Zika virus (ZIKV) is now in a post-pandemic period, for which the potential for re-emergence and future spread is unknown. Adding to this uncertainty is the unique capacity of ZIKV to directly transmit between humans via sexual transmission. Recently, we demonstrated that direct transmission of ZIKV between vertebrate hosts leads to rapid adaptation resulting in enhanced virulence in mice and the emergence of three amino acid substitutions (NS2A-A117V, NS2A- A117T, and NS4A-E19G) shared among all vertebrate-passaged lineages. Here, we further characterized these host-adapted viruses and found that vertebrate-passaged viruses also have enhanced transmission potential in mosquitoes. To understand the contribution of genetic changes to the enhanced virulence and transmission phenotype, we engineered these amino acid substitutions, singly and in combination, into a ZIKV infectious clone. We found that NS4A- E19G contributed to the enhanced virulence and mortality phenotype in mice. Further analyses revealed that NS4A-E19G results in increased neurotropism and distinct innate immune signaling patterns in the brain. None of the substitutions contributed to changes in transmission potential in mosquitoes. Together, these findings suggest that direct transmission chains could enable the emergence of more virulent ZIKV strains without compromising mosquito transmission capacity, although the underlying genetics of these adaptations are complex.

A promising candidate for arbovirus control and prevention relies on replacing arbovirus-susceptible Aedes aegypti populations with mosquitoes that have been colonized by the intracellular bacterium Wolbachia and thus have a reduced capacity to transmit arboviruses. This reduced capacity to transmit arboviruses is mediated through a phenomenon referred to as pathogen blocking. Pathogen blocking has primarily been proposed as a tool to control dengue virus (DENV) transmission, however it works against a range of viruses, including Zika virus (ZIKV). Despite years of research, the molecular mechanisms underlying pathogen blocking still need to be better understood. Here, we used RNA-seq to characterize mosquito gene transcription dynamics in Ae. aegypti infected with the wMel strain of Wolbachia that are being released by the World Mosquito Program in Medellín, Colombia. Comparative analyses using ZIKV-infected, uninfected tissues, and mosquitoes without Wolbachia revealed that the influence of wMel on mosquito gene transcription is multifactorial. Importantly, because Wolbachia limits, but does not completely prevent, replication of ZIKV and other viruses in coinfected mosquitoes, there is a possibility that these viruses could evolve resistance to pathogen blocking. Therefore, to understand the influence of Wolbachia on within-host ZIKV evolution, we characterized the genetic diversity of molecularly barcoded ZIKV virus populations replicating in Wolbachia-infected mosquitoes and found that within-host ZIKV evolution was subject to weak purifying selection and, unexpectedly, loose anatomical bottlenecks in the presence and absence of Wolbachia. Together, these findings suggest that there is no clear transcriptional profile associated with Wolbachia-mediated ZIKV restriction, and that there is no evidence for ZIKV escape from this restriction in our system.

A promising candidate for arbovirus control and prevention relies on replacing arbovirus-susceptible Aedes aegypti populations with mosquitoes that have been colonized by the intracellular bacterium Wolbachia and thus have a reduced capacity to transmit arboviruses. This reduced capacity to transmit arboviruses is mediated through a phenomenon referred to as pathogen blocking. Pathogen blocking has primarily been proposed as a tool to control dengue virus (DENV) transmission, however it works against a range of viruses, including Zika virus (ZIKV). Despite years of research, the molecular mechanisms underlying pathogen blocking still need to be better understood. Here, we used RNA-seq to characterize mosquito gene transcription dynamics in Ae. aegypti infected with the w Mel strain of Wolbachia that are being released by the World Mosquito Program in Medellín, Colombia. Comparative analyses using ZIKV-infected, uninfected tissues, and mosquitoes without Wolbachia revealed that the influence of w Mel on mosquito gene transcription is multifactorial. Importantly, because Wolbachia limits, but does not completely prevent, replication of ZIKV and other viruses in coinfected mosquitoes, there is a possibility that these viruses could evolve resistance to pathogen blocking. Therefore, to understand the influence of Wolbachia on within-host ZIKV evolution, we characterized the genetic diversity of molecularly barcoded ZIKV virus populations replicating in Wolbachia -infected mosquitoes and found that within-host ZIKV evolution was subject to weak purifying selection and, unexpectedly, loose anatomical bottlenecks in the presence and absence of Wolbachia . Together, these findings suggest that there is no clear transcriptional profile associated with Wolbachia -mediated ZIKV restriction, and that there is no evidence for ZIKV escape from this restriction in our system.