The family Caulimoviridae comprises viruses with semicircular double-stranded DNA genomes encapsulated into isometric or bacilliform particles, being divided into eight genera. The genus Badnavirus is the most important due to its high number of species reported infecting cultivated plants worldwide. The present study aimed to evaluate the taxonomic/phylogenetic positioning and population genetic variability into the genus Badnavirus. Initially, a data set comprising badnavirus full-length genome sequences was obtained from the non-redundant GenBank database. Multiple nucleotide sequence alignments were obtained for the data sets: complete genome; ORF III; full (1020pb) and partial (579pb) RT/RNaseH. Pairwise sequence comparisons, phylogenetic and recombination analyses were performed for all data sets. A total of 127 isolates were obtained, representing 53 badnavirus species. Nucleotide pairwise comparisons for the data sets RT/RNaseH and ORF III showed that a number of distinct badnavirus species shared up to 82,5% of identity, higher than the 80% threshold currently used for species demarcation in the genus. Bayesian phylogenetic trees showed four well supported clusters, with clusters 1 and 3 being sister groups comprising predominantly isolates infecting sugarcane and banana. Non-tree-like evolution evidenced a complex pattern of recombination, and at least 23 independent events were detected with recombination breakpoints occurring predominantly in the ORF III and in the intergenic region. By the analysis of nucleotide diversity of the partial RT/RNaseH region in 12 badnavirus population, a high genetic variability was observed. These results showed that mutation and recombination are important evolutionary mechanisms acting on the diversification of badnaviruses, and that the partial RT/RNaseH sequence is sufficient to determine the taxonomic placement of most viral species described in this genus. / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A família Caulimoviridae compreende fitovírus com genomas semicirculares de DNA de fita dupla encapsidados em partículas isométricas ou baciliformes, sendo dividida em oito gêneros. O gênero Badnavirus é o mais importante devido ao seu alto número de espécies relatadas infectando plantas cultivadas em todo o mundo. O presente estudo teve como objetivo avaliar a variabilidade genética de populações e o posicionamento taxonômico/filogenético de espécies de badnavírus. Inicialmente, sequências completas para o genoma das espécies de badnavírus foram obtidas a partir do banco de dados não-redundante GenBank. Alinhamentos múltiplos das sequências nucleotídicas foram obtidos para os seguintes conjuntos de dados: genoma completo, ORFIII, RT/RNaseH completa (1020nt) e parcial (579nt). Comparações pareadas de sequências, análises filogenéticas, de recombinação e variabilidade genética foram realizadas para os conjuntos de dados. Um total de 127 isolados foram obtidos, representando 53 espécies de badnavírus. As comparações de sequências de nucleotídeos para o conjunto de dados RT/RnaseH (completa e parcial) mostraram que algumas espécies de badnavírus relatadas como distintas apresentam entre 57,1-82,5% de identidade, superior ao limite de 80,0% atualmente utilizado para a demarcação de espécies em Badnavirus. Resultados similares foram observados para os dados da ORFIII e genoma completo, reforçando que sequências parciais do domínio RT/RNaseH são suficiente para determinar o posicionamento taxonômico da maioria das espécies descritas neste gênero. Quatro grupos (clusters 1-4) bem suportados foram observados nas árvores filogenéticas para genoma completo e ORFIII, com os cluster 1 e 3 formando grupos irmãos compreendendo espécies/isolados infectando predominantemente cana-de-açúcar (Saccharum spp.) e banana (Musa spp.). Análise de evolução em rede evidenciou alguns possíveis eventos de recombinação afetando a diversificação de espécies de badnavírus, com pelo menos 23 eventos independentes sendo detectados com pontos de recombinação ocorrendo predominantemente na ORFIII e região intergênica. Finalmente, altos índices de diversidade nucleotídica foram observados para a região RT/RnaseH parcial em populações de 12 espécies distintas de badnavírus. Estes resultados mostram que mutação e recombinação são mecanismos importantes atuando na evolução dos badnavírus.

