1. Michael Turell, Kirti Dave, Maria Mayda, Zahra Parker, Russell Coleman, and Daniel Strickman US Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD; VecTOR Test Systems, Inc., Thousand Oaks, CA; Walter Reed Army Institute of Research, Silver Spring, MD; US Army Medical Materiel Development Activity, Fort Detrick, MD; US Department of Agriculture, Agriculture Research Service, Beltsville, MD Evaluation of novel dipstick assays for the detection of Rift Valley fever virus (RVFV) in mosquitoes

2. Opinions, interpretations, conclusions, and recommendations are those of the author and are not necessarily endorsed by the U.S. Army.

3. Research was conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council, 1996. The facility where this research was conducted is fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International.

4. Overview of RVFV

5. POTENTIAL FOR BECOMING ESTABLISHED IN NORTH AMERICA Many North American mosquito species are potential vectors of RVF virus Ample supply of susceptible domestic vertebrate hosts (cattle, goat, sheep) Role of deer, horses, rodents, etc. is not known

6. AVAILABLE DIAGNOSTIC ASSAYS Research Laboratory based RT-PCR ELISA Infectious virus assays (cell culture) Commercially available None

8. Negative control Positive “Control” line “Test” line RVFV-infected mosquito

9. Schematic side view of test strip

10. <3 3.5 7.9 <3 6.1 8.1 7.0 8.1 Genome Equivalents

11. Sample numberDipstickCT ValueVirus titer 247029.40 250029.20 261029.30 262029.10 265028.90 270028.90 267028.21 253028.61.7 251+/-27.81.8 260030.22 269028.62.5 255027.82.8 266026.93 248+/-29.33.3 268027.93.3 264025.53.7 256025.43.8 257025.54 233024.84.2 240+/-23.44.2 243+23.04.3 254+/-23.74.3 235 ??24.04.5 236+27.14.6 252 ??25.24.7 258025.44.7 244025.35 246+25.25.1 239+++22.85.2 271+/-24.65.2 234+++22.35.3 241++21.95.3 242++25.35.5 238+++21.75.6 237+++21.15.7 249++21.25.8 263++20.26 259++21.36.2

12. Sensitivity of the dipstick based on the amount of infectious virus present Titer Number testedNegativePositive? >5.01009 (90%)1 4.5-5.0521 (20%)2 <4.522180 (0%)4

13. Sensitivity of the dipstick based on the CT (RT-PCR) value CT value Number testedNegativePositive? <2325223 (92%)0 23-25.31243 (25%)5 >25.335312 (6%)2

14. RVFD-1 RVF WNSLEVEEJED-2D-3D-4

15. Specificity Dengue 1-4 Japanese encephalitis La Crosse St. Louis encephalitis Venezuelan equine encephalitis West Nile Yellow fever

16. Strains of RVFV tested OS-1 Mauritania Rhod 2269/74 Zimbabwe Ar-B 1976 Central African Republic 21445 Kenya ZH501Egypt StrainWhere isolated

17. Summary The newly developed dipsticks for RVFV are about as sensitive as the VecTest ® West Nile virus dipstick

18. Summary The newly developed dipsticks for RVFV are about as sensitive as the VecTest ® West Nile virus dipstick The RVFV dipstick detected virus in nearly all samples containing 10 5 plaque-forming units of virus.

19. Summary The newly developed dipsticks for RVFV are about as sensitive as the VecTest ® West Nile virus dipstick The RVFV dipstick detected virus in nearly all samples containing 10 5 plaque-forming units of virus. There was a strong correlation between the infectious virus assay titer and the CT value