West Nile Virus Infection Alters Midgut Gene Expression In Culex pipiens quinquefasciatus Chelsea T. Smartt, Stephanie L. Richards, Sheri L. Anderson and Jennifer S. Erickson Florida Medical Entomology Laboratory University of Florida, IFAS
Culex Mosquito Culex spp. Transmitter
of WNV, SLEV, & EEEV Potential threat in Florida
Photographs by M. M. Cutwa-Francis
Female Mosquito Midgut
Pathogens enter the midgut in the blood meal. They must move through the midgut cell wall into the hemolymph in order to be passed into the next host.
Midgut Environment Digestive Enzymes Peritrophic Matrix Physical Barriers
May not be relevant to viruses
Research Objectives Investigate
the role mosquito midgut genes play in WNV infection. Gene expression alteration after WNV exposure.
Vector Competence Physical/structural barriers
Midgut factors Immune peptides Encapsulation reactions
Our Approach
Midgut gene expression changes Fluorescent
Differential Display analysis Semi-quantitative RTPCR Sequence analysis
Image by GenHunter Corporation
Experimental Design Blood feed mosquitoes -/+ WNV ( 6.8 logs/ml) Dissect midguts (4 days post-infection)
Extract RNA (Differential Display) Sequence analysis
-/+ WNV ( 6.8 logs/ml) Dissect midguts (0-9 hour and 1-10 days post-infection) Extract RNA (RT-PCR)
Differential Gene Expression 1 1,1’,2,2’
2
3
1,1’,2,2’ 1,1’,2,2’
4
5
1,1’,2,2’
1,1’,2,2’
Sample 1= uninfected Sample 2= WNV infected
6
7
1,1’,2,2’ 1,1’,2,2’
Preliminary Results
Differential display revealed ~23 PCR products with reproducible expression differences after feeding on WNV-infected blood. Eleven products with increases expression after WNV infection were cloned. Sequence analysis of one showed identity to immune response products (leucine-rich protein and Toll-like receptors).
100 bp
Expression Analysis 0h 3h 6h 9h 1d 2d 3d 4d 5d 6d 7d 8d 9d 10d
100 bp
MG RNA –WNV
0h 3h 6h 9h 1d 3d 4d 5d 6d 7d 8d 9d 10d
MG RNA +WNV
Conclusions
Gene expression studies revealed 23 PCR products with reproducible expression differences after feeding on WNV-infected blood. One clone shares identity to immune response elements of Culex quinquefasciatus and Aedes aegypti. Expression differences of our Cx. quinquefasciatus immune response product were detected in midguts pre- and post-WNV exposure.
Future Studies
Involvement in WNV infection Sequence analysis
Silence the genes (Gene Knockdown)
Acknowledgements This project is supported in part by funding and from the Florida Department of Agriculture and Consumer Services grants 00072154 and 00077263 to CTS.
Acknowledgements Collaborators at FMEL: Lab members: Dr. Stephanie Richards Shainnel Eans Jennifer Erickson Alessandra Guimaraes Sheri Anderson
