An accumulated mutation gained in mosquito cells enhances Zika virus virulence and fitness in mice.

Zika virus (ZIKV) remains a significant public health threat worldwide. A number of adaptive mutations have accumulated within the genome of ZIKV during global transmission, some of which have been linked to specific phenotypes. ZIKV maintains an alternating cycle of replication between mosquitoes and vertebrate hosts, but the role of mosquito-specific adaptive mutations in ZIKV has not been well investigated. In this study, we demonstrated that serial passaging of ZIKV in mosquito Aag2 cells led to the emergence of critical amino acid substitutions, including A94V in the prM protein and V153D and H401Y in the E protein. Further characterization via reverse genetics revealed that the H401Y substitution in the E protein did not augment viral replication in mosquitoes but significantly enhanced neurovirulence and lethality compared with those of the wild-type (WT) virus in mice. More importantly, the H401Y mutant maintained its virulence phenotype in mice after propagation in mosquitoes in mosquito-mouse cycle model. In particular, recombinant ZIKV harboring the H401Y substitution showed enhanced competitive fitness over WT ZIKV in various mammalian cells and mouse brains, but not in mosquito cells. Notably, the H401Y substitution in the ZIKV E protein has been detected in recent isolates derived from both mosquitoes and humans in Asia and the Americas. In summary, our findings not only identify a novel virulence determinant of ZIKV but also highlight the complexity of the relationship between the evolution of vector-borne viruses and their clinical outcome in nature. IMPORTANCE: Zika virus (ZIKV) is an important arbovirus with a global impact. Experimental evolution by serial passaging of ZIKV in susceptible cells has led to the identification of a panel of critical amino acid substitutions with specific functions. Herein, we identified a mosquito cell-derived substitution, H401Y, in the ZIKV E protein via experimental evolution. The H401Y substitution significantly enhanced viral virulence and fitness in mammal cells and mice. Notably, the H401Y substitution has been detected in recent mosquito and human isolates from regions spanning Asia to the Americas. Our work elucidates unrecognized virulence determinant in the ZIKV genome that warrants urgent attention. Moreover, the findings underscore the critical need for extensive molecular surveillance and rigorous clinical observation to establish the potential impact in natural circulation. These endeavors are crucial for unraveling the potential of mutation to act as a catalyst for future epidemics, thereby preempting the public health challenges it may pose.