ICTV Virus Taxonomy Profile: Caulimoviridae.

Caulimoviridae is a family of non-enveloped reverse-transcribing plant viruses with non-covalently closed circular dsDNA genomes of 7.1-9.8 kbp in the order Ortervirales. They infect a wide range of monocots and dicots. Some viruses cause economically important diseases of tropical and subtropical crops. Transmission occurs through insect vectors (aphids, mealybugs, leafhoppers, lace bugs) and grafting. Activation of infectious endogenous viral elements occurs in Musa balbisiana, Petunia hybrida and Nicotiana edwardsonii. However, most endogenous caulimovirids are not infectious. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Caulimoviridae, which is available at ictv.global/report/caulimoviridae.

Virus taxonomy and classification: naming of virus species.

Virus classification arranges viruses showing similar properties into groups and, even though this depends on choices of which specific properties have a value for classification, it does have certain important features. It aims to give a structured arrangement of viruses so that the human mind can comprehend them more easily. It helps with communication between virologists, and between virologists and non-virologists (e.g. regulators, advisers, other stakeholders etc.). It enables properties of new viruses to be predicted, and it could reveal possible evolutionary relationships. We need appropriate unambiguous names for virus species, which is the keystone taxon, howsoever these are defined. We react to the recent consultation paper [1] and suggest that, before deciding on a binomial (Latinized or non-Latinized) system for virus species names, the International Committee on Taxonomy of Viruses develops a 21st century virus classification system that handles the large numbers of new virus species expected from metagenomic studies. This system should be user-friendly for easy communication, especially between virologists and non-virologist stakeholders.

The phenotypic expression of a genotype: bringing muddy boots and micropipettes together.

Starting with the influences of having a father who was an agricultural plant pathologist, I sketch my career through university and research institute from field epidemiology, basic virus characterization to molecular biology. I note what I consider to be the highlights of my scientific career and the events that shaped the development of my thinking. These include secondment to teach in a university in Uganda, a sabbatical year in the University of California, Davis, where I became aware of the emerging DNA technology, studying the molecular biology of Cauliflower mosaic virus, rice tungro viruses, and Banana streak virus with the aim of developing diagnostics and approaches to control of viruses. Bringing these experiences together, I am now involved in facilitating the uptake of the application of biotechnology to crop improvement in developing countries. I conclude with some thoughts on opportunities for young plant pathologists over the next years of rapid change. As I am one of the few British scientists who have had the honor of writing such an article, I also note some of the vagaries of the British system.

Consensus statement: Virus taxonomy in the age of metagenomics.

The number and diversity of viral sequences that are identified in metagenomic data far exceeds that of experimentally characterized virus isolates. In a recent workshop, a panel of experts discussed the proposal that, with appropriate quality control, viruses that are known only from metagenomic data can, and should be, incorporated into the official classification scheme of the International Committee on Taxonomy of Viruses (ICTV). Although a taxonomy that is based on metagenomic sequence data alone represents a substantial departure from the traditional reliance on phenotypic properties, the development of a robust framework for sequence-based virus taxonomy is indispensable for the comprehensive characterization of the global virome. In this Consensus Statement article, we consider the rationale for why metagenomic sequence data should, and how it can, be incorporated into the ICTV taxonomy, and present proposals that have been endorsed by the Executive Committee of the ICTV.

Viral sequences integrated into plant genomes.

Sequences of various DNA plant viruses have been found integrated into the host genome. There are two forms of integrant, those that can form episomal viral infections and those that cannot. Integrants of three pararetroviruses, Banana streak virus (BSV), Tobacco vein clearing virus (TVCV), and Petunia vein clearing virus (PVCV), can generate episomal infections in certain hybrid plant hosts in response to stress. In the case of BSV and TVCV, one of the parents contains the integrant but is has not been seen to be activated in that parent; the other parent does not contain the integrant. The number of integrant loci is low for BSV and PVCV and high in TVCV. The structure of the integrants is complex, and it is thought that episomal virus is released by recombination and/or reverse transcription. Geminiviral and pararetroviral sequences are found in plant genomes although not so far associated with a virus disease. It appears that integration of viral sequences is widespread in the plant kingdom and has been occurring for a long period of time.

