Genetic variability of grapevine fabavirus variants and development of a broad-spectrum assay for their detection.

Complete RNA1 and RNA2 sequences of two and nearly complete genome sequences of six new variants of grapevine fabavirus found in Japan were compared to those of previously reported variants. Negative selection pressure was suggested, and no recombination events were detected in either RNA1 or RNA2. The first 18 nucleotides in both RNAs were predicted to form a stem-loop structure. The variants could be genetically divided into four groups based on RNA1 and two based on RNA2. A broad-spectrum reverse transcription polymerase chain reaction assay using a primer pair designed based on an RNA2 consensus sequence was able to detect all of the known variants.

High throughput sequencing reveals a novel fabavirus infecting sweet cherry.

The genus Fabavirus currently consists of five species represented by viruses that infect a wide range of hosts but none reported from temperate climate fruit trees. A virus with genomic features resembling fabaviruses (tentatively named Prunus virus F, PrVF) was revealed by high throughput sequencing of extracts from a sweet cherry tree (Prunus avium). PrVF was subsequently shown to be graft transmissible and further identified in three other non-symptomatic Prunus spp. from different geographical locations. Two genetic variants of RNA1 and RNA2 coexisted in the same samples. RNA1 consisted of 6,165 and 6,163 nucleotides, and RNA2 consisted of 3,622 and 3,468 nucleotides.

[Molecular identification and sequence analysis of broad bean wilt virus 2 isolates from atractylodes macrocephala Koidz].

To identity the pathogen that causes the mosaic and yellowing symptoms on Atractylodes macrocephala Koidz in Jiangxian, Shanxi province, biological inoculation, sequence-independent amplification (SIA),RT-PCR and other identification methods were used. The results showed that the chlorotic and necrosis symptoms occurred in the indicator plant Chenopodium quinoa after it was infected with the pathogen,and the same symptoms appeared after the reinoculation of healthy Atractylodes macrocephala Koidz; this reflected that the disease was likely to be caused by a virus. The results of SIA and sequencing showed that Broad bean wilt virus 2 (BBWV2) was present in severely mosaic Atractylodes macrocephala Koidz leaves. To further characterize the BBWV2 isolate from Atractylodes macrocephala (BBWV2-Am), the polyprotein partial gene encoded by BBWV2-Am RNA2 was cloned and sequenced. Sequence alignments showed that the nucleotide sequence identity of BBWV2-Am SCP and LCP genes ranged from 79.3% to 87.2% and from 80.1% to 89.2% compared to other BBWV2 strains,respectively; the deduced amino acid sequence similarities of the two gene products ranged from 91.2% to 95.7% and from 89.44 to 95.5%, respectively,compared to those of other BBWV2 strains. Phylogenetic comparisons showed that BBWV2-Am was most likely to be related to BBWV2-Rg,but formed an independent branch. This is the first report of BBWV2 in Atractylodes macrocephala Koidz.

Genetic variability and evolution of broad bean wilt virus 1: role of recombination, selection and gene flow.

Analysis of four genomic regions from 37 geographically diverse isolates of broad bean wilt virus 1 (BBWV-1) showed high genetic diversity in comparison to most plant viruses. Comparison of synonymous and nonsynonymous substitutions of the small coat protein gene (SCP) revealed negative selection for most amino acid positions. Phylogenetic analysis of SCP showed that some BBWV-1 isolates from distant geographical areas were genetically close, suggesting long-distance migration. Analysis of genetic differentiation revealed high gene flow between Spanish and Near Eastern subpopulations, which were separated from North-Central and South-Eastern European subpopulations. Finally, putative recombinant and reassortant genomes were also identified.

Detection and identification of Fabavirus species by one-step RT-PCR and multiplex RT-PCR.

The genus Fabavirus of the family Secoviridae comprises a group of poorly characterized viruses. To date, only five species have been described: Broad bean wilt virus 1 (BBWV-1), Broad bean wilt virus 2 (BBWV-2), Lamium mild mosaic virus (LMMV), Gentian mosaic virus (GeMV) and Cucurbit mild mosaic virus (CuMMV). The development is described of two RT-PCR procedures for the detection and identification of Fabavirus species: a one-step RT-PCR using a single pair of conserved primers for the detection of all fabaviruses, and a one-step multiplex RT-PCR using species-specific primers for the simultaneous detection and identification of the above-mentioned species of the genus Fabavirus. These methods were applied successfully to field samples and the results were compared with those obtained by molecular hybridization and ELISA. The combination of the two techniques enables rapid, sensitive and reliable identification of the five known fabavirus species, as well as the possibility of discovering new species of this genus.

