Abstract
The Variola virus is the causative agent of smallpox disease. Accurate identification of Variola and differential diagnosis between Variola and other vesicle forming (smallpox-like) pathogens are both technically challenging and of great importance. We compiled a list of vesicle forming pathogens that are prone to be misdiagnosed as smallpox. Some of the pathogens (members of the Orthopoxvirus genus) are also genetically highly similar to Variola.
We established an approach for genetic identification and differential diagnosis between the closely related Orthopoxvirus genus members. This approach couples multiplex PCR with DNA microarray as molecular means for specific and sensitive pathogen identification. It consists of PCR amplification of conserved gene fragments, followed by sequence-based specific identification on a DNA microarray platform utilizing the Arrayed Primer Extension (APEX) technique. Our prototype array – the ChiPox, is based on genes that harbor variable regions flanked by highly conserved Orthopoxvirus sequences; a pattern supporting APEX-based diagnostics. This novel approach was successfully applied for the differential identification of six members of the Orthopoxvirus genus. The ChiPox is a powerful and sensitive tool for discrimination between closely related Orthopoxvirus species, and is the first step in the establishment of a comprehensive assay for genetic discrimination of vesicle forming pathogens.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Arita, M. & Tagaya, I. (1977) Structural polypeptides of several strains of orthopoxvirus. Microbiol Immunol, 21(6), 343–6.
Becker, C., Kurth, A., Hessler, F., Kramp, H., Gokel, M., Hoffman, R., Kuczka, A. & Nitsche, A. (2009) Cowpox virus infection in pet rat owners. Deutsches Arzteblatt Int, 106(19), 329–34.
Esposito, J. J. & Knight, J. C. (1985) Orthopoxvirus DNA: a comparison of restriction profiles and maps. Virology, 143(1), 230–51.
Esposito, J. J., Sammons, S. A., Frace, A. M., Osborne, J. D., Olsen-Rasmussen, M., Zhang, M., Govil, D., Damon, I. K., Kline, R., Laker, M., Li, Y., Smith, G. L., Meyer, H., Leduc, J. W. & Wohlhueter, R. M. (2006) Genome sequence diversity and clues to the evolution of variola (smallpox) virus. Science, 313, 807–12.
Henderson, D. A. (2002) Countering the post eradication threat of smallpox and polio. Clin Infect Dis, 34(1), 79–83.
Henderson, D. A., Inglesby, T. V. & Barlett, J. G. (1999) Smallpox as a biological weapon: medical and public health management. Working group on Civilian Biodefense. JAMA, 281(22), 2127–37.
Ibrahim, S. M., Kulesh, D. A., Saleh, S. S., Damon, I. K., Esposito, J. J., Schmaljohn, A. L. & Jahrling, P. B. (2003) Real-time PCR assay to detect smallpox virus. J Clin Microbiol, 41, 3835–9.
Kulesh, D. A., Baker, R. O., Loveless, B. M., Norwood, D., Zwiers, S. H., Mucker, E., Hartmann, C., Herrera, R., Miller, D., Christensen, D., Wasieloski, L. P., Huggins, J. & Jahrling, P. B. (2004) Smallpox and pan-orthopox virus detection by real-time 3-minor groove binder TaqMan assays on the Roche LightCycler and the Cepheid smart Cycler platforms. J Clin Microbiol, 42, 601–9.
Kurg, A., Tonisson, I., Georgiou, J., Shumaker, J., Tollett, A. & Metspalu, A. (2000) Arrayed primer extension: solid-phase four-color DNA resequencing and mutation detection technology. Genetic Tests, 4, 1–7.
Laassri, M., Chizhikov, V., Mikheev, M., Shchelkunov, S. & Chumakov, K. (2003) Detection and discrimination of orthopoxviruses using microarrays of immobilized oligonucleotides. J Virol Methods, 112, 67–78.
Massung, R. F., Liu, L. I., Qi, J., Knight, J. C. T., Yuran, E. A., Kerlavage, R., Parsons, J. M., Venter, J. C. & Esposito, J. J. (1994) Analysis of the complete genome of smallpox variola major virus strain Bangladesh-1975. Virology, 201, 215–40.
Meyer, H., Osterrieder, N. & Pfeffer, M. (1993) Differentiation of species of the genus Orthopoxvirus in a dot blot assay using digoxigenin-labeled DNA probes. Vet Microbiol, 34, 333–44.
Moss, B. (1996) Genetically engineered poxviruses for recombinant gene expression, vaccination, and safety. Proc Natl Acad Sci USA, 93(21), 11341–8.
Nitsche, A., Ellerbrok, H. & Pauli, G. (2004) Detection of orthopoxvirus DNA by real-time PCR and identification of Variola virus DNA by melting analysis. J Clin Microbiol, 42(3), 1207–13.
Obijeski, J. F., Palmer, E. L., Gafford, L. G. & Randall, C. C. (1973) Plyacrylamide gel electrophoresis of fowlpox and Vaccinia virus proteins. Virology, 51(2), 512–6.
Pastinen, T., Raitio, M., Lindroos, K., Tainola, P., Peltonen, L. & Syvanen, A. C. (2000) A system for specific, high-throughput genotyping by allele-specific primer extension on microarrays.Genome Res, 10(7), 1031–42.
Podder, M., Ruan, J., Tripp, B. W., Chu, Z. E. & Tebbutt, S. J. (2008) Robust SNP genotyping by multiplex PCR and arrayed primer extension. BMC Med Genomics, 5(1) 165–70.
Ropp, S. L., Jin, Q., Knight, J. C., Massung, R. F. & Esposito, J. J. (1995) PCR strategy for identification and differentiation of smallpox and other orthopoxviruses. J Clin Microbiol, 33, 2069–76.
Syvanen, A. C. (2005) Toward genome-wide SNP genotyping. Nat Genet 37(Suppl.), S5–10
Tonisson, N., Kurg, A., Kaasik, K., Lohmussaar, E. & Metspalu, A. (2000) Unravelling genetic data by arrayed primer extension. Clin Chem Lab Med, 38(2), 165–70.
Tonisson, N., Zernant, J., Kurg, A., Pavel, H., Slavin, G., Roomere, H., Meiel, A., Hainaut, P. & Metspalu, A. (2002) Evaluating the arrayed primer extension resequencing assay of TP53 tumor suppressor gene. Proc Natl Acad Sci USA, 99, 5503–8.
Turner, P. C. & Moyer, R. W. (1990) The molecular pathogenesis of poxviruses. Curr Topics Microbiol Immunol, 163, 125–51.
Wenli, M., Yan, W., Hongmin, W. & Wenling, Z. (2004) An oligonucleotide microarray for the detection of vaccinia virus. Br J Biomed, 61, 142–5.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Springer Science+Business Media B.V.
About this paper
Cite this paper
Beth-Din, A. et al. (2010). Genetic Detection of Vesicle Forming Pathogens by Arrayed Primer Extension (APEX). In: Shafferman, A., Ordentlich, A., Velan, B. (eds) The Challenge of Highly Pathogenic Microorganisms. Springer, Dordrecht. https://doi.org/10.1007/978-90-481-9054-6_18
Download citation
DOI: https://doi.org/10.1007/978-90-481-9054-6_18
Published:
Publisher Name: Springer, Dordrecht
Print ISBN: 978-90-481-9053-9
Online ISBN: 978-90-481-9054-6
eBook Packages: Biomedical and Life SciencesBiomedical and Life Sciences (R0)