Subterranean Clover Stunt Virus Research Articles

Minimizing IP issues associated with gene constructs encoding the Bt toxin - a case study

BackgroundAs part of a publicly funded initiative to develop genetically engineered Brassicas (cabbage, cauliflower, and canola) expressing Bacillus thuringiensis Crystal (Cry)-encoded insecticidal (Bt) toxin for Indian and Australian farmers, we designed several constructs that drive high-level expression of modified Cry1B and Cry1C genes (referred to as Cry1BM and Cry1CM; with M indicating modified). The two main motivations for modifying the DNA sequences of these genes were to minimise any licensing cost associated with the commercial cultivation of transgenic crop plants expressing CryM genes, and to remove or alter sequences that might adversely affect their activity in plants.ResultsTo assess the insecticidal efficacy of the Cry1BM/Cry1CM genes, constructs were introduced into the model Brassica Arabidopsis thaliana in which Cry1BM/Cry1CM expression was directed from either single (S4/S7) or double (S4S4/S7S7) subterranean clover stunt virus (SCSV) promoters. The resulting transgenic plants displayed a high-level of Cry1BM/Cry1CM expression. Protein accumulation for Cry1CM ranged from 5.18 to 176.88 µg Cry1CM/g dry weight of leaves. Contrary to previous work on stunt promoters, we found no correlation between the use of either single or double stunt promoters and the expression levels of Cry1BM/Cry1CM genes, with a similar range of Cry1CM transcript abundance and protein content observed from both constructs. First instar Diamondback moth (Plutella xylostella) larvae fed on transgenic Arabidopsis leaves expressing the Cry1BM/Cry1CM genes showed 100% mortality, with a mean leaf damage score on a scale of zero to five of 0.125 for transgenic leaves and 4.2 for wild-type leaves.ConclusionsOur work indicates that the modified Cry1 genes are suitable for the development of insect resistant GM crops. Except for the PAT gene in the USA, our assessment of the intellectual property landscape of components presents within the constructs described here suggest that they can be used without the need for further licensing. This has the capacity to significantly reduce the cost of developing and using these Cry1M genes in GM crop plants in the future.

Subterranean clover stunt virus

This datasheet on Subterranean clover stunt virus covers Identity, Distribution, Hosts/Species Affected, Vectors & Intermediate Hosts, Further Information.

Expression of the Trichoderma reesei expansin-like protein, swollenin, in poplar results in biomass with improved sugar release by enzymatic hydrolysis

Ethanol production from lignocellulosic biomass has been considered an alternative to corn-based ethanol and fossil fuel. However, the secondary cell wall structure prevents efficient degradation of cellulose. Expression of cell wall degrading enzymes has been suggested as one solution to generate suitable feedstocks for lignocellulosic ethanol production. Swollenin (SWO), originating from Trichoderma reesei, disrupts cellulose structure with low-level hydrolytic activity to the glucan chain. Previously, characterization of T. reesei SWO has been carried out in multiple microorganisms and tobacco. This study heterologously overexpressed T. reesei SWO in hybrid poplar (Populus tremula × alba), a promising feedstock for biofuel production, under the control of either the constitutive Cauliflower Mosaic Virus 35S (CaMV 35S) promoter or the putative vascular tissue specific Subterranean Clover Stunt Virus S7 (SCSV) promoter. Transgenic poplar lines with high or moderate SWO expression level showed abnormal growth and reduced biomass production, along with increased glucose and reduced lignin contents. Increased cellulose crystallinity was observed in two transgenic events. This study also investigated the effect of in planta SWO expression on cellulose saccharification efficiency and found that transgenic events improved glucan conversion rates compared to wild type poplar. The results suggest SWO-overexpressing transgenic poplar lines could be a potential feedstock for ethanol production.

Subterranean Clover Stunt Virus Revisited: Detection of Two Missing Genome Components.

Subterranean clover stunt virus (SCSV) is a type species of the genus Nanovirus in the family Nanoviridae. It was the first single-stranded DNA plant virus with a multipartite genome, of which genomic DNA sequences had been determined. All nanoviruses have eight genome components except SCSV, for which homologs of two genome components present in all other nanovirus genomes, DNA-U2 and DNA-U4, were lacking. We analysed archived and more recent samples from SCSV-infected legume plants to verify its genome composition and found the missing genome components. These results indicated that SCSV also has eight genome components and is a typical member of the genus Nanovirus.

