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Detection of Crimean–Congo Hemorrhagic Fever, Hanta, and Sandfly Fever Viruses by Real-Time RT-PCR

  • Sofi M. Ibrahim
  • Mohamed Aitichou
  • Justin Hardick
  • Jamie Blow
  • Monica L. O’Guinn
  • Connie Schmaljohn
Part of the Methods in Molecular Biology book series (MIMB, volume 665)

Abstract

The development of sensitive and specific nucleic acid diagnostic assays for viral pathogens is essential for proper medical intervention. This chapter describes four fluorescence-based PCR assays to detect the Crimean–Congo Hemorrhagic Fever (CCHFV), Andes (ANDV), Hantaan (HANV), and Sandfly Fever Sicilian (SFSV) Viruses. These assays are based on species-specific hydrolysis probes targeting the nucleocapsid protein gene for CCHFV and SFSV and the glycoprotein gene for ANDV and HANV. All four assays were optimized for LightCycler 2.0 (Roche Diagnostics, Indianapolis, IN) or Ruggedized Advanced Pathogen Identification Device (R.A.P.I.D.; Idaho Technology Inc., Salt Lake City, UT). The assays were evaluated using the protocols described in the Subheading 3. The limits of detection were approximately 5, 2, 2, and 5 plaque-forming units (PFUs) for CCHFV, ANDV, HTNV, and SFSV assays, respectively. The sensitivity and specificity of the assays were evaluated with test panels that consisted of 20–60 known positive and 30–135 known negative samples, representing 7–34 genetically diverse bacterial and viral species. The CCHFV assay detected 59 out of the 60 positive samples and no false positives, resulting in 98.3% sensitivity at LOD of 5 PFU and 100% specificity. The ANDV and HTNV assays correctly identified all the positive samples with no false positive reactions; therefore, the sensitivity and specificity of these assays were determined to be 100% at LOD of 2 PFU. The SFSV assay missed three positive samples and cross-reacted with one of 48 negative samples, resulting in 95% sensitivity at LOD of 5 PFU and 98% specificity.

Key words

CCHF virus Sandfly fever virus Andes virus Hantaan virus Fluorescence-based real-time assay 

Notes

Acknowledgements

This work was supported by research program funds (Project No. 19499) managed by the Defense Threat Reduction Agency. We thank Katheryn Kenyon for reviewing the manuscript. The mention of materials or products in this article does not constitute endorsement by the Department of Defense or the United States government. The opinions, interpretations, conclusions, and recommendations contained herein are those of the authors and are not necessarily endorsed by the U.S. Army.

