Whole genome characterization of Potato virus Y isolates collected in the western USA and their comparison to isolates from Europe and Canada.

Potato virus Y (PVY) is a serious potato pathogen that affects potato seed and commercial production crops. In recent decades, novel PVY strains have been described that cause necrotic symptoms on tobacco foliage and/or potato tubers. The major PVY strains that affect potato include PVY(O) and PVY(N), which have distinct serotypes that can be differentiated by immunoassay. Other economically important strain variants are derived from recombination events, including variants that cause tuber necrotic symptoms (PVY(NTN)) and PVY(O) serotypes that cause tobacco veinal necrosis (PVY(N)-W, PVY(N:O)). Although the PVY(NTN) and PVY(N)-W variants were first reported in Europe, apparently similar strains have been appearing in North America. Confirmation of the existence of these recombinant strains in North America is important, as is whether they spread from a common source or were derived by independent recombination. Whole genome sequencing can be used to positively identify strain variants and begin to address the issue of origins. Symptomology, serology, RT-PCR, and partial sequencing of the coat protein region were used to identify isolates of the PVY(NTN), PVY(N), PVY(NA-N), and PVY(N:O) for whole-genome sequencing. Sequencing confirmed the presence of PVY(NTN) and PVY(N) isolates that were >99% identical to European sequences deposited in GenBank in the 1990's. Sequences of the PVY(NA-N) and PVY(N:O) types were 99.0% and 99.5% identical to known sequences, respectively. There was no indication that recombinant strains PVY(NTN) or PVY(N:O) had different parental origins than recombinant strains previously sequenced. This is the first confirmation by whole-genome sequencing that "European"-type strain variants of PVY(N) and PVY(NTN) are present in North America, and the first reported full-length sequence of a tuber necrotic isolate of PVY(N:O).

The Occurrence of PVYO, PVYN, and PVYN:O Strains of Potato virus Y in Certified Potato Seed Lot Trials in Washington and Oregon.

Totals of 960 and 286 certified potato seed lots from locations across North America were planted in trials in Washington and Oregon, respectively, in 2001 to 2003 and tested for strains of Potato virus Y (PVY). The incidence of PVYO-infected lots averaged 16.4 and 25.9% in the Washington and Oregon trials, respectively. There was a general trend of increasing incidence of the PVYO, PVYN:O, and PVYN strains during this period, as evidenced by more infected cultivars, sites of seed origin, and number of seed growers providing infected seed lots. In particular, there was a dramatic increase in seed lots with the PVYN:O strain from 2002 to 2003. PVYN:O, in contrast to PVYO, which only causes yield reduction, also causes internal and external damage to tubers, making them unmarketable. In 2003, PVYN:O occurred in seed lots originating in eight states and three Canadian provinces. The increased incidence of PVYN:O was likely due to the difficulty in differentiating this strain from PVYO. The prevalence of PVY in potato seed lots documented herein poses a threat to potato production in the United States and suggests that current measures to reduce the incidence of this virus are inadequate.

Wheat streak mosaic virus--structural parameters for a Potyvirus.

Wheat streak mosaic virus is a Tritimovirus, a member of the Potyviridae family, which includes the very large Potyvirus genus. We have examined wheat streak mosaic virus by electron microscopy and fiber diffraction from partially oriented sols, and analyzed the results to estimate the symmetry and structural parameters of the viral helix. The virions have an apparent radius of 63 +/- 5 A. The viral helix has a pitch of 33.4 A +/- 0.6 A. There appear to be 6.9 subunits per turn of the helix, although we cannot completely eliminate values of 5.9 or 7.9 for this parameter.

Isolation and Partial Nucleic Acid Characterization of a New Isolate of Potato virus V with Distinct Biological and Serological Properties.