Banana streak virus is very diverse in Uganda.

Banana streak virus (BSV) is a badnavirus that causes a viral leaf streak disease of banana and plantain (Musa spp.). Identified in essentially all Musa growing areas of the world, it has a deleterious effect on the productivity of infected plants as well as being a major constraint to Musa breeding programmes and germplasm dissemination. Banana is a staple food in Uganda which is, per capita, one of the worlds largest banana producers and consumers. BSV was isolated from infected plants sampled across the Ugandan Musa growing area and the isolates were analysed using molecular and serological techniques. These analyses showed that BSV is very highly variable in Uganda. They suggest that the variability is, in part, due to a series of introductions of banana into Uganda, each with a different complement of infecting viruses.

Detection of petunia vein-clearing virus: model for the detection of DNA viruses in plants with homologous endogenous pararetrovirus sequences.

A number of cases of plant virus sequence integration into host plant genome have been reported. In at least two cases, endogenous pararetrovirus sequences are correlated strongly with subsequent episomal virus infection and there is circumstantial evidence that this also occurs for Petunia vein-clearing virus (PVCV). The detection of viruses is a critical component of plant health and therefore, it is important to have diagnostic procedures that differentiate between the detection of encapsidated viral DNA and homologous sequences in the host genome. PCR-based detection methods targeted at PVCV DNA have been tested and particular attention was paid to design controls that would indicate the existence of host DNA in the reaction. The use of ion-exchange chromatography for the partial purification of plant viruses from other cellular components, including chromosomal DNA, is described. The methods tested for PVCV detection are used to illustrate general principles for the specific detection of virus infections in host plants that carry homologous virus sequences in their genomes.

DTREEv2, a computer-based support system for the risk assessment of genetically modified plants.

Risk assessment of genetically modified organisms (GMOs) remains a contentious area and a major factor influencing the adoption of agricultural biotech. Methodologically, in many countries, risk assessment is conducted by expert committees with little or no recourse to databases and expert systems that can facilitate the risk assessment process. In this paper we describe DTREEv2, a computer-based decision support system for the identification of hazards related to the introduction of GM-crops into the environment. DTREEv2 structures hazard identification and evaluation by means of an Event-Tree type of analysis. The system produces an output flagging identified hazards and potential risks. It is intended to be used for the preparation and evaluation of biosafety dossiers and, as such, its usefulness extends to researchers, risk assessors and regulators in government and industry.

pep2pro: a new tool for comprehensive proteome data analysis to reveal information about organ-specific proteomes in Arabidopsis thaliana.

pep2pro is a comprehensive proteome analysis database specifically suitable for flexible proteome data analysis. The pep2pro database schema offers solutions to the various challenges of developing a proteome data analysis database and because data integrated in pep2pro are in relational format, it enables flexible and detailed data analysis. The information provided here will facilitate building proteome data analysis databases for other organisms or applications. The capacity of the pep2pro database for the integration and analysis of large proteome datasets was demonstrated by creating the pep2pro dataset, which is an organ-specific characterisation of the Arabidopsis thaliana proteome containing 14 522 identified proteins based on 2.6 million peptide spectrum assignments. This dataset provides evidence of protein expression and reveals organ-specific processes. The high coverage and density of the dataset are essential for protein quantification by normalised spectral counting and allowed us to extract information that is usually not accessible in low-coverage datasets. With this quantitative protein information we analysed organ- and organelle-specific sub-proteomes. In addition we matched spectra to regions in the genome that were not predicted to have protein coding capacity and provide PCR validation for selected revised gene models. Furthermore, we analysed the peptide features that distinguish detected from non-detected peptides and found substantial disagreement between predicted and detected proteotypic peptides, suggesting that large-scale proteomics data are essential for efficient selection of proteotypic peptides in targeted proteomics surveys. The pep2pro dataset is available as a resource for plant systems biology at www.pep2pro.ethz.ch.

