Development of cell-based colorimetric immunodetection of zika virus

Abstract

Background: Zika virus (ZIKV) is a mosquito-borne flavivirus. ZIKV infection is a self-limiting febrile illness that showed symptoms that similar to those caused by other flaviviruses such as dengue virus (DENV). ZIKV infection diagnostic remains a challenge, as short viremic phase during infection makes it difficult to detect the viral components. The serologic assay, therefore, plays an important role for ZIKV diagnosis. Confirmation using Plaque Reduction Neutralization Test (PRNT) on IgM positive specimen is recommended to reduce potential false-positive results. The “gold standard” test, plaque forming assay (PFA), however, relies on formation of the cytopathic effects (CPE), which is time-consuming. In the current study, we developed a cell-based colorimetric immunodetection method or focus forming assay (FFA) for the detection and quantification of ZIKV. Methods and materials: Vero cells were seeded in a 24-well plate. ZIKV stock, diluted in 10-fold was added onto the Vero cells monolayer. After 1 h of virus adsorption, the virus inoculum was removed. Infected cells were kept at 37 °C incubator. For FFA, the cells were fixed at 2, 2.5, 3, 3.5 and 4 days after the infection. Subsequently, the cells were incubated with anti-flavivirus antibody and stained with DAB-peroxidase substrate solution. For the PFA, the cells were fixed at 3, 4, 5, 6, and 7 days after the infection. Subsequently, the cells were stained with crystal violet. Results: Microscopic evaluation of cells infected with ZIKV demonstrated that the ZIKV infection foci could be detected beginning on day 2 post-infection. In a 24-well plate format, the focus forming at 2.5 days after the infection was chosen. A complete plaque formation can be seen day-7 after the infection. The size of the plaque was huge; hence, overlapped plaques were observed in the 24 well plates. A PFA on a 6-well plate was performed and demonstrated that a 6-well plate format was more suitable to be used for PFA counting. Conclusion: Findings from the study demonstrated that both FFA and PFA were able to detect ZIKV detection. The newly developed FFA assay, however, is time-saving and suitable for higher throughput screening.