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

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Title: Adaptation of Chikungunya virus to Aedes albopictus mosquitoes: The role of mutations in the E1 AND E2 glycoproteins
Author: Konstantin Tsetsarkin
Abstract: 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
URI: http : / /hdl .handle .net /2152 .3 /206
Date: 2009-10-08


Adaptation of Chikungunya virus to Aedes albopictus mosquitoes: The role of mutations in the E1 AND E2 glycoproteins. Doctoral dissertation, The University of Texas Medical Branch. Available electronically from http : / /hdl .handle .net /2152 .3 /206 .

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