Parameters of Mosquito-Enhanced West Nile Virus Infection

Abstract

The arthropod-borne West Nile virus (WNV) emerged in New York State in 1999 and quickly spread throughout the United States. Transmission is maintained in an enzootic cycle in which infected mosquitoes transmit the virus to susceptible hosts during probing and feeding. Arthropod-derived components within the viral inoculum are increasingly acknowledged to play a role in infection of vertebrate hosts. We previously showed that Culex tarsalis mosquito saliva and salivary gland extract (SGE) enhance the in vivo replication of WNV. Here, we characterized the effective dose, timing, and proximity of saliva and SGE administration necessary for enhancement of WNV viremia using a mouse model. Mosquito saliva and SGE enhanced viremia in a dose-dependent manner, and a single mosquito bite or as little as 0.01 g of SGE was effective at enhancing viremia, suggesting a potent active salivary factor. Viremia was enhanced when SGE was injected in the same location as virus inoculation from 24 h before virus inoculation through 12 h after virus inoculation. These results were confirmed with mosquito saliva deposited by uninfected mosquitoes. When salivary treatment and virus inoculation were spatially separated, viremia was not enhanced. In summary, the effects of mosquito saliva and SGE were potent, long lasting, and localized, and these studies have implications for virus transmission in nature, where vertebrate hosts are fed upon by both infected and uninfected mosquitoes over time. Furthermore, our model provides a robust system to identify the salivary factor(s) responsible for enhancement of WNV replication. IMPORTANCEMosquito-borne viruses are a significant class of agents causing emerging infectious diseases. WNV has caused over 18,000 cases of neuroinvasive disease in the United States since its emergence. We have shown that Culex tarsalis mosquito saliva and SGE enhance the replication of WNV. We now demonstrate that saliva and SGE have potent, long-lasting, and localized effects. Our model provides a robust system to identify the salivary factor(s) and characterize the mechanism responsible for enhancement of WNV replication. These studies could lead to the identification of novel prophylactic or treatment options useful in limiting the spread of WNV, other mosquito-borne viruses, and the diseases that they cause.

Host inflammatory response to mosquito bites enhances the severity of arbovirus infection

“…(F and G) Neutrophil influx into bite sites (n R 6) was ascertained by assaying cutaneous CXCR2 expression (F) and the frequency of Ly6G (legend continued on next page) immunosuppressed mice are often used as an alternative, which precludes the experimental study of many aspects of host immune responses to these infections. Building on the work of other important studies (Conway et al, 2014;Cox et al, 2012;McCracken et al, 2014;Moser et al, 2015;Schneider et al, 2006;Styer et al, 2011), we suggest that the inclusion of mosquito bites, or their saliva, might sufficiently enhance viral replication to enable the study of these infections in wild-type mice.…”

“…When arboviruses are transmitted by mosquitoes, they replicate and disseminate more effectively to the blood, which may increase both their chance of onward transmission and their ability to cause more pronounced disease. The experimental deposition of uninfected mosquito saliva alone, in the absence of a bite, is sufficient to mediate this effect (Conway et al, 2014;Le Coupanec et al, 2013;Limesand et al, 2000;Moser et al, 2015;Styer et al, 2011). Although work has begun to define the factors within mosquito saliva that modulate arbovirus infection, the mechanistic basis that explains these observations is not known.…”

Preparation of Mosquito Salivary Gland Extract and Intradermal Inoculation of Mice

“…As an alternative, ‘spot feeding’ of uninfected female mosquitoes followed by intradermal inoculation of the pathogen via a needle mimics the natural deposition of saliva into mouse skin and delivers a defined dose of pathogen. The ‘spot feeding’ model has successfully been used to study Dengue virus (Cox et al ., 2012; McCracken et al ., 2014) and West Nile virus (Moser et al ., 2015) infection but still requires the concomitant use of live mosquitoes and mice, and cannot control for the amount of saliva delivered. To separately control for mosquito and mouse experiments, inserting the mosquito proboscis into a sucrose solution in a capillary tube can serve to collect mosquito saliva artificially.…”

“…To separately control for mosquito and mouse experiments, inserting the mosquito proboscis into a sucrose solution in a capillary tube can serve to collect mosquito saliva artificially. The saliva collected during sugar feeding, however, differs qualitatively from mosquito saliva that is inoculated into the host skin during natural blood feeding (Marinotti et al ., 1990; Moser et al ., 2015). …”

“…This method allows for independent handling of live mosquitoes and mice between collaborators and can more easily be standardized between assays and research groups. Use of SGE has proven useful to study infection with West Nile virus (Schneider et al ., 2006; Moser et al ., 2015), Dengue virus (Conway et al ., 2014; Schmid et al ., 2016), Rift Valley fever virus (Le Coupanec et al , 2013), and Sindbis virus (Schneider et al ., 2004). Injection of SGE does not precisely mimic inoculation via the mosquito proboscis and likely contains non-secreted components of salivary glands.…”