Melon Yellow Spot Virus Research Articles

Biological and molecular characterization of tospoviruses in Thailand

Twenty-eight isolates of tospoviruses associated with tomato, pepper, cucurbits, peanut, and Physalis plants collected from fields in different regions of Thailand were characterized. On the basis of N gene and protein sequence relationships, three tospoviruses were identified, namely Watermelon silver mottle virus (WSMoV), Capsicum chlorosis virus (CaCV), and Melon yellow spot virus (MYSV). CLUSTAL analysis of selected N protein sequences showed different isolates of CaCV in three distinct clades. Based on necrosis symptoms on tomato and their 93% identity to CaCV isolates in the other two clades, CaCV-TD8, CaCV-AIT and CaCV-KS16-Thailand tomato tospovirus were designated as CaCV-tomato necrosis strain. A phylogenetic tree based on the 413-amino-acid Gc fragment of the CaCV-Pkk isolate supported the existence of three distinct CaCV clades. Vigna unguiculata produced concentric rings useful for discriminating the Thai CaCV peanut isolates from tomato or pepper isolates. By using reverse transcription polymerase chain reaction with species-specific primers, the three tospoviruses could be detected in mixed infections in watermelon and Physalis, as well as in the bodies of thrips vectors, Thrips palmi and Scirtothrips dorsalis, collected from fields.

プライマーカクテルを用いたＲＴ‐ＰＣＲによるキュウリおよびメロンに感染する８種ウイルスの検出法

Multiplex primers were calculated using a web-based program, muPlex in order to detect eight cucurbit-infecting viruses: Watermelon mosaic virus (WMV), Zucchini yellow mosaic virus (ZYMV), Cucumber mosaic virus (CMV), Kyuri green mottle mosaic virus (KGMMV), Papaya ringspot virus (PRSV), Melon yellow spot virus (MYSV), Cucumber green mottle mosaic virus (CGMMV), and Beet pseudo-yellows virus (BPYV). The predicted length of the amplified fragments ranged from 283 by to 1, 144 bp, and were distinct from each other. RT-PCR conducted using a mixture of all the primer pairs (primer cocktail) amplified the appropriate fragments corresponding to each virus species. Non-specific fragments were not observed. Two fragments were simultaneously amplified when RNA mixtures containing two distinct viral species were used as templates. However, if the concentration of one viral RNA was too high, the other virus could not be detected.

Characterization of Tomato fruit yellow ring virus: a new Tospovirus species infecting tomato in Iran

During surveys for Tomato spotted wilt virus (TSWV) in the major tomato production areas of Iran, tomato fruits showing a bright yellow ring pattern were observed, from which a tospovirus was isolated by mechanical transmission to herbaceous hosts. The virus originated from the Varamin area where >30% of the tomato crop was infected with TSWV. Although typical tospovirus virions were found by electron microscopy, a panel of antibodies used for detection and identification of tospoviruses by ELISA failed to react with this isolate. An antiserum against virus nucleoprotein preparations (DSMZ-AS0526) reacted specifically with plants infected with the new isolate, but not with other tospoviruses. Analysis of the S RNA genome comprising the N-protein gene (275 aa; GenBank accession number AJ493270) and a segment of the L gene (GenBank accession number AJ493271), to determine the taxonomic position of the virus, confirmed that this is a distinct tospovirus species, sharing 72–75% nucleoprotein amino acid identity with isolates of Iris yellow spot virus (IYSV), its closest relative. A 42–45% N-protein similarity with N-gene sequences of tospoviruses from Asia (Watermelon silver mottle virus, Melon yellow spot virus and Watermelon bud necrosis virus), and only 28–30% amino acid sequences in common with tospoviruses from Europe–Africa (TSWV, Tomato chlorotic spot virus and Groundnut ringspot virus), further confirmed its taxonomic position. The absence of any detectable serological relationships, and the limited sequence similarity with IYSV and other tospoviruses, justify the classification of this new virus as a distinct species of the genus Tospovirus for which the name Tomato fruit yellow ring virus (TFYRV) is proposed. This virus has an extensive host range (Ghotbi et al., 2005), but is found most often in tomato and in mixed virus infections with TSWV.

