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Construction of a full-length infectious cDNA clone of Cowpea mild mottle virus

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Abstract

Infectious cDNA clones are an important tool to study the molecular and cellular process of RNA virus infection. In vitro and in vivo transcription systems are the two main strategies used in the generation of infectious cDNA clones for RNA viruses. This study describes the first generation of a full-length infectious cDNA clone of Cowpea mild mottle virus (CPMMV), a Carlavirus. The full-length genome was synthesized by Overlap Extension PCR of two overlapping fragments and cloned in a pUC-based vector under control of the SP6 RNA polymerase promoter. After in vitro run-off transcription, the produced RNA was mechanically inoculated into soybean plants cv. CD206. The systemic infection was confirmed by RT-PCR and further sequencing of amplified cDNA fragments. To simplify the transfection process, the complete genome was subcloned into a binary vector under control of the 35S promoter of cauliflower mosaic virus by the Gibson Assembly protocol. The resulting clones were inoculated by particle bombardment onto soybean seedlings and the recovery of the virus was confirmed 2 weeks later by RT-PCR. Our results indicate the constructs of the full-length cDNA of CPMMV are fully infectious in both in vitro and in vivo transcription strategies.

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Acknowledgments

We are grateful to Dr. John Lindbo and Dr. Massimo Turina for providing the plasmid vector pJL89. We are also grateful to Sam Elliot for checking the English. This study was funded by FAPEMIG, Vale S.A., CAPES and INCT in Plant-Pest Interactions. CCM is in receipt of a CNPq productivity grant. We are grateful to two anonymous referees for their evaluations of this paper.

Authors’ contribution

Conception or design of the work: Silvia L. Carvalho, Tatsuya Nagata, Claudine M. Carvalho; Data collection: Silvia L. Carvalho, Tatsuya Nagata, Bruna R. Junqueira, Larissa G. Zanardo and Ana C. S. Paiva; Data analysis and interpretation: Silvia L. Carvalho, Tatsuya Nagata and Bruna R. Junqueira. Drafting the manuscript: Silvia L. Carvalho. Critical revision of the manuscript: Tatsuya Nagata and Claudine M. Carvalho. Final approval of the version to be published: Silvia L. Carvalho, Tatsuya Nagata, Bruna R. Junqueira, Larissa G. Zanardo, Ana C. S. Paiva and Claudine M. Carvalho.

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Authors and Affiliations

  1. Department of Phytopathology, Universidade Federal de Viçosa, Av. P. H. Rolfs, s/n Campus Universitário, Viçosa, MG, 36570-900, Brazil

    Silvia L. Carvalho, Larissa G. Zanardo, Ana C. S. Paiva & Claudine M. Carvalho

  2. Department of Cell Biology, Universidade de Brasília, Campus Universitário Darcy Ribeiro, Brasília, DF, 70910-900, Brazil

    Tatsuya Nagata & Bruna R. Junqueira

Authors
  1. Silvia L. Carvalho
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  2. Tatsuya Nagata
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  3. Bruna R. Junqueira
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  4. Larissa G. Zanardo
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  5. Ana C. S. Paiva
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  6. Claudine M. Carvalho
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Corresponding author

Correspondence to Claudine M. Carvalho.

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Edited by Thomas Hohn.

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Carvalho, S.L., Nagata, T., Junqueira, B.R. et al. Construction of a full-length infectious cDNA clone of Cowpea mild mottle virus . Virus Genes 53, 137–140 (2017). https://doi.org/10.1007/s11262-016-1395-x

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  • DOI: https://doi.org/10.1007/s11262-016-1395-x

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