Genus Nanovirus Research Articles

Nanoviruses, small plant viruses: similarities and differences with geminiviruses

Nanoviridae family comprises genus Nanovirus (viruses infecting legume plants) and Babuvirus (viruses infecting banana plants). Nanovirus genomes consist of multiple circular single-stranded DNAs of about 1 kb each. They are encapsidated individually in small icosahedral particles. Each DNA molecule encodes only one protein. Nanoviruses replicate in the nucleus of infected plants by a rolling-circle replication mechanism initiated by viral Rep protein. Multiple DNAs encoding similar but clearly distinct Rep proteins have been described for each nanovirus. All Rep proteins of a given nanovirus are functional and each initiates replication of the DNA by which it is encoded (auto-replication). Only the Master Rep protein is able to initiate replication of other genome components (trans-replication). Induction of nanovirus disease by cloned genome components was established only for Faba bean necrotic yellows virus.

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.

First report of Faba bean necrotic yellows virus (FBNYV) infecting chickpea (Cicer arietinum) and faba bean (Vicia faba) crops in Sudan

A total of 45 faba bean samples ( Vicia faba ) and 130 chickpea samples ( Cicer arietinum ) were collected from plants with symptoms that suggested a viral infection (leaf rolling, yellowing and stunting) during the period 1999– 2001 at the Shambat and Hudeiba Research Stations, Sudan. These samples were tested, using the tissue-blot immunoassay procedure (Makkouk & Comeau, 1994), at the Virology Laboratory of ICARDA, Syria. The antisera used included both monoclonal and polyclonal antibodies. The monoclonal antibodies were ATCC PVAS647 (American Type Culture Collection, Manassas, VA, USA), used to test for Beet western yellows virus (BWYV, genus Polerovirus , family Luteoviridae ); ATCC PVAS650, used to test for Soybean dwarf virus (SbDV, family Luteoviridae ); 4B10, used to test for Bean leaf roll virus (BLRV, family Luteoviridae ; Katul, 1992); and 3-2E9 and 2-5H9, used to test for Faba bean necrotic yellows virus (FBNYV, genus Nanovirus , family Nanoviridae ) – the latter monoclonal is FBNYV-specific (Franz et al ., 1996). A polyclonal antibody (provided by H.J. Vetten, BBA, Braunschweig, Germany) was used to test for Chickpea chlorotic dwarf virus (CpCDV, genus Mastrevirus , family Geminiviridae ). Laboratory tests showed that CpCDV, a leafhopper-transmitted virus, was the most abundant virus in chickpea; however, FBNYV, an aphid-transmitted virus, was the most common virus in faba bean samples. FBNYV has been reported to infect food legumes in many countries in western Asia and North Africa, and reaches epidemic levels in some countries, including Egypt (Makkouk et al ., 1994). CpCDV was detected in 77·0% of chickpea and 15·5% of faba bean samples tested, whereas FBNYV was detected in 95·5% of faba bean and 6·2% of chickpea samples tested. Only two chickpea samples tested positive for BWYV, while SbDV and BLRV were not detected in any of the samples. Based on the reaction with the FBNYV-specific monoclonal (2-5H9) and the characteristic symptoms of leaf rolling, stunting and yellowing of leaves, with necrosis of the leaf periphery in faba bean and yellowing and stunting in chickpea, this is the first record of FBNYV affecting faba bean and chickpea in Sudan.

First record of Chickpea chlorotic dwarf virus (CpCDV) affecting faba bean (Vicia faba) crops in Egypt