Nucleotide sequence of melon yellow spot virus M RNA segment and characterization of non-viral sequences in subgenomic RNA

The nucleotide sequence of melon yellow spot virus (MYSV) M RNA segment was determined. The M RNA segment contains one open reading frame (ORF) encoding 308 amino acids (aa) in the sense orientation and another ORF encoding 1,127 aa in the complementary orientation, which were homologous to the NSm protein and G1/G2 glycoprotein precursor (Gp) protein, respectively. Amino acid sequences identities with the other tospovirus suggested that MYSV is closely related to groundnut bud necrosis virus and watermelon silver mottle virus. To analyze subgenomic RNA of the M RNA segment, RNA transcripts corresponding to the NSm and Gp genes were specifically amplified, and the nucleotide sequence of the 5' terminal region was determined. Sequence analysis of the NSm and Gp transcripts showed that they had a non-viral sequence 12-18 and 10-18 nucleotides long, respectively. Although these sequences varied considerably, in more than half of the cases, a cytosine residue was observed at the 3' end of the non-viral leader sequence, which suggests that the viral transcriptase prefers certain cap-donor sequences harboring a 3'CA dinucleotide.

A One-Step Reverse Transcription-Polymerase Chain Reaction System for the Simultaneous Detection and Identification of Multiple Tospovirus Infections

ABSTRACT A one-step reverse transcription-polymerase chain reaction (RT-PCR) method has been developed for the simultaneous detection and identification of multiple tospoviruses that infect plants. The RT-PCR system is composed of six primers in a single tube: a universal degenerate primer and five virus species-specific primers. Amplifications resulted in an 848-bp PCR product for Watermelon silver mottle virus, 709-bp for Tomato spotted wilt virus, 589-bp for Impatiens necrotic spot virus, 511-bp for Melon yellow spot virus, and a 459-bp amplicon for Iris yellow spot virus. This system enables the simultaneous detection of at least three types of tospovirus infections, in addition to their species identities, from five possible tospoviruses studied, on the basis of their S RNA combinations. This multiplex RT-PCR system was applied to the detection of tospovirus in ornamental crops cultivated in fields and shows potential for epidemiological studies.

メロン黄化えそウイルスのＬ ＲＮＡ分節の全塩基配列

Melon yellow spot virus causes yellow spots or necrosis on melon (Cucumis melo) and cucumber (C. sativas) plants in Japan. This virus belongs to the genus Tospovirus, which has a tripartite RNA genome. The complete nucleotide sequence of the largest RNA segment (L RNA) of an MYSV isolate was determined and analyzed. The L RNA segment was 8, 918 nucleotide (nt) long and had one open reading frame (ORF) in the complementary sense. Deduced amino acid sequence of the ORF was 2, 870 amino acids and contained motifs of RNA-dependent RNA polymerase. Comparison of the nucleotide and deduced amino acid sequences with those of other tospoviruses revealed that MYSV was closer to Groundnut bud necrosis virus (71.0% identity) and Watermelon silver mottle virus (70.6% identity) than other tospoviruses. The 19-nt terminal sequences in the 5' and 3' untranslated regions were incomplete inverted complementary sequences. They were not similar to those in the S RNA of MYSV, but were similar to those of the L RNA of known tospovirus species.

MELON YELLOW SPOT VIRUS AND WATERMELON SILVER MOTTLE VIRUS: OUTBREAK OF CUCURBIT INFECTING TOSPOVIRUS IN JAPAN

Melon yellow spot virus (MYSV) and Watermelon silver mottle virus (WSMoV) are distinct virus species belonging to the genus Tospovirus in the Family Bunyaviridae. They have quasi-spherical virions with lipid envelopes where three single-stranded RNA segments are contained, and are persistently transmitted by Thrips palmi. WSMoV was first identified in 1982, and it has been causing problems on watermelon, wax gourd, melon, cucumber and bitter gourd productions in Japan and Taiwan. MYSV was first identified in 1992, and it has been causing severe damages on melon and cucumber productions in Japan. Nucleotide sequences of the smallest RNA segments of WSMoV and MYSV were analyzed to examine genetic relationships. Identities of the nucleocapsid protein genes of WSMoV or MYSV with other tospovirus species suggested that MYSV and Physalis severe mottle virus (PYSV) were considerably similar. Identities of the nucleocapsid protein genes and the non-structural genes of two isolates of MYSV from cucumber plants with those of reported MYSV with those with PSMV were more than 96%. Therefore, PSMV and MYSV should be classified as the same species. Comparison of Japanese isolates and Taiwanese isolates of WSMoV suggested that they are genetically related each other, except for a 20 nucleotide insertion sequence was found in Japanese isolates. RT-PCR assay with specific primers of WSMoV or MYSV were examined. RT-PCR with WSMoV specific primers WN5 (5' -GCACACACATAGGTATAACT-3') and WN3 (5' -TCCTTTGTAAACACCATG-3') detected approximately 1.1 kb DNA fragment from WSMoV-infected samples. RT-PCR with MYSV specific primers MN5 (5' TATTTCATTCAACTAGTTAA3') and MN3 (5' ATTCAACATCAGCAAGTCAA3') detected approximately 1.0 kb DNA fragment only from MYSV-infected samples. No significant amplification was detected from other tospovirus -infected samples or healthy samples.