Genetic improvement of subterranean clover (Trifolium subterraneum L.). 2. Breeding for disease and pest resistance

Subterranean clover (Trifolium subterraneum L.) is the most widely sown pasture legume in southern Australia and resistance to important diseases and pests has been a major plant-breeding objective. Kabatiella caulivora, the cause of clover scorch, is the most important foliar fungal pathogen, and several cultivars have been developed with resistance to both known races. Screening of advanced breeding lines has been conducted to prevent release of cultivars with high susceptibility to other important fungal foliar disease pathogens, including rust (Uromyces trifolii-repentis), powdery mildew (Oidium sp.), cercospora (Cercospora zebrina) and common leaf spot (Pseudopeziza trifolii). Several oomycete and fungal species cause root rots of subterranean clover, including Phytophthora clandestina, Pythium irregulare, Aphanomyces trifolii, Fusarium avenaceum and Rhizoctonia solani. Most breeding efforts have been devoted to resistance to P. clandestina, but the existence of different races has confounded selection. The most economically important virus diseases in subterranean clover pastures are Subterranean clover mottle virus and Bean yellow mosaic virus, while Subterranean clover stunt virus, Subterranean clover red leaf virus (local synonym for Soybean dwarf virus), Cucumber mosaic virus, Alfalfa mosaic virus, Clover yellow vein virus, Beet western yellows virus and Bean leaf roll virus also cause losses. Genotypic differences for resistance have been found to several of these fungal, oomycete and viral pathogens, highlighting the potential to develop cultivars with improved resistance. The most important pests of subterranean clover are redlegged earth mite (RLEM) (Halotydeus destructor), blue oat mite (Penthaleus major), blue-green aphid (Acyrthosiphon kondoi) and lucerne flea (Sminthurus viridis). New cultivars have been bred with increased RLEM cotyledon resistance, but limited selection has been conducted for resistance to other pests. Screening for disease and pest resistance has largely ceased, but recent molecular biology advances in subterranean clover provide a new platform for development of future cultivars with multiple resistances to important diseases and pests. However, this can only be realised if skills in pasture plant pathology, entomology, pre-breeding and plant breeding are maintained and adequately resourced. In particular, supporting phenotypic disease and pest resistance studies and understanding their significance is critical to enable molecular technology investments achieve practical outcomes and deliver subterranean clover cultivars with sufficient pathogen and pest resistance to ensure productive pastures across southern Australia.

Occurrence of viruses within Tasmanian vegetable crops and identification of a novel Polerovirus infecting pea

Tasmanian vegetable crops (broccoli, carrots, cauliflower, common bean, and pea) were tested for virus infection with Potyviruses (all crops), members of the Luteoviridae (all except carrot), Alfalfa mosaic virus (all except brassicas), Cucumber mosaic virus (all except carrot and cauliflower), Subterranean clover stunt virus (legumes only), Tomato spotted wilt virus (legumes only) and Cauliflower mosaic virus (brassicas only) over three seasons (2007–2010). Low (and sporadic) incidence of each virus (or virus group) was found in pea crops. Only Luteovirid and SCSV infections were found in bean crops at low frequency and incidence. Virus infection was not detected in carrot crops. Broccoli crops recorded generally low incidence of all four viruses or groups tested for, whilst cabbage crops recorded very low incidence of CaMV only. In the pea and broccoli crops members of the Luteoviridae were the most prevalent viruses detected. Samples that tested positive for generic Potyvirus and Luteoviridae tests were retested using virus specific antiserum and/or RT-PCR. Potyvirus detections (from pea crops in the first year of testing) were due to infection with Pea seed-borne mosaic virus (PSbMV). These samples were all sourced from a single imported seed line. Subsequent testing over the next two seasons failed to show evidence of PSbMV which may have been eliminated with destruction of the seed source. Molecular analyses of a selection of Luteovirid isolates revealed most infections were due to Turnip yellows virus with five isolates from pea belonging to a putative novel Polerovirus species with closest homology to Cucurbit aphid-borne yellows virus.

