392
October 2002
Family: Secoviridae
Genus: Fabavirus
Species: Broad bean wilt virus 2
Acronym: BBWV-2


Broad bean wilt virus 2

Xueping Zhou
Institute of Biotechnology, Zhejiang University, Hangzhou 310029, P R China

Contents

Introduction
Main Diseases
Geographical Distribution
Host Range and Symptomatology
Strains
Transmission by Vectors
Transmission through Seed
Transmission by Grafting
Transmission by Dodder
Serology
Nucleic Acid Hybridization
Relationships
Stability in Sap
Purification
Properties of Particles
Particle Structure
Particle Composition
Properties of Infective Nucleic Acid
Molecular Structure
Genome Properties
Satellites
Relations with Cells and Tissues
Ecology and Control
Notes
References
Acknowledgements
Figures

Introduction

Described by Stubbs (1947, 1960); Taylor and Stubbs (1972)

Synonyms
Broad bean wilt virus (Taylor and Stubbs, 1972)
Plantago II virus (Uyemoto and Provvidenti, 1974)
Patchouli mild mosaic virus (Natsuaki et al., 1994)

An RNA-containing virus with isometric particles about 25nm in diameter. Virus particle preparations contain two ssRNA species of 6.0 and 3.6 kb, each is encapsidated separately in particles composed of two distinct polypeptides of 22 kDa and 44 kDa. The virus is readily transmissible by mechanical inoculation of sap and by aphids in a nonpersistent manner, and infects a wide range of plants worldwide.

Main Diseases

Vein clearing, mosaic, stunting and wilt of broad bean (Fig 1 & Fig 2); mosaic and stunting of pea; mosaic of cowpea, chickpea, Phaseolus beans and soybean (Xu et al., 1988); blight of spinach (Schroeder & Provvidenti, 1970); mosaic of lettuce (Provvidenti et al., 1984); necrotic spots or streaks on leaves and stems with stunting and apical necrosis of Capsicum annuum (Lee et al., 2000); mosaic of Capsicum frutescens (Imoto, 1975); and distortion of Megakepasma erythrochlamys (Koh et al., 2001). Also found naturally in Solanum melongena and Raphanus sativus (Wu et al., 1999), Brassica pekinensis and Brassica oleracea (Zhou et al., 1996), Bouvardia spp, Catharanthus roseus, Cornus florida, Holleborus vesicarius and Plantago lanceolata (Lisa & Boccardo, 1996), Eustoma russellianum and Gentiana scabra (Kobayashi et al., 1999), and other ornamentals, wild plants and weeds (Lisa & Boccardo, 1996). BBWV is an economically important virus in China (Qi et al., 2000b).

Geographical Distribution

Probably occurs worldwide. It is reported from Europe, North and South America, Asia, Africa and Australia (Lisa & Boccardo, 1996; Sutic et al., 1999).

Host Range and Symptomatology

The virus has a wide host range, and can infect 177 species in 39 families including many dicotyledonous and some monocotyledonous plants (Edwardson & Christie, 1991). Symptoms on field-infected plants range from mottle, mosaic, ringspots, distortion, wilting, apical necrosis, to stem necrosis; in others infection is symptomless (Lisa & Boccardo, 1996).

Diagnostic species

Vicia faba (broad bean). Systemic mosaic (Fig.1) and leaflet malformation, stunting, apical necrosis, general wilting and often death (Fig.2).

Chenopodium quinoa. Chlorotic lesions (up to 4mm diameter) in inoculated leaves, followed by systemic chlorotic mottling (Fig.3), leaf epinasty, and apical necrosis.

Chenopodium amaranticolor. Numerous small (usually < 1 mm diameter) chlorotic local lesions with whitish necrotic centres, systemic mosaic and leaf distortion (Fig.4).

Nicotiana glutinosa. Chlorotic ringspots in inoculated leaves, that become necrotic; systemic mosaic, chlorotic ringspots and line-patterns (Fig.5).

Petunia hybridum. Veinal necrosis and ringspots in inoculated leaves (Fig.6); systemic chlorotic mottling and brown ringspots.

Propagation species

Chenopodium quinoa is a good propagation host for virus purification. Broad bean, pea and Nicotiana clevelandii are suitable hosts for maintaining cultures.

Assay species

Vigna sinensis (cowpea) is a good local lesion host. Reddish-brown local lesions develop 3-4 days after inoculation (Fig.7). C. amaranticolor and C. quinoa are also sensitive local lesion hosts.

Strains

No strains of BBWV-2 have been distinguished. The two serotypes of BBWV described previously are now regarded as two distinct viruses (BBWV-1 and BBWV-2) (Wellink et al., 2000). Patchouli mild mosaic virus (PatMMV), once recognized as a species in the genus Fabavirus (Wellink et al., 2000), is now regarded as a strain of BBWV-2 (Qi et al., 2000b; Ikegami et al., 2001).

