Detection of Brevipalpus-transmitted viruses in their mite vectors by RT–PCR

Kubo, Kosuke; Novelli, Valdenice M.; Bastianel, Marinês; Locali, E. C.; Antonioli-Luizon, R.; Machado, Marcos Antônio; Freitas-Astúa, Juliana

doi:10.1007/s10493-011-9425-9

Cited by 24 publications

(14 citation statements)

References 14 publications

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“…Attempts to detect CiLV-C in individual mites that were feeding on sweet orange leaf lesions were unsuccessful, probably because the virus titer is extremely low. Even RT-PCR, which is a more sensitive test, requires at least 5-10 mites for consistent positive result (Kubo et al, 2011).…”

Section: Discussionmentioning

confidence: 99%

Polyclonal antibodies to the putative coat protein of Citrus leprosis virus C expressed in Escherichia coli: production and use in immunodiagnosis

Calegario¹,

Locali²,

Stach-Machado³

et al. 2013

Trop. plant pathol.

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This work reports the in vitro expression of Citrus leprosis virus C (CiLV-C) putative coat protein (p29) and the production of a polyclonal antibody to be used as a tool for serological diagnosis of citrus leprosis. The ORF2/RNA1, corresponding to p29, was cloned in pET28a and transformed into Escherichia coli cells (BL21). Expression of p29 was induced in vitro and the protein was purified and used for immunization of rabbits to produce the polyclonal antibody. The anti p29 serum was shown to be highly specific to CiLV-C detection by immunological methods (Western blot, PTA-ELISA, tissue blot and in situ immunolocalization), without cross reaction with healthy citrus plants or other cytoplasmic and nuclear viruses transmitted by Brevipalpus mites. These results demonstrate that the antibody against CiLV-C p29 protein is highly specific for CiLV-C detection. In situ immunogold labeling assays on thin sections of sweet orange leaf cells infected by CiLV-C demonstrated that short, bacilliform particles present within cisternae of the endoplasmic reticulum were specifically labeled, confirming their viral nature. The dense cytoplasmic viroplasm was also heavily labeled indicating that it represents a site of p29 accumulation.

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Section: Discussionmentioning

confidence: 99%

Polyclonal antibodies to the putative coat protein of Citrus leprosis virus C expressed in Escherichia coli: production and use in immunodiagnosis

Calegario¹,

Locali²,

Stach-Machado³

et al. 2013

Trop. plant pathol.

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“…3 and 7). It is of particular interest whether a similar interaction between the N and P proteins can be observed for other candidate dichorhabdoviruses, including the citrus leprosis nuclear type, coffee ringspot, and Clerodendrum chlorotic spot viruses (54)(55)(56)(57).…”

Section: Figmentioning

confidence: 99%

Orchid Fleck Virus Structural Proteins N and P Form Intranuclear Viroplasm-Like Structures in the Absence of Viral Infection

