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Objective <p>This study aimed the standardization of detection, isolation, titration, replication kinetics, and virus neutralization assay protocols for DENV-2 (New Guinea C strain) with the C6/36 mosquito-derived cell line.</p> Results <p>DENV-2 was isolated, titrated by TCID₅₀, and quantified via molecular assays using serially diluted viral RNA to generate a standard curve using the C6/36 cell line. The serum neutralization assay was optimized in 96-well plates based on visual scoring of cytopathic effect (CPE). Optimized serum dilutions effectively reduced nonspecific cytotoxicity caused by residual complement, preventing misinterpretation and enabling reliable detection of serum neutralizing activity in C6/36 cells. RT-qPCR assays calibrated using a TCID₅₀-titrated DENV-2 viral stock showed high amplification efficiency (90–100%) with R² ≥ 0.98. The results demonstrate that the standardized workflow enables proof-of-principle DENV-2 neutralization testing in C6/36 cells, yielding neutralization patterns consistent with those observed in Vero cells, and supports the use of this platform as an alternative for resource-limited laboratories.</p> Graphical Abstract <p></p>

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Standardization of C6/36 cells for DENV-2 virological assays: isolation, TCID₅₀ and serum neutralization

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年3月6日

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  • Elmo José dos Santos,
  • Amanda da Rocha Santos,
  • Mayanna Moreira Costa Fogaça,
  • Naiara Soares da Silva,
  • Milena Silva Souza,
  • Ruth Dálety da Silva Brito,
  • Paloma Oliveira Vidal,
  • Samuel Santos Pereira,
  • Jéssica Pires Farias,
  • Alexander Birbrair,
  • Rúbens Prince dos Santos Alves,
  • Wilson Barros Luiz,
  • Luís Carlos de Souza Ferreira,
  • Jaime Henrique Amorim

摘要

Objective

This study aimed the standardization of detection, isolation, titration, replication kinetics, and virus neutralization assay protocols for DENV-2 (New Guinea C strain) with the C6/36 mosquito-derived cell line.

Results

DENV-2 was isolated, titrated by TCID₅₀, and quantified via molecular assays using serially diluted viral RNA to generate a standard curve using the C6/36 cell line. The serum neutralization assay was optimized in 96-well plates based on visual scoring of cytopathic effect (CPE). Optimized serum dilutions effectively reduced nonspecific cytotoxicity caused by residual complement, preventing misinterpretation and enabling reliable detection of serum neutralizing activity in C6/36 cells. RT-qPCR assays calibrated using a TCID₅₀-titrated DENV-2 viral stock showed high amplification efficiency (90–100%) with R² ≥ 0.98. The results demonstrate that the standardized workflow enables proof-of-principle DENV-2 neutralization testing in C6/36 cells, yielding neutralization patterns consistent with those observed in Vero cells, and supports the use of this platform as an alternative for resource-limited laboratories.

Graphical Abstract