In vitro cultures of potato (Solanum tuberosum cv. Papa Amarillo) were imported into the United States and were subjected to routine pathogen screening for newly introduced varieties. These cultures indexed positive for several of the six viruses tested and thus were treated using standard methods for eliminating viruses in in vitro cultures. Although the therapy apparently freed the potato of the tested viruses, some of the cultures continued to exhibit mosaic symptoms when planted in the greenhouse. When examined by electron microscopy, these plants contained flexuous rods. Tested samples did not index positive with any potato virus-specific polyclonal antisera but reacted with the "universal" potyvirus monoclonal antibody. These results indicated that these samples were likely infected with an as yet undescribed potyvirus. Further investigation indicated that this virus is a new strain of Potato virus V (PVV) that is serologically distinct from the common strain. This is the first description of a distinct strain of PVV. This particular strain has diverged sufficiently from other isolates of PVV to no longer be detectable by some commonly used antisera, and therefore is a concern for the future restriction of PVV spread into regions of the world where it is not currently present.

Host Range and Characterization of Sunflower mosaic virus.

ABSTRACT Sunflower mosaic is caused by a putative member of the family Potyviridae. Sunflower mosaic virus (SuMV) was characterized in terms of host range, physical and biological characteristics, and partial nucleotide and amino acid sequence. Cells infected with SuMV had cytoplasmic inclusion bodies typical of potyviruses. Of 74 genera tested, only species in Helianthus, Sanvitalia, and Zinnia, all Asteraceae, were systemic hosts. Commercial sunflower hybrids from the United States, Europe, and South Africa were all equally susceptible. The mean length of purified particles is approximately 723 nm. The virus was transmitted by Myzus persicae and Capitphorus elaegni, and also was seedborne in at least one sunflower cultivar. Indirect enzyme-linked immunosorbent assay tests with a broad-spectrum potyvirus monoclonal antibody were strongly positive. SuMV-specific polyclonal antisera recognized SuMV and, to a lesser extent, Tobacco etch virus (TEV). When tested against a panel of 31 potyvirus-differentiating monoclonal antibodies, SuMV was distinct from any potyvirus previously tested. SuMV shared four epitopes with TEV, but had a reaction profile more similar to Tulip breaking virus (TBV). SuMV did not possess epitopes unique only to TBV. The predicted coat protein had a molecular weight of 30.5 kDa. The 3' end of the virus genome was cloned and sequenced. Phylogenetic analysis of the coat protein amino acid sequence revealed that SuMV is a distinct species within the family Potyviridae, most closely related to TEV.

First Report of the Necrotic Strain of Potato virus Y (PVYN) on Potatoes in the Northwestern United States.

More than 50 isolates of Potato virus Y (PVY) with characteristics of strains that cause tobacco veinal necrosis (PVYN) were obtained from potatoes (Solanum tuberosum L.) grown in the northwestern United States. These isolates are being characterized at the biological and molecular levels. Isolate RR1 was obtained from leaves of potato cv. Ranger Russet showing distinct mottling and leaf deformity, which is in contrast to the leaf-drop and necrosis usually observed with ordinary strains of PVY (PVYO) in this variety. Isolate AL1 was obtained from tubers of potato cv. Alturas showing distinct internal light brown rings and blotches. When RR1 and AL1 were transmitted to tobacco (Nicotiana tabacum L. cvs. Samsun NN and 423), they caused systemic veinal necrosis, including stem and petiole lesions typical of PVYN strains (2). Symptoms induced by RR1 and AL1 on tobacco appeared 9 to 11 days after inoculation, whereas some other isolates caused delayed veinal necrosis. All isolates that produced veinal necrosis on tobacco were detectable with PVY polyclonal antisera. Potato virus X was not detected by enzyme-linked immunosorbent assay in tobacco plants showing veinal necrosis. Some isolates, including AL1, failed to react in serological tests using PVYN-specific monoclonal antibodies obtained from three commercial sources. Other isolates, including RR1, were detectable with these monoclonal antibodies. Reverse transcription-polymerase chain reaction (RT-PCR) products obtained with primers specific for the coat protein (CP) open reading frame (ORF) were cloned and sequenced. AL1 possesses a CP more closely related to PVYO type isolates, which would account for its failure to react with PVYN monoclonal antibodies. In this regard, AL1 is similar to the PVYN-Wilga isolate (1). Other isolates that are detectable with the PVYN monoclonal antibodies possess a CP more consistent with N strains of the virus. Results of RT-PCR tests using primers derived from the P1 ORF sequence (3), and the restriction enzyme analysis and sequencing of the RT-PCR products, all suggest that AL1 and RR1 are related to European-type members of PVY tuber necrotic (NTN) or N strains. However, other isolates under investigation appear to be more closely related to previously reported North American NTN types (3). The symptomatology of these viruses on tobacco and potato, and the serological and molecular data clearly show that at least two distinct variants of PVYN have been found for the first time in a major potato production area of the United States, and pose a potential threat to the potato industry. References: (1) B. Blanco-Urgoiti et al. Eur. J. Plant Pathol. 104:811, 1998. (2) J. A. de Bokx and H. Huttinga. Potato virus Y. Descriptions of Plant Viruses. No. 242, CMI/AAB, Surrey, England, 1981. (3) R. P. Singh et al. Can J. Plant Pathol. 20:227, 1998.