Mechanical inoculation of plant viruses.

This technique is for the mechanical inoculation of viruses to plants. It is used to diagnose a virus by its reactions in a variety of plant species, to test the infectivity of virus samples using local lesion hosts, and to propagate viruses. The virus preparation is rubbed onto the surface of the leaf in such a way as to break the surface cells without causing too much mechanical damage. The preparation of the virus sample, its application to the leaf, and the care of the plants before and after inoculation are described.

Propagation of major plant-virus hosts.

Plant viruses are propagated in host plants, which are usually grown in glasshouses, screen houses, or growth cabinets. In most cases, the plants are grown from seed; in some cases, they are propagated as cuttings. This unit describes the basic techniques of growing suitable plants from seed and cuttings.

Genome-scale proteomics reveals Arabidopsis thaliana gene models and proteome dynamics.

We have assembled a proteome map for Arabidopsis thaliana from high-density, organ-specific proteome catalogs that we generated for different organs, developmental stages, and undifferentiated cultured cells. We matched 86,456 unique peptides to 13,029 proteins and provide expression evidence for 57 gene models that are not represented in the TAIR7 protein database. Analysis of the proteome identified organ-specific biomarkers and allowed us to compile an organ-specific set of proteotypic peptides for 4105 proteins to facilitate targeted quantitative proteomics surveys. Quantitative information for the identified proteins was used to establish correlations between transcript and protein accumulation in different plant organs. The Arabidopsis proteome map provides information about genome activity and proteome assembly and is available as a resource for plant systems biology.

Nucleic acid binding property of the gene products of rice stripe virus.

GST fusion proteins of the six gene products from RNAs 2,3 and 4 of the tenuivirus, Rice stripe virus (RSV), were used to study the nucleic acid binding activities in vitro. Three of the proteins, p3, pc3 and pc4, bound both single- and double-stranded cDNA of RSV RNA4 and also RNA3 transcribed from its cDNA clone, while p2, pc2-N (the N-terminal part of pc2) nor p4 bound the cDNA or RNA transcript. The binding activity of p3 is located in the carboxyl-terminus amino acid 154-194, which contains basic amino acid rich beta-sheets. The acidic amino acid-rich amino-terminus (amino acids 1-100) of p3 did not have nucleic acid binding activity. The related analogous gene product of the tenuivirus, Rice hoja blanca virus, is a suppressor of gene silencing and the possibility of the nucleic acid binding ability of RSV p3 being associated with this property is discussed. The C-terminal part of the RSV nucleocapsid protein, which also contains a basic region, binds nucleic acids, which is consistent with its function. The central and C-terminal regions of pc4 bind nucleic acid. It has been suggested that this protein is a cell-to-cell movement protein and nucleic acid binding would be in accord with this function.

Detection and localization of Rice stripe virus gene products in vivo.

