Step I: White Paper Application
Application Guidelines
1. The application should be submitted electronically per requirements via the web site of any of the
NIAID Genome Sequencing Centers for Infectious Diseases. Include all attachments, if any, to the
application.
2. There are no submission deadlines; white papers can be submitted at anytime.
3. GSC personnel at any of the three Centers can assist / guide you in preparing the white paper.
4. Investigators can expect to receive a response within 4-6 weeks after submission.
5. Upon approval of the white paper, the NIAID Project Officer will assign the project to a NIAID GSC
to develop a management plan in conjunction with the participating scientists.
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White Paper Application
Project Title: Surveillance of dengue virus in Florida mosquitoes
Authors: Sharon Isern, Ph.D. and Scott F. Michael, Ph.D.
Primary Investigator Contact:
Name
Sharon Isern, Ph.D.
Position
Associate Professor
Institution
Florida Gulf Coast University
Address
10501 FGCU Blvd. South, Fort Myers
State
FL
ZIP Code
33965
Telephone
239-590-7438
Fax
239-590-7200
E-Mail
sisern@fgcu.edu
1. Executive Summary (Please limit to 500 words.)
After a 75-year absence, dengue virus has been re-introduced and is being actively
transmitted in at least three locations in Southern Florida. The causative agent of dengue
fever, dengue hemorrhagic fever and dengue shock syndrome, dengue virus has emerged in
the past several decades as the most important mosquito borne viral disease with an
estimated 2.5 billion people living in areas at risk for epidemic transmission and 50-100
million people infected annually worldwide. The two major mosquito species that transmit
dengue are already well established throughout the South Atlantic and Gulf Coasts of the
US. There is no approved vaccine against dengue and there are no specific treatments.
In 2010, 65 cases of locally acquired dengue were reported in Florida including one
case in Broward, one case in Miami-Dade, and 63 cases in Monroe (Key West) counties.
In addition, 129 dengue cases were reported in Florida from individuals with recent travel
histories to dengue endemic countries. Therefore, the current situation presents a clear
public health threat to the residents of Florida and the entire Southeastern US if control
measures fail. The situation in Puerto Rico can serve as an example of the consequences
of uncontrolled dengue transmission. In 2010, more than 12,000 cases of dengue were
reported in Puerto Rico, including 28 cases of dengue hemorrhagic fever, and 31 deaths.
Florida Gulf Coast University is uniquely positioned to take a leading role due to its
existing dengue virus expertise, state-of-the art biosafety facilities, geographic proximity to
the outbreak, and existing collaborations with other laboratories and mosquito control
districts in Southern Florida.
An important objective for an effective response against dengue fever in Florida
and the US is the surveillance of infected mosquito populations to identify areas where
transmission is occurring in order to focus mosquito control efforts. Our group is currently
leading this effort by collaborating with mosquito control districts and conducting testing
of collected mosquitoes from counties in Southern Florida. The specific objectives of this
effort are to relate the distribution of infected mosquitoes and infected people with
environmental factors to determine how the outbreak spreads; provide rapid turn around
data on infected mosquitoes to enable local mosquito control districts to focus their
eradication efforts on infected populations; and isolate and genetically characterize the
specific strains of dengue virus involved in order to determine the source and time of
origin as well as any adaptive mutations that may be related to their presence in the
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continental US. A recent episode of “This Week in Virology”, an international netcast
about viruses, highlights these efforts (“TWiV 111: Live at Florida Gulf Coast University”
http://www.twiv.tv/).
From our surveillance so far, three dengue virus positive pools from Key West
have been identified, and E protein sequence data from mosquito RNA extracts of two of
the pools has been acquired. We would like to request that the NIAID consider
sequencing the full-length genomes of these RNA extracts and future Florida isolates
through its program with the Broad Institute Genome Sequencing Center.
2. Justification
Globally, dengue virus is the most important mosquito-transmitted viral pathogen.
In 2009, evidence for locally transmitted dengue in Key West, Florida was discovered.
