During the 2015–2016 Zika virus (ZIKV) epidemic in the Americas, serological cross-reactivity with other flaviviruses and relatively high costs of nucleic acid testing in the region hindered the capacity for widespread diagnostic testing. In such cases where individual testing is not feasible, wastewater monitoring approaches may offer a means of community-level public health surveillance. To inform such approaches, we characterized the persistence and recovery of ZIKV RNA in experiments where we spiked cultured ZIKV into surface water, wastewater, and a combination of both to examine the potential for detection in open sewers serving communities most affected by the ZIKV outbreak, such as those in Salvador, Bahia, Brazil. We used reverse transcription droplet digital PCR to quantify ZIKV RNA. In our persistence experiments, we found that the persistence of ZIKV RNA decreased with increasing temperature, significantly decreased in surface water versus wastewater, and significantly decreased when the initial concentration of virus was lowered by one order of magnitude. In our recovery experiments, we found higher percent recovery of ZIKV RNA in pellets versus supernatants from the same sample, higher recoveries in pellets using skimmed milk flocculation, lower recoveries of ZIKV RNA in surface water versus wastewater, and lower recoveries from a freeze thaw. We also analyzed samples collected from Salvador, Brazil during the ZIKV outbreak (2015–2016) that consisted of archived samples obtained from open sewers or environmental waters thought to be contaminated by sewage. Although we did not detect any ZIKV RNA in the archived Brazil samples, results from these persistence and recovery experiments serve to inform future wastewater monitoring efforts in open sewers, an understudied and important application of wastewater monitoring.

2015-2016年寨卡病毒（ZIKV）在美洲流行期间，与其他黄病毒的血清学交叉反应以及该地区相对较高的核酸检测成本阻碍了广泛诊断检测的能力。在个体检测不可行的情况下，废水监测方法可以提供社区级公共卫生监测的一种手段。为了为这些方法提供信息，我们在实验中表征了 ZIKV RNA 的持久性和恢复，其中我们将培养的 ZIKV 添加到地表水、废水以及两者的组合中，以检查在受 ZIKV 爆发影响最严重的社区服务的开放下水道中检测的潜力，例如萨尔瓦多、巴伊亚、巴西等地。我们使用逆转录液滴数字 PCR 来定量 ZIKV RNA。在我们的持久性实验中，我们发现 ZIKV RNA 的持久性随着温度的升高而降低，与废水相比，地表水中的持久性显着降低，并且当病毒的初始浓度降低一个数量级时，ZIKV RNA 的持久性也显着降低。在我们的回收实验中，我们发现，与同一样品的上清液相比，颗粒中 ZIKV RNA 的回收率更高，使用脱脂牛奶絮凝的颗粒中 ZIKV RNA 的回收率更高，地表水中 ZIKV RNA 的回收率与废水中的回收率更低，冻融时的回收率更低。我们还分析了 ZIKV 爆发期间（2015-2016 年）从巴西萨尔瓦多收集的样本，其中包括从露天下水道或被认为受到污水污染的环境水域中获取的存档样本。 尽管我们没有在巴西存档样本中检测到任何 ZIKV RNA，但这些持久性和恢复实验的结果可为未来露天下水道中的废水监测工作提供信息，这是废水监测的一项尚未得到充分研究的重要应用。