The genome of the Tenuivirus, Rice stripe virus (RSV) comprises four RNAs, the smallest three of which each contain two open reading frames (ORFs) arranged in an ambisense manner. The expression of the ORFs from RNAs 2-4 in plants and the insect vector, Laodelphax striatellus, was studied using antisera raised against the gene products. In Western blotting of the proteins from infected plants, the molecular masses of p2, p3, pc3 (nucleocapsid protein, N) and p4 (major non-structural protein, NCP) were as expected; that of pc4 appeared larger than expected. Antisera to the N- and C-terminal parts of the complementary ORF on RNA 2, analogous to that encoding glycoproteins on genomes of bunyaviruses and tospoviruses, revealed banding patterns suggestive of processing of the product; the possible processing is discussed. Four types of inclusion bodies were identified by immunofluorescent and immunogold microscopy of thin sections of infected leaves. Most electron-dense amorphous semi-electron-opaque inclusion bodies (dASO) contained only p4 while some contained at least p2, pc2-N, p3, pc3 as well as p4. A ring-like structure containing at least pc2-N, p4 and pc4 was also identified in infected plant cells. Fibrillar amorphous semi-electron-opaque inclusion bodies (fASO) contained only p4. Filamentous electron-opaque inclusion bodies (FEO), which consist of pc2-N(.)and p4, were found both in infected plant cells and in the mid-gut lumen and mid-gut epithelial cells of L. striatellus. This suggests an interaction between p4 and pc2-N and a function of pc2-N distinct from that of its-homologue in Bunyaviridae. Our results confirm the in vivo ambisense coding strategy of Tenuivirus RNA 2 and provide further evidence that RSV does not produce enveloped virions in infected rice plants.

Banana contains a diverse array of endogenous badnaviruses.

Banana streak disease is caused by several distinct badnavirus species, one of which is Banana streak Obino l'Ewai virus. Banana streak Obino l'Ewai virus has severely hindered international banana (Musa spp.) breeding programmes, as new hybrids are frequently infected with this virus, curtailing any further exploitation. This infection is thought to arise from viral DNA integrated in the nuclear genome of Musa balbisiana (B genome), one of the wild species contributing to many of the banana cultivars currently grown. In order to determine whether the DNA of other badnavirus species is integrated in the Musa genome, PCR-amplified DNA fragments from Musa acuminata, M. balbisiana and Musa schizocarpa, as well as cultivars 'Obino l'Ewai' and 'Klue Tiparot', were cloned. In total, 103 clones were sequenced and all had similarity to open reading frame III in the badnavirus genome, although there was remarkable variation, with 36 distinct sequences being recognized with less than 85 % nucleotide identity to each other. There was no commonality in the sequences amplified from M. acuminata and M. balbisiana, suggesting that integration occurred following the separation of these species. Analysis of rates of non-synonymous and synonymous substitution suggested that the integrated sequences evolved under a high degree of selective constraint as might be expected for a living badnavirus, and that each distinct sequence resulted from an independent integration event.

Characterization of a protein from Rice tungro spherical virus with serine proteinase-like activity.

The RNA genome of Rice tungro spherical virus (RTSV) is predicted to be expressed as a large polyprotein precursor (Shen et al., Virology 193, 621-630, 1993 ). The polyprotein is processed by at least one virus-encoded protease located adjacent to the C-terminal putative RNA polymerase which shows sequence similarity to viral serine-like proteases. The catalytic activity of this protease was explored using in vitro transcription/translation systems. Besides acting in cis, the protease had activity in trans on precursors containing regions of the 3' half of the polyprotein but did not process a substrate consisting of a precursor of the coat proteins. The substitution mutation of Asp(2735) of the RTSV polyprotein had no effect on proteolysis; however, His(2680), Glu(2717), Cys(2811) and His(2830) proved to be essential for catalytic activity and could constitute the catalytic centre and/or substrate-binding pocket of the RTSV 3C-like protease.

Coevolution of viruses with hosts and vectors and possible paleontology.

The coevolution of viruses with their hosts and vectors depends on the evolution of the hosts and vectors coupled with factors involved in virus evolution. The long-term perspective involves the origin of life forms, the evolution of host and vector (especially arthropods) kingdoms and families, and changes in biological diversity induced mainly by the last five great extinctions. In the medium term, the diversification of hosts and vectors is important, and in the short term, recent events, especially humans, have had a great impact on virus coevolution. As there are few, if any, examples of conventional fossils of viruses, evidence for their evolution related to host and vector evolution is being found from other sources, especially virus-induced cellular structures and recent developments in molecular biology. Recognizing these other sources is becoming important for paleontologists gaining an understanding of the influence that viruses have had on the development of higher organisms.