1. A Plan for a Long Term Investigation of Human Exposure to West Nile Virus in Fremont County, Wyoming
Joy Watkins, Kaylan Schilling, Adam Conner, Shanda Barlow, Grant Hosking and Kelvin Kinyatta with Steven McAllister
Division of Health Science and Public Safety
Central Wyoming College

2. Virus Classification:
Discovery: Uganda, Detected in U.S. 1999
Family: Flaviviridae Other members: Dengue Fever, Tick Borne Encephalitis, Yellow Fever, and Zika Virus
Arbovirus (arthropod borne)
West Nile Virus was first discovered in Uganda in 1937, and detected in the U.S. in 1999 rapidly migrating to the East coast over the course of ten years. The virus is part of the persistently emerging family of Flaviviridae. As illustrated above, West Nile virus is a 50nm arbovirus that is maintained through an enzootic mosquito to bird cycle with horses and humans serving as incidental hosts. The Culex tarsalis mosquito being the most common vector endemic to our area that feeds on members of the Corvidae and Thrush family is categorized by its large size, blunt abdomen, and presence of white striations on its tarsals and proboscis. Other main mosquito vectors are the Culex pipiens, and Culex quinquefasciatus species. As a mosquito takes a blood meal from a human the mosquito saliva negatively affects vascular constriction, coagulation, platelet aggregation, angiogenesis and immunity creating inflammation,(1) This is increasingly advantageous to WNV, and other viruses part of the Flaviviridae complex with mosquito born vectors (2)(3)(4)(5) as global warming conditions may increase immature mosquito development.

3. There was an increase of total cases in WY reported to the CDC in 2003, 2007 and then again in 2013 a spike near a quarter of what was seen in 2007.

4. Symptoms: Complications: Fever Meningitis Malaise Encephalitis
Skin rash
Lymphadenopathy
Meningitis
Encephalitis
Polio-like paralysis
The symptoms of WNV include fever, headache, nausea, malaise, skin rash, and lymphadenophy. However according to the World Health Organization approximately 80% of people who are infected will not show any symptoms, and it is estimated that only 20% of those infected with the virus will develop complications. If further complications do take place they may lead to meningitis, encephalitis, and polio-like paralysis. Meningitis is an acute inflammation of the proactive membranes covering the brain and spinal cord (dura mater, arachnoid, pia mater). Important diagnostic tools include ramp testing of mosquitoes and birds, neutralization assays, RT-PCR, and ELISA testing for immunoglobulin M and G with IgM appearing first in response to initial exposure of the virus and normally class-switching to IgG within 2-3 months of initial exposure. (Murray 2013) Both Ramp testing of mosquitos and ELISA testing has been performed by the CWC lab with the results indicating the presence and prevalence of WNV in Fremont county.

5. Humoral Function: Ig’s produced by B-cells (after infection)
Normally 1-3m class switch
Direct neutralization
Opsonization
Agglutination

6. Now, in 2011, of the 85 subjects tested approximately 16
Now, in 2011, of the 85 subjects tested approximately 16.5% were IgG positive and 9.4% IgM positive. Then again, In 2012, of the 95 subjects tested approximately 10.5% were IgG positive and 5.3% IgM positive. Now, in order to decrease the margin of error it is CWC labs’ intentions to increase the sample size of the population being tested.

7. Title and Content Layout with Chart
IgM positive 5.66 or greater, IgM positive 3 or greater. In 2011 and 2012 CWC’s lab tested 180 human sera samples for the presence of West Nile virus IgG and IgM antibodies by an ELISA assay at 450nm. While the results indicated that a majority of the population, from 83.5% %, had not been exposed to WNV, It is of great interest that anywhere from 1% to 10% of cases reported in 2012 were new cases indicated by IgM levels, 4 of the subjects tested in 2012 reported exposure at least five years prior to testing and continued to exhibit high titers of IgM antibody.