Kondō

Chiba

Andika

et al. 2013

J Virol

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bOrchid fleck virus (OFV) has a unique two-segmented negative-sense RNA genome that resembles that of plant nucleorhabdoviruses. In infected plant cells, OFV and nucleorhabdoviruses induce an intranuclear electron-lucent viroplasm that is believed to be the site for virus replication. In this study, we investigated the molecular mechanism by which OFV viroplasms are produced in vivo. Among OFV-encoded proteins, the nucleocapsid protein (N) and the putative phosphoprotein (P) were present in nuclear fractions of OFV-infected Nicotiana benthamiana plants. Transient coexpression of N and P, in the absence of virus infection, was shown to be sufficient for formation of an intranuclear viroplasm-like structure in plant cells. When expressed independently as a fluorescent protein fusion product in uninfected plant cells, N protein accumulated throughout the cell, while P protein accumulated in the nucleus. However, the N protein, when coexpressed with P, was recruited to a subnuclear region to induce a large viroplasm-like focus. Deletion and substitution mutagenesis demonstrated that the P protein contains a nuclear localization signal (NLS). Artificial nuclear targeting of the N-protein mutant was insufficient for formation of viroplasm-like structures in the absence of P. A bimolecular fluorescence complementation assay confirmed interactions between the N and P proteins within subnuclear viroplasm-like foci and interactions of two of the N. benthamiana importin-␣ homologues with the P protein but not with the N protein. Taken together, our results suggest that viroplasm formation by OFV requires nuclear accumulation of both the N and P proteins, which is mediated by P-NLS, unlike nucleorhabdovirus viroplasm utilizing the NLS on protein N. R habdoviruses (family Rhabdoviridae, order Mononegavirales) have a broad range of hosts, including humans, livestock, fish, plants, and insects (1). They have bacilliform or bulletshaped virions containing a nonsegmented negative-sense RNA [(Ϫ)RNA] as the genome (2). All of the well-characterized animal rhabdoviruses replicate in the cytoplasm (1). However, the plant rhabdoviruses are divided into two groups, depending on their sites of replication and morphogenesis. Members of the genus Cytorhabdovirus replicate and undergo morphogenesis in the cytoplasm, whereas members of the genus Nucleorhabdovirus multiply in plant cell nuclei and their virions bud through the inner nuclear envelopes (3, 4).Orchid fleck virus (OFV), a Brevipalpus (Acari: Tenuipalpidae) mite-transmitted virus, has a unique two-segmented (Ϫ)RNA genome that resembles that of plant rhabdoviruses (5, 6). OFV is distributed worldwide and infects many kinds of orchids (5,7,8). OFV RNA1 (GenBank accession number AB244417; 6,413 nucleotides [nt]) contains five genes in the order 3=-N-P-3-M-G-5=, in which N encodes the nucleocapsid protein (open reading frame 1 [ORF1]), P encodes the phosphoprotein (ORF2), gene 3 encodes the candidate movement protein (ORF3), M encodes the matrix protein (ORF4), and G encodes the can...

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“…Its genome is bipartite (5 and 9 kb) and of positive-sense single-stranded (ss)RNA. Two other BrTV-Ns, CoRSV (17) and CICSV (26), have been partially sequenced, exhibiting sequence similarities with OFV, and may belong to the same genus as other, as-yet-uncharacterized BrTV-Ns. Parts of the genomes of PFGSV (1) and SvRSV (11) have been sequenced, showing sequence similarities with that of CiLV-C. LigRSV and HGSV (23) have not been molecularly characterized yet but their cytopathology indicates that they are BrTV-C.…”

Section: Discussionmentioning

confidence: 99%

Common Bean: Experimental Indicator Plant for Citrus leprosis virus C and Some Other Cytoplasmic-Type Brevipalpus-Transmitted Viruses

et al. 2013

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Garita, L. C, Tassi, A. D., Calegario, R. F., Kitajima, E. W., Carbonell, S. A. M., and Freitas-Astúa, J. 2013. Common bean: Experimental indicator plant for Citrus leprosis virus C and some other cytoplasmic-type Srev;/>a//JMi-transmitted viruses. Plant Dis. 97:1346-1351.Citrus leprosis (CL) caused by Citrus leprosis virus C (CiLV-C) is present in Latin America from Mexico to Argentina, where citrus plants are grown. CiLV-C is transmitted by the tenuipalpid mite, Brevipalpus phoenicis, causing localized lesions on citrus leaves, fruit, and stems. One limitation to study of the virus-vector-host relationship in this pathosystem is the lack of a suitable assay plant. On Citrus spp. used as susceptible hosts, symptoms may take weeks or months to appear after experimental inoculation by viruliferous mites. Common bean (Phaseolus vulgaris) was found to respond with localized necrotic lesions after inoculation with viruliferous B. phoenicis in 5 days. Thus far, 113 tested common bean varieties and lines and some recent accessions of varied genetic background behaved in a similar way.

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Detection of Brevipalpus-transmitted viruses in their mite vectors by RT–PCR

Cited by 24 publications

References 14 publications

Polyclonal antibodies to the putative coat protein of Citrus leprosis virus C expressed in Escherichia coli: production and use in immunodiagnosis

Polyclonal antibodies to the putative coat protein of Citrus leprosis virus C expressed in Escherichia coli: production and use in immunodiagnosis

Orchid Fleck Virus Structural Proteins N and P Form Intranuclear Viroplasm-Like Structures in the Absence of Viral Infection

Common Bean: Experimental Indicator Plant for Citrus leprosis virus C and Some Other Cytoplasmic-Type Brevipalpus-Transmitted Viruses

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