An infectious full-length cDNA clone of potato virus Y(NTN-NW), a recently reported strain of PVY that causes potato tuber necrotic ringspot disease.

A full-length infectious cDNA clone of potato virus Y (PVY) was constructed based on an isolate of the PVY(NTN-NW) strain, SYR-II-2-8, which is able to cause potato tuber necrotic ringspot disease (PTNRD). Silent point mutations were introduced to modify sequences similar to the prokaryotic promoter elements detected at the 5' terminus of the P3 coding region of SYR-II-2-8. This, along with modification of the growing conditions of E. coli, led to the successful construction of a stable full-length infectious cDNA clone, named p2-8C3. The biological properties of p2-8C3 were identical to those of SYR-II-2-8, both of which induced PTNRD in potato, veinal necrosis in tobacco, and similar symptoms in the potato cultivars inoculated.

The simultaneous differentiation of Potato virus Y strains including the newly described strain PVY(NTN-NW) by multiplex PCR assay.

New recombinant strain and genotype of PVY, designated as PVY(NTN-NW) and SYR-III, respectively, shared properties with PVY(NTN) and PVY(N)W has been reported recently. PVY(NTN-NW) predominated in potato fields in Syria and was able to induce potato tuber necrotic ringspot disease (PTNRD). Due to the rapid spread of the recombinant strains of PVY which might be the case of PVY(NTN-NW), a specific and reliable detection method is an essential step to control this strain and minimize its spread. The shared properties of PVY(NTN-NW) and SYR-III with PVY(NTN) and PVY(N)W, however, complicate their identification involving multiple detection methods. Therefore, a multiplex polymerase chain reaction (PCR), that relies on a combination of previously published and newly designed primers was developed for the detection and identification of PVY(NTN-NW) and SYR-III in single or mixed infections with the main PVY strains, PVY(O), PVY(N), PVY(NTN) and PVY(N)W. In addition, the present PCR assay was able to detect the recombination points in the P1 region enabling the differentiation of the variable genotypes of the recombinant strains PVY(NTN-NW), PVY(NTN) and PVY(N)W. The reliability of this PCR assay was confirmed using a significant number of well characterized PVY isolates collected from Syria and Japan including those of PVY(NTN-NW), SYR-III, PVY(O), NA-PVY(N), PVY(N)W and PVY(NTN).

Molecular phylogenetic analysis of Barley yellow mosaic virus.

Complete nucleotide sequences of three strains (I, III, and IV) of Barley yellow mosaic virus (BaYMV) isolated in Japan were determined. The length of the genome was the same among the three strains; RNA1 was 7,642 nt and RNA2 was 3,585 nt. The molecular phylogenetic analysis showed that strain I was most closely related to the Chinese isolate, and these two strains formed one cluster with European isolates. Strains II, III, and IV, and the Korean isolate formed another cluster. Amino acid sequences of each viral gene product were compared among strains. The sequences of the VPg protein showed less identity among almost strains (less than 92%) than the sequences of other proteins (more than 93%). VPg is thought to be involved in interactions with host factors, especially initiation factor 4E (eIF4E) or eIF(iso)4E, and infection. Therefore, the relationship between amino acid substitutions and infection of host plants is analyzed.

Complete nucleotide sequence of a Japanese isolate of Chrysanthemum virus B (genus Carlavirus).

The complete nucleotide sequence of a Chrysanthemum virus B isolate from Japan (CVB-S) has been determined. The genomic RNA of CVB-S is 8,990 nucleotides long, excluding the poly(A) tail and, like that of other carlaviruses, contains six open reading frames (ORFs). Multiple alignment and phylogenetic analyses indicated that the phylogenetic relationship among members of the genus Carlavirus is very diverse, with phlox virus S being the closest relative of CVB. In aphid transmission tests, CVB-S was transmitted at a very low rate by Aphis gossypii, a new vector of the virus.

