Arabis Mosaic Virus Isolates Research Articles

Metagenomic analysis of nepoviruses: diversity, evolution and identification of a genome region in members of subgroup A that appears to be important for host range

Data mining and metagenomic analysis of 277 open reading frame sequences of bipartite RNA viruses of the genus Nepovirus, family Secoviridae, were performed, documenting how challenging it can be to unequivocally assign a virus to a particular species, especially those in subgroups A and C, based on some of the currently adopted taxonomic demarcation criteria. This work suggests a possible need for their amendment to accommodate pangenome information. In addition, we revealed a host-dependent structure of arabis mosaic virus (ArMV) populations at a cladistic level and confirmed a phylogeographic structure of grapevine fanleaf virus (GFLV) populations. We also identified new putative recombination events in members of subgroups A, B and C. The evolutionary specificity of some capsid regions of ArMV and GFLV that were described previously and biologically validated as determinants of nematode transmission was circumscribed in silico. Furthermore, a C-terminal segment of the RNA-dependent RNA polymerase of members of subgroup A was predicted to be a putative host range determinant based on statistically supported higher π (substitutions per site) values for GFLV and ArMV isolates infecting Vitis spp. compared with non-Vitis-infecting ArMV isolates. This study illustrates how sequence information obtained via high-throughput sequencing can increase our understanding of mechanisms that modulate virus diversity and evolution and create new opportunities for advancing studies on the biology of economically important plant viruses.

Genetic diversity and molecular evolution of arabis mosaic virus based on the CP gene sequence.

Arabis mosaic virus (ArMV) is a virus with a wide host range. In this study, the genetic diversity of ArMV and the molecular mechanisms underlying its evolution were investigated using the coat protein (CP) sequence. Of the 33 ArMV isolates studied, three were found to be recombinants. The other 30 recombination-free ArMV isolates could be separated into two major lineages with a significant F ST value (0.384) and tended to cluster according to their geographical origin. Different evolutionary constraints were detected for the two linages, pointing to a role of natural selection in the differentiation of ArMV.

Short communication. Molecular analysis of the genomic RNAs 1 and 2 of the first Arabis mosaic virus isolate detected in Spanish grapevines

The Arabis mosaic virus (ArMV) is one of the causative agent of the grapevine fanleaf disease, one of the most widespread and damaging viral diseases of grapevine. Recently, the ArMV has been detected in Spanish vineyards, and its determination and molecular characterization was undertaken. To this aim, the nucleotide sequence of the genomic RNAs 1 and 2 of the first isolate of ArMV infecting grapevine detected in Spain (ArMV-DU13) has been determined. The ArMV-DU13 genomic sequences were compared to the corresponding sequences of other isolates of ArMV, or nepoviruses. The most divergent genes among ArMV isolates were the X1 and VPg genes on the RNA 1, and the 2A gene on the RNA 2, with identity levels at the amino acid level of 78% (X1 and VPg) or 69% (2A) between the most distant isolates. Interestingly, the VPg genes were identical between the two grapevine isolates ArMV-Du13 and –NW, suggesting a possible implication of the host. The phylogenetic analysis of the RNA 2 showed that the Spanish isolate was close to Grapevine fanleaf virus isolates. The analysis of the full length RNA 2 suggests a recombination event between ArMV-DU13 and GFLV-GHu isolates between nucleotides 54 and 586 in the ArMV-DU13 isolate. Altogether, these results confirm the high variability between isolates of ArMV, and will be helpful to design more appropriate and reliable molecular diagnostic techniques for the control of this emerging virus in Spain.

Complete nucleotide sequence of a South African isolate of Grapevine fanleaf virus

The complete sequences of RNA1 and RNA2 have been determined for a South African isolate of Grapevine fanleaf virus (GFLV-SAPCS3). The two RNAs are, respectively, 7,342 and 3,817 nucleotides in length, excluding the poly(A) tails. RNA1 has a large open reading frame (ORF) of 6,852 nucleotides and a 5'-UTR and a 3'-UTR of 243 and 244 nucleotides, respectively. RNA2 encodes for an ORF of 3,330 nucleotides and has the highest nucleotide identity (90.4 %) with GFLV-F13. The full length nucleotide sequence of GFLV-SAPCS3 RNA1 had the highest nucleotide identity (86.5 %) to the French isolate GFLV-F13. The 5'- and 3'-UTRs of GFLV-SAPCS3 RNA2 are 272 nucleotides and 212 nucleotides (nt) in length, respectively. The GFLV-SAPCS3 RNA2 5'-UTR is 32-53 nt longer compared to other GFLV isolates. The GFLV-SAPCS3 RNA2 5'-UTR is also more closely related to GFLV-GHu and Arabis mosaic virus (ArMV) isolates than to other GFLV isolates. Putative intra- and interspecies recombination events between GFLV and ArMV isolates involving GFLV-SAPCS3 RNA1 and RNA2 were investigated. Recombination analysis software has indicated that the GFLV-SAPCS3 5'-UTR might have evolved from a recombinational event between GFLV-F13-type and ArMV-Ta-type isolate.

