Combination of the Focus-Forming Assay and Digital Automated Imaging Analysis for the Detection of Dengue and Zika Viral Loads in Cultures and Acute Disease
Optimized methods for the detection of flavivirus infections in hyperendemic areas are still needed, especially for working with patient serum as a starting material. The focus-forming assay (FFA) reveals critical aspects of virus-host interactions, as it is a quantitative assay to determine viral l...
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ftdoajarticles:oai:doaj.org/article:33c2315771c54549ab72b2a4a8fcb3fa 2023-05-15T15:14:44+02:00 Combination of the Focus-Forming Assay and Digital Automated Imaging Analysis for the Detection of Dengue and Zika Viral Loads in Cultures and Acute Disease Sara Bolívar-Marin Irene Bosch Carlos F. Narváez 2022-01-01T00:00:00Z https://doi.org/10.1155/2022/2177183 https://doaj.org/article/33c2315771c54549ab72b2a4a8fcb3fa EN eng Hindawi Limited http://dx.doi.org/10.1155/2022/2177183 https://doaj.org/toc/1687-9694 1687-9694 doi:10.1155/2022/2177183 https://doaj.org/article/33c2315771c54549ab72b2a4a8fcb3fa Journal of Tropical Medicine, Vol 2022 (2022) Arctic medicine. Tropical medicine RC955-962 article 2022 ftdoajarticles https://doi.org/10.1155/2022/2177183 2022-12-31T00:47:20Z Optimized methods for the detection of flavivirus infections in hyperendemic areas are still needed, especially for working with patient serum as a starting material. The focus-forming assay (FFA) reveals critical aspects of virus-host interactions, as it is a quantitative assay to determine viral loads. Automated image analysis provides evaluations of relative amounts of intracellular viral protein at the single-cell level. Here, we developed an optimized FFA for the detection of infectious Zika virus (ZIKV) and dengue virus (DENV) viral particles in cell cultures and clinical serum samples, respectively. Vero-76 cells were infected with DENV-2 (16681) or ZIKV (PRVA BC59). Using a panel of anti-DENV and anti-ZIKV NS1-specific monoclonal antibodies (mAbs), the primary mAbs, concentration, and the optimal time of infection were determined. To determine whether intracellular accumulation of NS1 improved the efficiency of the FFA, brefeldin A was added to the cultures. Focus formation was identified by conventional optical microscopy combined with CellProfiler™ automated image analysis software. The FFA was used with spike assays for ZIKV and clinical specimens from natural infection by DENV-1 and DENV-2. mAb 7744-644 for ZIKV and mAb 724-323 for DENV used at a concentration of 1 μg/ml and a time of 24 hours postinfection produced the best detection of foci when combining conventional counting and automated digital analysis. Brefeldin A did not improve the assessment of FFUs or their digitally assessed intensity at single-cell level. The FFA showed 95% ZIKV recovery and achieved the detection of circulating DENV-1 and DENV-2 in the plasma of acutely ill patients. The combination of the two techniques optimized the FFA, allowing the study of DENV and ZIKV in culture supernatants and clinical specimens from natural infection in hyperendemic areas. Article in Journal/Newspaper Arctic Directory of Open Access Journals: DOAJ Articles Arctic Journal of Tropical Medicine 2022 1 11 |
institution |
Open Polar |
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Directory of Open Access Journals: DOAJ Articles |
op_collection_id |
ftdoajarticles |
language |
English |
topic |
Arctic medicine. Tropical medicine RC955-962 |
spellingShingle |
Arctic medicine. Tropical medicine RC955-962 Sara Bolívar-Marin Irene Bosch Carlos F. Narváez Combination of the Focus-Forming Assay and Digital Automated Imaging Analysis for the Detection of Dengue and Zika Viral Loads in Cultures and Acute Disease |
topic_facet |
Arctic medicine. Tropical medicine RC955-962 |