Complete nucleotide sequence and the genome organization of Patchouli mild mosaic virus RNA1.

The nucleotide sequence of the RNA1 of Patchouli mild mosaic virus (PatMMV) has been determined. It contains 5,957 nucleotides excluding the 3'-terminal poly(A) tail and contains a single long open reading frame (ORF) of 5,613 nucleotides extending from nucleotide 235 to 5847. The predicted polyprotein encoded by the long ORF is 1,870 amino acids in length with a molecular weight of 210 kD. The conserved residues of RNA-dependent RNA polymerase, cysteine protease, purine NTP-binding domain and a cofactor for viral protease are present in a 210-kD polyprotein. As PatMMV RNA showed high sequence identity (81-97%) with BBWV-2 RNA, PaMMV may be one strain of BBWV-2.

[Current spread of HPN and HEN: issue for making choice in home care].

Recently a number of patients have received home enteral nutrition (HEN) as well as home parenteral nutrition (HPN) in Terumo's home healthcare network 'Home-Joint'. This study was undertaken to compare the patients who received HEN with those who received HPN. 1. HPN patients in 2000. Number: 1,789 (45% females), age: 0-101 (mean: 66 years old, 48% over 70 years old), diagnosis: cancer 54%, therapeutic periods: 0-916 days (mean: 80 days). 2. HEN patients in 2000. Number: 736 (40% females), age: 1-101 (mean: 50 years old, 33% over 70 years old), diagnosis: Crohn's disease 44%, therapeutic periods 0-1,618 days (mean: 183 days). 3. Adoption of HPN and HEN in home care agents. HPN only 66%, HEN only 24%, both HPN and HEN 10%. It suggested that following two issue take place against making choice of HPN or HEN in home nutrition care. 1) Adoption of both HPN and HEN in more clinics. 2) Establishment of the guidelines for systematic HPN/HEN choice.

[Current spread of HPN and its forecast--especially in the dual therapy of HPN and HOT].

In 1999, Terumo's home healthcare network 'Home-Joint' managed more than 1,000 patients who underwent home parenteral nutrition (HPN). This spread of HPN led some patients to undergo another home medical treatment during the same period. This report evaluates the patients who underwent dual home therapy (HPN and HOT: home oxygen therapy) in comparison with patients who underwent HPN only. 1. Single HPN patients in 1999. Number: 1,034 (male 57%), age: 1-99 (mean 65, 43% over 70), diagnosis: cancer 52%, therapeutic periods: 1-437 days (mean 81). 2. Dual HPN-HOT patients in 1999. Number: 27 (male 70%), age: 44-94 (mean 75, 70% over 70), diagnosis: cancer 59%, therapeutic periods: HPN 1-388 days (mean 91), HOT 1-827 days (mean 99). 3. Adoption of dual HPN-HOT therapy. It is suggested that the dual HPN-HOT therapy may be adopted for at least the following two types of patients. 1) Pulmonary failure with HPN, 2) Malnutrition on HOT.

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.

Sequence variation of the rDNA ITS regions within and between anastomosis groups in Rhizoctonia solani.

Sequence analysis of the rDNA region containing the internal transcribed spacer (ITS) regions and the 5.8s rDNA coding sequence was used to evaluate the genetic diversity of 45 isolates within and between anastomosis groups (AGs) in Rhizoctonia solani. The 5.8s rDNA sequence was completely conserved across all the AGs examined, whereas the ITS rDNA sequence was found to be highly variable among isolates. The sequence homology in the ITS regions was above 96% for isolates of the same subgroup, 66-100% for isolates of different subgroups within an AG, and 55-96% for isolates of different AGs. In neighbor-joining trees based on distances derived from ITS-5.8s rDNA sequences, subgroups IA, IB and IC within AG-1 and subgroups HG-I and HG-II within AG-4 were placed on statistically significant branches as assessed by bootstrap analysis. These results suggest that sequence analysis of ITS rDNA regions of R. solani may be a valuable tool for identifying AG subgroups of biological significance.