Bean leafroll virus is widespread in subterranean clover (Trifolium subterraneum L.) seed crops and can be persistently transmitted by bluegreen aphid (Acyrthosiphon kondoi Shinji)

In the mid 2000s subterranean clover (Trifolium subterraneum) seed producers in South Australia reported symptoms of a red-leaf disease in fields with reduced seed yields. The red-leaf symptoms resembled those caused by several clover-infecting viruses. A set of molecular diagnostic tools were developed for the following viruses which are known to infect subterranean clover: Alfalfa mosaic virus; Bean leafroll virus (BLRV); Beet western yellows virus; Bean yellow mosaic virus; Cucumber mosaic virus; Pea seed-borne mosaic virus; Soybean dwarf virus and Subterranean clover stunt virus. Surveys of subterranean clover seed production fields in 2008 in the south-east of South Australia and western Victoria identified Bean leafroll virus, Alfalfa mosaic virus and Cucumber mosaic virus as present, with BLRV the most widespread. Surveys of pasture seed production fields and pasture evaluation trials in 2009 confirmed that BLRV was widespread. This result will allow seed producers to determine whether control measures directed against BLRV will overcome their seed losses. Bluegreen aphid (Acyrthosiphon kondoi) was implicated as a potential vector of BLRV because it was observed to be colonising lucerne plants adjacent to subterranean clover seed production paddocks with BLRV, and in a glasshouse trial it transmitted BLRV from an infected lucerne plant to subterranean clover in a persistent manner.

First Report of a Nanovirus Disease of Pea in Germany.

During the growing season of 2009, a disease consisting of leaf rolling, top yellows, and plant stunting affected pea (Pisum sativum) in fields near Aschersleben, Saxony-Anhalt, Germany. Samples from symptomatic plants collected in July 2009 were analyzed at the JKI in Braunschweig for infections by various legume viruses by ELISA, immunoelectron microscopy, and transmission assays by sap and aphids. Of 23 samples, 9 were shown to contain Pea enation mosaic virus and three samples each contained Bean leafroll virus and Soybean dwarf virus. From two further samples that had tested negative for the aforementioned viruses, we succeeded in transferring a disease agent to faba bean (Vicia faba) seedlings by giving 50 to 100 individuals of the pea aphid (Acyrthosiphon pisum) acquisition and inoculation access feedings each of ~48 h. Following vector transmission, the agent caused severe yellowing and stunting in pea and faba bean, sometimes followed by necrosis. Attempts at mechanical transmission of the agent failed, and isolation of double-stranded RNA from infected tissue was not successful. Therefore, we considered the possible presence of a nanovirus (4). When using polyclonal antibodies (PAbs) against Faba bean necrotic yellows virus (FBNYV) for double-antibody sandwich (DAS)-ELISA analysis of the two isolates of the disease agent we observed weak but clearly positive reactions. To confirm these weak DAS-ELISA reactions, we used all available monoclonal antibodies (MAbs) raised against FBNYV (1) and faba bean necrotic stunt virus (FBNSV) (3) individually in triple-antibody sandwich (TAS)-ELISA in combination with the FBNYV PAbs for plate coating. Six of 26 MAbs reacted from weak to strong with the two pea isolates, with MAbs FBNYV-3-1F7 and FBNSV-5-1G8 giving the strongest reactions and none of the MAbs giving a differential reaction with the two pea isolates. Employing rolling circle amplification of total DNA extracted from symptomatic leaves of one of the pea isolates yielded a substantial amount of high molecular weight DNA, whereas little or no amplification occurred when using DNA from noninoculated pea leaves. Restriction of the amplified DNA in a nanovirus iteron-specific manner by AatII endonuclease yielded a predominant and abundant product of ~1 kb (3). Sequence comparisons of eight cloned DNAs of 1,002 nucleotides long unequivocally identified them as complete DNA-R component of a new member of the genus Nanovirus (2,4). Its DNA-R sequence (GenBank No. GU553134) is nearly equidistant from the DNA-R sequences of FBNYV (Y11405), FBNSV (GQ150778), Milk vetch dwarf virus (MDV) (AB027511) and Subterranean clover stunt virus (SCSV) (AJ290434), sharing with them respective sequence identities of 79, 78, 79, and 73%. Moreover, it is more distinct from the DNA-R sequences of FBNYV, FBNSV, and MDV than the three latter are from each other (86 to 91%). This together with the serological data relating to the capsid protein properties of this virus strongly suggest that it is distinct from the hitherto described nanoviruses FBNYV, MDV, FBNSV, and SCSV. Therefore, we propose the name pea necrotic yellow dwarf virus (PNYDV) for this new nanovirus naturally infecting pea in Germany.

