K. L. Druffel

Phylogenetic analysis of the Potyviridae with emphasis on legume-infecting potyviruses

SummaryThe 3′-terminal nucleotide sequences of thirteen authenticated strains of bean common mosaic virus (BCMV) and one strain of bean common mosaic necrosis virus (BCMNV) were obtained. The regions sequenced included the coat protein coding sequence and 3′-end non-coding region. These data, combined with sequence information from other legume-infecting potyviruses and the Potyviridae were used for phylogenetic analysis. Evidence is provided for delineation of BCMNV as distinct from BCMV and the inclusion of azuki mosaic, dendrobium mosaic, blackeye cowpea mosaic, and peanut stripe viruses as strains of BCMV. This relationship defines the members of the BCMV and BCMNV subgroups. These data also provide a basis upon which to define virus strains, in combination with biological data. Other aspects and implications of legume-infecting potyvirus phylogenetics are discussed.

NL-3 K Strain Is a Stable and Naturally Occurring Interspecific Recombinant Derived from Bean common mosaic necrosis virus and Bean common mosaic virus.

ABSTRACT A strain of Bean common mosaic necrosis virus (BCMNV) from Idaho was identified by enzyme-linked immunosorbent assay using monoclonal antibodies and determined to be similar to the NL-3 D strain (of Drifjhout) by reaction of differential bean cultivars. However, this BCMNV strain (designated NL-3 K) caused earlier and more severe symptoms on bean plants representing host groups 0, 4, and 5. The nucleotide sequence encoding the predicted polyprotein of NL-3 K was 9,893 nucleotides (nt) in length, yielding a peptide with a molecular size of 362.1 kDa compared with a 9,626-nt, 350.9-kDa polyprotein for NL-3 D. Sequence analysis of the putative P1 protein suggests that the NL-3 K strain is a recombinant between NL-3 D and the Russian strain (RU1) of Bean common mosaic virus. The P1 protein of NL-3 K consisted of 415 amino acids compared with 317 for NL-3 D. The first 114 predicted amino acids of the NL-3 K P1 region were 98% identical with RU1. The remaining 301 amino acids of the protein shared only 34% identity with RU1 but were 98% identical with NL-3 D. Primers were designed that flanked the recombination point in the P1 coding sequence of NL-3 K. An amplicon of the expected size was produced by reverse-transcriptase polymerase chain reaction of total nucleic acid extracts of bean plants inoculated with NL-3 K, but not from those with NL-3 D or RU1. The increased symptom severity on selected common bean lines induced by NL-3 K suggests that the P1 gene may play a significant role in pathogenicity and virulence.

A new and distinct species in the genus Caulimovirus exists as an endogenous plant pararetroviral sequence in its host, Dahlia variabilis.

Viruses in certain genera in family Caulimoviridae were shown to integrate their genomic sequences into their host genomes and exist as endogenous pararetroviral sequences (EPRV). However, members of the genus Caulimovirus remained to be the exception and are known to exist only as episomal elements in the infected cell. We present evidence that the DNA genome of a new and distinct Caulimovirus species, associated with dahlia mosaic, is integrated into its host genome, dahlia (Dahlia variabilis). Using cloned viral genes as probes, Southern blot hybridization of total plant DNA from dahlia seedlings showed the presence of viral DNA in the host DNA. Fluorescent in situ hybridization using labeled DNA probes from the D10 genome localized the viral sequences in dahlia chromosomes. The natural integration of a Caulimovirus genome into its host and its existence as an EPRV suggests the co-evolution of this plant-virus pathosystem.