A field visit was conducted in March 2002 to identify viruses infecting the faba bean ( Vicia faba ) crop in four governorates (Fayoum, Beni Suef, El-Nobareia and Bihera) in Egypt. A total of 71 faba bean samples with symptoms of viral infection (leaf rolling, yellowing and mosaic) were collected. These were tested at the Virology Laboratory of ICARDA, Syria, for seven viruses using the tissue-blot immunoassay procedure (Makkouk & Comeau, 1994). Specific rabbit polyclonal antisera were used to test for Chickpea chlorotic dwarf virus (CpCDV, genus Mastrevirus , family Geminiviridae ; provided by H. J. Vetten, BBA, Braunschweig, Germany); for Broad bean stain virus (BBSV, genus Comovirus , family Comoviridae ); for Bean yellow mosaic virus (BYMV, genus Potyvirus , family Potyviridae ) and for Pea seed borne mosaic virus (PSbMV, genus Potyvirus , family Potyviridae ). In addition, three specific monoclonal antibodies were used to detect Faba bean necrotic yellows virus (FBNYV, genus Nanovirus , family Nanoviridae ) (Franz et al ., 1996); Bean leaf roll virus (BLRV, family Luteoviridae ) (4B10; Katul, 1992) and Beet western yellows virus (BWYV, genus Polerovirus , family Luteoviridae ) (ATCC PVAS-647; American Type Culture Collection, Manassas, VA, USA). Serological tests showed that BYMV, an aphid-transmitted and seedborne virus that was identified in 89% of samples tested, was the most common virus. In most of the fields surveyed, BYMV symptoms were noted to occur at high levels (80–100% infection). PSbMV was detected in nine samples (one from Fayoum and eight from Beni Suef); FBNYV was detected in 11 samples (all from Beni Suef). CpCDV, a leafhopper-transmitted virus, was, by contrast, detected in only two samples collected from the El-Nobareia governorate showing leaf rolling, yellowing and stunting. BWYV, BLRV and BBSV were not detected in any of the samples tested. In Egypt, FBNYV, BYMV and PSbMV have previously been reported in faba bean (Makkouk et al ., 1994), but this is the first record of CpCDV affecting faba bean in Egypt. CpCDV is the only member of the Geminivirus that is reported to naturally infect faba bean.

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Purified faba bean necrotic yellows virus (FBNYV; genus Nanovirus) alone is not transmissible by its aphid vector, Acyrthosiphon pisum, regardless of whether it is acquired from artificial diets or directly microinjected into the aphid's hemocoel. The purified virus contains all of the genetic information required for its infection cycle as it readily replicated in cowpea protoplasts and systemically infected Vicia faba seedlings that were biolistically inoculated using gold particles coated with intact virions or viral DNA. The bombarded plants not only developed the typical disease syndrome, thus indicating that FBNYV is the sole causal agent of the disease, but also served as a source from which the virus was readily acquired and transmitted by A. pisum. The defect of the purified virus in aphid transmissibility suggests that FBNYV requires a helper factor (HF) for its vector transmission that is either nonfunctional or absent in purified virus suspensions. The requirement for an HF was confirmed in complementation experiments using two distinct isolates of the virus. These experiments revealed that aphids transmitted the purified virus isolate from artificial diets only when they had fed previously on plants infected with the other FBNYV isolate. Also, microinjected FBNYV, which persisted to the same extent in A. pisum as naturally acquired virus, was transmissible when aphids had acquired the HF from infected plants. This suggests that one of the functions of the HF in the transmission process is to facilitate virus transport across the hemocoel–salivary gland interface.

Polyclonal Antibodies to the Bacterially Expressed Coat Protein of Faba Bean Necrotic Yellows Virus

