum size amplifiable by RCA.