Classification and identification of geminiviruses using sequence comparisons

comparison

The use of agroinfectious clones to investigate recombination between distinct maize streak virus strains

Bibliography: pages 199-213.The infectivity of the replicative form (RF) DNAs of MSV-Kom, MSV-Set and PanSV-Kar contained in the plasm ids pKom500, pSet100 and pPS100 was established by agroinoculating susceptible Jubilee sweetcorn with partial homodimeric Agrobacterium tumefaciens (C58C1) clones of RF-DNAs. Biological characteristics typical of Mastreviruses; such as, the appearance and leafhopper transmissibility of streak symptoms on infected plants, the presence of 18x30nm geminate particles in electron micrographs of leaf-dip preparations, and the presence of single-stranded and double-stranded DNA in Southern blot tests of infected plant DNA extracts, indicated that the RF-DNAs in pKom500, pSet100and PanSV-Kar represent the entire genomes of MSV-Kom, MSV-Set and PanSV-Karrespectively. The complete nucleotide (nt) sequence of the genome of MSV-Set was determined and characterised, and compared with those of MSV-Kom and PanSV-Kar. The genome sizes of MSV-Kom, MSV-Set and PanSV-Kar are 2701, 2690 and 2705 nt respectively, and all share Mastreviral genomic features. Phylogenetic analyses on the nt sequences and the putative amino acid sequences of the movement, coat and replication-associated proteins (MP, CP and Rep respectively) indicate that MSV-Set is grouped with, yet distinct from the MSV group of viruses isolated from maize. MSV-Set shares a 78% nt sequence identity with MSV-Kom which shares a >96% nt sequence identity with other MSVs. The PanSV-Kar genome shares a 60% nt sequence identity with the MSV group and89% with the Kenyan PanSV-Ken. PanSV-Kar causes mild non-persistent streak in Jubilee sweetcorn. MSV-Kom (previously isolated from maize in Komatipoort, Mpumalanga) and MSV-Set (previously isolated from a Setaria species in Mt. Edgecombe, Kwazulu/Natal) have different pathogenicities, and have overlapping, but non-identical, host ranges. Leafhopper transmission tests determined that MSV-Kom and MSV-Set generally cause severe and moderate streak in maize cultivars, or mild and severe streak in wheat cultivars respectively

Molecular identification and sequence analysis of bipartite Begomovirus infecting Horsegram legume in India

The molecular diversity of Begomovirus infecting Horsegram yellow Mosaic viruses (HgYMV1 and HgYMV2), from two main horsegram growing farms near Bangalore, Karnataka State, South India was investigated. The viral DNA was amplified from horsegram plants exhibiting mild and severe symptoms by polymerase chain reaction, and complete genome of the HgYMV were identified by their sequence analysis. Isolates of HgYMV1 and HgYMV2 were found to be associated with severe symptom phenotype from HgYMV. HgYMV was most closely related to Mungbean yellow mosaic indian viruse (MYMIV) and Mungbean yellow mosaic virus (MYMV) at 81.8 to 84.8 % nucleotide identity, based on DNA-A and DNA-B component sequences. HgYMV was distantly related to Dolicos yellow mosaic virus from Asia (DoYMV-Ban and DoYMV-DB) and partially to Cowpea golden mosaic virus from Nigeria (CPGMV-[NG]) at 64 and 62 % DNA nucleotide identity. Analysis of the DNA-B sequence of HgYMV revealed a DNA-B component identical to those of Bean golden yellow mosaic virus BGYMV isolates described. Furthermore, the DNA-B component for extant BGYMV isolates and strains were also the closest relatives for the HgYMV1 DNA-B components at 48.7 % nucleotide identity. Therefore, HgYMV could be considered to be a new species of the genus Begomovirus (family Geminiviridae).Keywords: Begomovirus, Horsegram, yellow Mosaic viruses, DNA sequencin

Molecular studies on the sweet potato virus disease and its two causal agents

The studies presented in this thesis contribute to an increased understanding of the molecular aspects, variability and interaction of the two most important viral pathogens of sweet potato (Ipomoea batatas L): Sweet potato feathery mottle virus (SPFMV) and Sweet potato chlorotic stunt virus (SPCSV), which cause the severe sweet potato virus disease (SPVD) when co-infecting sweet potato plants. SPVD is the most important disease affecting sweet potato in Africa, and may be the most important virus disease of sweet potato globally. The coat protein gene sequences of several African SPFMV isolates were determined and compared by phylogenetic analyses. Results showed that East African SPFMV isolates were genetically distinct. They could furthermore be divided into two serotypes which differed in their ability to systemically infect the sweet potato cultivar Tanzania. The aetiology of SPVD was studied in sweet potato plants co-infected with SPFMV and SPCSV using nucleic acid hybridisation, bioassays, tissue printing and thin section immunohistochemistry. Resistance to SPFMV in East African sweet potato cultivars was found to be due to inhibition of virus replication rather than movement and resistance was suppressed by infection with SPCSV, resulting in a ca. 600-fold increase in titres of SPFMV. Furthermore, in SPVD affected plants SPFMV is detected outside of the phloem, whereas SPCSV is detected only inside the phloem, which suggests novel as yet unknown mechanisms how SPCSV synergises SPFMV. The genomic sequence of SPCSV was determined. It was composed of two RNA molecules (9407 and 8223 nucleotides), representing the second largest (+)ssRNA genome of plant viruses. The genomic organization of SPCSV revealed novel features for the genus Crinivirus, such as i) the presence of a gene putatively encoding an ribonuclease III-like protein, ii) near-identical, 208 nucleotides long 3’-sequences on both viral RNAs, and iii) the placement of the SHP gene at a new position on the genome of SPCSV relative to other closteroviridae. Northern analyses showed the presence of several sub-genomic RNAs, of which the accumulation was temporally regulated in infected tissues. The 5’-ends of seven sub-genomic RNAs were determined using a PCR based method, which indicated that the sgRNAs were capped