The family Caulimoviridae comprises viruses with semicircular double-stranded DNA genomes encapsulated into isometric or bacilliform particles, being divided into eight genera. The genus Badnavirus is the most important due to its high number of species reported infecting cultivated plants worldwide. The present study aimed to evaluate the taxonomic/phylogenetic positioning and population genetic variability into the genus Badnavirus. Initially, a data set comprising badnavirus full-length genome sequences was obtained from the non-redundant GenBank database. Multiple nucleotide sequence alignments were obtained for the data sets: complete genome; ORF III; full (1020pb) and partial (579pb) RT/RNaseH. Pairwise sequence comparisons, phylogenetic and recombination analyses were performed for all data sets. A total of 127 isolates were obtained, representing 53 badnavirus species. Nucleotide pairwise comparisons for the data sets RT/RNaseH and ORF III showed that a number of distinct badnavirus species shared up to 82,5% of identity, higher than the 80% threshold currently used for species demarcation in the genus. Bayesian phylogenetic trees showed four well supported clusters, with clusters 1 and 3 being sister groups comprising predominantly isolates infecting sugarcane and banana. Non-tree-like evolution evidenced a complex pattern of recombination, and at least 23 independent events were detected with recombination breakpoints occurring predominantly in the ORF III and in the intergenic region. By the analysis of nucleotide diversity of the partial RT/RNaseH region in 12 badnavirus population, a high genetic variability was observed. These results showed that mutation and recombination are important evolutionary mechanisms acting on the diversification of badnaviruses, and that the partial RT/RNaseH sequence is sufficient to determine the taxonomic placement of most viral species described in this genus. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A família Caulimoviridae compreende fitovírus com genomas semicirculares de DNA de fita dupla encapsidados em partículas isométricas ou baciliformes, sendo dividida em oito gêneros. O gênero Badnavirus é o mais importante devido ao seu alto número de espécies relatadas infectando plantas cultivadas em todo o mundo. O presente estudo teve como objetivo avaliar a variabilidade genética de populações e o posicionamento taxonômico/filogenético de espécies de badnavírus. Inicialmente, sequências completas para o genoma das espécies de badnavírus foram obtidas a partir do banco de dados não-redundante GenBank. Alinhamentos múltiplos das sequências nucleotídicas foram obtidos para os seguintes conjuntos de dados: genoma completo, ORFIII, RT/RNaseH completa (1020nt) e parcial (579nt). Comparações pareadas de sequências, análises filogenéticas, de recombinação e variabilidade genética foram realizadas para os conjuntos de dados. Um total de 127 isolados foram obtidos, representando 53 espécies de badnavírus. As comparações de sequências de nucleotídeos para o conjunto de dados RT/RnaseH (completa e parcial) mostraram que algumas espécies de badnavírus relatadas como distintas apresentam entre 57,1-82,5% de identidade, superior ao limite de 80,0% atualmente utilizado para a demarcação de espécies em Badnavirus. Resultados similares foram observados para os dados da ORFIII e genoma completo, reforçando que sequências parciais do domínio RT/RNaseH são suficiente para determinar o posicionamento taxonômico da maioria das espécies descritas neste gênero. Quatro grupos (clusters 1-4) bem suportados foram observados nas árvores filogenéticas para genoma completo e ORFIII, com os cluster 1 e 3 formando grupos irmãos compreendendo espécies/isolados infectando predominantemente cana-de-açúcar (Saccharum spp.) e banana (Musa spp.). Análise de evolução em rede evidenciou alguns possíveis eventos de recombinação afetando a diversificação de espécies de badnavírus, com pelo menos 23 eventos independentes sendo detectados com pontos de recombinação ocorrendo predominantemente na ORFIII e região intergênica. Finalmente, altos índices de diversidade nucleotídica foram observados para a região RT/RnaseH parcial em populações de 12 espécies distintas de badnavírus. Estes resultados mostram que mutação e recombinação são mecanismos importantes atuando na evolução dos badnavírus.

No description available.

No description available.

Various research efforts have focused on Banana streak badnavirus (BSV), the causal agent of banana streak disease (BSD), since the discovery of endogenous sequences of the virus in the nuclear genome of several Musa (banana and plantain) species. In vitro propagation of Musa was identified as one of the main activation triggers of integrated BSV sequences to cause systemic (episomal) BSD. This was especially observed in B genome-containing tetraploid hybrids. Although, the South African banana industry is based on Cavendish varieties, some plantations with tetraploid hybrids have been established. In order to investigate the occurrence of episomally expressed BSV, a survey was done in the Kiepersol area of South Africa and episomal BSV was detected in six out of seven locations sampled. No episomal BSV was detected in the Cavendish cultivars sampled in close proximity to BSV infected cultivars. To determine the risk of vector-assisted spread of endogenous BSV, which has become episomally activated after tissue culture, transmission studies with local mealybug species (Planococcus citri (Risso), P. ficus (Signoret), Dysmicoccus brevipes (Cockerell) and Pseudococcus longispinus (Targioni-Tozzetti)) were conducted under controlled conditions. Virus-free FHIA-21 was multiplied in vitro and resulting progeny with, putatively episomally activated BSV, served as sources for mealybug-assisted transmissions to Cavendish. Activated, episomal BSV was transmitted by three mealybug species to Cavendish. Transmission with P. ficus was demonstrated for the first time. Limited antiserum stocks against BSV occur worldwide and detection of the virus remains crucial for the safe movement of Musa germplasm between continents. Antiserum is needed in order to detect the episomal form of the virus that causes BSD. Using conventional immunization methodology, antisera against a wide spectrum of BSV isolates were produced. Twenty diverse BSV isolates were characterized by IC-PCR and selected as sources for the production of the polyclonal antiserums in two animal species. An effective triple antibody sandwich (TAS) enzyme linked immunosorbent assay (ELISA) system; able to detect various serologically different species of BSV was developed. BSV was screened with a synthetically manufactured phage displayed antibody library; however, no satisfactory polyclonal or monoclonal antibodies were obtianed in using this approach. / Dissertation (MSc (Microbiology))--University of Pretoria, 2007. / Microbiology and Plant Pathology / unrestricted

