Zika virus (ZIKV) is an emerging flavivirus that causes congenital abnormalities and Guillain-Barré syndrome. ZIKV infection also results in severe eye disease characterized by optic neuritis, chorioretinal atrophy, and blindness in newborns and conjunctivitis and uveitis in adults. We evaluated ZIKV infection of the eye by using recently developed mouse models of pathogenesis. ZIKV-inoculated mice developed conjunctivitis, panuveitis, and infection of the cornea, iris, optic nerve, and ganglion and bipolar cells in the retina. This phenotype was independent of the entry receptors Axl or Mertk, given that Axl(-/-), Mertk(-/-), and Axl(-/-)Mertk(-/-) double knockout mice sustained levels of infection similar to those of control animals. We also detected abundant viral RNA in tears, suggesting that virus might be secreted from lacrimal glands or shed from the cornea. This model provides a foundation for studying ZIKV-induced ocular disease, defining mechanisms of viral persistence, and developing therapeutic approaches for viral infections of the eye.

The recent emergence of both chikungunya and Zika viruses in the Americas has significantly expanded their distribution and has thus increased the possibility that individuals may become infected by more than one Aedes aegypti-borne virus at a time. Recent clinical data support an increase in the frequency of coinfection in human patients, raising the likelihood that mosquitoes could be exposed to multiple arboviruses during one feeding episode. The impact of coinfection on the ability of relevant vector species to transmit any of these viruses (that is, their vector competence) has not been determined. Thus, we here expose Ae. aegypti mosquitoes to chikungunya, dengue-2 or Zika viruses, both individually and as double and triple infections. Our results show that these mosquitoes can be infected with and can transmit all combinations of these viruses simultaneously. Importantly, infection, dissemination and transmission rates in mosquitoes are only mildly affected by coinfection.

Chikungunya virus (CHIKV) is the mosquito-borne alphavirus that is the etiologic agent of massive outbreaks of arthralgic febrile illness that recently affected millions of people in Africa and Asia. The only CHIKV vaccine that has been tested in humans, strain 181/clone 25, is a live-attenuated derivative of Southeast Asian human isolate strain AF15561. The vaccine was immunogenic in phase I and II clinical trials; however, it induced transient arthralgia in 8% of the vaccinees. There are five amino acid differences between the vaccine and its parent, as well as five synonymous mutations, none of which involves cis -acting genome regions known to be responsible for replication or packaging. To identify the determinants of attenuation, we therefore tested the five nonsynonymous mutations by cloning them individually or in different combinations into infectious clones derived from two wild-type (WT) CHIKV strains, La Reunion and AF15561. Levels of virulence were compared with those of the WT strains and the vaccine strain in two different murine models: infant CD1 and adult A129 mice. An attenuated phenotype indistinguishable from that of the 181/clone 25 vaccine strain was obtained by the simultaneous expression of two E2 glycoprotein substitutions, with intermediate levels of attenuation obtained with the single E2 mutations. The other three amino acid mutations, in nsP1, 6K, and E1, did not have a detectable effect on CHIKV virulence. These results indicate that the attenuation of strain 181/clone 25 is mediated by two point mutations, explaining the phenotypic instability observed in human vaccinees and also in our studies.

In 2015, Zika virus (ZIKV; Flaviviridae; Flavivirus) emerged in the Americas, causing millions of infections in dozens of countries. The rapid spread of the virus and the association with disease outcomes such as Guillain-Barré syndrome and microcephaly make understanding transmission dynamics essential. Currently, there are no reports of vector competence (VC) of American mosquitoes for ZIKV isolates from the Americas. Further, it is not clear whether ZIKV strains from other genetic lineages can be transmitted by American Aedes aegypti populations, and whether the scope of the current epidemic is in part facilitated by viral factors such as enhanced replicative fitness or increased vector competence. Therefore, we characterized replication of three ZIKV strains, one from each of the three phylogenetic clades in several cell lines and assessed their abilities to be transmitted by Ae. aegypti mosquitoes. Additionally, laboratory colonies of different Culex spp. were infected with an American outbreak strain of ZIKV to assess VC. Replication rates were variable and depended on virus strain, cell line and MOI. African strains used in this study outcompeted the American strain in vitro in both mammalian and mosquito cell culture. West and East African strains of ZIKV tested here were more efficiently transmitted by Ae. aegypti from Mexico than was the currently circulating American strain of the Asian lineage. Long-established laboratory colonies of Culex mosquitoes were not efficient ZIKV vectors. These data demonstrate the capacity for additional ZIKV strains to infect and replicate in American Aedes mosquitoes and suggest that neither enhanced virus replicative fitness nor virus adaptation to local vector mosquitoes seems likely to explain the extent and intensity of ZIKV transmission in the Americas. The mechanisms contributing to the explosive nature of the current ZIKV outbreak in the Americas are poorly understood. Therefore, we characterized the replication of three strains, one from each phylogenetic clade of ZIKV and evaluated virus strain differences in transmission efficiency by American mosquitoes. Our results suggest that the strain currently circulating in the Americas does not have unusually high infectivity for American Ae. aegypti as compared to the African strains used in this study. Colonized Culex mosquitoes were inefficient vectors. In vitro data suggested slower replication and decreased fitness for the currently circulating American strain compared to African strains isolated decades ago. Therefore, viral adaptation to local mosquitoes does not appear, at present, to be driving the current ZIKV outbreak in the Americas.

Chikungunya virus (CHIKV) is a reemerging mosquito-borne pathogen that has recently caused devastating urban epidemics of severe and sometimes chronic arthralgia. As with most other mosquito-borne viral diseases, control relies on reducing mosquito populations and their contact with people, which has been ineffective in most locations. Therefore, vaccines remain the best strategy to prevent most vector-borne diseases. Ideally, vaccines for diseases of resource-limited countries should combine low cost and single dose efficacy, yet induce rapid and long-lived immunity with negligible risk of serious adverse reactions. To develop such a vaccine to protect against chikungunya fever, we employed a rational attenuation mechanism that also prevents the infection of mosquito vectors. The internal ribosome entry site (IRES) from encephalomyocarditis virus replaced the subgenomic promoter in a cDNA CHIKV clone, thus altering the levels and host-specific mechanism of structural protein gene expression. Testing in both normal outbred and interferon response-defective mice indicated that the new vaccine candidate is highly attenuated, immunogenic and efficacious after a single dose. Furthermore, it is incapable of replicating in mosquito cells or infecting mosquitoes in vivo. This IRES-based attenuation platform technology may be useful for the predictable attenuation of any alphavirus. Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that has reemerged since 2004 to cause millions of cases of severe and often persistent arthralgia. Because no licensed vaccine exists to prevent this disease, we utilized an attenuation approach to produce a live CHIKV vaccine candidate that elicits a robust, protective immune response yet causes no detectable disease in mice. It is also incapable of infecting mosquito vectors, an important safety feature for a live virus vaccine that may be used in nonendemic locations to immunize travelers or laboratory personnel. This vaccine approach, which exploits the attenuating effect of altering the expression of the alphavirus structural proteins with a picornavirus IRES, may be broadly applicable to other alphaviruses that cause important febrile diseases as well as encephalitis.