Prediction and Comparative Analysis of MHC Binding Peptides and Epitopes in Nanoviridae Nano-organisms

Nanoviridae is a family of single stranded DNA viruses which infect the plants through their phloem tissues. The few members of these viruses are now turning towards animals. It includes two genera, Nanovirus and Babuvirus. Nanovirus includes three species namely Faba beans necrotic yellows virus (FBNYV), Milk vetch dwarf virus (MVDV) and Subterranean clover stunt virus (SCSV) while Babuvirus have two species accounted yet, namely Abaca bunchy top virus (ABTV) and Banana bunchy top virus (BBTV). The viral coat proteins are likely to be responsible for many diseases in plants as well as animals. The intra-genomic changes or post translational modifications with in SCSV have decreased the length of the sequence, but increased the antigenic potential and numbers of antigenic binding regions on the surface of the protein. We have predicted the most probable responsible antigenic determinants and MHC binders within the viruses in Nanoviridae family. MHC molecules play a crucial role in host immune response. The nonamers of antigenic determinants in Nanoviridae family viral coat proteins are highly sensitive to H-2Kd and I-Ag7 molecules. The species within particular genera shows their common epitopes along with diverse colleague epitopes. These common antigenic peptides can be used as their identifi ers. These identifi ers along with their diverse colleagues could be most informative for antidotes production against themselves. Also, these are important for synthetic peptide vaccine production against the relevant viruses.

First Report of Faba bean necrotic yellows virus Affecting Legume Crops in Azerbaijan.

A total of 482 chickpea (Cicer arietinum L.), 182 lentil (Lens culinaris Medik.), 12 vetch (Vicia sativa L.), 5 field pea (Pisum sativum L.), and 3 faba bean (Vicia faba L.) samples were collected from plants with symptoms suggestive of a viral infection (leaf rolling, yellowing, and stunting) from the major legume-production areas of Azerbaijan in the 2007 and 2008 growing seasons. All samples were tested by the tissue-blot immunoassay (3) at the Virology Laboratory of ICARDA, Syria using 11 specific legume virus antisera including a monoclonal antibody (2-5H9) (1) for Faba bean necrotic yellows virus (FBNYV). Laboratory tests showed that FBNYV was detected in 73, 61, 11, 3, and 2 samples of chickpea, lentil, vetch, field pea, and faba bean, respectively. Total DNA was extracted from six FBNYV-positive samples (two chickpea, two lentil, and two vetch) and tested by PCR with the following four primer sets (FBNYV, Milk vetch dwarf virus [MDV], Subterranean clover stunt virus [SCSV], and nanovirus DNA-R primers [F103 and R101]) (2). All six Azeri samples as well as the reference nanovirus isolates (SCSV-Australia, MDV-Japan, and FBNYV-Syria) generated amplicons of the expected size (~770 bp) using the nanovirus DNA-R primers (F103 & R101). In addition, Azeri samples and FBNYV-Syria yielded a PCR amplicon of the expected size (666 bp) with the FBNYV primer pair. The MDV- and SCSV-specific primers did not generate amplicons with these six samples. Sequence analysis of the FBNYV amplicons from two isolates (AzL 282-07 from lentil [GenBank Accession No. GQ351600] and AzV 277-07 from vetch [GenBank Accession No. GQ371215]) showed that they were 99% identical with each other. Comparing the sequence of AzL 282-07 with that of other nanoviruses revealed identities of 97% (FBNYV-Spain; DQ830990), 96% (FBNYV-Iran; AM493900), 92% (FBNYV-Syria; Y11408), 92% (FBNYV-Egypt; AJ132183), 78% (MDV; AB044387) and 69% (SCSV-Australia; U16734). FBNYV has been reported to infect food legumes in many countries in West Asia and North Africa and cause economic losses on faba bean in Egypt, Jordan, and Syria. To our knowledge, this is the first record of FBNYV infecting legume crops in Azerbaijan.

