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Project 1:  The Role of Biological and Environmental Factors in Vector Competence           

            The following project is being carried out in collaboration with Dr. Cynthia Lord (clord@ufl.edu) and Dr. Walter Tabachnick (wjt@ufl.edu).

            Presented at the annual meeting of The American Society of Tropical Medicine and Hygiene in 2008, click here to view presentation.

            Although biological and environmental factors influence mosquito vector competence for arboviruses, there has been little work to determine how such factors may interact with one another. These interactions must be characterized in order to understand how vector competence variation in nature influences the epidemiology of arbovirus cycles. This project focuses on Culex pipiens quinquefasciatus populations and the influence of biological and environmental factors on their susceptibility to infection and ability to transmit West Nile and St. Louis encephalitis viruses (Flavivirus:  Flaviviridae). Our initial studies show that complex environmental and biological effects must be taken into account when comparing laboratory studies of vector competence. This is especially important when comparing results of laboratory studies to field populations where the extent of biological and environmental variability is unknown.

West Nile Virus
(Photo: CDC/P.E. Rollin)

This work is funded by a National Institutes of Health grant (no. 42164) to Cynthia C. Lord, Walter J. Tabachnick, Jonathan F. Day, George F. O'Meara, and C. Roxanne Connelly.

Project 2:  Mechanistic Causes of Differential Vector Competence for West Nile Virus and St. Louis Encephalitis Virus

            The following project is being carried out in collaboration with the molecular biology lab of Dr. Chelsea Smartt (ctsmart@ufl.edu).

            We are currently investigating mechanistic causes of differential arthropod vector competence focusing on Culex mosquitoes and the extent to which these factors are affected by biological and environmental factors. This is the first work to examine the extent to which biological and environmental factors affect mechanisms of West Nile and St. Louis encephalitis virus (Flavivirus:  Flaviviridae) infection in the midgut of Culex pipiens quinquefasciatus.  This information is critical to begin further exploration of these processes that will ultimately be needed to assess their impact on vector competence. The midgut is the first formidable barrier to virus entry in a mosquito. Virions likely bind to receptors in midgut epithelial cells and must overcome midgut infection and dissemination barriers for dissemination to salivary glands and subsequent transmission to occur.

            We have shown West Nile virus-binding proteins in Culex pipiens quinquefasciatus midguts from Central and Eastern Florida colonies (Figure 1). Proteins from Culex pipiens quinquefasciatus were electrophoresed on a gel and showed ≥ 15 proteins with apparent molecular weights from 14-200 kDa (Fig. 2a). Western blot analysis with West Nile virus overlays was conducted at 25°C and showed that ≥ 8 of the 15 proteins ranging from 38-200 kDa bound West Nile virus (Fig. 2b).

This work is funded by a National Institutes of Health grant (no. 42164) to Cynthia C. Lord, Walter J. Tabachnick, Jonathan F. Day, George F. O'Meara, and C. Roxanne Connelly.

Project 3:  West Nile Virus Alters Gene Expression in Culex pipiens quinquefasciatus

            The following project is being carried out in collaboration with the molecular biology lab of Dr. Chelsea Smartt (ctsmart@ufl.edu).

            Presented at the annual meeting of The Florida Mosquito Control Association in 2008, click here to view presentation.

            Alterations in gene expression in the midgut of female Culex pipiens quinquefasciatus exposed to blood meals containing 6.7 logs plaque-forming units/mL West Nile virus (WNV) were investigated by Fluorescent Differential Display. One clone that was upregulated in response to infection, CQ G12A2, shares identity (85%) with a leucine-rich repeat-containing protein from Culex pipiens quinquefasciatus and shares 42% identity to Toll-like receptors from Aedes aegypti. This is the first cDNA clone isolated from female Culex pipiens quinquefasciatus midgut tissue whose protein product may interact with West Nile virus and play a role in disease transmission.

Figure 1. Semi-quantitative RT-PCR analyses of DD clone CQ G12A2 in Culex pipiens quinquefasciatus mosquito midgut RNA isolated from West Nile virus-infected females (Panel A). Panel B shows the agarose gel of the qRT-PCR analysis of WNV titer in the same RNA used in panel A.