Establishment of an agroinoculation system for broad bean wilt virus 2.

We determined the complete nucleotide sequence of a broad bean wilt virus 2 (BBWV-2) isolate from gentian in Japan. The full-length RNA1 and RNA2 sequences, excluding poly(A) tails, were 5955 and 3600 nucleotides long, respectively. Analysis indicated that, in contrast to other BBWV-2 isolates, the 5' end of both RNA1 and RNA2 starts with a GUU sequence. We successfully inoculated Nicotiana benthamiana with BBWV-2 by infiltrating a mixed suspension of two Agrobacterium tumefaciens clones carrying binary vectors with the full-length RNA1 and RNA2 sequences. This is the first report on the efficient, easy and high-throughput use of agroinoculation for generating BBWV-2 infections.

Complete genomic sequence analysis reveals a novel fabavirus infecting cucurbits in China.

The complete genome sequence of a cucurbit-infecting fabavirus was determined. Sequence analysis revealed that it had a genomic organization typical of fabaviruses, with genome segment sizes of 5870 nt (RNA-1) and 3294 nt (RNA-2). It shared CP and Pro-Pol amino acid sequence identities of 52.0-58.9% with those of reported fabaviruses. ELISA and western blots gave no cross-reactions between this cucurbit virus and broad bean wilt viruses 1 and 2. Based on molecular and serological criteria for species demarcation in the genus Fabavirus, the virus represents a distinct species, for which the species name Cucurbit mild mosaic virus (CuMMV) is proposed.

Detection and absolute quantitation of Broad bean wilt virus 1 (BBWV-1) and BBWV-2 by real time RT-PCR.

Broad bean wilt virus 1 (BBWV-1) and BBWV-2 are the two most significant viruses in the genus Fabavirus, causing damage to many economically important agricultural crops worldwide. A quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR) procedure using two TaqMan(®)MGB probes was developed for sensitive and specific detection and quantitation of BBWV-1 and BBWV-2. Primers and probes were designed from conserved sequence stretches to detect all isolates of each virus. Standard curves using RNA transcripts identical to both TaqMan(®)MGB probes enabled absolute quantitation, with a wide dynamic range and high sensitivity (10(3)-10(10) RNA molecules). RT-qPCR was assayed with genetically divergent BBWV-1 and BBWV-2 isolates from different plant hosts and countries, and was used to evaluate the temporal accumulation of BBWV-1 RNA in two plant hosts.

Antipredator behaviour of Myzus persicae affects transmission efficiency of Broad bean wilt virus 1.

Biological control has the potential to limit the population growth of arthropod vectors and consequently may be expected to reduce plant virus spread in a crop. However, reduction of vector abundance is not the only effect of biological control. Natural enemies might induce antipredator behaviour that affects feeding and dispersal of vectors, and therefore virus spread. Here we test the effect of two natural enemies on dispersal of the aphid vector Myzus persicae and transmission of Broad bean wilt virus 1 (BBWV-1), genus Fabavirus, which is non-persistently transmitted by aphids. One of the predators tested, the syrphid Sphaerophoria rueppellii, is considered to induce low disturbance in aphid colonies, whereas the other, the coccinellid Adalia bipunctata, is assumed to induce high disturbance. Natural enemies enhanced aphid dispersal, but not virus transmission as compared to the control treatment without predators. However, transmission efficiency of BBWV-1 was higher in the presence of coccinellid adults than with syrphids. The behavioural observations of predators and the reactions of aphids to their presence indicate that a stronger antipredator behaviour is induced by coccinellid adults than by syrphids. The different antipredator behaviour displayed by aphids towards coccinellid adults and syrphids might explain the differences found in the rate of virus spread in their presence.

Genome sequence and characterization of a new virus infecting Mikania micrantha H.B.K.

The complete RNA genomic sequence of a new virus infecting Mikania micrantha, designated as Mikania micrantha wilt virus (MMWV), has been determined. The genomic sequence and the predicted gene products of MMWV were similar to those of the other viruses of the genus Fabavirus. The MMWV nucleotide sequence showed 75.6% identity to that of gentian mosaic virus, 56.6 and 57% identity to those of two Broad bean wilt virus 1 isolates, and between 55.7 and 58% identity to those of seven Broad bean wilt virus 2 isolates. Our results suggested that MMWV represents a distinct isolate of the candidate species Gentian mosaic virus.

New molecular methods for identification of broad bean wilt virus 1.

