Characterization of the molecular and biological determinants of viral dissemination from the mosquito midgut: Yellow feber virus in Aedes aegypti

In order for a mosquito-borne virus to be transmitted from an infected mosquito to a vertebrate host, virus must disseminate from the mosquito midgut- the initial site of infection following uptake of an infectious bloodmeal- to infect the salivary glands and enter the saliva. The genetic determinants of viral dissemination from the midgut were characterized using the flavivirus yellow fever virus (YFV) in the Aedes aegypti mosquito as a model system. Fifteen chimeric viruses were generated between infectious clones of genotypically and phenotypically distinct YFV strains: the wild-type, disseminating Asibi strain, and the attenuated, non-disseminating 17D vaccine strain. These viruses were characterized in vitro in Vero green monkey kidney cells and C6/36 Ae. albopictus mosquito cells and in vivo for virus production, infection and dissemination in orally infected Ae. aegypti. Data from these studies map the YFV genetic determinants of dissemination from the midgut to position 36 in the membrane (M) structural protein gene, domain III of the envelope (E) protein gene, non-structural protein gene 2A (NS2A), and position 95 in non-structural protein gene 4B (NS4B). \r\nThe virus distribution and tissue tropisms Asibi, 17D, and a chimera 17D/Asibi M-E were evaluated in whole-sectioned Ae. aegypti by immunohistochemistry to characterize the temporal and spatial aspects of YFV dissemination. Data from these studies suggest the following sequence of events takes place after infection of the posterior and anterior midgut: virus travels through the basal lamina underlying the midgut epithelium to the hemocoel and infects the abdominal and thoracic fat body tissues. Virus then may infect the salivary glands following amplification in the fat body tissues or infect and amplify in the intussuscepted foregut, cardia, and thoracic ganglia and return to the hemocoel to infect the salivary glands. Successful completion of these events is dependent on efficient receptor binding, replication, and virus packaging, maturation and release from infected cells, events which are mediated by the M, E, NS2A, and NS4B protein genes. This information contributes to our fundamental understanding of mosquito-virus interactions and may be used in the rational design of live attenuated flavivirus vaccines which are not transmitted by mosquitoes.\r\n