Identification and genome analysis of tomato chlorotic spot virus and dsRNA viruses from coinfected vegetables in the Dominican Republic by high-throughput sequencing.

The Tomato chlorotic spot virus (TCSV) was first reported in the 1980s, having its occurrence limited to Brazil and Argentina. Due to an apparent mild severity in the past, molecular studies concerning TCSV were neglected. However, TCSV has disseminated over the USA and Caribbean countries. In Dominican Republic TCSV has been recently reported on important cultivated crops such as pepper and beans. In this work, we provide the first complete genome of a TCSV isolate from symptomatic plants in Dominican Republic, which was obtained by high-throughput sequencing. In addition, three dsRNA viruses from different virus families were identified coinfecting these plants Bell pepper endornavirus (BPEV), Southern tomato virus (STV) and Pepper cryptic virus 2 (PCV-2). Phylogenetic analysis showed that the Dominican Republic TCSV isolate has a close relationship with other TCSV isolates and a reassortant isolate between TCSV and Groundnut ringspot virus (GRSV), all found in USA. BPEV, STV and PCV-2 isolates from Dominican Republic were close related to corresponding American isolates. The possible biological implications of these virus-mixed infections are discussed.

The complete genome of the tospovirus Zucchini lethal chlorosis virus.

BACKGROUND: Zucchini lethal chlorosis virus (ZLCV) causes significant losses in the production of cucurbits in Brazil. This virus belongs to the genus Tospovirus (family Bunyaviridae) and seems to be exclusively transmitted by Frankliniella zucchini (Thysanoptera). Tospoviruses have a tripartite and single-stranded RNA genome classified as S (Small), M (Medium) and L (Large) RNAS. Although ZLCV was identified as a member of the genus Tospovirus in 1999, its complete genome had not been sequenced until now. FINDINGS: We sequenced the full-length genome of two ZLCV isolates named ZLCV-SP and ZLCV-DF. The phylogenetic analysis showed that ZLCV-SP and ZLCV-DF clustered with the previously reported isolate ZLCV-BR09. Their proteins were closely related, except the non-structural protein (NSm), which was highly divergent (approximately 90 % identity). All viral proteins clustered similarly in our phylogenetic analysis, excluding that these ZLCV isolates have originated from reassortment events of different tospovirus species. CONCLUSION: Here we report for the first time the complete genome of two ZLCV isolates that were found in the field infecting zucchini and cucumber.

Analysis of the coding-complete genomic sequence of groundnut ringspot virus suggests a common ancestor with tomato chlorotic spot virus.

Groundnut ringspot virus (GRSV) and tomato chlorotic spot virus (TCSV) share biological and serological properties, so their identification is carried out by molecular methods. Their genomes consist of three segmented RNAs: L, M and S. The finding of a reassortant between these two viruses may complicate correct virus identification and requires the characterization of the complete genome. Therefore, we present for the first time the complete sequences of all the genes encoded by a GRSV isolate. The high level of sequence similarity between GRSV and TCSV (over 90 % identity) observed in the genes and proteins encoded in the M RNA support previous results indicating that these viruses probably have a common ancestor.

First complete genome sequence of a tomato spotted wilt virus isolate from the United States and its relationship to other TSWV isolates of different geographic origin.

We report the first complete nucleotide sequence of a tomato spotted wilt virus (genus Tospovirus, family Bunyaviridae) isolate from the United States. The tripartite genome of PA01 consisted of L, M and S RNAs of 8914, 4765 and 2984 nt, respectively. Similarity percentages in nucleotide and amino acid sequence among PA01 and previously characterized TSWV isolates are provided here. Phylogenetic analysis on the RNA-dependent RNA polymerase (RdRp) gene placed PA01 in a different clade from an isolate from Hawaii that was partially characterized previously. Evidence of two putative reassortment events in the M segment, among PA01 and isolates from South Korea, Italy and Brazil, was found by phylogenetic and recombination analysis, further supporting a role for genetic exchange among isolates of different geographic origin in TSWV evolution.

Pepper necrotic spot virus, a new tospovirus infecting solanaceous crops in Peru.

Two virus isolates, T1 and T2, causing necrotic spots on leaves and stems of pepper and tomato, respectively, were isolated in the La Joya valley, Arequipa, Peru, in 2010. These two isolates were inoculated to differential hosts for tospoviruses and showed differential fitness: T1 induced necrotic local lesions in Vigna unguiculata, whereas T2 produced only chlorotic spots. The complete nucleotide sequence of the small (S) RNA from T2 and 1863 bp of the S RNA from T1 were determined. The deduced N protein sequence showed high amino acid identity (97%) between the isolates, indicating that the T1 and T2 are isolates of the same virus. Sequence comparisons indicated that the amino acid sequence of the N protein shared 53.49-87.98% identity with known American tospoviruses. Phylogenetic analysis of both the NSs and N proteins revealed that this new tospovirus belongs to the American group. We conclude that this tospovirus should be considered a member of a new species. The name Pepper necrotic spot virus (PNSV) is proposed.

Complete genome sequence of a tomato spotted wilt virus isolate from China and comparison to other TSWV isolates of different geographic origin.

Tomato spotted wilt virus (TSWV) is well established in most countries worldwide, while it is rarely reported in China. In this report, we have determined the complete nucleotide sequence of a TSWV isolate named TSWV-YN infecting tomato in Yunnan province in southwestern China. The tripartite genome of TSWV-YN was found to consist of L, M and S RNAs of 8910, 4773 and 2970 nt, respectively. The complete genome sequence and the sequence of each genomic region of TSWV-YN from China were compared to those of four other TSWV isolates from Brazil and Korea. The phylogenetic relationship of the Chinese TSWV-YN isolate to other TSWV isolates of different geographic origin, based on the nucleotide sequences of the glycoprotein (GP) and nucleocapsid (N) genes, was also analyzed in this study.

