Zika virus (ZIKV) is a mosquito-borne flavivirus that predominantly circulates between humans and Aedes mosquitoes. The recent ZIKV epidemics in South America caused millions of infected cases by the end of 2017. Many studies have demonstrated that several neurological complications, such as Guillain-Barré syndrome in adults and microcephaly in neonates, are associated with ZIKV infection. Hence, the ZIKV epidemic poses an important challenge to public health worldwide.

The classic transmission cycle of ZIKV is established between a vertebrate host and an invertebrate vector. Mosquitoes acquire the virus via biting an infected patient and then transmit ZIKV to a healthy person when the infectious viral particles are released into the saliva of the mosquitoes (Fig. 1). 

                                                 Fig. 1 The classic transmission cycle of ZIKV

Previous studies have indicated that the peak of ZIKV viremia in infected patients is low and rapidly elapses due to the host immune response. Intriguingly, infectious ZIKV particles are persistent and exist longer in other body fluids, especially in the semen and urine. One mosquito engorges 1 to 2 μl blood via a successful blood meal. Usually, only hundred or fewer infectious particles would be ingested by one mosquito at a time. Within the mosquito, these viral particles first endure rigorous conditions in the mosquito midgut until they successfully invade the gut epidermal cells to initiate infection in the mosquito, which might make ZIKV infection through this classic transmission route inefficient. Therefore, how did ZIKV dramatically sweep through South American in such a short time? We wondered whether there are some alternative transmission approaches for viral dissemination in nature. With this question, we began our exploration.

First, we noticed that ZIKV genomic RNA can exist in infected human urine for up to several weeks. In addition, several independent studies claimed that infectious ZIKV particles were detected and isolated from patients’ urine samples. The life span of the mosquito includes an immature aquatic larval stage and a pupal period before they emerge as an adult mosquito. Hence, we can image a scenario in the ZIKV outbreak region. Many infected individuals may intensively discharge a large volume of infectious urine into a restricted aquatic habitat with immature mosquitoes,and the mosquito larva and pupa may directly contact the viral particles and become infected (Fig. 2).

                        Fig. 2. The relationship between the mosquito life span and ZIKV infection

To demonstrate this, we first collected hundreds of urine samples from healthy people and found that ZIKV can survive in human urine, and that a pH value above 6.5 is an essential determinant for ZIKV survival. Subsequently, the mosquito larvae and pupae were reared in ZIKV-infected urine, and the emerged adult mosquitoes were collected for ZIKV detection. As expected, up to 4% of the mosquitoes that emerged from the ZIKV-infected urine were infected, and some of them successfully transmitted ZIKV to animals by biting AG6 mice. Furthermore, to mimic the natural scenario, we collected several sewage samples to rear the mosquito pupae, and then urine containing a low titer of ZIKV was continuously added to the sewage sample every 3 hours, which mimicked the urination of ZIKV patients. In this sewage condition, mosquitoes also successfully acquired ZIKV and obtained the ability to transmit viruses to animals.

Our results indicate that mosquitoes breeding in a water system contaminated by ZIKV-containing human urine can carry and transmit ZIKV. This new potential transmission approach may accelerate the dissemination of ZIKV in nature and might have contributed to the recent ZIKV epidemic in South America (Fig. 3). In particular, cesspits are still commonly used in many areas all over the world, especially in some undeveloped countries and regions. Their poor public sanitary infrastructure may significantly increase the risk of mosquito-borne flavivirus epidemics.

                                                         Fig. 3. ZIKV transmission approaches

Overall, this study offers laboratory evidence that ZIKV could be transmitted to mosquitoes via virus-contaminated human urine, emphasizing the importance of improving the sanitary system in mosquito-borne disease epidemic regions and providing some new possible avenues for interrupting the mosquito-borne flavivirus transmission cycle in nature.