Nobumichi Sako

Potato Tuber Necrotic Ringspot Disease Occurring in Japan: Its Association with Potato virus Y Necrotic Strain

Unfamiliar necrotic symptoms on or within potato tubers of cultivars Nishiyutaka and Dejima were observed in Nagasaki prefecture, Japan, in 1992. Symptoms were typically on the surface of the tuber, which either protruded at first and then became sunken, or showed necrotic spots, with necrosis within the tubers. Symptoms sometimes appeared at harvesting but more often appeared after storage for several months. Investigations revealed that the causal agents of the disease were isolates of Potato virus Y necrotic strain (PVYNTN) and the disease was potato tuber necrotic ringspot disease (PTNRD), previously reported in Europe and Lebanon. Five potato cultivars were inoculated with an isolate from necrotic tubers. The highest percentage of progeny tubers showing PTNRD was found in cv. Nishiyutaka (23.3%). In contrast, cvs. Shima-bara, Mayqueen, and Danshaku showed a low percentage of PTNRD. Additional potato tubers with PTNRD were also observed after storage of the tubers. To investigate the relatedness between isolates of PVYNTN and of necrotic strain PVYN, previously isolated in Japan, Nishiyu-taka was inoculated with an isolate of PVYN, which also induced PTNRD. Nucleotide sequences of coat protein (CP) genes of six PVYNTN isolates were determined. The CPs were 267 amino acids in length, with a substitution of one or no amino acid among each of the six isolates. The phylogenetic relationship based on nucleotide sequences of CP genes showed that these six PVYNTN isolates clustered together with PVYN isolates. This is the first report of PTNRD caused by PVYNTN isolates in Asia.

Different helper factors associated with aphid transmission of some potyviruses

Myzus persicae transmitted watermelon mosaic virus (WMV) after acquiring it through artificial membranes from a solution of purified virus mixed with the soluble fraction from infected leaf extracts or by prefeeding on the soluble fraction before acquiring purified virus. WMV-induced helper factor assisted M. persicae in transmitting purified turnip mosaic virus (TuMV) but not potato virus Y (PVY). TuMV-induced helper factor from infected leaves was ineffective for the transmission of purified WMV or PVY. PVY-induced helper factor from infected leaves was capable of helping the transmission of TuMV but not that of WMV. The results indicate that there are at least three distinct helper factors with different specificity associated with potyviruses.

The complete nucleotide sequence of turnip mosaic virus RNA Japanese strain.

SummaryThe complete nucleotide sequence of the RNA genome of turnip mosaic virus Japanese strain (TuMV-J) has been determined from five overlapping cDNA clones and by direct sequencing of viral RNA. The RNA sequence was 9833 nucleotides in length, excluding a 3′ terminal poly(A) tail. An AUG triplet at position 130–132 was assigned as the initiation codon for the translation of the genome size viral polyprotein which would consist of 3164 amino acid residues. Interestingly, a different amino acid sequence (continuous twenty amino acids) within the cytoplasmic inclusion protein between TuMV-J and Canadian strain of TuMV was observed, caused by an insertion and a deletion of nucleotides.

Nucleotide sequences of the helper component-proteinase genes of aphid transmissible and non-transmissible isolates of turnip mosaic virus

SummaryWe have compared nucleotide sequences of the helper component-proteinase (HC-Pro) coding region of aphid transmissible (isolate 1) and non-transmissible (isolate 31) isolates of turnip mosaic virus (TuMV). HC-Pro coding regions of both TuMV isolates 1 and 31 were 1,374 nucleotide long. The nucleotide sequence homology between these isolates was 93.5%, with 89 nucleotides substitution. The nucleotides of HC-Pro regions of two isolates of TuMV genomes encoded 458 amino acids of Mr 51,746 (isolate 1) and Mr 51,764 (isolate 31). The deduced amino acid sequence homology between these isolates was 98.7% with six different amino acids. These amino acids appeared to regulate the activity of HC-Pro needed for aphid transmissibility of TuMV.

Complete nucleotide sequence and synthesis of infectious in vitro transcripts from a full-length cDNA clone of a rakkyo strain of tobacco mosaic virus

SummaryThe complete nucleotide sequence of the genome of a rakkyo strain of tobacco mosaic virus (TMV-R), which exhibits distinct host range differences from the common strain of TMV, was determined. The overall nucleotide sequence homology with TMV-U1 (a common strain of TMV) is 94.2%. The amino acid sequence homologies of the four encoded proteins (180K, 130K, 30K, coat protein) are from 95.9% to 98.0% compared with TMV-U1. To facilitate the analysis of the novel host range of TMV-R, a full-length clone of the genome containing a bacteriophage T7 RNA polymerase promoter was assembled from two cDNA clones and designated pRF3. In vitro transcripts derived from pRF3 were highly infectious. The infections of RF3, wild-type TMV-R, and U3/12-4 (derived from pU3/12-4, an infectious clone of TMV-U1) were compared onNicotiana tabacum cv. Bright Yellow (BY) plants. No systemic mosaic symptoms were observed on plants inoculated with RF3 and TMV-R, while BY plants inoculated with U3/12-4 developed distinct mosaic symptoms on the upper leaves 8–9 days post-inoculation. The green fluorescent protein (GFP) gene was introduced into pRF3 and pU3/12-4 by replacing the coat protein gene to get two GFP expressing chimeric virus clones: pR-GFP or pU1-GFP. Transcripts from pU1-GFP produced strong fluorescence when inoculated onto BY leaves, while those from pR-GFP produced only very faint fluorescence.