Detection of geminiviruses from tropical countries by a double monoclonal antibody ELISA using antibodies to African cassava mosaic virus

Using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) tests, monoclonal antibodies (mAbs), which were prepared against particles of a Nigerian isolate of African cassava mosaic virus (ACMV), differentiated African geminiviruses (tomato yellow leaf curl virus, TYLCV; ACMV from West and East Africa; and okra leaf curl virus) from those originating in other countries of the world (euphorbia mosaic virus; Indian cassava mosaic virus; and TYLCV (Indian isolate)). All these viruses belong to the whitefly-transmitted geminivirus subgroup III. One mAb reacted with all 7 tested viruses and isolates. For detecting TYLCV from Senegal, it proved necessary to use a reducing agent in extraction buffer. A diagnostic DAS-ELISA was developed. This relies only on the use of mAbs and is useful for large-scale field screening.Des anticorps monoclonaux (AcMc) dirigés contre le virus de la mosaïque africaine du manioc («ACMV») (isolat du Nigéria) et utilisés dans un test DAS-ELISA («double antibody sandwich-ELISA») permettent de distinguer (à l’intérieur du sous-groupe III des geminivirus), les geminivirus originaires dAfrique (ACMV souche Ouest et Est ; virus de l’enroulement jaunissant de la tomate : «TYLCV» ; virus de l’enroulement du gombo) de ceux provenant des autres continents du monde (virus de la mosaïque de l’euphorbe ; virus indien de la mosaïque du manioc ; TYLCV de l’Inde). L’un de ces AcMc réagit avec les 7 geminivirus testés. Néanmoins, la détection du TYLCV (isolat du Sénégal) nécessite un tampon d’extraction spécifique, contenant un agent réducteur. Un test de diagnostic ou de détection utilisant seulement des AcMc, et par conséquent applicable à grande échelle, a été mis au point

Investigations into the use of maize streak virus as a gene vector

Bibliography: pages 190-214.This thesis describes investigations into the potential use of the Subgroup I geminivirus, maize streak virus (MSV), as a gene vector. These involved testing MSV-based replicons in transgenic cell lines, in transient expression assays in maize cells and in an infectious gene expression system in plants. MSV vectors which contained three different versions of a bar (bialaphos resistance) expression cassette in place of the viral movement and coat protein genes were used to generate transformed maize cell lines. A high proportion of these contained MSV-based episomes at high copy number. However, embryogenic maize tissue of the Hill line was not regenerable when an MSV-based replicon was present, possibly due to toxicity of the viral replication associated protein. In non-regenerable Black Mexican sweetcorn cell lines some of the MSV-bar episomes, which ranged in size from 3.15 kb to 4.78 kb, replicated for periods of over two years, and appeared structurally stable. The cellular levels of the bar gene product, phosphinothricin acetyl transferase (PAT), were significantly enhanced in lines where the gene was amplified by linkage to an MSV replicon in comparison with lines where the same gene was not amplified. Northern blot analysis also showed that higher levels of bar mRNA were produced in lines where the gene was amplified. However, the 3- to 5-fold enhancement in gene expression was less than was anticipated, based on results from similar Subgroup ill geminivirus-based transgene amplification systems. Several mutants of the MSV genome were generated to investigate the extent to which genome amplification contributes to the expression of the viral coat protein gene. The introduction of an Ncol restriction site at the start of the coat protein gene facilitated fusion of the gus marker gene with the coat protein upstream transcription and translation regulatory sequences. In one viral construct the plus strand origin of replication was inactivated by insertion of a short oligonucleotide; in another, the viral rep gene was inactivated by a frameshift mutation. These constructs were used to show that the MSV coat protein promoter has low, but measurable constitutive activity in the absence of genome amplification, but that viral replication enhances coat protein expression about 45-fold. I found no evidence for Rep-mediated transactivation of the coat protein promoter

Molecular studies on the interactions between African cassava mosaic virus (ACMV) and East African cassava mosaic virus (EACMV-UG) infecting cassava (Manihot esculenta Crantz)

[no abstract