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.

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

The 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.

The complete nucleotide sequence of apple mosaic virus RNA-3.

The complete nucleotide sequence of apple mosaic ilarvirus (ApMV) RNA-3 has been determined from cloned viral cDNAs. The 5' terminus of RNA-3 was determined by direct RNA sequencing, while the 3' end was determined by polyadenylation of genomic RNA and sub-cloning using oligo dT. ApMV RNA-3 is 2056 bases in length and encodes at least two open reading frames. It is similar in size and genome organization to the RNA-3 of other members of the Bromoviridae, which includes ilarviruses. The CP gene is in the 3' half of the molecule, and another large open reading frame is upstream of the CP gene and can potentially encode a protein of 32,400 daltons. This peptide is the same size and shows limited sequence homology to an open reading frame located at the 5' end of RNA 3 in tobacco streak and prune dwarf ilarviruses and alfalfa mosaic virus, which is postulated to be the viral movement protein. The nucleic acid sequence was not homologous to tobacco streak virus, prune dwarf virus, alfalfa mosaic virus or other members of the Bromoviridae. The 5'-non-coding region of ApMV RNA-3 contains a 15 base palindromic sequence which encloses a sequence resembling the ICR-2 regions of eukaryotic tRNA gene promoters.

The nucleotide sequence of apple mosaic virus coat protein gene has no similarity with other Bromoviridae coat protein genes.

A double-stranded cDNA was synthesized from in vitro polyadenylated apple mosaic virus (ApMV) RNA 3 using oligo(dT) and sequence-specific primers, and was cloned into plasmid vectors. A set of overlapping cDNA clones was used to determine the nucleotide sequence of RNA 4. ApMV RNA 4 was found to contain an open reading frame (ORF) of 666 nucleotides, which was flanked by 5' and 3' non-translated sequences of 55 and 264 nucleotides, respectively. The ORF encoding the coat protein was identified by comparing the predicted amino acid sequence with that obtained from direct protein microsequencing of the native viral coat protein. The ORF encodes a protein with an M(r) of 25,056. The nucleotide sequence of the ApMV coat protein gene showed no similarity to those of alfalfa mosaic virus, tobacco streak virus (TSV), brome mosaic virus or cucumber mosaic virus. The predicted amino acid sequence of the amino-terminal region of the ApMV coat protein is basic, rich in cysteine residues and may contain a zinc finger motif similar to that found in TSV.

The nucleotide sequence of satellite St. Augustine decline virus.

The complete nucleotide sequence of the satellite virus of St. Augustine decline virus (sSADV) has been determined. It comprises 824 nucleotides and contains a single, large open reading frame that corresponds to the coat protein of the satellite virus, as confirmed by direct amino acid sequencing. The genome contains two other potential open reading frames, similar to the organization of satellite Panicum mosaic virus (sPMV). These two satellite viruses share significant sequence homology at both the nucleotide and the amino acid levels; there are 36 nucleotide differences, resulting in five amino acid changes. Other than the close homology with sPMV, there is no homology with the known satellite viruses at the nucleotide or amino acid level, while there is some similarity in the hydropathy profiles of the coat proteins of these viruses.