RT-PCR for detecting five distinct Tospovirus species using degenerate primers and dsRNA template

RT-PCR procedures for detection of multiple species of tospovirus from plant tissues were investigated. Downstream primers were designated from the 3′ untranslated sequences of the S RNA. An upstream primer was designated from the degenerated sequences of the nucleocapsid protein. Approximately 450 bp DNA fragments were detected when Tomato spotted wilt virus (TSWV)- or Impatiens necrotic spot virus (INSV)- infected tissues were examined. Approximately 350 bp DNA fragments were detected when Watermelon silver mottle virus (WSMoV)- or Melon yellow spot virus (MYSV)-infected tissues were examined. Template RNA was extracted using CF 11 cellulose powder, and nonspecific amplification became unnoticeable when double-stranded RNA was used. The amplified fragments of WSMoV were differentiated from those of MYSV by AluI or TaqI digestion. The amplified fragments of TSWV were differentiated from those of INSV by DraI or HindIII digestion. An alstroemeria plant that was infected with an unidentified tospovirus was also examined, and a 350 bp fragment that was 97% identical to Iris yellow spot virus was detected. This method, therefore, detected at least five distinct Tospovirus species.

メロン黄化えそウイルス（Ｍｅｌｏｎ ｙｅｌｌｏｗ ｓｐｏｔ ｖｉｒｕｓ）によるキュウリ（Ｃｕｃｕｍｉｓ ｓａｔｉｖｕｓ）の黄化えそ病

Cucumber plants (Cucumis sativus) with necrotic spots, yellowing and mosaic symptoms were found in Kochi Prefecture, Japan in 1995. The causal agent was identified as Melon yellow spot virus (MYSV) on the basis of host reactions, stability in crude sap, particle morphology, transmission by Thrips palmi, serological relationships and the identity of amino acid sequence of the N protein. This is the first report of the occurrence of MYSV on cucumber, and the name “potted wilt”s was proposed for the disease.

Melon yellow spot virus: A Distinct Species of the Genus Tospovirus Isolated from Melon

ABSTRACT A tospovirus-like virus recovered from netted melon was transmitted by Thrips palmi in a persistent manner but had different cytopathological features from tospoviruses previously reported. Viral nucleocapsid (N) was purified with two protective reagents, 2-mercaptoethanol and L-ascorbic acid, and RNA extracted from the viral nucleocapsid was used for genomic analysis. The virus had a genome consisting of three single-stranded RNA molecules. The open reading frame on the viral complementary strand, located at the 3' end of the viral S RNA, encoded the N protein. The 3' terminus of this RNA also contained an eight-nucleotide sequence similar to the conserved sequence at the 3' end of genomic RNA molecules of tospoviruses. These features of the viral genome are identical to those of tospoviruses; therefore, this virus is considered to belong to the genus Tospovirus. Its N protein comprised 279 amino acids and had a molecular mass of 31.0 kDa. Comparisons of its amino acid sequence with those of known tospoviruses revealed less than 60% identity. This melon virus is concluded to be a distinct species in the genus Tospovirus, and the name Melon yellow spot virus is proposed.

メロン黄化えそウイルスのＳ ＲＮＡの特性

The full nucleotide sequence of the S RNA segment of Melon yellow spot virus (MYSV) was determined to be 3232 nucleotides long and to have every feature of the S RNAs of tospoviruses reported so far. From an amino acid sequence comparison of nonstructural proteins (NSs), MYSV is most closely related to two tospoviruses in serogroup IV. MYSV had nucleotide and amino acid sequence identities over 96% with Physalis severe mottle virus in all regions of S RNA except in an intergenic region and in deduced open reading frames, including that on complementary strand RNA.