8. Serosurvey Conducted with IgM and IgG Titers
Researcher: IgM Persistance: RT-PCR: Patients:
A Papa, years post No 10 of 26
K Murray, ,6, 8 years post No 42, 34, 23%
K Murray, Yes, 6.7 years Yes, + 5 of 25
K Gibney, Baty 2010 Yes, 6 years Yes, - 40
Urine Collection Assessing Viral Presence
Researcher: IgM/IgG Positive: RT-PCR:
Papa- 6 of 10 neuroinvasive, Murray % of whole pop tested, Gibney had 32 of 63 neuroinvasive

9. K murray, 2013, Percentages of participants (N = 163) with detectable IgM or undetectable IgG by year post-infection.
 Interestingly, the percentage of study participants with detectable IgM antibodies seems to decline over years 1–4 but then increases again from years 5 to 8. Anti-WNV IgG antibodies steadily declined over the study period.  (P/N)= The optical density of the patients serum divided by the optical density of the negative control.  A negative result was defined as a P/N < 2. An equivocal result was defined as a P/N ≥ 2 and < 3. A positive result was defined as a P/N ≥ 3.  IgM was assessed by a MAC (IgM antibody capture) ELISA at 450nm, and IgG was assessed by an indirect ELISA at 405nm. Covalescence= 1, 6, and 8 years post. IgM+=42% 1 year post, 34% 6 years post, 23% 8 years post, IgG-=4%, 44%, and 46%.  Interestingly, 7 of 11 (64%) and 4 of 5 (80%) of those participants with detectable IgM were found to be negative for IgG antibodies at 6 and 8 years, respectively. Their study population consisted of 62% males and 83% whites. The average age at acute disease onset was 52 years (range = 9–88 years). The study population was comprised of 56% neuroinvasive disease cases (38% encephalitis and 18% meningitis) and 44% non-neuroinvasive disease cases (28% febrile and 16% asymptomatic).

10. RT-PCR in combination with ELISA
prM Protein
E Protein
Primers for Protein Genes:
prM, and M: pre-membrane and membrane proteins
E: envelope protein
C: capsid protein
Capsid Protein
Viral RNA
Lipid Membrane

11. CWC Longitudinal Study
Identify and track subjects infected or previously infected with WNV
Assess additional subjects expressing high titration of IgM long after exposure
Increase sample population and testing time frame
Test individuals for cryptic infection through reverse transcriptase PCR
Investigate if IgM and IgG are elevated in tandem with virus replication

12. Acknowledgements:
This project is supported in part by a grant from the National Institute of General Medical Sciences (2P20GM103432) from the National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
We thank Dr. Scott Seville and the University of Wyoming INBRE network for their support in this research.

13. Works Cited
 1. Baty S, Gibney K, Staples J, et al. Evaluation for West Nile virus (WNV) RNA in urine of patients within 5 months of WNV infection. J Infect Dis  2012; 205:
2.Busch, M. P., Wright, D. J., Custer, B., Tobler, L. H., Stramer, S. L., Kleinman, S. H., Glynn, S. A. (2006). West Nile Virus Infections Projected from Blood Donor Screening Data, United States, 2003. Emerging Infectious Diseases, 12(3), 395–
3. Busch, M. P., Kleinman, S. H., Tobler, L. H., Kamel, H. T., Norris, P. J., Walsh, I., Matud, J., Prince, H., Lanciotti, R., Wright, D. J., Linnen, J., Caglioti S. (2008), Virus and Antibody Dynamics in Acute West Nile Virus Infection. J Infect Dis; 198 (7): doi: /591467
4.  Gibney K, Lanciotti R, Sejvar J, et al. West Nile virus not detected in urine of 40 people tested 6 years after acute West Nile virus disease. J Infect Dis 2010; 203:344–7
5. Murray K, Melissa N. Garcia, Chris Yan, Rodion Gorchakov (2013) Persistence of Detectable Immunoglobulin M Anitbodies Up to 8 Years After Infection with West Nile Virus Nov 6; 89(5): 996–1000.
6. Papa, A., Anastasiadou, A., & Delianidou, M. (2015). West Nile virus IgM and IgG antibodies three years post- infection. Hippokratia, 19(1), 34–36.
7.Huhn, G. D., Sejvar, J. J., Montgomery, S. P., Dworkin, M. S. (2003). West Nile Virus in the United States: An Update on an Emerging Infectious Disease.  American Academy of Family Physicians; 68 (4):