Seed Transmission of Dahlia mosaic virus in Dahlia pinnata

Dahlia is an economically important ornamental crop in the United States and several other countries in the world. Among the viral diseases that affect dahlia, Dahlia mosaic virus (DMV) is considered to be the most widespread and to have the greatest impact on flower production. Using grow-out tests followed by polymerase chain reaction (PCR)-based testing of the seedlings, dahlia seed obtained from three different sources were shown to contain DMV. Additionally, the distribution of DMV in various parts of the dahlia seed was determined by PCR. Growout tests revealed a high rate of seed transmission. DMV was detected in cotyledons and, rarely, in the seed coat. The virus also was detected in pollen collected from infected plants. In addition to vegetative propagation, seedborne infection could be contributing to the spread of DMV in dahlia. Use of virus-free seed and vegetative material would result in reduced incidence of the disease.

Molecular characterization of two potato virus S isolates from late-blight-resistant genotypes of potato (Solanum tuberosum)

Potato virus S (PVS) belongs to the family Flexiviridae [1], genus Carlavirus. The virus is transmitted by aphids (Aphis fabae, A. nasturtii, Myzus persicae and Rhopalosiphum padi) in a non-persistent manner. It is transmissible through vegetative propagation and also mechanically transmissible to several members of the Solanaceae and Chenopodiaceae. Most PVS isolates induce inconspicuous symptoms in many potato cultivars. However, in susceptible cultivars, some isolates cause deepening of the veins, rugosity of the leaf surface, mottling of the leaves and early drop of the lower leaves [3]. There are two major virus strains of PVS recognized by their reactions on Chenopodium quinoa. The PVS Andean strain induces systemic symptoms on C. quinoa; however, the PVS Ordinary strain does not [6]. The members of the genus Carlavirus contain a singlestranded, positive-sense RNA genome of 8.4–8.5 kb [8] packaged in a filamentous particle of 610–710 9 10–15 nm [4, 8, 12]. The RNA genome contains a 50 cap structure, six open reading frames (ORFs), and a polyadenylated tail at the 30 terminus. Although PVS has a widespread distribution, only two complete nucleotide sequences of PVS isolates have been published so far: the Leona isolate from Germany (GenBank accession number AJ863509) and the Vltava isolate from the Czech Republic (AJ863510) [9]. Our recent survey of potato fields in Washington State has shown that PVS is widely prevalent. Additionally, PVS isolates from late-blight-resistant (LBR) potato cultivars and genotypes [13, 16] showed high susceptibility to PVS. To better understand this phenomenon and to characterize PVS at the biological and molecular levels, the complete genomic sequence of two PVS isolates from LBR potato genotypes were determined in this study.

In vitro destabilization of plant viruses and cDNA synthesis

DNA copies of a wide range of RNA viruses can be made by the direct addition of appropriately treated, purified virus particles to a reverse transcription reaction. Therefore, many problems associated with RNA isolation can be circumvented. Virus particles can be sufficiently destabilized by adjustments of salt content, buffer, pH or by the use of physical force supplied by a freeze/thaw cycle so that RNA in sufficient quantity and physical condition is available for the synthesis of in some cases, full length cDNAs. cDNAs have been made of viruses in the bromo-, poty-, carla-, ilar-, potex-, tobra and tobamovirus groups. Reported here are experiments with cowpea chlorotic mottle virus and bean common mosaic virus.

Complete nucleotide sequences and genome organization of a cherry isolate of cherry leaf roll virus

The complete nucleotide sequence of cherry leaf roll virus (CLRV, genus Nepovirus) from a naturally infected cherry tree (Prunus avium cv. Bing) in North America was determined. RNA1 and RNA2 consist of 7,893 and 6,492 nucleotides, respectively, plus a poly-(A) tail. Each RNA encodes a single potential open reading frame. The first 657 nucleotides of RNA1 and RNA2 are 99% identical and include the 5′-UTR and the first 214 deduced amino acids of the polyproteins following the first of two in-frame start codons. Phylogenetic analysis reveals close relationships between CLRV and members of subgroup C of the genus Nepovirus.

