Adaptation of Chikungunya virus to Aedes albopictus mosquitoes: The role of mutations in the E1 AND E2 glycoproteins



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Chikungunya virus (CHIKV) is a positive sense single-stranded RNA virus in the family Togaviridae that between 2005 and 2007 caused its largest outbreak/epidemic in documented history, affecting parts of Africa, the Indian Ocean islands, India, and Europe. An unusual feature of this epidemic was the involvement of the previously unrecognized vector of CHIKV: Ae. (Stegomyia) albopictus mosquitoes. It was postulated that genetic changes in the virus might have contributed to the scale of these epidemics by facilitating CHIKV transmission by Ae. albopictus mosquitoes. In order to characterize genetic factors that might influence the ability of CHIKV to be transmitted by Ae. albopictus, I developed full-length infectious clone (i.c.) (pCHIKV-LR i.c.) and an i.c. that expressed enhanced green fluorescent protein (eGFP) from either a 3’ or 5’ additional sub-genomic promoters (pCHIKV-LR 3’GFP and pCHIKV-LR 5’GFP respectively) based on a CHIKV strain isolated during 2005-2006 epidemic on Reunion Island (LR2006 OPY1). The viruses produced from these i.c. were characterized in cell culture and in Ae. aegypti and Ae. albopictus mosquitoes. I concluded that, pCHIKV-LR i.c. and pCHIKV-LR 5’GFP infectious clones are suitable for investigation of the genetic factors influencing CHIKV fitness in the mosquito and vertebrate hosts. \r\nPrevious phylogenetic analysis had demonstrated that the 2005-2006 epidemic on Reunion Island was associated with a strain of CHIKV with a mutation in the E1 glycoprotein (E1-A226V). Using viral infectious clones of Reunion and West African strains of CHIKV, into which either the E1-226 A or V residues were engineered, I demonstrated that the E1-A226V mutation was directly responsible for a significant increase in CHIKV infectivity for Ae. albopictus, and led to more efficient viral dissemination into mosquito secondary organs and transmission to suckling mice. I also demonstrated that increased CHIKV infectivity of Ae. albopictus midgut cells associated with the E1-A226V mutation is directly responsible for more efficient virus replication in the mosquito, more rapid dissemination of the virus into salivary glands and more efficient transmission. Interestingly, this mutation caused a marginal decrease in the ability of CHIKV to infect the Ae. (Stegomyia) aegypti midgut, but had no effect on viral dissemination, and was associated with a slight increase in transmission by Ae. aegypti. These findings demonstrate that the E1-A226V mutation confers CHIKV adaptation to transmission by Ae. albopictus, and provide a plausible explanation of how this mutant virus caused an epidemic in a region lacking the typical vector.\r\nI also demonstrated that the E1-A226V mutation is associated with an increase in cholesterol-dependency of CHIKV for growth and entry into C6/36 cells, and is responsible for increase in the pH dependency of CHIKV fusion reaction. However, analysis of viruses with specific mutations at position E1-226, and at other CHIKV genomic regions that modulate cholesterol dependency of CHIKV, demonstrated that there is no clear mechanistic correlation between dependency for cholesterol and increased infectivity to Ae. albopictus mosquitoes. Also no correlation was observed between pH dependency of CHIKV fusion and infectivity to Ae. albopictus mosquitoes. Based on these data, I conclude that the E1-A226V mutation probably acts at different steps of the CHIKV life cycle, affecting multiple functions of the virus.\r\nUsing i.c. of Reunion and Ugandan strains of CHIKV, I demonsrated that mutations at positions 60 and 211 of the E2 glycoprotein of CHIKV are responsible for modulating the effect of the E1-A226V mutation on CHIKV infectivity for Ae. albopictus. Analysis of the effect of the mutations at E2-211 on CHIKV replication in cell culture and on CHIKV binding to the brush border membrane proteins of Ae.albopictus midgut cells, indicated that different residues at E2-211 might differentially affect the ability of CHIKV to interact with specific proteins expressed on the surface of midgut epithelial cells. I hypothesized, that after internalization by endocytosis, these interactions might determine the particular location within endosomal compartments where the CHIKV membrane fusion and release of virus nucleocapsid occur.\r\nThe information from the present study provides insight into the processes of CHIKV adaptation to a new vector species, which would determine the potential threat for spreading and establishment of CHIKV in tropical and temperate regions populated with Ae. albopictus mosquitoes.\r\n



Chikungunya virus, aegypti, Aedes albopictus