description |
Optimized methods for the detection of flavivirus infections in hyperendemic areas are still needed, especially for working with patient serum as a starting material. The focus-forming assay (FFA) reveals critical aspects of virus-host interactions, as it is a quantitative assay to determine viral loads. Automated image analysis provides evaluations of relative amounts of intracellular viral protein at the single-cell level. Here, we developed an optimized FFA for the detection of infectious Zika virus (ZIKV) and dengue virus (DENV) viral particles in cell cultures and clinical serum samples, respectively. Vero-76 cells were infected with DENV-2 (16681) or ZIKV (PRVA BC59). Using a panel of anti-DENV and anti-ZIKV NS1-specific monoclonal antibodies (mAbs), the primary mAbs, concentration, and the optimal time of infection were determined. To determine whether intracellular accumulation of NS1 improved the efficiency of the FFA, brefeldin A was added to the cultures. Focus formation was identified by conventional optical microscopy combined with CellProfiler™ automated image analysis software. The FFA was used with spike assays for ZIKV and clinical specimens from natural infection by DENV-1 and DENV-2. mAb 7744-644 for ZIKV and mAb 724-323 for DENV used at a concentration of 1 μg/ml and a time of 24 hours postinfection produced the best detection of foci when combining conventional counting and automated digital analysis. Brefeldin A did not improve the assessment of FFUs or their digitally assessed intensity at single-cell level. The FFA showed 95% ZIKV recovery and achieved the detection of circulating DENV-1 and DENV-2 in the plasma of acutely ill patients. The combination of the two techniques optimized the FFA, allowing the study of DENV and ZIKV in culture supernatants and clinical specimens from natural infection in hyperendemic areas. |
format |
Article in Journal/Newspaper |
author |
Sara Bolívar-Marin Irene Bosch Carlos F. Narváez |
author_facet |
Sara Bolívar-Marin Irene Bosch Carlos F. Narváez |
author_sort |
Sara Bolívar-Marin |
title |
Combination of the Focus-Forming Assay and Digital Automated Imaging Analysis for the Detection of Dengue and Zika Viral Loads in Cultures and Acute Disease |
title_short |
Combination of the Focus-Forming Assay and Digital Automated Imaging Analysis for the Detection of Dengue and Zika Viral Loads in Cultures and Acute Disease |
title_full |
Combination of the Focus-Forming Assay and Digital Automated Imaging Analysis for the Detection of Dengue and Zika Viral Loads in Cultures and Acute Disease |
title_fullStr |
Combination of the Focus-Forming Assay and Digital Automated Imaging Analysis for the Detection of Dengue and Zika Viral Loads in Cultures and Acute Disease |
title_full_unstemmed |
Combination of the Focus-Forming Assay and Digital Automated Imaging Analysis for the Detection of Dengue and Zika Viral Loads in Cultures and Acute Disease |
title_sort |
combination of the focus-forming assay and digital automated imaging analysis for the detection of dengue and zika viral loads in cultures and acute disease |
publisher |
Hindawi Limited |
publishDate |
2022 |
url |
https://doi.org/10.1155/2022/2177183 https://doaj.org/article/33c2315771c54549ab72b2a4a8fcb3fa |
geographic |
Arctic |
geographic_facet |
Arctic |
genre |
Arctic |
genre_facet |
Arctic |
op_source |
Journal of Tropical Medicine, Vol 2022 (2022) |
op_relation |
http://dx.doi.org/10.1155/2022/2177183 https://doaj.org/toc/1687-9694 1687-9694 doi:10.1155/2022/2177183 https://doaj.org/article/33c2315771c54549ab72b2a4a8fcb3fa |
op_doi |
https://doi.org/10.1155/2022/2177183 |
container_title |
Journal of Tropical Medicine |
container_volume |
2022 |
container_start_page |
1 |
op_container_end_page |
11 |
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1766345162020093952 |