Students Nick Pondelis and Julie Tamanini collaborate with Museum Director, Heather Lerner, to test bird specimens for West Nile Virus

Abstract

West Nile virus has become endemic to the United States since its introduction in 1999. The virus can cause a number of severe symptoms including fever, neuroinvasive disease, and encephalitis, among other serious complications. West Nile spreads primarily through mosquito vectors, and passerine birds are the primary host reservoirs. Understanding the historic distribution and prevalence of West Nile virus is important in controlling continued outbreaks of the disease, including in Indiana. In 2012 there were 62 cases of West Nile virus, 43 in Indiana, including one in Wayne county, contributing to one of the worst U.S. epidemics to date. Implications for human health are numerous, due to the potential for zoonotic transmission from carrier mosquitoes. The Joseph Moore Museum is a valuable resource for investigating trends in animal populations. Utilizing RT-PCR techniques on avian specimens from the Joseph Moore Museum’s collection, we investigated historic and current distributions of West Nile virus in Indiana. We examined 12 fresh tissues from 11 different species and 5 different orders, especially Passerines. The Passerine family Corvidae is especially vulnerable to WNV infection, which makes them particularly useful for monitoring outbreaks. Half of the specimens tested in this study were positive for West Nile Virus, including a crow, two cuckoos, a flicker, a robin and a hermit thrush. Results from these tests allow for analysis of the rapid spread of an important modern disease in Indiana bird populations.

Objectives

West Nile Virus is a rapidly emerging disease with great potential impact on human health, and avian populations. Dead bird monitoring is an effective method of predicting the presence of WNV in bird populations. Birds are the primary host of WNV, and a high viremia in bird populations predicts human outbreaks (Guptill et al. 2003). Due to budget restrictions there is no current system in place for the monitoring of West Nile virus in deceased birds in Indiana (Price, personal communication). Our goal was to investigate the prevalence of WNV in the Joseph Moore Museum tissue collection, and provide evidence that a dead-bird monitoring program is essential for West Nile virus quantification and outbreak prediction.

Materials and Methods

Tissue specimens from the Joseph Moore Museum tissue collection were chosen based on likelihood of West Nile virus infection. Passerines, especially corvids, were first priority.

Samples of muscle tissue, from the breast and/or heart of each specimen were taken from frozen birds, thawed for removal, then refrozen, with one exception.  A tissue size of 40 mg per sample was chopped on ice with a razor blade, then suspended in 900 ul Qiazol lysis reagent, or divided into two tubes with 450 ul Qiazol in each tube.  Three individuals (G016, G024, and G025) were homogenized using alternating rounds of vortexing, further razor blade slicing, and grinding with a melted micropipette tip.  All other samples were lysed by 10 seconds of sonication using a QSonica with cup horn at 50% amplification following slicing with a razor blade.  Sonication was followed by five minutes on ice and vortexing.  The 10 second-five minute ice/vortex cycle was repeated 11-28 times.  A cold water bath was cycled through the sonicator to help maintain low temperatures during processing.  Samples that were visually homogenized with no obvious pellet were removed from further rounds and frozen. Sample G045 (Corvus brachyrhynchos) was comprised of blood rather than muscle tissue, and homogenization was limited to vortexing. G045 was not frozen after removal from the crow. Positive samples were provided by Bryan Price of the Indiana State Department of Health in the form of RNA isolated from infected mosquitos (primary vector).

RNA extraction was performed using the Qiagen RNeasy Mini Kit and extracted RNA was frozen before rt-PCR. Nested primers were used from Shi et al. (2001). We followed the rt-PCR method outlined in Shi et al. (2001) using 40 cycles and an annealing time of 30 s. PCR products were visualize on Invitrogen 2% agarose E-Gels with SYBR Safe.

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Results

  • Half of all tested tissue samples were infected with West Nile virus.
  • Infections were found in a crow, two cuckoos, a flicker, a robin and a hermit thrush.
  • Half of the songbirds (Order Passeriformes) tested were positive for West Nile Virus infection
  • The death of the crow sample coincides with the onset of 2012 West Nile Virus Mosquito Infections

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Conclusion

In conclusion, rt-PCR of both muscle tissue and blood is an effective way to determine West Nile virus infection in dead birds. Of 12 birds sampled, six were infected (50%). While this large proportion of infected specimens may be in part due to selected sampling, as we targeted birds likely to have WNV, it does show that WNV is prevalent in local bird populations. Because of the high risk to human health WNV poses and the prevalence of WNV in Indiana, testing birds for WNV should be an important part of state monitoring procedures. Museum collections, including the Joseph Moore Museum collection, can serve as an important source of information that can be used in monitoring disease outbreak and epidemiology of zoonotic pathogens such as West Nile virus.

References

Eidson M, J Miller, L Kramer, B Cherry, Y Hagiwara, West Nile Virus Bird Mortality Analysis Group. 2001. Dead crow densities and human cases of West Nile virus, New York State, 2000. Emerging Infectious Diseases 7(4):662-664.

Fitzgerald, S. D., J. S. Patterson, M. Kiupel, H. A. Simmons, S. D. Grimes, C. F. Sarver, R. M. Fulton, B. A. Steficek, T. M. Cooley, J. P. Massey, and J. G. Sikarskie. 2003. Clinical and pathologic features of West Nile virus infection in native North American owls (family Strigidae).  Avian Diseases 47: 602-610.

Guptill SC, KG Julian, GL Campbell, SD Price, and AA Marfin. 2003. Early-season avian deaths from West Nile virus as warnings of human infection. Emerging Infectious Diseases 9:483-484.

Hackett SJ, RT Kimball, S Reddy, RCK Bowie, EL Braun, MJ Braun, JL Chojnowski, WA Cox, KL Han, J Harshman, CJ Huddleston, BD Marks, KJ Miglia, WS Moore, FH Sheldon, DW Steadman, CC Witt, and T Yuri. 2008. A phylogenomic study of birds reveals their evolutionary history. Science 320:1763-1768.

Shi, P-Y., E. B. Kauffman, P. Ren, A. Felton, J. H. Tai, A. P. Dupuis II, S. A. Jones, K. A. Ngo, D. C. Nicholas, J. Maffei, G. D. Ebel, K. A. Bernard, and L. D. Kramer. 2001. High-throughput detection of West Nile virus RNA. Journal of Clinical Microbiology 39: 1265-1271.

Yaremych, S. A., R. E. Warner, P. C. Mankin, J. D. Brawn, A. Raim, and R. Novak. 2004. West Nile virus and high death rate in American crows. Emerging Infectious Diseases 10.

U.S. Department of Interior, U.S. Geological Survey. 2013. West Nile virus – Mosquito infections by week – Indiana 2012. http://diseasemaps.usgs.gov/wnv_in_mosquito.html

Acknowledgements

This research was generously funded by grants from the Earlham College’s Annual Research Conference and the Joseph Moore Museum Cope Research Fund. In addition, we thank those who facilitated our research process, including Peter Blair, Deanna McCartney, Mark Glazier (Indiana State Department of Health), Bryan Price (Indiana State Department of Health), John Iverson, and Mic and Sandy Jackson.

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