A single amino acid substitution at N-terminal region of coat protein of turnip mosaic virus alters antigenicity and aphid transmissibility

SummaryThe antigenic activity of the N-terminal region of coat protein of turnip mosaic virus (TuMV) aphid transmissible strain 1 and non-transmissible strain 31 was examined by using a panel of monoclonal antibodies (MAbs) raised against the two virus strains as well as antisera raised against several synthetic peptides from the N-terminal region of the protein. The reactivity of these antibodies was tested in ELISA and in a biosensor system (BIAcore Pharmacia) using virus particles, dissociated coat protein and synthetic peptides as antigens. Substitution of a single amino acid at position 8 in the coat protein of TuMV strain 1 abolished any cross-reactivity between MAbs to strain 1 and the substituted peptide (strain 31) in ELISA although some cross-reactivity was apparent in BIAcore inhibition experiments. In reciprocal tests with MAbs to strain 31 no cross-reactivity with the heterologous peptide was detected in either type of assay. The amino acid residue present at position 8 appears to play a critical role in the binding capacity of MAbs specific for the N-terminal region of TuMV. Antiserum to a synthetic peptide corresponding to residues 1–14 of the protein of TuMV strain 1 was found to react strongly with dissociated coat protein and intact virus particles and was able to inhibit the aphid transmission of the virus. Antiserum to the corresponding peptide of strain 31 did not have this capacity.

Nucleotide sequences and expression in Escherichia coli of the coat protein genes from two strains of melon necrotic spot virus.

SummaryThe nucleotide sequences of coat protein (CP) genes of two Japanese strains of melon necrotic spot virus·(MNSV-NH and MNSV-S) were determined. The size of the CP genes of both strains was 1170 nucleotides. Comparisons of the deduced amino acid sequences among MNSV strains showed more than 95% homology, and those among other carmoviruses showed 31–34% homology. cDNAs of MNSV CP genes were cloned into anEscherichia coli expression fusion vector and MNSV-NH and MNSV-S CPs were successfully produced. Furthermore, synthetic oligonucleotide primers were used for differentiating MNSV strains by reverse transcription-polymerase chain reaction method.

Characterization of epitopes recognized by monoclonal antibodies to aphid transmissible and non-transmissible strains of turnip mosaic virus

SummaryFourteen monoclonal antibodies (MAbs) were prepared against two strains of turnip mosaic virus (TuMV) differing in aphid transmissibility. Serological specificity of fourteen MAbs against the two strains was tested by indirect ELISA. Three MAbs were able to distinguish aphid transmissible TuMV strain 1 from non-aphid transmissible strain 31 while four MAbs reacted only with strain 31. No cross-reactivity between the two strains was found using these specific MAbs. Based upon the ability of Mab to inhibit the reaction of other MAbs, antibody competition test indicated that fourteen MAbs recognized six different epitopes on the virus particle; MAbs specific to strain 1 recognized two epitopes while MAbs specific to strain 31 also recognized two epitopes. The remaining two epitopes are common. Since the six amino acid differences between the coat proteins of the two strains were found at the N-terminal regions, MAbs specific to strain 1 or 31 bound to the different epitopes on the N-terminal regions in coat proteins of the two strains.

Production of avian antibodies to three potyviruses in coturnix quail

Avian antibodies against three potyviruses were produced in a small bird, coturnix quail (Coturnix coturnix japonica Temminck et Schlegel), with 15-60 micrograms of purified virus preparations. Intramuscular injections of immunogen with Freunds incomplete or complete adjuvant into the birds did not result in higher titer of antibody compared to that of control birds given intravenous injections. Quail egg yolk antibody was as useful as hen antibody for indirect-ELISA and allowed virus to be detected in purified preparation (10-50 ng/ml) and in crude extracts (10(-6)-10(-7) dilution). The advantages of using quail to produce avian antibodies are discussed.

Cross-reactive and major virus-specific epitopes are located at the N-terminal halves of the cylindrical inclusion proteins of turnip mosaic and zucchini yellow mosaic potyviruses.

Summary. To investigate the antigenic nature of cylindrical inclusion proteins (CIPs) of the potyviruses Turnip mosaic virus (TuMV) and Zucchini yellow mosaic virus (ZYMV), monoclonal antibodies (MAbs) against the two CIPs were produced and epitopes on the CIPs were localized using Escherichia coli-expressed CIP fragments in Western blot analysis. All 23 MAbs against ZYMV CIP reacted only with ZYMV CIP. In contrast out of the 18 MAbs produced against TuMV CIP, 14 MAbs were TuMV CIP-specific while the remaining four MAbs cross-reacted with both CIPs. The four cross-reactive MAbs recognized two distinct epitopes in the N-terminal half of TuMV CIP corresponding to amino acid residues 103–119 and 224–237. Thirteen out of 14 TuMV CIP-specific MAbs recognized two distinct epitopes within residues 1–102 and 120–214, while the other one recognized an epitope within residues 301–644. On the other hand, 21 out of 23 ZYMV CIP-specific MAbs recognized epitopes within residues 1–118, while the remaining two recognized epitopes within residues 301–522. These results suggest that cross-reactive and major virus-specific epitopes are located at the N-terminal half of the respective CIPs.