Characterization of alfalfa virus F, a new member of the genus Marafivirus.

Viral infections of alfalfa are widespread in major cultivation areas and their impact on alfalfa production may be underestimated. A new viral species, provisionally named alfalfa virus F (AVF), was identified using a virion-associated nucleic acid (VANA) metagenomics-based approach in alfalfa (Medicago sativa L.) samples collected in Southern France. The nucleotide sequence of the viral genome was determined by de-novo assembly of VANA reads and by 5'/3' RACE with viral RNA extracted from enriched viral particles or with total RNA, respectively. The virus shares the greatest degree of overall sequence identity (~78%) with Medicago sativa marafivirus 1 (MsMV1) recently deduced from alfalfa transcriptomic data. The tentative nucleotide sequence of the AVF coat protein shares ~83% identity with the corresponding region of MsMV1. A sequence search of the predicted single large ORF encoding a polyprotein of 235kDa in the Pfam database resulted in identification of five domains, characteristic of the genus Marafivirus, family Tymoviridae. The AVF genome also contains a conserved "marafibox", a 16-nt consensus sequence present in all known marafiviruses. Phylogenetic analysis of the complete nucleotide sequences of AVF and other viruses of the family Tymoviridae grouped AVF in the same cluster with MsMV1. In addition to 5' and 3' terminal extensions, the identity of the virus was confirmed by RT-PCRs with primers derived from VANA-contigs, transmission electron microscopy with virus-infected tissues and transient expression of the viral coat protein gene using a heterologous virus-based vector. Based on the criteria demarcating species in the genus Marafivirus that include overall sequence identity less than 80% and coat protein identity less than 90%, we propose that AVF represents a distinct viral species in the genus Marafivirus, family Tymoviridae.

Genetic Diversity, Reassortment, and Recombination in Alfalfa mosaic virus Population in Spain.

The variability and genetic structure of Alfalfa mosaic virus (AMV) in Spain was evaluated through the molecular characterization of 60 isolates collected from different hosts and different geographic areas. Analysis of nucleotide sequences in four coding regions--P1, P2, movement protein (MP), and coat protein (CP)--revealed a low genetic diversity and different restrictions to variation operating on each coding region. Phylogenetic analysis of Spanish isolates along with previously reported AMV sequences showed consistent clustering into types I and II for P1 and types I, IIA, and IIB for MP and CP regions. No clustering was observed for the P2 region. According to restriction fragment length polymorphism analysis, the Spanish AMV population consisted of seven haplotypes, including two haplotypes generated by reassortment and one involving recombination. The most frequent haplotypes (types for P1, MP, and CP regions, respectively) were I-I-I (37%), II-IIB-IIB (30%), and one of the reassortants, II-I-I (17%). Distribution of haplotypes was not uniform, indicating that AMV population was structured according to the geographic origin of isolates. Our results suggest that agroecological factors are involved in the maintenance of AMV genetic types, including the reassortant one, and in their geographic distribution.

Complete nucleotide sequence of Alfalfa mosaic virus isolated from alfalfa (Medicago sativa L.) in Argentina.

The complete nucleotide sequence of an Alfalfa mosaic virus (AMV) isolate infecting alfalfa (Medicago sativa L.) in Argentina, AMV-Arg, was determined. The virus genome has the typical organization described for AMV, and comprises 3,643, 2,593, and 2,038 nucleotides for RNA1, 2 and 3, respectively. The whole genome sequence and each encoding region were compared with those of other four isolates that have been completely sequenced from China, Italy, Spain and USA. The nucleotide identity percentages ranged from 95.9 to 99.1 % for the three RNAs and from 93.7 to 99 % for the protein 1 (P1), protein 2 (P2), movement protein and coat protein (CP) encoding regions, whereas the amino acid identity percentages of these proteins ranged from 93.4 to 99.5 %, the lowest value corresponding to P2. CP sequences of AMV-Arg were compared with those of other 25 available isolates, and the phylogenetic analysis based on the CP gene was carried out. The highest percentage of nucleotide sequence identity of the CP gene was 98.3 % with a Chinese isolate and 98.6 % at the amino acid level with four isolates, two from Italy, one from Brazil and the remaining one from China. The phylogenetic analysis showed that AMV-Arg is closely related to subgroup I of AMV isolates. To our knowledge, this is the first report of a complete nucleotide sequence of AMV from South America and the first worldwide report of complete nucleotide sequence of AMV isolated from alfalfa as natural host.

Effects of the number of genome segments on primary and systemic infections with a multipartite plant RNA virus.