Specific rabbit polyclonal antibodies against bacterially expressed coat protein of Faba bean necrotic yellows virus (FBNYV, genus Nanovirus) were produced using a recombinant DNA approach. The FBNYV capsid protein (CP) gene located on component 5 was cloned in an expression vector pQE‐9 (Qiagen, QIAGEN Inc., Chatswortch, CA91311, USA). Expression of the CP with an N‐terminal hexahistidine tag in Escheri‐ chia coli M15 cells was induced by adding isopropyl‐3‐D‐1‐thiogalactoside (IPTG) to a final concentration of 2 mM. About 8 mg of bacterially expressed CP (BE‐CP) was purified from 1 litre of bacterial liquid culture using a Ni‐NTA resin column (Qiagen). The expressed CP which migrated as a protein of approximately 23 kDa in sodium dodecyl sulphate (SDS)‐polyacrylamide gel electrophoresis (PAGE) was identified by its strong reaction with polyclonal antibodies produced against FBNYV particles and 2‐5H9 FBNYV‐monoclonal in Western blots. Expressed and purified CP (SDS‐PAGE 23 kDa band) was injected into a white rabbit, using seven intramuscular injections at weekly intervals. The antiserum produced was evaluated for FBNYV detection in double antibody sandwich (DAS)‐enzyme‐linked immunosorbent assay (ELISA), triple antibody sandwich (TAS)‐ELISA, tissue blot immunoassay (TBIA), dot blot, Western blot and goat antimouse coating (GAMC)‐ELISA using 13 different FBNYV monoclonal antibodies. The antiserum raised against the BE‐CP gave strong FBNYV‐specific TBIA reactions and very weak background reactions with non‐infected tissue, similar to those produced by monoclonal antibodies. Furthermore, BE‐CP polyclonal antibody reacted weakly with FBNYV‐infected tissue and strongly with BE‐CP in DAS‐ELISA, but not with FBNYV‐infected tissue in TAS‐ELISA when 13 detecting monoclonal antibodies were used. In addition, BE‐CP polyclonal antibody reacted strongly with BE‐CP in TAS‐ELISA only when 2‐5H9 detecting monoclonal was used. When monoclonals were used as primary antibody and BE‐CP polyclonal as detecting antibody (GAMC‐ELISA), FBNYV‐infected tissue gave moderate reactions with 2‐5H9 and strong reactions with 3‐2E9 monoclonal, whereas BE‐CP gave equally strong reactions with both monoclonals. These results showed that the BE‐CP polyclonal antibody is useful for the detection of FBNYV in infected tissue by TBIA and dot blot tests.

Reduction of incidence of three persistently transmitted aphid-borne viruses affecting legume crops by seed-treatment with the insecticide imidacloprid (Gaucho ®)

The effectiveness of using seed treatment with imidacloprid (Gaucho ®) to reduce the incidence of Bean leaf roll virus (BLRV, family Luteoviridae), Faba bean necrotic yellows virus (FBNYV, genus Nanovirus) and Soybean dwarf virus (SbDV, family Luteoviridae) in faba bean and lentil was investigated, using artificial inoculation with an aphid vector, Acyrthosiphon pisum (Harris). Imidacloprid was applied as a seed dressing at the rate of 0.0, 0.35, 0.7, 1.4 and 2.8 g a.i./kg of seeds. Incidence of BLRV, FBNYV and SbDV in faba bean was reduced from 92, 28 and 98% in untreated plots to 23, 2 and 85% in plots treated with 1.4 g a.i./kg and 13, 1 and 85% in plots treated with 2.8 g a.i./kg, respectively. Moreover, the yield loss was reduced from 80, 37 and 56% in untreated plots to 14, 0 and 39% in plots treated with 1.4 g a.i./kg, and to 14, 0 and 34% in plots treated with 2.8 g a.i./kg, for the above three viruses, respectively. Results indicated that imidacloprid at 2.8 g a.i./kg reduced virus incidence in faba bean plants when they were inoculated with BLRV two months after sowing from 91.6% in untreated plots to 0%, but no effect was detected when virus inoculation was done three months after sowing. Moreover, imidacloprid seed treatment significantly improved lentil yields of moderately resistant and susceptible lentil genotypes, but had no effect on the yield of resistant genotypes. In addition, seed treatment was more effective in increasing seed yield of BLRV and FBNYV-inoculated plots compared to SbDV-inoculated plots. Based on these results, seed treatment with imidacloprid at 1.4 g a.i./kg of seeds can effectively reduce the incidence of two persistently transmitted aphid-borne viruses, BLRV and FBNYV, affecting faba bean and lentil crops.

Activities associated with the putative replication initiation protein of coconut foliar decay virus, a tentative member of the genus Nanovirus.