Transmission by Vectors

The virus is transmitted by aphids in a nonpersistent manner, with minimum acquisition and transmission access feeding times varying from 15 sec to 10 min (Lisa & Boccardo, 1996) and 3min to 2hr respectively, with no obvious latent period. Myzus persicae, Aphis craccivora and Acyrthosiphon pisum are the most efficient vectors.

Transmission through Seed

Generally, no seed transmission in broad bean and pea is reported (Taylor & Stubbs, 1972) but, a Syrian isolate was transmitted through the seed of broad bean at a very low frequency (0.4-0.6%) (Makkouk et al., 1990).

Transmission by Dodder

The virus is not transmitted by Cuscuta californica, C. campestris or C. subinclusa (Taylor & Stubbs, 1972).

Serology

The virus is strongly immunogenic. Titres of rabbit antiserum of 1/1024-1/2048 are reported (Taylor et al., 1968). Monoclonal antibodies with the titres of ascitic fluids ranged from 1/256,000 to 1/640,000 are reported to react with the 44 kDa large coat protein (Qing et al., 2000). In gel double diffusion tests, a single precipitin line forms with purified virus preparations, and this test is useful for differentiating BBWV-2 and BBWV-1 based upon spur formation (Uyemoto & Provvidenti, 1974). Weak precipitin lines are obtained with infected broad bean sap unless leaves are ground in buffer containing a reducing agent ( 0.05 M phosphate buffer + 0.1% thioglycollate + 0.1 M DIECA, pH 7.6) (Taylor & Stubbs, 1972). ELISA and ISEM are also useful for detecting the virus.

Relationships

BBWV-2 and BBWV-1 are distinguishable serologically in ELISA and by immunoblotting with monoclonal antibodies, and the two viruses do not cross-react (Qing et al., 2000; Qi et al., 2000c). BBWV-2 isolates are reported more commonly from Asia, Australia and North America, while BBWV-1 seems more prevalent in Europe. All isolates of BBWV-2 are closely related serologically and indistinguishable in gel double diffusion tests. BBWV-2 is closely related serologically to PatMMV in ISEM (Natsuaki et al., 1994), but Lamium mild mosaic virus (LMMV) appears to differ from any BBWV-2 isolates (Lisa et al., 1982).

Complete and partial nucleotide sequences of BBWV-2 and BBWV-1 are reported (Nakamura et al., 1998; Kobayashi et al., 1999; Lee et al., 2000; Qi et al., 2000a; 2000b; 2000c; Koh et al., 2001). Comparisons show that the large and small coat proteins of isolates of BBWV-2 share 86-98% amino acid sequence identities, but only 58-66% amino acid sequence identity with isolates of BBWV-1 (Kobayashi et al. 1999; Qi et al., 2000b). High amino acid sequence identities of the RNA-1 and RNA-2 encoded polyproteins (up to 98.6% and 93.8% respectively) with those of PatMMV indicate that PatMMV is a strain of BBWV-2.

BBWV-2 has similar physicochemical and physical properties, and genomic organization to comoviruses and nepoviruses. However, BBWV-2 is transmitted by aphids, whereas comoviruses are transmitted by beetles, and several nepoviruses by nematodes. Definitive nepoviruses differ from BBWV-2 in having only one coat protein species, whereas BBWV-2 has two (Wellink et al., 2000). Genomic sequence comparisons and phylogenetic analyses show that BBWV-2 has a relatively low sequence homology to RNAs of comoviruses and nepoviruses (Koh et al., 2001; Zhou et al., 2001).

Stability in Sap

In sap of broad bean and C. quinoa, a typical isolate lost infectivity after diluting 10-4, heating for 10 min at 60°C, and storage for 4 days at 22°C.

Purification

C. quinoa is the best propagation host for purification and may yield up to 15 mg virus per 100g tissues. Purification may be troublesome for some isolates because of aggregation but this can be decreased by the addition of sucrose to the extraction buffer to a final concentration of 25% and clarification of crude leaf extracts with Triton X-100 (Zhou & Li, 1996).

Harvest locally and systemically infected C. quinoa leaves 8-10 days after inoculation and homogenize in 0.5 M potassium phosphate buffer, pH 7.5 containing 0.1% 2-mercaptoethanol, 25% sucrose and 0.01% Triton X-100. Keep the homogenate for 1 hr at 4°C and homogenize again. After centrifugating at 6000g for 20 min, mix the supernatant fluid with 2.5% Triton X-100, 6% polyethylene glycol (mol. wt 6000) and 0.1 M NaCl and stir overnight at 4°C. Centrifuge at 8500g for 15 min and resuspend the pellets in 0.01M potassium phosphate buffer, pH 7.5 containing 0.01% Triton X-100. Centrifuge at 2200g for 15 min and centrifuge the supernatant fluid at 78000g for 2 hr. Resuspend the pellets in 0.01 M potassium phosphate buffer and purify further by centrifuging in sucrose density gradients (Zhou & Li, 1996).