Three aphid vectors of faba bean (Vicia faba) viruses in northern New South Wales and occurrence ofAcyrthosiphon pisum-transmitted isolates ofSoybean dwarf virus

Aphid-transmitted virus diseases are a constraint on production of faba bean (Vicia faba) and other cool season grain legumes in northern New South Wales (NSW), Australia. A field study in 2004 identified potentially important aphid vectors of viruses that infect faba bean. Individual alatae were trapped on vertical nets in four field situations and placed immediately on bioassay plants (faba bean seedlings) to identify viruses transmitted in persistent and non-persistent manners. Viruses were identified by nine virus-specific tissue blot immunoassays (TBIAs). Out of a total of 447 individual alatae comprising 14 species, 10 individuals comprising three aphid species transmitted a virus: five Acyrthosiphon pisum (two that transmitted Bean leafroll virus, two Bean yellow mosaic virus, and one Soybean dwarf virus, SbDV), four Aphis craccivora (all Beet western yellows virus, BWYV), and one Myzus persicae (BWYV). No transmissions were detected for the other 11 aphid species, including the faba bean colonising species Acyrthosiphon kondoi, although virus-like symptoms were observed in some cases. None of the alatae transmitted Subterranean clover stunt virus, Alfalfa mosaic virus, Cucumber mosaic virus, Pea seed-borne mosaic virus, or Turnip mosaic virus. In separate tests in which acquisition fed apterae were used to inoculate faba bean, A. pisum was able to transmit four additional isolates of SbDV from chickpea or subterranean clover from Queensland, Tasmania, and NSW. The potential importance of A. pisum, A. craccivora, and M. persicae as vectors is discussed. Of these, A. pisum appears the most likely to cause crop losses in faba bean based on propensity as a vector, numbers trapped, efficient colonisation, and severity of virus isolates transmitted.

Transcripts encoding the nanovirus master replication initiator proteins are terminally redundant

The multicomponent single-stranded DNA plant nanoviruses encode unique master replication initiator (Rep) proteins. We have mapped the 5' and 3' termini of the corresponding polyadenylated mRNAs from faba bean necrotic yellows virus (FBNYV) and subterranean clover stunt virus and found that these are terminally redundant by up to about 160 nt. Moreover, the origin of viral DNA replication is transcribed into RNA that is capable of folding into extended secondary structures. Other nanovirus genome components, such as the FBNYV DNA encoding the protein Clink or an FBNYV DNA encoding a non-essential para-Rep protein, are not transcribed in such a unique fashion. Thus, terminally redundant mRNAs and the resulting transcription of the replication origin appear to be restricted to nanovirus master Rep DNAs. We speculate that this may be a way to regulate the expression of the essential master Rep protein.

Birth-and-death evolution of protein-coding regions and concerted evolution of non-coding regions in the multi-component genomes of nanoviruses

Genomes of the four plant viruses of the genus Nanovirus consist of multiple circular single-stranded DNA components, each of which encodes a single protein. Protein phylogenies supported the hypothesis that faba bean necrotic yellows virus (FBNYV) and milk vetch disease virus (MDV) are sister taxa; that subterranean clover stunt virus (SCSV) branched next; and that banana bunchy top virus (BBTV) is an outgroup to the three other species. The phylogeny of replication (Rep) proteins indicate that this small viral multi-gene family has evolved by a process of duplication and subsequent loss of Rep-encoding genome components, analogous to the “birth-and-death” process of evolution which has been described in eukaryotic multi-gene families. By contrast, repeated recombinational events between components were found to have homogenized the non-coding portions of several components encoding unrelated components. For example, as result of recent recombination a portion of the non-coding region is virtually identical among SCSV components 1, 3, 4, 5, and 7. Thus, there is a process of concerted evolution of non-coding regions of Nanovirus genome components, which raises the possibility that certain non-coding regions are subject to functional constraint.

