Species: Broad bean wilt virus 2
Institute of Biotechnology, Zhejiang University, Hangzhou 310029, P R China
Host Range and Symptomatology
Transmission by Vectors
Transmission through Seed
Transmission by Grafting
Transmission by Dodder
Nucleic Acid Hybridization
Stability in Sap
Properties of Particles
Properties of Infective Nucleic Acid
Relations with Cells and Tissues
Ecology and Control
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.
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).
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.
Chenopodium quinoa is a good propagation host for virus purification. Broad bean, pea and Nicotiana clevelandii are suitable hosts for maintaining cultures.
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.
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).
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).
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).
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).
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).
Systemic mosaic in Vicia faba leaves.
Apical necrosis and wilting in Vicia faba.
Systemic chlorotic mottle in Chenopodium quinoa leaves.
Systemic mosaic and distortion in Chenopodium amaranticolor leaves.
Systemic chlorotic ringspots and line-patterns in a Nicotiana glutinosa leaf.
Veinal necrosis in an inoculated Petunia hybrida leaf.
Reddish-brown local lesions in an inoculated Vigna sinensis leaf.
Purified virus particles negatively stained with phosphotungstic acid, pH 7.0. Bar represents 100 nm.
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.
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.
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.
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.
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.