The complete genome of the tospovirus Zucchini lethal chlorosis virus
© The Author(s). 2016
Received: 7 March 2016
Accepted: 28 June 2016
Published: 7 July 2016
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.
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.
Here we report for the first time the complete genome of two ZLCV isolates that were found in the field infecting zucchini and cucumber.
KeywordsZucchini Tospovirus Illumina Genome ZLCV
Body of text
Zucchini lethal chlorosis virus (ZLCV) is a member of the genus Tospovirus, family Bunyaviridae . Although some tospovirus species are notorious for their broad-host range , ZLCV mainly infects cucurbits and is only known to be transmitted by Frankliniella zucchini (Thysanoptera: Thripidae) . So far ZLCV has just been reported in Brazil, naturally infecting sponge gourd, West Indian gherkin, cucumber, watermelon and several species of squash [4, 5]. Infection by ZLCV abrogate fruit production in zucchini plants and the only resistant cultivar is Cucurbita maxima cv. Exposição , making ZLCV one of the most economically important viral pathogen for cucurbits in Brazil.
Tospoviruses have a tripartite single-stranded RNA genome and each segment is named according to its size. The L (large) RNA has a negative polarity and encodes a RNA-dependent RNA polymerase (RdRp) . The ambisense M (medium) RNA encodes the precursor of two viral glycoproteins (Gn/Gc) and a non-structural protein (NSm) involved in viral cell-to-cell movement . The S (small) RNA, which is also ambisense, encodes another non-structural protein (NSS) with RNA silencing suppression activity and the nucleocapsid (NP) protein .
There are currently 11 approved and 18 tentative tospovirus species, but only a small number of species were completely sequenced. Genome sequence data has the potential to solve key questions in tospovirus evolution, epidemiology and physiology, such as the occurrence and importance of interspecific reassortment  and the presence of potential undescribed genes . Even though ZLCV was described in 1999  and some genes of one isolate has been sequenced [12–14], its complete genome is still unknown. Here, we report the complete genome of two ZLCV isolates found infecting zucchini (Cucurbita pepo cv. Caserta) and cucumber (Cucumis sativus L.) in Brazil and compared them to other tospoviruses.
In 2010, a virus isolate (hereafter ZLCV-SP) from zucchini was found in a commercial field in São Paulo state and transmitted to Datura stramonium L. by F. zucchini as previously described . Then, virus particles were propagated in D. stramonium by mechanical inoculation and infected leaves were used for ribonucleoprotein (RNP) purification following the protocol of De Avila et al. . Moreover, cucumber plants showing typical ZLCV symptoms were collected in Planaltina, Federal District, in 2015. Viral particles were semi-purified from leaves as previously described . Briefly, 40 g of leaf material were homogenized in PBS-EDTA plus 0.2 % 2-mercaptoethanol. The plant extract was then filtered and centrifuged through a sucrose cushion at 33,000 x g for 2 h and the pellet resuspended in PBS. Genomic RNA was extracted from purified RNPS of both isolates as previously described by De Oliveira et al.  and sequenced at Macrogen (South Korea) using Illumina HiSeq 2000 platform. The resulting paired-end reads were filtered and assembled de novo using CLC Genomics Workbench version 6.0.3. The contigs related to ZLCV were selected using Blastx against a RefSeq virus database. To determine if the entire length of each segment was included in the assembled contigs, the reads were mapped back to the ZLCV related contigs. All contigs from both isolates presented the consensus sequences AGAGCAAU and AUUGCUCU at the 5’- and 3’-terminal ends, but some contigs presented distal terminal bases that were trimmed off. These palindromic sequences are conserved among all tospoviruses. Moreover, the ZLCV segments derived from cucumber samples (hereafter ZLCV-DF) presented some unresolved gaps (L segment: 1 gap of 13 nucleotide and 1 of 2 nucleotide). The genome of both isolates were annotated and submitted to NCBI GenBank under the accession numbers no. KU641378-KU641380 (ZLCV-SP) and KU681010-KU681012 (ZLCV-DF).
Genome comparison of ZLCV isolates
L RNA full length (nt)
L gene ORF (nt)
L protein (aa)
2877 (330.85 kDa)
2877 (331.16 kDa)
M RNA full length (nt)
NSm gene ORF (nt)
NSm protein (aa)
303 (34.4 kDa)
303 (34,37 kDa)
GPs gene ORF (nt)
GP protein (aa)
1136 (127.58 kDa)
1136 (127.73 kDa)
S RNA full length (nt)
NSs gene ORF (nt)
NSs protein (aa)
468 (53.06 kDa)
468 (53.01 kDa)
NP gene ORF (nt)
NP protein (aa)
261 (29.22 kDa)
261 (29.24 kDa)
Considering the high prevalence of tospoviruses worldwide, the complete genome of the ZLCV isolates is important for future surveillance and research. Additional investigations in important plant crops should keep being performed to extend the number of characterized species.
aa, amino acids; GP, glycoproteins precursor; IR, Intergenic region; kDa, kilodaltons; nt, nucleotides; RdRp, RNA-dependent RNA polymerase; RNP, ribonucleoprotein; ZLCV, Zucchini lethal chlorosis virus
This work was supported by CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), and FAPDF (Fundação de Apoio à Pesquisa do Distrito Federal).