Formation of multimers of cucumber mosaic virus satellite RNA.

Double-stranded RNA multimers of cucumber mosaic virus (CMV) satellite RNA were detected in CMV-infected plants. RT-PCR showed that plus-sense and minus-sense monomers and plus-sense multimers of satellite RNA were present. Multimeric minus-sense RNA was not present except in the form of multimeric dsRNA. Sequence analysis of 52 cloned junction regions in head-to-tail repeats of unit-length satellite RNA indicated that about 35% of the junction sequences were precise fusions of monomer units, 56% lacked sequence of the 5' component, and 10% lacked sequence of both 3' and 5' components. No junction contained additional nucleotides. Deletions at the junction regions may have accumulated during CMV multiplication in inoculated plants. These data suggest that replicase is not released from the template during synthesis of multimeric molecules of satellite RNA.

The nucleotide sequence of RNA-1 of raspberry bushy dwarf virus.

Raspberry bushy dwarf virus (RBDV) has isometric, 33 nm diameter particles and a bipartite RNA genome. Sequencing of the larger component (RNA-1) showed that it consists of 5449 nucleotides and contains one large open reading frame encoding a putative translation product with a calculated M(r) of 190,000. Comparisons of this polypeptide with non-structural proteins of other plant viruses revealed significant homologies with those of alfalfa mosaic virus (AlMV), brome mosaic virus (BMV), cucumber mosaic virus (CMV) and tobacco mosaic virus (TMV). Thus RBDV belongs to the supergroup of 'Sindbis-like' plant viruses. The translation product of RBDV RNA-1 contains motifs characteristic of proteins with polymerase, methyltransferase and helicase activities, suggesting that this protein is involved in the replication of the viral RNA. Thus in RBDV, as in TMV, all three functional domains are combined in the single protein, whereas in AlMV, BMV and CMV these domains are distributed over the proteins encoded by RNA-1 and RNA-2. These findings support the idea that RBDV should be placed in a distinct virus genus for which the name idaeovirus has been proposed.

Nucleotide sequence of raspberry bushy dwarf virus RNA-2: a bicistronic component of a bipartite genome.

Northern blot analysis with cDNA probes to RNA-3 (1 kb) of raspberry bushy dwarf virus (RBDV) revealed extensive sequence homology with RBDV RNA-2 (2.2 kb). Nucleotide sequencing showed that RNA-2 contains two large open reading frames (ORFs), of 1074 (5' ORF) and 822 (3' ORF) bases. The 3' ORF is virtually identical in sequence to RNA-3, which encodes the Mr 30509 (30K) coat protein. The 5' ORF encodes an Mr 38860 (39K) protein which slightly resembles the 32K protein encoded by RNA-3 of alfalfa mosaic virus (AlMV). RBDV RNA-2 resembles AlMV RNA-3 in being bicistronic and encoding a coat protein at the 3' end. Comparing RNA-1 and RNA-2 of RBDV, only the 18 3'-terminal nucleotides are identical in sequence but the 3'-terminal 71 nucleotides of each RNA species have the potential to form similar stem-loop structures.

Relationships between the cryptic and temperate viruses of alfalfa, beet and white clover.

Small isometric virus particles containing double-stranded RNA have been independently reported in Europe and Japan from apparently healthy alfalfa (Medicago sativa), beet (Beta vulgaris), and white clover (Trifolium repens). They have been called cryptic viruses in Europe and temperate viruses in Japan. Serological comparison using immunoelectron microscopy, and polyacrylamide gel electrophoresis of the RNAs indicate that alfalfa cryptic and temperate viruses are the same, beet cryptic virus is probably a mixture of two different viruses, one of which is similar to or the same as beet temperate virus, and white clover temperate virus is a mixture of at least three different viruses, two of them indistinguishable from white clover cryptic viruses 1 and 2, respectively, and the third very likely the same as white clover cryptic virus 3.