Sugarcane bacilliform virus (SCBV) is an economically important pathogen of sugarcane in Australia which limits access to foreign sugarcane germplasm. Although SCBV is present in the major cane growing regions worldwide, very little is known about its variability, virulence and the yield losses resulting from infection. The limited information on SCBV has resulted in quarantine measures being introduced to protect the Australian sugarcane industry, with a major consequence being restricted access to imported sugarcane germplasm for breeding programs. Foreign sugarcane germplasm plays an important role in breeding of new commercial varieties for the Australian sugar industry and is essential for the long term productivity, profitability and sustainability of the sugar industry. This study was aimed at characterising Australian isolates of SCBV to enable the development of reliable and robust molecular and/or antibody-based diagnostic tests which could be used to not only assess the impact of SCBV on the Australian sugarcane industry, but could also be used to screen imported sugarcane germplasm for the virus. SCBV virions (SCBV-IM) were purified from the sugarcane accession "Ireng Maleng" and the dsDNA genome was cloned and sequenced. The genome of SCBV-IM comprised 7687 bp with an organisation typical of other badnaviruses. When the entire nucleotide sequence of SCBV-IM was compared to that of the Moroccan SCBV isolate (SCBV-Mo), less than 75% similarity was present. Within the coding regions, ORF I, ORF II and ORF III had 83%, 71% and 73% nucleotide similarity to SCBV-Mo, respectively. At the amino acid level, ORFs I, II and III from SCBV-IM showed 91%, 84% and 85% similarity to the equivalent regions in SCBV-Mo, respectively. To further investigate the level of sequence variability within Australian SCBV isolates, virions were purified from three further sugarcane accessions and a 220 bp fragment of the reverse transcriptase-coding region was amplified. Five clones from each sub-population were selected and sequenced. Analysis of these sequences revealed considerable variability in the virus population with variability within one plant as great as it was between isolates. However, since the use of specific primers could also be selecting for a sub-population of SCBV sequences, it was possible that the variability may actually be greater than that reported. These results indicated that SCBV isolates are complex and variable and may represent a continuum of genetic variability. High molecular weight DNA species larger than the SCBV 7.6 kbp unit-length genome were found in DNA extracted from purified SCBV-IM virions. We confirmed that these high molecular weight nucleic acids were virus-specific and open circular in conformation. Using field inversion gel electrophoresis (FIGE), the SCBV-IM DNA was separated into four discrete bands with sizes ranging from between 1 to 4 genome copies. The DNA was shown to comprise overlapped individual genome-length molecules and not covalently-bonded continuous DNA strands. We presume that these DNA molecules are concatamers formed during replication as a result of a terminal overlap on the sense strand. The presence of these concatamers within virions may explain the observation of particles with lengths corresponding to one, two or three times the modal length of 130 nm. Four SCBV-infected Saccharum officinarum plants were examined for the presence of integrated viral DNA. Southern blot analysis of viral DNA and total DNA extracted from the same plant source were compared with, or without, restriction digestion. The resulting restriction patterns from viral and total DNA were almost identical suggesting that there were no integrated SCBV sequences in the sugarcane cultivars tested. Although larger-than-single-genome copy bands were detected in both the viral and the total DNA samples, this was probably due to the presence of genomic concatamers. SCBV integration studies using Southern analyses were further complicated by high sequence variability which precluded the restriction digestion of all viral DNA species. As such, some of the SCBV DNA species remain as concatamers which appear as larger-than-unit-length SCBV products. An antiserum derived from a mixture of purified SCBV isolates has been used routinely in the past to screen for SCBV infection, but the heterogeneity reported for badnaviruses has cast doubt on the ability of this antiserum to detect all SCBV isolates. We attempted to determine whether antiserum generated against proteins other than the viral capsid could be used to detect SCBV infections, thus improving the reliability and robustness of SCBV diagnosis. The complete coding regions of SCBV ORF I and ORF II were bacterially expressed and used as antigens for antiserum production. Both ORF I and II proteins were found to be highly immunogenic and generated high-titre antisera, designated AS-I and AS-II, respectively. The diagnostic utility of both antisera to detect SCBV in six different infected sugarcane plants was tested using both immunosorbent electron microscopy (ISEM) and western blots. The currently used SCBV antiserum (AS-V), generated against a mixture of purified SCBV isolates, was included for comparison. In western analyses, neither AS-I nor AS-V was able to conclusively detect SCBV in any of the six infected plants due to reactivity with numerous non-specific proteins. In contrast, AS-II reacted specifically with a protein of the expected size (~13.5 kDa) in 2/6 infected plants. When compared using ISEM, AS-V, AS-I and AS-II trapped virions from 6/6, 6/6 and 2/6 SCBV-infected plants, respectively. However, the number of virions trapped using AS-V was approximately 30-fold more than that trapped using either AS-I or AS-II. These results highlight the variability between SCBV isolates and suggest that ISEM with antisera raised against mixtures of viral proteins may be a useful tool for the detection of viral isolates.