             Future expanded studies addressing realistic public health risk scenarios would include: 1) Evaluating effects of gene silencing on the extent to which target genes affect vector competence for West Nile virus, 2) What variation exists in these genes in geographically distinct subpopulations of Culex pipiens quinquefasciatus or other Culex species and how does this influence vector competence? 3) Does similar gene expression occur in other Culex-virus systems? 4) Do other environmental conditions affect the capacity of virus-binding proteins and controlling genes? The final solutions to these questions will provide a realistic way to assess vector competence of Culex spp. for West Nile virus, gauge the danger posed by specific populations under different environmental conditions, and provide opportunities to mitigate Culex-borne epidemics through genetic and/or environmental strategies, or targeted vector control to the most dangerous Culex populations.

This work is funded by grants from the Florida Department of Agriculture and Consumer Services (no. 72514 and 77263) to Chelsea T. Smartt and the National Institutes of Health (no. 42164) to Cynthia C. Lord, Walter J. Tabachnick, Jonathan F. Day, George F. O'Meara, and C. Roxanne Connelly.

Project 4:  The Relationship of Chikungunya Virus with Aedes aegypti and Aedes albopictus

            The following project is being carried out under Catherine Westbrook of the L. Philip Lounibos Laboratory (lounibos@ufl.edu).

            Presented at the annual meeting of The Entomological Society of America in 2008, click here to view presentation.

            Presented at the annual meeting of The American Society of Tropical Medicine and Hygiene in 2009, click here to view poster.

             Many biotic and abiotic features of the environment of the aquatic larvae of holometabolous insects influence life history traits of their terrestrial adults, such as age at maturity, size, fecundity, and life span. However, less is known about how the larval environment affects the physical and physiological characteristics of adults in ways that may alter the susceptibility of an insect vector to a pathogen resulting in changes in its transmissibility.  This research investigates the link between mosquito larval ecology and the capacity of adult mosquitoes to vector pathogens, with specific emphasis on how multiple environmental factors interact to change the characteristics of emerging infectious disease.

             The invasive container mosquitoes Aedes aegypti and A. albopictus have sympatric distributions in many parts of the world, and co-occurrence of their immature stages in the same container is common. Therefore, this system provides an excellent model to study how abiotic and biotic factors impinging on the juvenile life stage of a holometabolous insect affects vector-virus interactions in the adult stage. Aedes mosquito-vectored viruses, such as chikungunya, dengue, and yellow fever, threaten the health of millions of people worldwide each year. This research specifically focuses on the relationship of chikungunya virus, a recently emerging and re-emerging human arboviral pathogen with the two Aedes species.

Project 5:  Effects of West Nile Virus Dose on Temporal Infection of the Culex pipiens quinquefasciatus Midgut  

            The following project is being carried out in collaboration with Dr. Chelsea Smartt (ctsmart@ufl.edu) and Dr. Walter Tabachnick (wjt@ufl.edu).           

            Presented at the annual meeting of The American Society of Tropical Medicine and Hygiene in 2008, click here to view poster.

            Culex pipiens quinquefasciatus were fed blood meals containing either 6.7 logs plaque-forming units (PFU)/mL or 4.8 logs PFU/mL of West Nile virus.  Midguts and legs from five mosquitoes per dose were collected every other day from 0-12 days post-infection (dpi) to investigate spatiotemporal midgut infection and dissemination patterns.  Midgut infection was assessed with immunofluorescent staining and virus dissemination to the legs was determined with qRT-PCR.  Dissemination was calculated as the percentage of midgut infected mosquitoes with infected legs.  Sixty percent of mosquitoes in the high-dose group had infected midguts as early as 2 dpi and 75% of mosquitoes with infected midguts had disseminated infections by 4 dpi.  For the low-dose group, 20% of mosquitoes had infected midguts at 4 dpi and 100% of the midgut infected mosquitoes had disseminated infections by 8 dpi.  The midgut infection rate at 4 dpi was significantly different between the high (75%) and low dose (20%) groups (p<0.05).  The disseminated infection rate was also significantly different between doses at 4 dpi (75% high-dose and 0% low-dose) and 6 dpi (80% high-dose and 0% low-dose) (p<0.05).  By 10 and 12 dpi, 100% of mosquitoes had infected midguts and 80% of mosquitoes with infected midguts had disseminated infections, suggesting the presence of a midgut escape barrier, regardless of initial virus dose.  Our results indicate that virus dose had greatest effect on midgut infection and dissemination 4 and 6 dpi.  We demonstrate variation in spatiotemporal patterns of midgut infection and dissemination between doses that may help explain variation vector competence resulting from differences in the extrinsic incubation period of mosquito vectors.