A RT/PCR-partial restriction enzymatic mapping (PREM) method for the molecular characterisation of the large satellite RNAs of Arabis mosaic virus isolates

The satellite RNA of the grapevine isolate NW of Arabis mosaic virus (ArMV) was cloned and sequenced, and showed 75% identity at the nucleotide level to the satellite RNA of the lilac isolate of ArMV. In order to survey ArMV isolates from various geographical origins and natural hosts for the presence of large satellite RNAs and analyse their degree of variability, a RT/PCR-partial restriction enzymatic mapping (PREM) method was developed. The method is based on the incorporation of 5-methyl-dCTP in the RT/PCR reaction, and the subsequent digestion of the RT/PCR products by methyl-sensitive restriction enzymes. Satellites RNAs were detected by RT/PCR in eight isolates out of 47, six of them originating from grapevine, one from hop and one from lilac. The partial restriction digestion patterns allowed to distinguish six different types of satellites. Cloning and sequencing of the different satellites confirmed these results, the PREM proving able to discriminate sequences with 96% identity. The sizes of the different satellites varied between 1092 and 1139 nucleotides, their encoded proteins between 338 and 360 amino acids. Conserved domains were found in the amino and carboxy-termini between the sequences of the proteins encoded by the satellites of the different isolates of ArMV.

Size and sequence variability of the Arabis mosaic virus protein 2A.

The RNA 2 of the nepovirus Arabis mosaic virus (ArMV) encodes a polyprotein from which protein 2A is released by proteolytic cleavage at the N-terminus. The 2A gene of 19 ArMV isolates from different geographical origin and 9 distinct natural hosts was amplified by RT/PCR and subsequently cloned and sequenced. These 19 isolates and those from databanks were classified into four groups based on the size of the protein 2A which ranged from 233 to 280 amino acids, and sequence identities. Sequence variability was mainly located in the N-terminus of the proteins, whereas the core region and the C-terminus were conserved.

Complete nucleotide sequences of the RNAS 2 of German isolates of Grapevine fanleaf and Arabis mosaic nepoviruses

The RNAs 2 of an Arabis mosaic virus (ArMV) and a grapevine fanleaf virus (GFLV) isolate, originating from South West of Germany near Neustadt an der Weinstrasse (NW), were sequenced. They are 3820 and 3775 nucleotides long respectively, and both contain one open reading frame encoding a polypeptide of 1110 amino acids. Their 5′ non-coding regions contain conserved and repeated sequences, which are able to form stem-loop structures. Nucleotide sequence comparisons between the full-length RNAs 2 revealed homology levels of 84 and 82% between the ArMV-NW and the ArMV-L and -U, respectively, 90% between GFLV-NW and GFLV-F13, and 72% between ArMV-NW and GFLV-NW. Amino acid sequence comparisons showed that the greatest difference was found between the 2A proteins of the different ArMV isolates, the 2A protein of the ArMV-NW showing more similarity to the 2A protein of GFLV-NW than to those of ArMV-L2 or -U2.

The 119 kDa and 124 kDa polyproteins of arabis mosaic nepovirus (isolate S) are encoded by two distinct RNA2 species.

Arabis mosaic virus (ArMV) is a nepovirus that is serologically distantly related to grapevine fanleaf virus (GFLV). Both ArMV and GFLV induce grapevine degeneration disease. Several ArMV isolates, unlike isolates of GFLV, produce upon in vitro translation of RNA2 a polyprotein (P2) that forms a double band in polyacrylamide-SDS gels. Cloning of full-length copies of RNA2 of an ArMV isolate from grapevine (ArMV-S) revealed that this isolate contained two RNA2s of different length, called RNA2-U and RNA2-L. The two species were not readily separated by electrophoresis of the virion RNA under denaturing gel electrophoresis conditions but could be distinguished by analysis of primer extension and in vitro translation products. The size difference of the two RNA2s is due mostly if not exclusively to differences in their coding regions. The 124 kDa RNA2-U-encoded polyprotein P2' and the 119 kDa RNA2-L-encoded polyprotein P2", which co-migrate, respectively, with the upper and lower polyprotein bands produced by RNA2 of ArMV-S, were more than 95% identical except in their N-terminal domains. In vitro maturation experiments and sequence comparisons indicate that the N-terminal products of P2' and P2" have a molecular mass of 31 kDa and 26 kDa. The genomic organization proposed is similar to that of GFLV RNA2.

Characterization and Detection of Grapevine Fanleaf Virus by Using cDNA Probes

Complementary DNA copies to the RNAs of grapevine fanleaf virus (GFLV) strain F13 were cloned in Escherichia coli plasmid pUC9 and used after nick-translation labeling as probes to detect and to characterize several GFLV isolates by molecular hybridization. Picogram amounts of viral RNA were detected specifically in RNAs extracted from plants with 32 P probes. Cross-hybridizations indicating nucleotide sequence homologies were obtained between GFLV probes and RNAs from arabis mosaic virus (ArMV). When a probe specific to the GFLV-F13 satellite RNA was used, it was possible to reveal the presence of satellite RNA associated with different GFLV and ArMV isolates, thus indicating strong homologies between these large satellite RNAs (...)