References

  1. 1.
    Elliott, R.M. (1990) Molecular biology of the Bunyaviridae. J Gen Virol 71, 501–522.PubMedCrossRefGoogle Scholar
  2. 2.
    Bouloy, M. (1991) Bunyaviridae: genome organization and replication strategies. Adv Virus Res 40, 235–275.PubMedCrossRefGoogle Scholar
  3. 3.
    Schmaljohn, C.S. and Hooper, J.W. (2001) Bunyaviridae: the viruses and their replication. In: Fields, B.N., Knipe, D.M., Howley, P.M., et al. Eds. Fields Virology, 4th Edn. Lippincott Williams & Wilkins, Philadelphia, PA, pp. 1581–1602.Google Scholar
  4. 4.
    Centers for Disease Control and Prevention (2006) Hantavirus pulmonary syndrome-five states, 2006. Morb Mortal Wkly Rep 55, 627–629.Google Scholar
  5. 5.
    Muranyi, W., Bahr, U., Zeier, M., and van der Woude, F.J. (2005) Hantavirus infection. J Am Soc Nephrol 16, 3669–3679.PubMedCrossRefGoogle Scholar
  6. 6.
    Lopez, N., Padula, P., Rossi, C., Lazaro, M.E., and Franze-Fernandez, M.T. (1996) Genetic identification of a new hantavirus causing severe pulmonary syndrome in Argentina. Virology 220, 223–226.PubMedCrossRefGoogle Scholar
  7. 7.
    Peters, C.J. and Khan, A.S. (2002) Hantavirus pulmonary syndrome: the new American hemorrhagic fever. Clin Infect Dis 34, 1224–1231.PubMedCrossRefGoogle Scholar
  8. 8.
    Schmaljohn, A.L., Li, D., Negley, D.L., Bressler, D.S., Turell, M.J., Korch, G.W., Ascher, M.S., and Schmaljohn, C.S. (1995) Isolation and initial characterization of a newfound hantavirus from California. Virology 206, 963–972.PubMedCrossRefGoogle Scholar
  9. 9.
    Williams, R.J., Bryan, R.T., Mills, J.N., Palma, R.E., Vera, I., De Velasquez, F., Baez, E., Schmidt, W.E., Figueroa, R.E., Peters, C.J., Zaki, S.R., Khan, A.S., and Ksiazek, T.G. (1997) An outbreak of hantavirus pulmonary syndrome in western Paraguay. Am J Trop Med Hyg 57, 274–82.PubMedGoogle Scholar
  10. 10.
    PAHO (2004) Number of cases of hantavirus pulmonary syndrome (HPS) (region of the Americas, 1993–2004). http://www.paho.org/English/AD/DPC/CD/hantavirus-1993-2004.htm.
  11. 11.
    Weidmann, M., Sanchez-Seco, M.P., Sall, A.A., Ly, P.O., Thiongane, Y., Lo, M.M., Schley, H., and Hufert, F.T. (2007) Rapid detection of important pathogenic phleboviruses. J Clin Virol 41, 138–142. doi: 10.1016/j.jcv.2007.10.001.PubMedCrossRefGoogle Scholar
  12. 12.
    Verani, P., Nicoletti, L., Ciufolini, M.G., and Balducci, M. (1991) Viruses transmitted by sand flies in Italy. Parasitologia 33, 513–518.Google Scholar
  13. 13.
    Tesh, R.B., Boshell, J., Young, D.G., Morales, A., Ferra de Carrasquilla, C., Corredor, A., Modi, G.B., Travassos da Rosa, A.P., McLean, R.G., de Rodriguez, C., et al. (1989) Characterization of five new phleboviruses recently isolated from sand flies in tropical America. Am J Trop Med Hyg 40, 529–533.PubMedGoogle Scholar
  14. 14.
    Drosten, C., Gottig, S., Schilling, S., Asper, M., Panning, M., Schmitz, H., and Gunther, S. (2002) Rapid detection and quantification of RNA of Ebola and Marburg viruses, Lassa virus, Crimean–Congo hemorrhagic fever virus, Rift Valley fever virus, dengue virus, and yellow fever virus by real-time reverse transcription-PCR. J Clin Microbiol 40, 2323–2330.PubMedCrossRefGoogle Scholar
  15. 15.
    Yapar, M., Aydogan, H., Pahsa, A., Besirbellioglu, B.A., Bodur, H., Basustaoglu, A.C., Guney, C., Avci, I.Y., Sener, K., Setteh, M.H., and Kubar, A. (2005) Rapid and quantitative detection of Crimean–Congo hemorrhagic fever virus by one-step real-time reverse transcriptase-PCR. Jpn J Infect Dis 58, 358–362.PubMedGoogle Scholar
  16. 16.
    Duh, D., Saksida, A., Petrovec, M., Dedushaj, I., and Avsic-Zupanc, T. (2006) Novel one-step real-time RT-PCR assay for rapid and specific diagnosis of Crimean–Congo hemorrhagic fever encountered in the Balkans. J Virol Methods 133, 175–179.PubMedCrossRefGoogle Scholar
  17. 17.
    Garrison, A.R, Alakbarova, S., Kulesh, D.A., Shezmukhamedova, D., Khodjaev, S., Endy, T.P., and Paragas, J. (2007) Development of a Taqman minor groove binding protein assay for the detection and quantification of Crimean–Congo hemorrhagic fever virus. Am J Trop Med Hyg 77, 514–520.PubMedGoogle Scholar
  18. 18.
    Aitichou, M., Saleh, S.S., McElroy, A.K., Schmaljohn, C., and Ibrahim, M.S. (2005) Identification of Dobrava, Hantaan, Seoul, and Puumala viruses by one-step real-time RT-PCR. J Virol Methods 124, 21–26.PubMedCrossRefGoogle Scholar
  19. 19.
    Evander, M., Eriksson, I., Pettersson, L., Juto, P., Ahlm, C., Olsson, G.E., Bucht, G., and Allard, A. (2007) Puumala hantavirus viremia diagnosed by real-time reverse transcriptase PCR using samples from patients with hemorrhagic fever and renal syndrome. J Clin Microbiol 45, 2491–2597.PubMedCrossRefGoogle Scholar
  20. 20.
    Jakab, F., Sebok, J., Ferenczi, E., Horvath, G., and Szucs, G. (2007) First detection of Dobrava hantavirus from a patient with severe haemorrhagic fever with renal syndrome by SYBR Green-based real time RT-PCR. Scand J Infect Dis 39, 902–906.PubMedCrossRefGoogle Scholar
  21. 21.
    Breslauer, K.J., Frank, R., Blöcker, H., and Marky, L.A. (1986) Predicting DNA duplex stability from the base sequence. Proc Natl Acad Sci USA 83, 3746–3750.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Sofi M. Ibrahim
    • 1
  • Mohamed Aitichou
    • 1
  • Justin Hardick
    • 1
  • Jamie Blow
    • 2
  • Monica L. O’Guinn
    • 1
  • Connie Schmaljohn
    • 1
  1. 1.The United States Army Medical Research Institute of Infectious DiseasesFrederickUSA
  2. 2.The Armed Forces Pest Management BoardSilver SpringUSA

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