Assessment of flavivirus RNA stability and infectivity in various water environments

Abstract

Introduction: Flaviviruses such as dengue virus (DENV), Zika virus (ZIKV), Japanese encephalitis virus (JEV), and Yellow fever virus (YFV) are mosquito-borne RNA viruses causing major public health threats in major parts of the world. While DENV and ZIKV have been detected in urine samples, data on the presence and stability of flaviviruses in the water environment are limited.

Methods: In this study, we determined the stability and infectivity of flavivirus in different water environments by utilizing RT-qPCR and plaque assay to explore the feasibility of environmental detection and surveillance of flaviviruses.

Results: Viral RNA could be detected for up to 49-days, at 4 °C, 25 °C and 37 °C temperatures, and infectious particles could be detected for up to 7 days. While our findings showed that flaviviral RNA has higher stability and better detection rates at lower temperatures, the infectious capacity of flaviviruses was comparatively short at 7 days.

Conclusions: Our results indicate that flaviviruses retains limited infectivity in general water environments and highlight the feasibility of detection and surveillance in various epidemiologic and environmental settings.

Keywords: Dengue; Environment; Japanese encephalitis; Orthoflavivirus; RNA virus; Surveillance; Water; Yellow fever; Zika.

Fig. 1

Viral RNA genome stability of DENV-1, JEV, YFV and ZIKV in aquatic waters and EMEM for each temperature. Viral genome copy number in each specimen is shown as mean ± standard deviation log10 genome copies/μl. RT-qPCR assays were performed in triplicate. A, B, D, E, G Viral RNA copy number of DENV-1 in aquatic waters and EMEM. C, F Decrease rate of DENV-1 against EMEM in aquatic waters. H, J Viral RNA copy number of JEV in aquatic waters and EMEM. I, K Decrease rate of JEV against EMEM in aquatic waters. L, N Viral RNA copy number of YFV in aquatic waters and EMEM. M, O Decrease rate of YFV against EMEM in aquatic waters. P, R Viral RNA copy number of ZIKV in aquatic waters and EMEM. Q, S Decrease rate of ZIKV against EMEM in aquatic waters. N.D. indicates not detected. N.T. indicates not tested

Fig. 2

Viral infectivity of DENV-1, JEV, YFV and ZIKV in aquatic waters and EMEM for each temperature. Viral titers are shown as mean ± standard deviation log10 PFU/ml. Plaque assays were performed in a triplicate. Dash line indicates detection limits (1.0 × 10² PFU/ml). Some bars below detection limit without N.D. labels mean virus was detected in several wells but not all wells in the triplex wells. A, C, E Virus titer of DENV-1 in aquatic waters and EMEM. B, D Decrease rate of DENV-1 against EMEM in aquatic waters. F, H Virus titer of JEV in aquatic waters and EMEM. G, I Decrease rate of JEV against EMEM in aquatic waters. J, L Virus titer of YFV in aquatic waters and EMEM. K, M Decrease rate of YFV against EMEM in aquatic waters. N, P Virus titer of ZIKV in aquatic waters and EMEM. O, Q Decrease rate of ZIKV against EMEM in aquatic waters. N.D. indicates not detected. N.T. indicates not tested