Properties of Particles

Purified preparations contain three centrifugal components: top (T), consisting of empty protein shells without RNA; middle (M), containing a single molecule of RNA-2; and bottom (B), containing a single molecule of RNA-1.

Sedimentation coefficients, s20,w (svedbergs) ranged from 56 to 63 S(T), 93 to 100 S (M), and 113 to 126 S (B) for different BBWV-2 isolates (Lisa & Boccardo, 1996).

In CsCl without detergent, the buoyant density values were 1.30 (T), 1.38 (M), and 1.46 g.cm-3(B) (Lisa & Boccardo, 1996).

A260/A280 ratio 1.32 (T), 1.64 (M), and 1.75 (B) (Taylor & Stubbs, 1972).

Purified preparations of the virus contain two electrophoretic components that move towards the anode (Zhou & Li, 1996).

Particle Structure

The virus particles are isometric with a diameter of about 25 nm (Fig.8). Particles appear to be angular, but the surface capsomer arrangement is not obvious.

Particle Composition

Nucleic acid: Virus particles contain two species of ssRNA, which are present in equimolar amounts. M component contains 25-26% RNA (RNA-2) and the B component contains 35% RNA (RNA-1), both RNA species are necessary for infectivity. The complete nucleotide sequence of several isolates has been determined (Nakamura et al., 1998; Kobayashi et al., 1999; Lee et al., 2000; Qi et al., 2000a; 2000b; 2000c; Koh et al., 2001). The total genome size is 9.6 kb. The base composition of the RNA species is 28% A, 17% C, 26% G and 29% U for RNA-1, and 28% A, 19% C, 24% G and 29% U for RNA-2. Each RNA is polyadenylated at its 3' terminus and may possess a genome-linked protein (Vpg) at its 5' terminus (Qi et al., 2000a, 2000c). A Vpg, if present in the virus, is not required for infectivity (Lisa & Boccardo, 1996).

Protein: Virus particles contain two coat protein species with mol. wts of 44,000 and 22,000, as estimated by SDS-PAGE and immunoblotting (Lee et al.2000; Qi et al., 2000c).

Genome Properties

The genome comprises RNA-1 and RNA-2. The complete nucleotide sequences (Accession numbers for RNA-1 and RNA-2 respectively in parenthesis) have been determined for isolates MB7 (AB013615 and AB013616; Nakamura et al., 1998), B935 (AF149425 and AJ132844; Qi et al., 2000a, 2000c), K (AF144234 and AF104335; Lee et al., 2000), ME (AF225953 and AF225954; Koh et al., 2001), IA (AB051386 and AB032403) and IP (AB023484 and AB018698). RNA-1 and RNA-2 are 5951 to 5989nt and 3569 to 3607nt in length respectively, excluding the 3' terminal poly (A) tail. The 5'-NCR of RNA-1 shares 56.6% nucleotide sequence identity with that of the 3'-NCR, with the highest identities being within the first 95 nucleotides (89%) and nucleotides 149 to 192 (97%) (Zhou et al., 2001); no significant homology was observed between the 3'-NCRs of RNA-1 and RNA-2 (Qi et al., 2000a). Both the 5' and 3' NCR of RNA-1 and RNA-2 contain relatively few G+C residues (29-33%) but more abundant U residues (34-43%) (Qi et al., 2000a; 2000c). The repeated motif (AAACAGCUUUC) is present in the 5'-NCR of each RNA (Zhou et al., 2001). RNA-1 encodes a 210 kDa polyprotein, that may be proteolytically cleaved to yield a putative protease cofactor (Co-Pro, 38kDa), a nucleotide triphosphate (NTP)-binding protein (NTBM, 67kDa), a viral genome-linked protein (Vpg, 3kDa), a protease (Pro, 23kDa) and an RNA-dependent RNA polymerase (RdRp, 79kDa) (Fig.9) (Koh et al., 2001; Zhou et al., 2001). In rabbit reticulocyte lysate, RNA-1 produced a large polypeptide of approximately 200 kDa (Nakamura et al., 1998; Zhou et al., 2001).

