Establishment of quantitative and recovery method for detection of dengue virus in wastewater with noncognate spike control

Wastewater-based epidemiology (WBE) represents an efficient approach for public pathogen surveillance as it provides early warning of disease outbreaks; however, it has not yet been applied to dengue virus (DENV), which might cause endemics via mosquito spread. In this study, a working platform was established to provide direct virus recovery and qPCR quantification from wastewater samples that were artificially loaded with target DENV serotypes I to IV and noncognate spike control viral particles. The results showed qPCR efficiencies of 91.2 %, 94.8 %, 92.6 % and 88.7 % for DENV I, II, III, and IV, respectively, and a broad working range over 6 orders of magnitude using the preferred primer sets. Next, the results revealed that the ultrafiltration method was superior to the skimmed milk flocculation method for recovering either DENV or control viral particles from wastewater. Finally, DENV-2 was loaded simultaneously with the noncognate spike control and could be recovered at comparable levels either in PBS or in wastewater, indicating the applicability of noncognate spike control particles to reflect the efficiency of experimental steps. In conclusion, our data suggest that DENV particles in wastewater could be recovered and quantitatively detected in absolute amounts, indicating the feasibility of DENV surveillance using the WBE approach.