A suite of novel promoters and terminators for plant biotechnology. II. The pPLEX series for use in monocots

A suite of plant expression vectors (pPLEX), constructed from the gene regulation signals from subterranean clover stunt virus (SCSV) genome, has previously been used in dicot transformation for a variety of applications in plant biotechnology. To assess their use for the transformation of monocots, a number of modifications were made to the basic vector series and assessed in rice. In their unmodified forms, the SCSV promoters directed low levels of gene expression, however, insertion of an intron between the promoter and the transgene open reading frame (analogous to the rice actin and maize ubiquitin promoter systems) increased transgene expression 50-fold. The expression patterns from the intron-modified SCSV (segments 4 and 7) promoters were very similar to those directed by the actin or ubiquitin promoters. All promoter systems investigated directed expression that appeared to be constitutive within leaf tissue, and localised to the epidermal and vascular tissues of the root. The pPLEX vectors described here are an important counterpart to the dicot pPLEX series and have the potential to be useful in monocot research and biotechnology.

A suite of novel promoters and terminators for plant biotechnology.

The gene regulation signals from subterranean clover stunt virus (SCSV) were investigated for their expression in dicot plants. The SCSV genome has at least eight circular DNA molecules. Each circular DNA component contains a promoter element, a single open reading frame and a terminator. The promoters from seven of the segments were examined for their strength and tissue specificity in transgenic tobacco (Nicotiana tabacum L.), potato (Solanum tuberosum L.) and cotton (Gossypium hirsutum L.) using a GUS reporter gene assay system. While the promoters of many of the segments were poorly expressed, promoters derived from segments 4 and 7 were shown to direct high levels of expression in various plant tissues and organs. The segment 1 promoter directs predominantly callus-specific expression and, when used to control a selectable marker gene, facilitated the transformation of all three species (tobacco, potato and cotton). From the results, a suite of plant expression vectors (pPLEX) derived from the SCSV genome were constructed and used here to produce herbicide- and insect-resistant cotton, demonstrating their utility in the expression of foreign genes in dicot crop species and their potential for use in agricultural biotechnology.

The 3' non-coding region of a C4 photosynthesis gene increases transgene expression when combined with heterologous promoters.

The Me1 gene of the dicot Flaveria bidentis encodes NADP malic enzyme, which catalyses the decarboxylation reaction of C4 photosynthesis in bundle sheath cells. We have previously shown that the 3' non-coding region (Me1 3') controls quantitative expression of the gene. We wondered whether Me1 3' can increase expression when combined with heterologous promoters. We tested a highly expressed, constitutive promoter, the S4 promoter from subterranean clover stunt virus, and a highly expressed, leaf-specific promoter, the light-harvesting chlorophyll a/b-binding protein gene 3 (Lhcb3) promoter of Arabidopsis thaliana. Promoter-3'-end combinations were tested in transgenic C4 Flaveria plants and C3 tobacco. We found that Me1 3' increased expression of the gusA reporter gene several-fold in leaves of both species in combination with either of the promoters. In both cases Me1 3' does not alter the expression pattern for either promoter. We conclude that Me1 3' can be used as a transcription terminator to increase transgene expression in C3 dicot plants.

The Master Rep Concept in Nanovirus Replication: Identification of Missing Genome Components and Potential for Natural Genetic Reassortment

Faba bean necrotic yellows virus (FBYNV), Milk vetch dwarf virus (MDV), and Subterranean clover stunt virus (SCSV) are nanoviruses that infect leguminous plants. From MDV- and SCSV-infected tissue we identified viral DNAs that encode a replication initiator protein (Rep), essential for replication of the multiple circular single-stranded DNAs of these viruses. These previously undescribed Rep proteins of MDV and SCSV are strikingly similar in sequence and functionally equivalent to the master Rep protein of FBYNV. Moreover, we demonstrated that the master Rep proteins of the three viruses are able to trigger replication of heterologous nanovirus DNAs. Such cross-species replication may reflect a considerable potential for genetic reassortment among nanoviruses in nature and be of significance for their evolution.

Sequences of ten circular ssDNA components associated with the milk vetch dwarf virus genome.