Broad bean wilt virus 1 (BBWV-1) causes damages in economically important plant crops such as pepper, bean, spinach, etc. Fast, cheap and reliable diagnostic tools are crucial to limit or control the disease. In this work, tissue blot immunoassay (TBIA), dot-blot (DB) and tissue-print (TP)-hybridization were developed for BBWV-1 diagnosis and evaluated for sensitivity, specificity and reliability in plants of several host species grown in the greenhouse or in the field, in comparison with ELISA and RT-PCR. RT-PCR followed by DB-hybridization provided the most sensitive and efficient diagnostic, but the virus was also detected in most samples by ELISA. Detection by TBIA or by TP-hybridization avoided sample processing, but they were less consistent and greatly depended on host species and tissue. DB-hybridization with probes corresponding to different genomic regions allowed universal detection of BBWV-1 and discrimination between genetically distant isolates.

Detection and identification of species of the genus Fabavirus by RT-PCR with a single pair of primers.

The genus Fabavirus includes three species: Broad bean wilt virus 1 (BBWV-1), BBWV-2 and Lamium mild mosaic virus (LMMV), but a new candidate species, Gentian mosaic virus (GeMV), has been proposed. Analysis of the complete nucleotide sequences of fabaviruses was used to design a pair of conserved primers for specific detection of members of this genus. These primers encompassed the 5'-terminal non-translatable region (NTR) , whose size for BBWV-1, BBWV-2 and GeMV was different. RT-PCR, with this pair of primers, is a rapid and sensitive procedure for diagnosis of fabavirus infections, that also allows identification of distinct species involved in single or mixed infections, based on the size of the amplification products. Moreover, it might allow future discovery of potential new species of this genus.

The complete sequence of a Spanish isolate of Broad bean wilt virus 1 (BBWV-1) reveals a high variability and conserved motifs in the genus Fabavirus.

The genome of a Spanish isolate of Broad bean wilt virus-1 (BBWV-1) was completely sequenced and compared with available sequences of other isolates of the genus Fabavirus (BBWV-1 and BBWV-2). This consisted of two RNAs of 5814 and 3431 nucleotides, respectively, and their organization was similar to that of other members of the family Comoviridae. Its mean nucleotide identity with a BBWV-1 American isolate was 81.5%, and between 59.8 and 63.5% with seven BBWV-2 isolates. Our analysis showed sequence stretches in the 5' non-coding regions which are conserved in both genomic RNAs and in BBWV-1 and BBWV-2 isolates.

Complete nucleotide sequence and infectious cDNA clone of the RNA1 of a Chinese isolate of broad bean wilt virus 2.

The nucleotide sequence of the RNA1 of broad bean wilt virus 2 (BBWV2) isolate B935 has been determined from overlapping cDNA clones. It contains 5956 nucleotides in length excluding the 3' terminal poly(A) tail and contains a single long open reading frame (ORF) of 5613 nucleotides extending from nucleotide 234 to 5846. A repeated motif has been found in the 5' non-coding region. The predicted polyprotein encoded by the long ORF is 1870 amino acid in length with a molecular weight of 210 K. Amino acid sequence comparisons between portions of the BBWV2 RNA1-encoded polyprotein and proteins encoded by several species in Comoviridae revealed the putative functions of BBWV2 RNA1-encoded proteins and the same general genetic organization as that of comoviruses and nepoviruses. Based on the determined sequence, full-length cDNA clone of RNA1 designated as pU1FL was constructed. Together with transcripts from full-length cDNA clone of RNA2 (pU2FL), transcripts from pU1FL infected Chenopodium quinoa successfully.

Complete nucleotide sequence of the genome organization of RNA2 of patchouli mild mosaic virus, a new favavirus.

The complete nucleotide sequence and the genome organization of the RNA 2 of a patchouli mild mosaic virus (PaMMV) was determined. The sequence consists of 3591 nucleotides and contains a single long open reading frame sufficient to code for 118 K protein. Three proteins of 52 K, 44 K and 22 K could be encoded by the PaMMV RNA 2 genome. Our analysis of the N-terminal sequences of two species of coat protein (CP) allowed precise location of the CP cistrons within the polyprotein. 44 K and 22 K proteins are the coat proteins. The positions of the cleavage sites are Gln/Ala between 44 K and 22 K coat proteins and Gln/Gly between 52 K and 44 K proteins. Comparison of PaMMV RNA 2 with comoviral and nepoviral RNA 2 showed no sequence similarity. These results as well as previous serological studies strongly suggest that PaMMV is a member in the genus Fabavirus.