FLM, MOL, RNL, ROR and TN conceived and designed the experiments. MOL and TN performed the experiments. ASO, FLM, RB and RNL analyzed the data. FLM, ROR and TN contributed reagents/materials/analysis tools. ASO, RNL and FLM wrote the paper. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Bezerra IC, Resende RD, Pozzer L, Nagata T, Kormelink R, De Avila AC. Increase of tospoviral diversity in Brazil with the identification of two new tospovirus species, one from chrysanthemum and one from zucchini. Phytopathology. 1999;89:823–30.View ArticlePubMedGoogle Scholar
- Pappu HR, Jones RAC, Jain RK. Global status of tospovirus epidemics in diverse cropping systems: Successes achieved and challenges ahead. Virus Res. 2009;141:219–36.View ArticlePubMedGoogle Scholar
- Nakahara S, Monteiro RC. Frankliniella zucchini (Thysanoptera : Thripidae), a new species and vector of tospovirus in Brazil. Proc Entomol Soc Wash. 1999;101:290–4.Google Scholar
- Yuki VA, Rezende JAM, Kitajima EW, Barroso PAV, Kuniyuki H, Groppo GA, Pavan MA. Occurrence, distribution, and relative incidence of five viruses infecting cucurbits in the state of Sao Paulo, Brazil. Plant Dis. 2000;84:516–20.View ArticleGoogle Scholar
- Camelo-García VM, Lima EFB, Rezende JAM. Identification of natural hosts of Zucchini lethal chlorosis virus. Tropical Plant Pathology. 2015;40:345–9.View ArticleGoogle Scholar
- Giampan JS, Rezende JAM, Piedade SMS. Yield loss caused by Zucchini lethal chlorosis virus (ZLCV) on zucchini squash ‘Caserta’. Summa Phytopathol. 2009;35:223–5.View ArticleGoogle Scholar
- De Haan P, Kormelink R, Resende RD, Vanpoelwijk F, Peters D, Goldbach R. tomato spotted wilt virus-l rna encodes a putative rna-polymerase. J Gen Virol. 1991;72:2207–16.View ArticlePubMedGoogle Scholar
- Kormelink R, Storms M, Vanlent J, Peters D, Goldbach R. Expression and subcellular location of the nsm protein of tomato spotted wilt virus (tswv), a putative viral movement protein. Virology. 1994;200:56–65.View ArticlePubMedGoogle Scholar
- Takeda A, Sugiyama K, Nagano H, Mori M, Kaido M, Mise K, Tsuda S, Okuno T. Identification of a novel RNA silencing suppressor, NSs protein of Tomato spotted wilt virus. Febs Letters. 2002;532:75–9.View ArticlePubMedGoogle Scholar
- Webster CG, Reitz SR, Perry KL, Adkins S. A natural M RNA reassortant arising from two species of plant- and insect-infecting bunyaviruses and comparison of its sequence and biological properties to parental species. Virology. 2011;413:216–25.View ArticlePubMedGoogle Scholar
- Firth AE. Mapping overlapping functional elements embedded within the protein-coding regions of RNA viruses. Nucleic Acids Res. 2014;42:12425–39.View ArticlePubMedPubMed CentralGoogle Scholar
- Silva MS, Martins CR, Bezerra IC, Nagata T, de Avila AC, Resende RO. Sequence diversity of NS(M) movement protein of tospoviruses. Arch Virol. 2001;146:1267–81.View ArticlePubMedGoogle Scholar
- Nagata T, Carvalho KR, Sodre RDA, Dutra LS, Oliveira PA, Noronha EF, Lovato FA, Resende RDO, De Avila AC, Inoue-Nagata AK. The glycoprotein gene of Chrysanthemum stem necrosis virus and Zucchini lethal chlorosis virus and molecular relationship with other tospoviruses. Virus Genes. 2007;35:785–93.View ArticlePubMedGoogle Scholar
- Hallwass M, Leastro MO, Lima MF, Inoue-Nagata AK, Resende RO. Sequence determination and analysis of the NSs genes of two tospoviruses. Arch Virol. 2012;157:591–6.View ArticlePubMedGoogle Scholar
- Deavila AC, Dehaan P, Smeets MLL, Resende RD, Kormelink R, Kitajima EW, Goldbach RW, Peters D. Distinct levels of relationships between tospovirus isolates. Arch Virol. 1993;128:211–27.View ArticleGoogle Scholar
- Silva KN, Melo FL, Orilio AF, Nagata T, Silva MS, Fernandes CD, Fragoso RR, Dessaune SN, Resende RO. Biological and molecular characterization of a highly divergent Johnsongrass mosaic virus isolate from Pennisetum purpureum. Arch Virol. 2016; Apr 21. [Epub ahead of print].Google Scholar
- de Oliveira AS, Melo FL, Inoue-Nagata AK, Nagata T, Kitajima EW, Resende RO. Characterization of bean necrotic mosaic virus: a member of a novel evolutionary lineage within the Genus Tospovirus. PLoS ONE. 2012;7:e38634.View ArticlePubMedPubMed CentralGoogle Scholar
- van Knippenberg I, Goldbach R, Kormelink R. Tomato spotted wilt virus S-segment mRNAs have overlapping 3 '-ends containing a predicted stem-loop structure and conserved sequence motif. Virus Res. 2005;110:125–31.View ArticlePubMedGoogle Scholar
- Singh P, Indi SS, Savithri HS. Groundnut Bud Necrosis Virus Encoded NSm Associates with Membranes via Its C-Terminal Domain. Plos One. 2014;9:e99370.View ArticlePubMedPubMed CentralGoogle Scholar
- Katoh K, Standley DM. MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability. Mol Biol Evol. 2013;30:772–80.View ArticlePubMedPubMed CentralGoogle Scholar
- Price MN, Dehal PS, Arkin AP. FastTree 2 - Approximately maximum-likelihood trees for large alignments. Plos One. 2010;5:e9490.View ArticlePubMedPubMed CentralGoogle Scholar