Milk vetch dwarf virus (MDV) is a member of the proposed genus Nanovirus, and its genome is composed of multiple, circular ssDNA components of about 1 kb. We have cloned and sequenced ten ssDNA components and designated them MDV-C1 to C10. Each DNA component contains one potential major open reading frame, and contains a putative stem-loop structure in the non-coding region. Notably, four components (C1, C2, C3 and C10) encode distinct replication-associated (Rep) proteins of 33 kDa, which show only limited (42-57%) amino acid identity. The six other components encode proteins with calculated molecular masses ranging from 12.7 to 19.7 kDa. Comparison of the sequences with those of other nanoviruses reveals that MDV is closely related to faba bean necrotic yellows virus (FBNYV) and subterranean clover stunt virus (SCSV). Six putative MDV genome products, including one Rep and five non-Rep proteins, show high (70.4-90.9%) amino acid identity to the corresponding six FBNYV proteins, whereas two other Rep proteins encoded by MDV-C2 and C3 are 82.3% and 73.0% identical to those encoded by SCSV-C2 and C6, respectively. These results indicate that MDV, FBNYV and SCSV have diverged from a common origin, which had multiple Rep components. In addition, the putative proteins encoded by MDV-C4 and its homologues contain a consensus retinoblastoma-binding motif, suggesting that they may be involved in controlling the host cell cycle.

Ten distinct circular ssDNA components, four of which encode putative replication-associated proteins, are associated with the faba bean necrotic yellows virus genome.

Four further circular ssDNA components (C7-C10), about 1 kb in size and structurally similar to the previously described components (C1-C6) found associated with a Syrian (Sy) isolate of faba bean necrotic yellows virus (FBNYV), have been identified. Similar to C1 and C2, two of the new components (C7 and C9) encode putative replication-associated (Rep) proteins of 33.2 and 32.7 kDa, respectively, the former of which is 90% identical to the C10 Rep protein of milk vetch dwarf virus (MDV). C8 encodes a putative protein (17.4 kDa) whose function is unknown, but which is highly conserved between FBNYV and the other nanoviruses MDV, subterranean clover stunt virus and banana bunchy top virus. The putative protein (19.7 kDa) encoded by C10 contains an LXCXE motif, which is also present in the homologues of its relatives, suggesting that they may all interact with plant retinoblastoma-like proteins. Sequence information for seven components of an Egyptian (Eg) FBNYV isolate indicated that six of them share > 96% identity with FBNYV-Sy. However, the C1 Rep protein of FBNYV-Eg was only 63.5% identical to that of FBNYV-Sy, but was 88.3 % identical to the MDV-C2 Rep protein. We conclude that the FBNYV genome consists of seven to ten components, six of which encode non-Rep proteins. All ten components of the FBNYV genome, except the Rep components C2 and C9, had closely related counterparts in the MDV genome. In spite of this similarity, FBNYV and MDV appear to be distinct virus species.

Psittacine Beak and Feather Disease Virus Nucleotide Sequence Analysis and Its Relationship to Porcine Circovirus, Plant Circoviruses, and Chicken Anaemia Virus

Cloning and sequencing of the circular, single-stranded DNA of one isolate of psittacine beak and feather disease virus (BFDV) demonstrate a genome composed of a circular molecule of 1993 nucleotide bases. An analysis of the assembled replicative form demonstrated seven open reading frames (ORFs) (three in the virion strand and four in the complementary strand), potentially encoding seven viral proteins of >8.7 kDa. High amino acid sequence similarity was demonstrated between a potential 33.3-kDa protein product of ORF1 of BFDV and the replicase-associated protein of porcine circovirus (PCV), subterranean clover stunt virus, and faba bean necrotic yellows virus. However, significant similarity in nucleotide or amino acid sequences was not present between BFDV and chicken anaemia virus. A potential stem–loop structure similar to that found in PCV and plant circoviruses was present in the putative encapsidated strand of the BFDV genome. At the top of this structure, a nonanucleotide motif (TAGTATTAC) similar to that of PCV, plant circoviruses, and geminiviruses also was recognised. Comparison of the deduced amino acid sequences of ORF2 of BFDV and PCV demonstrated 29.1% identity, and in both viruses, ORF2 is located on the complementary strand, beginning close to or within the hairpin stem. Our findings provide further evidence of a close relationship among BFDV, PCV, and plant circoviruses but not chicken anaemia virus.