Outside of sporadic transmission along the Texas-Mexico border, this represents the first
evidence of dengue transmission in the continental United States in 75 years. Knowledge
of the genetic composition of dengue strains circulating in Key West will be invaluable in
understanding the origin of this virus as well as any selective forces that have contributed
to its invasion of the U.S.
There are numerous other dengue virus sequences available from Genbank and the
NIAID currently supports a dengue virus sequence databank. The availability of these
resources will allow for comparison of newly acquired sequences to identify origins and
evolutionary changes.
3. Rationale for Strain Selection
We propose to sequence the genomes of dengue viruses from mosquitoes collected
in the Florida Keys. These viruses are unique in being the only dengue viruses with local
transmission in Florida.
4a. Approach to Data Production: Data Generation
We plan to generate whole genome sequences (approximately 10,000 nt each) from
three currently identified mosquito pools as well as from other future dengue positive
mosquito pools as required to understand the origins and evolution of the virus in Florida.
4b. Approach to Data Production: Data Analysis
Analysis of the sequence data generated is expected to include phylogenetic
analysis, molecular clock analysis, as well tests for recombination and selective pressure.
5. Community Support and Collaborator Roles:
Sequence data from dengue virus strains circulating locally in Florida will be of
great interest to U.S. health agencies as the mosquito vectors (Aedes aegypti and Aedes
albopictus) are now established along the entire Gulf Coast as well as extensive portions of
the Atlantic Coast. If the virus currently circulating in the Florida Keys were to spread to
other areas, it may impact a substantial area of the Southeastern U.S. At this point this is
of primary interest to Florida mosquito control districts and the Florida Department of
Health as well the Centers for Disease Control and Prevention.
The current collaborators on this project are investigators at Florida Gulf Coast
University, Florida Keys Mosquito Control District, and Miami-Dade Mosquito Control.
Funding for mosquito collection in the Keys is provided by Monroe County, Florida.
Other aspects of dengue virus research at Florida Gulf Coast University are currently
funded by the Defense Threat Reduction Agency.
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6. Availability & Information of Strains:
We currently have three reverse transcriptase PCR confirmed dengue positive
mosquito pools from Key West, Florida, and we are screening more mosquito pools as we
receive them. We have successfully amplified 1.7kb reverse transcriptase PCR fragments
from two of these pools. We are in the process of isolating and growing virus stocks from
these pools. Based on conversations with other investigators with extensive experience
isolating dengue viruses from mosquitoes, we do not expect to recover and grow virus
stocks from the majority of samples. We have date and GPS coordinates for each of the
mosquito pools.
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7. Compliance Requirements:
7a. Review NIAID’s Reagent, Data & Software Release Policy:
NIAID supports rapid data and reagent release to the scientific community for all sequencing and
genotyping projects funded by NIAID GSC. It is expected that projects will adhere to the data and
reagent release policy described in the following web sites.
http://www3.niaid.nih.gov/LabsAndResources/resources/mscs/data.htm
http://grants.nih.gov/grants/guide/notice-files/NOT-OD-08-013.html
Once a white paper project is approved, NIAID GSC will develop with the collaborators a detailed data
and reagent release plan to be reviewed and approved by NIAID.
Accept
Decline
7b. Public Access to Reagents, Data, Software and Other Materials:
We plan to make all sequence data publically available according to the guidelines on the
NIAID GSC website:
http://www.niaid.nih.gov/LABSANDRESOURCES/RESOURCES/GSC/Pages/data.aspx
We also plan on making any isolated virus stocks available through BEI, although we have
not yet successfully grown virus from any of the dengue positive mosquito pools.
7c. Research Compliance Requirements
Upon project approval, NIAID review of relevant IRB/IACUC documentation is required prior to
commencement of work. Please contact the GSC Principal Investigator(s) to ensure necessary
documentation are filed for / made available for timely start of the project.
n/a
Investigator Signature:
Investigator Name: Sharon Isern, Ph.D.
Date: March 3, 2011
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