RNA-2 encodes a 119 kDa or 104 kDa polyprotein, that is cleaved at Q/G residues and Q/A residues, to release three mature proteins: a N-terminal 53 or 37kDa protein (designated as VP53 and VP37), a large coat protein (44kDa) and a small coat protein (22kDa) (Fig.10) (Zhou et al., 2001). The VP 53 and VP37 proteins are C-coterminally overlapping proteins resulting from two potential translation initiation sites on RNA-2. The VP37 protein was found to accumulate to high level in infected C. quinoa leaves, and its ability to bind ssRNA and ssDNA suggests that the protein may play a role similar to movement proteins (Qi et al., 2002). Defective RNAs were found in BBWV-2-M-infected pepper (Capsicum annuum) plants showing severe necrotic symptoms (Lee et al., 2000).

Relations with Cells and Tissues

In mesophyll cells of C. quinoa and Pisum sativum, a large number of crystalline virus aggregates (Fig.11) and proliferating membranous inclusions were found (Fig.12). Tubular arrays of virus particles were also founded in epidermal cells (Fig.13). Dissociation of the endomembrane and accumulation of starch was found in chloroplasts in these plant hosts. BBWV-2 infection does not appear to alter cell organelles in specific ways (Lisa & Boccardo, 1996).

Ecology and Control

The virus has a wide natural host range, including herbaceous annuals, woody perennials and perennial ornamentals. Consequently, natural hosts, including many of wild plants, play an important role in the epidemiology of the virus. Virus control in crops is difficult because of its wide natural host range and the nonpersistent mode of transmission by aphids. Roguing of weeds, application of insecticides and mineral oils, altering the sowing date of the crop to avoid aphid vectors and covering fields with silver-lustred plastic films have been suggested as ways to control the virus in broad bean. In some crops, such as lettuce, spinach and French bean, BBWV-2-resistant or tolerant genotypes have been found (Lisa & Boccardo, 1996).

Notes

The physico-chemical characters of BBWV-2 resemble closely those of viruses in the genus Comovirus. However, it is serologically unrelated to comoviruses and is transmitted by aphids, but not beetles, and has a relatively low nucleotide sequence homology with comoviruses. BBWV-2 is indistinguishable from BBWV-1 in host range and symptoms, in transmission by aphids, and in particle properties, but the two viruses are distinguished easily by immunodiffusion serological tests, ELISA, immunoblotting, and nucleotide sequence. The complete nucleotide sequence of PatMMV has been determined (accession numbers AB050782, RNA-1; AB011007, RNA-2) (Ikegami et al., 1998; Ikegami et al., 2001) and high amino acid sequence identities (up to 98.6%) of the RNA-1 and RNA-2 encoded proteins occur with BBWV-2 isolates (Ikegami et al., 2001; Kuroda et al., 2000; Koh et al., 2001; Qi et al., 2000b; Zhou et al., 2001). Phylogenetic trees, constructed using the sequences of the CPs (Zhou et al., 2001) confirm that PatMMV should be regarded as a strain of BBWV-2, rather than a distinct virus in the genus Fabavirus.

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Figure 1

Systemic mosaic in Vicia faba leaves.

Figure 2

Apical necrosis and wilting in Vicia faba.

Figure 3

Systemic chlorotic mottle in Chenopodium quinoa leaves.

Figure 4

Systemic mosaic and distortion in Chenopodium amaranticolor leaves.

Figure 5

Systemic chlorotic ringspots and line-patterns in a Nicotiana glutinosa leaf.

Figure 6

Veinal necrosis in an inoculated Petunia hybrida leaf.

Figure 7

Reddish-brown local lesions in an inoculated Vigna sinensis leaf.

Figure 8

Purified virus particles negatively stained with phosphotungstic acid, pH 7.0. Bar represents 100 nm.

Figure 9

Genome map of BBWV-2 RNA-1. The line indicates the noncoding sequence and boxes represent the proteins cleaved from the polyprotein encoded by the single ORF. The possible cleavage sites of their proteins are indicated above the boxes.

Figure 10

Genome map of BBWV-2 RNA-2. The line indicates the noncoding sequence and boxes represent the proteins cleaved from the polyprotein encoded by the single ORF. The cleavage sites of their proteins are indicated above the boxes.

Figure 11

Electron micrograph of an ultrathin section of a Chenopodium quinoa leaf cell infected with BBWV-2, showing rafts of crystallized virus particles. Bar represents 500 nm.

Figure 12

Electron micrograph of an ultrathin section of a Chenopodium quinoa leaf cell infected with BBWV-2, showing inclusions of proliferating membranes in the cytoplasm. Bar represents 200 nm.

Figure 13

Electron micrograph of an ultrathin section of a Pisum sativum leaf cell infected with BBWV-2, showing tubular arrays (arrow) of virus particles. CH represents chloroplast. M represents mitochondria. Bar represents 1000 nm.