An RNA-dependent RNA polymerase gene of a distinct Brazilian tospovirus.

The tospoviral RNA-dependent RNA polymerases (RdRp), or L proteins, perform several conserved functions during virus replication in host cells. In this study, an L segment sequence of 9,040 bp from a new tospovirus (family Bunyaviridae) naturally infecting bean (Phaseolus vulgaris L.) plants was characterized. It encodes the largest RdRp gene known yet for this genus, with deduced 2932aa and a molecular mass of approximately 336 kDa. A Lysine-rich C-terminal extension was found, which apart from our isolate, was only recognized in another recently discovered tospovirus infecting Fabaceae, Soybean vein necrosis associated virus (SVNaV). Due to its distinct biological features and L protein-based phylogenetic analysis showing an almost equidistant position in comparison to Eurasian and American Tospovirus groups, as well as the clustering with SVNaV, we suggest the tentative name Bean necrotic mosaic virus for this unique isolate.

A distinct tospovirus causing necrotic streak on Alstroemeria sp. in Colombia.

A tospovirus causing necrotic streaks on leaves was isolated from Alstroemeria sp. in Colombia. Infected samples reacted positively with tomato spotted wilt virus (TSWV) antiserum during preliminary serological tests. Further analysis revealed a close serological relationship to tomato chlorotic spot virus (TCSV) and groundnut ringspot virus (GRSV). A major part of the S-RNA segment, encompassing the nucleocapsid (N) protein gene, the 5' untranslated region and a part of the intergenic region 3' of the N gene, was cloned and sequenced. The deduced N protein sequence showed highest amino acid identity (82%) to that of TCSV, indicating that the virus represents a new tospovirus species, for which the name Alstroemeria necrotic streak virus (ANSV) is coined. Phylogenetic analysis based on the N protein sequence revealed that this Alstroemeria-infecting tospovirus clustered with tospoviruses from the American continent. Frankliniella occidentalis was identified as potential vector species for ANSV.

Evidence for plant viruses in the region of Argentina Islands, Antarctica.

This work focused on the assessment of plant virus occurrence among primitive and higher plants in the Antarctic region. Sampling occurred during two seasons (2004/5 and 2005/6) at the Ukrainian Antarctic Station 'Academician Vernadskiy' positioned on Argentina Islands. Collected plant samples of four moss genera (Polytrichum, Plagiatecium, Sanionia and Barbilophozia) and one higher monocot plant species, Deschampsia antarctica, were further subjected to enzyme-linked immunosorbent assay to test for the presence of common plant viruses. Surprisingly, samples of Barbilophozia and Polytrichum mosses were found to contain antigens of viruses from the genus Tobamovirus, Tobacco mosaic virus and Cucumber green mottle mosaic virus, which normally parasitize angiosperms. By contrast, samples of the monocot Deschampsia antarctica were positive for viruses typically infecting dicots: Cucumber green mottle mosaic virus, Cucumber mosaic virus and Tomato spotted wilt virus. Serological data for Deschampsia antarctica were supported in part by transmission electron microscopy observations and bioassay results. The results demonstrate comparatively high diversity of plant viruses detected in Antarctica; the results also raise questions of virus specificity and host susceptibility, as the detected viruses normally infect dicotyledonous plants. However, the means of plant virus emergence in the region remain elusive and are discussed.

Sequence analysis of the glycoproteins of Tomato chlorotic spot virus and Groundnut ringspot virus and comparison with other tospoviruses.

The tospoviruses Tomato chlorotic spot virus (TCSV) and Groundnut ringspot virus (GRSV) cause high economic losses in several vegetable crops in Brazil. The glycoprotein precursor coding sequence was still not available for these two viruses. In this study, the 3' 4 kb M RNA of TCSV and GRSV genome was cloned and sequenced. The sequences were compiled with the available 5' region sequence (NSM gene and 5' UTR) of the same isolates. The M RNA of TCSV was deduced as formed by 4,882 nucleotides, while of GRSV by 4,855 nucleotides. Both M RNA comprised two ORFs in an ambisense arrangement. The vcRNA ORF coded for viral glycoprotein (G1/G2) precursor of TCSV (128.46 kDa) and for glycoprotein precursor of GRSV (128.16 kDa). Comparison of the TCSV and GRSV glycoprotein precursor proteins with those of other tospoviruses showed the highest identity with Tomato spotted wilt virus (81 and 79%, respectively). The amino acid sequence comparison of glycoprotein precursor between TCSV and GRSV revealed a high identity of 92%. However, the nucleotide sequence of the M RNA intergenie region showed only 78%. Phylogenetic analysis was done based on glycoprotein precursor and on M RNA intergenic region of tospoviruses and parameters on tospovirus taxonomic classification were discussed.

Detection of Tospovirus species by RT-PCR of the N-gene and restriction enzyme digestions of the products.

Tomato spotted wilt is a serious disease that affects several economically important crops. From the epidemiological point of view and for the development of a successful plan for transgenic resistance plants, the four known Tospovirus species must be discriminated at the molecular level. A RT-PCR assay using primers complementary to the N gene was used to detect and differentiate fourteen Argentinian isolates of Tospovirus from different crops and geographical areas. Extracts were reverse transcribed using a thermo-resistant reverse transcriptase and PCR reactions were performed for 30 min in a capillar thermo-cycler. The products were digested with restriction enzymes and three of the four described species were identified. Additionally, the results were confirmed by DAS-ELISA. The method described here is rapid and reliable.