The etiology of hop chlorotic disease

SUMMARYHop chlorotic disease was first described in England in 1930, but it has since been seldom seen and its etiology has remained unknown. In 1983 a patch of plants with the disease occurred in a large area of hops (Humulus lupulus) cv. Bramling Cross planted at Yalding, Kent in 1967. All plants in a rectangular area enclosing the disease outbreak were infected with hop mosaic, hop latent and prunus necrotic ringspot viruses; the diseased plants were additionally infected with arabis mosaic virus (AMV). The disease was also associated with seed‐transmitted AMV, and was induced in hop seedlings inoculated with partially purified preparations of AMV originating from chlorotic disease‐affected hops prepared from Chenopodium quinoa. The disease appears to be caused by AMV, but AMV isolates from hops with chlorotic disease were serologically indistinguishable from AMV isolates from hops with symptoms of bare‐bine and/or nettlehead and showed similar pathogenicity in diagnostic hosts. The basis of the difference between isolates in their pathogenicity in hop remains unknown.

Characterization of antigenic structures on arabis mosaic virus with monoclonal antibodies

Seven different epitopes on arabis mosaic virus (ArMV) were discerned. Neo-, crypto-, and epitopes exposed on the virion and isolated coat protein were differentiated by their reactivity with monoclonal antibodies in an indirect enzyme-linked immunosorbent assay. The monoclonal antibodies, produced in vitro and in vivo, were of IgGl, IgM and IgA isotype. No epitope exclusively specific for one isolate was found. One epitope was specific for ArMV isolates only. With the common epitopes an operational antigenic map was devised. An immunological relationship between nepoviruses of different serological subgroups was demonstrated. Grapevine fanleaf virus, a member of the ArMV subgroup could not be shown to expose crossreactive epitopes. For serotyping ArMV isolates were discriminated by comparing the reactivity of two or more monoclonal antibodies specific for different epitopes.

The isolation and identification of rhubarb viruses occurring in Britain

SUMMARYVirus‐like symptoms were common in British crops of rhubarb. All plants tested of the three main varieties, ‘Timperley Early’, ‘Prince Albert’ and ‘Victoria’, were virus‐infected. Turnip mosaic virus and a severe isolate of arabis mosaic virus (AMV) were obtained from ‘Timperley Early’; and ‘Prince Albert’ contained turnip mosaic virus, cherry leaf roll virus (CLRV), a mild isolate of AMV and, infrequently, cucumber mosaic virus (CMV). The main commercial variety ‘Victoria’ contained turnip mosaic virus, CLRV, a mild isolate of AMV and, infrequently, strawberry latent ringspot virus (SLRV). All the viruses were identified serologically. The rhubarb isolates did not differ markedly from other isolates of these viruses in herbaceous host reactions, properties in vitro or particle size and shape. A rhubarb isolate of CLRV was distinguished serologically from a cherry isolate of the virus.Turnip mosaic virus, CLRV and SLRV, were transmitted with difficulty, but AMV isolates were readily transmitted by mechanical inoculation. Turnip mosaic virus was also transmitted to rhubarb by Myzus persicae and Aphis fabae. CLRV was transmitted in 6–8% of the seed of infected ‘Prince Albert’ and ‘Victoria’ rhubarb and in 72% of the seed of infected Chenopodium amaranticolor. Mild isolates of AMV were also transmitted in 10–24% of the seed of infected ‘Prince Albert’ and ‘Victoria’ plants.

Arabis mosaic and Prunus necrotic ringspot viruses in hop (Humulus lupulus L.)

SUMMARYPurified virus preparations made from nettlehead‐diseased hop plants, or from Chenopodium quinoa, to which the virus was transmitted by inoculation of sap, contained polyhedral virus particles of 30 mμ diameter which were identified serologically as arabis mosaic virus (AMV). There were serological differences between AMV isolates from hop and from strawberry, and also differences in host range and in symptoms caused in C. quinoa and C. amaranticolor. AMV was always associated with nettlehead disease. The nematode Xiphinema diversicaudatum occurred in small numbers in most hop gardens, but was numerous where nettlehead disease was spreading rapidly.Preparations from nettlehead‐affected hops also contained a second virus, serologically related to Prunus necrotic ringspot virus (NRSV), in mild and virulent forms which infected the same range of test plants but showed some serological differences. Mild isolates did not protect C. quinoa plants against infection by virulent isolates. Hop seedlings inoculated with virulent isolates of NRSV developed symptoms indistinguishable from those of split leaf blotch disease. Latent infection with NRSV was prevalent in symptomless hop plants. Nettlehead disease is apparently associated with dual infection of AMV and virulent isolates of NRSV.An unnamed virus with rod‐shaped particles 650 mμ long was common in hop and was transmitted by inoculation of sap to herbaceous plants. Cucumber mosaic virus was obtained from a single plant of Humulus scandens Merr.