The putative replication initiation protein (Rep) of Coconut foliar decay virus (CFDV) was expressed as a 6x His recombinant protein in E. coli and in recombinant baculovirus. Purified 6x His-Rep protein was demonstrated to possess sequence non-specific RNA- and ssDNA-binding activities as well as magnesium-dependent ATPase/GTPase activity. The yeast two-hybrid system revealed that CFDV Rep could interact with itself. Subcellular distribution of the CFDV Rep was studied by fractionation of insect cells infected with recombinant baculovirus expressing the 6x His-Rep protein and by laser scanning confocal microscopy of Nicotiana benthamiana epidermal cells bombarded with a construct encoding CFDV Rep fused to GFP. It was shown that CFDV Rep associated predominantly with nuclei and membranes of infected/transfected cells. These activities of CFDV-encoded Rep are very similar to those reported for Reps of geminiviruses.

Survey of Faba Bean ("Vicia faba" L.) Virus Diseases in Ethiopia

Surveys conducted in 1996 and 1997 to assess the status of virus diseases affecting faba bean in the major growing areas of Ethiopia indicated that leaf yellowing, rolling, necrosis and stunting were the most common disease symptoms. The highest visually-observed disease incidence in a field was 85%, recorded in the Wello region (north-eastern Ethiopia). When 3049 symptomatic samples collected from 211 fields from all over Ethiopia were tested by the tissue blot immunoassay (TBIA) for 14 viruses, 1592 samples (52.2%) were found to be infected with at least one virus. Faba bean necrotic yellows virus (FBNYV, genus Nanovirus) was the most frequent (63.2%), followed by luteoviruses (28.5%) [such as Beet western yellows virus (BWYV, genus Polerovirus, family Luteoviridae) and Bean leaf roll virus (BLRV, family Luteoviridae)] and Chickpea chlorotic dwarf virus (CCDV, genus Mastrevirus, family Geminiviridae) (3.1%). Mosaic/mottling symptoms were observed in some fields but incidence was always very low (<1%). The mosaic/mottling causing viruses identified were Broad bean stain virus (BBSV) and Broad bean true mosaic virus (BBTMV) (genus Comovirus, family Comoviridae), Pea seed-borne mosaic virus (PSbMV) and Bean yellow mosaic virus (BYMV) (genus Potyvirus, family Potyviridae). Testing of 3035 random samples collected in 1997 from 37 fields in the Shewa and Arsi regions revealed 1.6% luteovirus and 0.6% FBNYV infection, indicating that the incidence of these viruses on faba bean is very low in central Ethiopia. The survey suggested that virus diseases are currently of economic importance on faba bean in north-eastern Ethiopia and potentially important in the other areas, with FBNYV and luteoviruses being the most important.

Faba Bean Necrotic Yellows Virus (Genus Nanovirus) Requires a Helper Factor for Its Aphid Transmission

Purified faba bean necrotic yellows virus (FBNYV; genus Nanovirus) alone is not transmissible by its aphid vector, Acyrthosiphon pisum, regardless of whether it is acquired from artificial diets or directly microinjected into the aphid's hemocoel. The purified virus contains all of the genetic information required for its infection cycle as it readily replicated in cowpea protoplasts and systemically infected Vicia faba seedlings that were biolistically inoculated using gold particles coated with intact virions or viral DNA. The bombarded plants not only developed the typical disease syndrome, thus indicating that FBNYV is the sole causal agent of the disease, but also served as a source from which the virus was readily acquired and transmitted by A. pisum. The defect of the purified virus in aphid transmissibility suggests that FBNYV requires a helper factor (HF) for its vector transmission that is either nonfunctional or absent in purified virus suspensions. The requirement for an HF was confirmed in complementation experiments using two distinct isolates of the virus. These experiments revealed that aphids transmitted the purified virus isolate from artificial diets only when they had fed previously on plants infected with the other FBNYV isolate. Also, microinjected FBNYV, which persisted to the same extent in A. pisum as naturally acquired virus, was transmissible when aphids had acquired the HF from infected plants. This suggests that one of the functions of the HF in the transmission process is to facilitate virus transport across the hemocoel–salivary gland interface.

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.