Incidence of disease due to dengue (DENV), chikungunya (CHIKV) and yellow fever (YFV) viruses is increasing in many parts of the world. The viruses are primarily transmitted by Aedes aegypti, a highly domesticated mosquito species that is notoriously difficult to control. When transinfected into Ae. aegypti, the intracellular bacterium Wolbachia has recently been shown to inhibit replication of DENVs, CHIKV, malaria parasites and filarial nematodes, providing a potentially powerful biocontrol strategy for human pathogens. Because the extent of pathogen reduction can be influenced by the strain of bacterium, we examined whether the wMel strain of Wolbachia influenced CHIKV and YFV infection in Ae. aegypti. Following exposure to viremic blood meals, CHIKV infection and dissemination rates were significantly reduced in mosquitoes with the wMel strain of Wolbachia compared to Wolbachia-uninfected controls. However, similar rates of infection and dissemination were observed in wMel infected and non-infected Ae. aegypti when intrathoracic inoculation was used to deliver virus. YFV infection, dissemination and replication were similar in wMel-infected and control mosquitoes following intrathoracic inoculations. In contrast, mosquitoes with the wMelPop strain of Wolbachia showed at least a 104 times reduction in YFV RNA copies compared to controls. The extent of reduction in virus infection depended on Wolbachia strain, titer and strain of the virus, and mode of exposure. Although originally proposed for dengue biocontrol, our results indicate a Wolbachia-based strategy also holds considerable promise for YFV and CHIKV suppression.

O'nyong-nyong virus (ONNV) and chikungunya virus (CHIKV) are closely related alphaviruses that cause human disease in Africa and Asia. Like most alphaviruses, CHIKV is vectored by culicine mosquitoes. ONNV is considered unusual as it primarily infects anopheline mosquitoes; however, there are relatively few experimental data to support this. In this study, three strains of ONNV and one strain of CHIKV were evaluated in Anopheles gambiae and Aedes aegypti mosquitoes and in four cell lines. As predicted, CHIKV was not infectious to An. gambiae, and we observed strain-variability for ONNV with respect to the ability of the virus to infect An. gambiae and Ae. aegypti. The species specificity in vivo was reflected by in vitro experiments using culicine and anopheline-derived cell lines.

Live-attenuated virus vaccines are key components in controlling arboviral diseases, but they must not disseminate in or be transmitted by mosquito vectors. Although the cycles in which many mosquito-borne viruses are transmitted are well understood, the role of viral genetics in these processes has not been fully elucidated. Yellow fever virus (YFV) is an important arbovirus and the prototype member of the family Flaviviridae. Here, YFV was used in Aedes aegypti mosquitoes as a model to investigate the genetic basis of infection and dissemination in mosquitoes. Viruses derived from infectious clones and chimeric viruses with defined sequential manipulations were used to investigate the influence of specific sequences within the membrane and envelope structural protein genes on dissemination of virus from the mosquito midgut. Substitution of domain III of the envelope protein from a midgut-restricted YFV into a wild-type YFV resulted in a marked decrease in virus dissemination, suggesting an important role for domain III in this process. However, synergism between elements within the flavivirus structural and non-structural protein genes may be necessary for efficient virus escape from the mosquito midgut. INTRODUCTIONMosquito-borne flaviviruses such as Yellow fever virus (YFV), Dengue virus (DENV) (serotypes 1-4), Japanese encephalitis virus (JEV) and West Nile virus (WNV) cause millions of infections and tens of thousands of deaths each year (Petersen & Marfin, 2005; WHO, 2003). The lack of effective antiviral measures and the failure of mosquitocontrol programmes in many affected areas make vaccination the primary means of prevention and, in the case of YFV, of control of human outbreaks of these diseases. Considerable effort is being directed towards the development of live virus vaccines against DENV 1-4 and WNV and an improved vaccine for JEV (Hombach et al., 2005;Monath et al., 2005). It is imperative that live-attenuated vaccine viruses should not replicate in, disseminate in or be transmitted by mosquito vectors. Our goal in these studies was to identify flavivirus genes or partial genes that, when modified appropriately, would block dissemination and transmission of virus by mosquitoes. These regions can then be targeted in the rational design of future live flavivirus vaccine candidates.Little is known about the viral genetic factors that mediate the processes of infection, replication and dissemination in mosquito vectors, despite approximately a century of study on the ecology and epidemiology of mosquito-borne viruses. A number of steps must be completed successfully for transmission of a mosquito-borne virus to occur. Following uptake of an infectious blood meal by a female mosquito, virus must infect the midgut epithelium and replicate. To develop a disseminated infection, virus must then escape the midgut into the haemocoel and infect secondary tissues such as the salivary glands. This step is critical in allowing transmission of virus from a mosquito to a susceptible host. Transmission may then...

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In addition to modulating blood meal digestion and protecting the midgut epithelial cells from mechanical and chemical damage, a biological function attributed to the mosquito type I peritrophic matrix (PM) is preventing or reducing pathogen invasion, especially from Plasmodium spp. Previously, we demonstrated that chitin is an essential component of the PM and is synthesized de novo in response to blood feeding in Aedes aegypti. Therefore, knocking down chitin synthase expression by RNA interference severely disrupts formation of the PM. Utilizing this artificial manipulation, we determined that the absence of the PM has no effect on the development of Brugia pahangi or on the dissemination of dengue virus. However, infectivity of Plasmodium gallinaceum is lower, as measured by oocyst intensity, when the PM is absent. Our findings also suggest that the PM seems to localize proteolytic enzymes along the periphery of the blood bolus during the first 24 hours after blood feeding. Finally, the absence of the PM does not affect reproductive fitness, as measured by the number and viability of eggs oviposited.