The complete nucleotide sequences of bean common mosaic necrosis virus strains NL-5, NL-8 and TN-1

Bean common mosaic virus (BCMV) and bean common mosaic necrosis virus (BCMNV) belong to the genus Potyvirus and are probably the most economically important viruses affecting common bean (Phaseolus vulgaris L.). Of the two viruses, BCMNV has been the most important in common bean production in North America, East Africa and Europe due to significant yield losses incurred from infected fields [10]. BCMV and BCMNV are transmitted in a non-persistent manner by several aphid species. All known strains of each virus can be highly seed-transmitted in bean and are distributed worldwide, in large part as a result of movement of contaminated seed [4, 5]. Drijfhout [4] originally separated the two viruses and their associated strains into eight different pathogroups based on host reaction to infection under specific temperature regimes. Strains NL-5 and NL-3 from Africa were originally assigned to the BCMV pathogroup VI by Drijfhout [4], and NL-8 was assigned to pathogroup III according to host responses in 12 different common bean host groups. The TN-1 strain from Tanzania was later assigned to pathogroup VI by Silbernagel [12]. Prior to 1992, all strains of BCMNV were designated as BCMV, at which time Vetten et al. [14] designated stains NL-3, NL-5 and NL-8 as serogroup A (BCMNV). All other known strains were assigned to serogroup B (BCMV) based on serological reactions to two highly specific monoclonal antibodies, and this was further supported by molecular analysis and ultrastructural comparisons [14]. Currently, several isolates of strain NL-3 have been sequenced and are available in GenBank; however, the sequences of strains NL-5, NL-8 and TN-1 are unavailable. Therefore, we report here the complete genome sequences of these three strains of BCMNV. Common bean seeds (variety ‘Dubbele Witte’) infected with NL-5, NL-8 and TN-1, respectively, were germinated in pots containing a mixture of peat moss and perlite (Sun Gro Horticulture Canada Ltd.) in the greenhouse, and plants were observed for symptoms. Tissues from symptomatic seedlings were evaluated for infection with BCMNV by direct ELISA using monoclonal antibodies specific for serogroup A, and also to serogroup B [11] to verify that there was no contamination with any strain of BCMV. Total nucleic acids were extracted from plant tissues, and cDNA was produced by reverse transcription using MMLV reverse transcriptase (Promega Corp., Madison, WI). Thermocycling parameters were optimized, and a final profile was employed that consisted of a single cycle of 2 min at 94 C; 25 cycles of 30 s at 94 C, 30 s at 50 C, and 3 min at 72 C; and a final extension for 7 min at 72 C. The initial reactions included oligo-dT24 as the reverse primer and a degenerate forward primer, 5’-GAA YAG CAA TGC NAT AG-3’, that produced a fragment approximately 3425 bp in length. The degenerate primer begins at nucleotide 6206, located in the NIa protein region. Subsequent reactions to complete the genome sequence were carried out using custom primer combinations based on the partial nucleotide sequence data obtained. The 5’-proximal region of the genomic sequence R. C. Larsen (&) USDA-ARS, 24106 N. Bunn Rd., Prosser, WA 99350, USA e-mail: richard.larsen@ars.usda.gov

Molecular characterization of a new tymovirus from Diascia ornamental plants.

Two tymoviruses were identified in plants of Diascia × hybrida ‘Sun Chimes™ Coral’ that exhibited chlorotic mottling and reduced growth. A strain of Nemesia ring necrosis virus (NeRNV) designated NeRNV-WA was detected in symptomatic plants; the deduced amino acid sequence is virtually identical to that of the previously reported NeRNV-Nf from Nemesia fruticosa. Sequence analysis also revealed the presence of a new tymovirus, and the entire genomic sequence of this virus was determined. The genome of 6,290 nucleotides was organized into three potential open reading frames (ORFs) typical of viruses in the genus Tymovirus. Based on sequence identity to tymovirus sequences, ORFs I to III encoded the replicase, movement protein and coat protein, respectively. Amino acid sequence identities to those of NeRNV-Nf were 84.8, 50.3 and 94.8%, respectively. The 5′-untranslated region could potentially form four hairpin structures. Secondary structure analysis of the 3′-terminus showed that the RNA can form a transfer-RNA-like structure that has an anticodon specific for histidine. Only 77.9% nucleotide identity was found when complete genomic sequences of this tymovirus from diascia and NeRNV-Nf were compared. The name Diascia yellow mottle virus (DiaYMV) is proposed for this new tymovirus.