Multipartite plant viruses were discovered because of discrepancies between the observed dose response and predictions of the independent-action hypothesis (IAH) model. Theory suggests that the number of genome segments predicts the shape of the dose-response curve, but a rigorous test of this hypothesis has not been reported. Here, Alfalfa mosaic virus (AMV), a tripartite Alfamovirus, and transgenic Nicotianatabacum plants expressing no (wild type), one (P2), or two (P12) viral genome segments were used to test whether the number of genome segments necessary for infection predicts the dose response. The dose-response curve of wild-type plants was steep and congruent with the predicted kinetics of a multipartite virus, confirming previous results. Moreover, for P12 plants, the data support the IAH model, showing that the expression of virus genome segments by the host plant can modulate the infection kinetics of a tripartite virus to those of a monopartite virus. However, the different types of virus particles occurred at different frequencies, with a ratio of 116:45:1 (RNA1 to RNA2 to RNA3), which will affect infection kinetics and required analysis with a more comprehensive infection model. This analysis showed that each type of virus particle has a different probability of invading the host plant, at both the primary- and systemic-infection levels. While the number of genome segments affects the dose response, taking into consideration differences in the infection kinetics of the three types of AMV particles results in a better understanding of the infection process.

The complete nucleotide sequence of apple mosaic virus (ApMV) RNA 1 and RNA 2: ApMV is more closely related to alfalfa mosaic virus than to other ilarviruses.

The complete nucleotide sequences of apple mosaic virus RNA 1 and 2 have been characterized. Apple mosaic virus RNA 1 is 3476 nucleotides in length and encodes a single large open reading frame (ORF), whereas apple mosaic virus RNA 2 is 2979 nucleotides in length and also encodes a single ORF. The amino acid sequences encoded by RNA 1 and 2 show similarity to all of the other ilarviruses for which sequence data are available, but both are more closely related to alfalfa mosaic virus (AMV) than to other ilarviruses. Points of similarity include the absence of ORF 2b, present on the RNA 2 of all previously characterized ilarviruses. The close relationship to AMV also occurs in the movement protein, encoded by RNA 3, but not with the coat protein. These data suggest that the present taxonomy should be revised, and that AMV should be considered an aphid-transmissible ilarvirus.

Evidence for two distinct subgroups of alfalfa mosaic virus (AMV) from france and italy and their relationships with other AMV strains Brief report.

The nucleotide sequence of the putative coat protein open reading frame of seven previously uncharacterized AMV strains from Italy and France was determined and aligned with comparable sequences of other AMV strains (425 L, 425 M, YSMV, S, VRU, 15/64 and Da). The data set of AMV sequences was used to determine phylogenetic relationships by both a stochastic (stationary Markov model) and a deterministic method (maximum-parsimony) of analysis. The topology of the trees obtained with the two methods was essentially the same showing that all AMV strains clustered in two monophyletic groups. Close clustering of Italian strains in subgroup I and of French strains in subgroup II seems to suggests the effect of geographic distinctiveness of evolutionary dynamics of these AMV strains. This separation did not correlate with differences in host range or symptoms (necrotic or non necrotic) induced in tomato but rather it reflected variations in the amino acid sequence of their CP, which might be related to structural properties of virus particles. A simple and rapid procedure based on the reverse transcriptase-polymerase chain reaction (RT-PCR) followed by ezymatic digestion (RFLP) was developed to identify and classify AMV isolates into the two subgroups. The method applied to a number of other AMV isolates from Italy and France supported their division in two distinct subgroups. This RT-PCR RFLP method may be useful way to investigate the dynamics of AMV populations in nature.

The sequence of RNA 1 and RNA 2 of tobacco streak virus: additional evidence for the inclusion of alfalfa mosaic virus in the genus Ilarvirus.

Here we describe the complete sequence of RNA 1 and 2 of the WC isolate of tobacco streak virus (TSV). These two sequences complete the information on the genome of TSV, the type member of the genus Ilarvirus, and are the first sequences described for the RNA 1 and RNA 2 of a member of subgroup 1 of this genus. The sequences have a similar organization to those reported for the corresponding RNAs of other ilarviruses. However, the putative translation products of these two molecules differ sufficiently from previously sequenced ilarviruses so that TSV should remain in a subgroup on its own. Phylogenetic comparison of sequence data for RNA 1 with that of other ilarviruses and alfalfa mosaic virus (AMV) reveals two distinct clusters (TSV, CiLRV, and SpLV) and (AMV, PDV, and ApMV). These data support the suggestion [16] based on data for RNA 3 of ilarviruses that AMV should be included as a true ilarvirus.

The complete nucleotide sequence of prune dwarf ilarvirus RNA-1.

The sequence of prune dwarf ilarvirus (PDV) RNA-1 has been determined; it consists of 3,374 nucleotides and contains a single open reading frame of 3,168 nucleotides. The putative translation product is 1,055 amino acids in length with a calculated molecular mass of 118.9 kDa. Both the nucleic acid and the translated amino acid sequences show stronger homology to the corresponding RNA-1 and ORF-1 of apple mosaic ilarvirus and alfalfa mosaic alfamovirus than to spinach latent mosaic ilarvirus or citrus leaf rugose ilarvirus. These findings are consistent with the inclusion of alfalfa mosaic virus in the ilarvirus genus. The reported sequence of PDV RNA-1 and its single ORF conform to the genomic organization typical of the Bromoviridae family.