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Specific antibody production using recombinant proteins to elucidate seed transmission and nuclear localization of Coguvirus citrulli and Coguvirus henanense in radicles of watermelon crop.
Kauffmann, Caterynne M; Vendramini, Marina; Batista, Amanda M V; Mota, Helena B S; Andrade, Ikaro A; Cárdenas, Stephanny B S; Queiroz, Paloma S; Silva, Bruno A; Correa, José R; Nagata, Tatsuya.
Afiliação
  • Kauffmann CM; Departamento de Fitopatologia, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF, 70910-900, Brazil.
  • Vendramini M; Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF, 70910-900, Brazil.
  • Batista AMV; Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF, 70910-900, Brazil.
  • Mota HBS; Departamento de Fitopatologia, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF, 70910-900, Brazil.
  • Andrade IA; Departamento de Biologia Microbiana, Universidade de Brasília, Brasília, DF, 70910-900, Brazil.
  • Cárdenas SBS; Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF, 70910-900, Brazil.
  • Queiroz PS; Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF, 70910-900, Brazil.
  • Silva BA; Departamento de Fitopatologia, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF, 70910-900, Brazil.
  • Correa JR; Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF, 70910-900, Brazil.
  • Nagata T; Departamento de Fitopatologia, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF, 70910-900, Brazil; Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF, 70910-900, Brazil; Departamento de Biologia Microbiana, Universidade de Brasília, Brasília, DF, 70910-
J Virol Methods ; 325: 114886, 2024 Apr.
Article em En | MEDLINE | ID: mdl-38246564
ABSTRACT
Watermelon crinkle leaf-associated virus 1 (WCLaV-1) and WCLaV-2, both belonging to the genus Coguvirus (family Phenuiviridae), have been identified in watermelon plants in Brazil. To study tissue tropism and the potential for seed transmission of these viruses, we initially planned to produce specific antibodies. However, difficulties in isolating and propagating the virus in host plants hindered the purified virus preparations. To overcome this problem, the nucleocapsid (N) proteins of WCLaV-1 and -2 were produced using the pepper ringspot virus vector. The N protein genes and the vector backbone were prepared by (RT-)PCR and ligated by Gibson assembly. The constructs were agro-infiltrated in Nicotiana benthamiana plants. The expressed N proteins were purified and used for polyclonal antibody production. The specificity of both antibodies was confirmed by antigen-coating ELISA, tissue-blot immunobinding assay and Western blot. By antigen-coating ELISA demonstrated that WCLaV-1 showed 93.1% of seed-transmission, while WCLaV-2 showed only 17.8%. The N protein of WCLaV-1 was detected in the cytoplasm of the seed tissues. It was also found in the nuclei of the radicle, as confirmed by confocal microscopy. We concluded that the antibodies exhibited both a high titer and sufficient specificity for use in ELISA-based diagnostics and for subcellular localization study.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Citrullus Idioma: En Revista: J Virol Methods Ano de publicação: 2024 Tipo de documento: Article País de afiliação: Brasil País de publicação: Holanda

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Citrullus Idioma: En Revista: J Virol Methods Ano de publicação: 2024 Tipo de documento: Article País de afiliação: Brasil País de publicação: Holanda