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.

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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

An Application of Immuno Peroxidase Staining Technique for the Titration of Dengue Virus in vitro

DHFおよびDSS患者から分離された野生株を用い, 哺乳マウス脳に連続的に継代を行い, 継代毎に病理組織学的観察を行い, 症状および脳内ウイルス量と合わせて比較検討した。マウス継代歴のない野生株の脳内接種では死亡率は低く, しかも脳の病理組織学的変化も軽微であったが脳内ウイルス量は比較的高値を示した。継代数が増すにつれて死亡率も上昇し, 病理組織変化もその程度は増強し, 血管周囲腔や脳実質に及ぶ炎症性変化が観察された。特にDHF患者由来株では脳実質内に著明な膿瘍形成が認められた。

Yukio Ohsugi

Papers

An Application of Immuno Peroxidase Staining Technique for the Titration of Dengue Virus in vitro

Pathological study of mouse brain infected with wild type strains of dengue virus type 2

Pathogenicity and Pathophysiology of Dengue Virus type 2 Strains in Mice