Genomic characterization of pea enation mosaic virus-2 from the Pacific Northwestern USA

Pea enation mosaic virus (PEMV) infects several legume crops, including chickpea (Cicer arietinum), faba bean (Vicia faba), lentil (Lens culinaris) and pea (Pisum sativum). The virus caused yield losses of food legumes in the Pacific Northwestern US during 1983, 1987 and 1990 [1]. Our recent surveys of pea and alfalfa fields in the states of Washington and Idaho, USA, have shown the prevalence of PEMV on pea. PEMV consists of a large (RNA-1 or PEMV-1) and a small (RNA-2 or PEMV-2) single-stranded positive-sense RNA, which are encapsidated separately into distinct isometric particles [2]. PEMV-2 is one of the seven distinct virus species in the genus Umbravirus. The other species of this genus are Carrot mottle virus (CMoV), Carrot mottle mimic virus (CMoMV), Groundnut rosette virus (GRV), Lettuce speckles mottle virus (LSMV), Tobacco mottle virus (TMoV) and Tobacco bushy top virus (TBTV) [3]. Members of this group lack the coat protein (CP) gene in their genomes and depend on a helper virus for survival [4]. PEMV-2 in association with PEMV-1 (genus Enamovirus), form a symbiotic bipartite virus complex referred to as PEMV. It has been suggested that RNA-1 and RNA-2 are essential for PEMV infection [4, 5]. The RNA-1 (5706 nucleotides) encodes five major ORFs (1-5) that have nucleotide and amino acid sequence similarities with subgroup II luteoviruses [5]. While PEMV-1 provides the necessary encapsidation and vector-transmission abilities for PEMV-2, the latter provides PEMV-1 with long-distance movement and mechanical transmission functions. As part of an ongoing project to determine the genetic diversity of PEMV, we recently characterized the genome of PEMV-1 from the Pacific Northwest [6]. So far, there are only two complete genomic sequences of PEMV-2 in GenBank (NC_003853 and AY714213). To better understand sequence diversity of PEMV-2, the genome structure and organization of two PEMV isolates, one from Idaho (PEMV-2-ID) and one from Washington (PEMV-2-WA), were determined in this study. The PEMV-2 genome (*4.2 kbp), like that of other umbraviruses, predominantly consists of four ORFs (1-4), which perform diverse functions [4, 7, 8]. RNA-2 lacks a polyadenylation signal at its 30 end and contains a large 50 genome-linked protein [9]. ORF-1, at the 50 end of the virus genome, initiates after a short, 20-nt non-coding region (NCR) and encodes a putative 33-kDa protein of unknown function. ORF-2 overlaps with ORF-1 at its 30 end and potentially encodes a protein of 65 kDa through a frameshift mechanism. ORF-2 contains sequence motifs characteristic of a viral RNA-dependent RNA-polymerase (RdRp). The presence of a polymerase cassette in RNA-2 also reveals its independent replicative capabilities. Also, due to the presence of an octanucleotide frameshift signal ‘‘GGATTTTT’’ immediately upstream of the stop codon of ORF-1, ORF-1, along with ORF-2, is expressed by a -1 frameshift mechanism as a minor translation product of 97 kDa [4]. ORFs 3 and 4 in the genome occur after a nonThe sequences described here were deposited in the GenBank database with the following accession numbers: JF713435 for PEMV2-WA and JF713436 for PEMV-2-ID.