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Appendix Variola Strains Used to Validate Diagnostic and Detection Assays (Table on following pages.) 

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Method for Detection of VAR from Other 0 VAR Strain (Origin) Viral DNA Probes/Targets OPVs? (Y/N) 6/58, S/6 Microarray VAR CL/BR, BR genesa Y Microarray CrmB geneb Y 12/62, S/12 (Tanzania) Microarray VAR C33L/B29R, B19R genesa Y MPCRc Five pairs of primersd Y Microarray CrmB geneb Y 22/62, S/22 Microarray VAR C33L/B29R, B19R genesa Y 102, SAF65-102 (Republic of South Africa) Real-Time PCR VAR HA genee N Real-Time PCR 14-kDa protein genef Y 103, SAF65-103, Tvaal, Nelspruit (Republic Real-Time PCR VAR HA genee N of South Africa) Sequencing GeneChips whole-genome sequences of 24 VAR strainsg N Real-Time PCR 14-kDa protein genef Y 7124 (India) Real-Time PCR VAR HA genee N Real-Time PCR 14-kDa protein geneh Y Real-Time PCR 14-kDa protein genef Y 7125 (India) Real-Time PCR VAR HA genee N Real-Time PCR 14-kDa protein genef Y 66-39, CDC v66-39, Sao (Brazil) Real-Time PCR VAR HA genee N Sequencing GeneChips whole-genome sequences of 24 VAR strainsg N Real-Time PCR 14-kDa protein geneh Y Real-Time PCR 14-kDa protein genef Y 68-59 (Benin) Real-Time PCR VAR HA genee N Real-Time PCR 14-kDa protein genef Y

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68-258 (Sierra Leone) Real-Time PCR VAR HA genee N Real-Time PCR 14-kDa protein geneh Y Real-Time PCR 14-kDa protein genef Y PCR, RFLP CrmB genei Y 70-222 (Indonesia) Real-Time PCR VAR HA genee N Real-Time PCR 14-kDa protein genef Y 70-228 (Indonesia) Real-Time PCR VAR HA genee N 14-kDa protein genef Y 72-119 (Syria) Real-Time PCR VAR HA genee N Real-Time PCR 14-kDa protein genef Y 72-143 (Botswana) Real-Time PCR VAR HA genee N Real-Time PCR 14-kDa protein genef Y 73-175 (Nepal) Real-Time PCR VAR HA genee N Real-Time PCR pan-E9L3, VAR B9R, VAR 10R genesj N 73-225, BOT73_225, CDC v73-225 Real-Time PCR VAR HA genee N (Botswana) Sequencing GeneChips whole-genome sequences of 24 VAR strainsg N Real-Time PCR 14-kDa protein genef Y 77-1252 (Somalia) Real-Time PCR VAR HA genee N Real-Time PCR 14-kDa protein genef Y 77-1605 (Somalia) Real-Time PCR VAR HA genee N Real-Time PCR pan-E9L3, VAR B9R, VAR B10R genesj Y  continued

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Method for Detection of VAR from Other  VAR Strain (Origin) Viral DNA Probes/Targets OPVs? (Y/N) 77-227 Real-Time PCR VAR HA genee N Variolator 4 (Afghanistan) Real-Time PCR VAR HA genee N Real-Time PCR 14-kDa protein geneh Y Real-Time PCR 14-kDa protein genef Y Real-Time PCR pan-E9L3, VAR B9R, B10R genesj Y Aslam, S/ASL (Pakistan) Microarray VAR C33L/B29R, B19R genesa Y MPCRc five pairs of primersd Y Microarray CrmB geneb Y Aziz, S/AZI Microarray VAR C33L/B29R, B19R genesa Y Microarray CrmB geneb Y Variola major-ABA (Bangladesh) Real-Time PCR VAR HA genek N Bangladesh-1975 (Bangladesh) Microarray VAR C33L/B29R, B19R genesa Y Real-Time PCR 14-kDa protein genef Y PCR, RFLP CrmB genei Y Bombay (India) Real-Time PCR VAR HA genee N Brazil 128, S/B128 (Brazil) Microarray VAR C33L/B29R, B19R genesa Y MPCRc five pairs of primersd Y Brazil 131, S/B131 (Brazil) Microarray VAR C33L/B29R, B19R genesa Y MPCRc five pairs of primersd Y

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Brazil Garcia (Brazil) Real-Time PCR VAR HA genee N Real-Time PCR 14-kDa protein geneh Y Microarray VAR C33L/B29R, B19R genesa Y Real-Time PCR 14-kDa protein genef Y Microarray CrmB geneb Y PCR, RFLP CrmB genei Y BSH Real-Time PCR VAR HA genee N BSH75_banu, CDC v75-550 (Bangladesh) Sequencing GeneChips whole-genome sequences of 24 VAR strainsg N Real-Time PCR pan-E9L3, VAR B9R, VAR B10R genesj Y Butler, S/BUT (United Kingdom) Real-Time PCR VAR HA genee N Microarray VAR C33L/B29R, B19R genesa Y MPCRc five pairs of primersd Y Real-Time PCR 14-kDa protein genef Y Microarray CrmB geneb Y China Horn, CHN48_horn (China) Sequencing GeneChips whole-genome sequences of 24 VAR strainsh N Real-Time PCR pan-E9L3, VAR B9R, VAR B10R genesj Y Congo (Congo) Real-Time PCR VAR HA genee N Real-Time PCR 14-kDa protein genef Y PCR, RFLP CrmB genei Y v70-46 (Congo) Real-Time PCR pan-E9L3, VAR B9R, VAR B10R genesj Y Variola major-CNG (Congo) Real-Time PCR VAR HA genek N Congo-2, S/CNG2 (Congo) Microarray VAR C33L/B29R, B19R genesa Y MPCRc five pairs of primersd Y Microarray CrmB geneb Y  continued

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Method for Detection of VAR from Other  VAR Strain (Origin) Viral DNA Probes/Targets OPVs? (Y/N) Microarray C23L/B29R genel Y Congo-9, v74-227, S/CNG9 (Congo) Microarray VAR C33L/B29R, B19R genesa Y Real-Time PCR 14-kDa protein geneh Y MPCRc five pairs of primersd Y Microarray CrmB geneb Y Ethiopia 16, ETH72-16, R14-IX-72, Addis Sequencing GeneChips whole-genome sequences of 24 VAR strainsg N (Ethiopia) Real-Time PCR 14-kDa protein genef Y Ethiopia 17, ETH72-17 (Ethiopia) Real-Time PCR VAR HA genee N Sequencing GeneChips whole-genome sequences of 24 VAR strainsg N Real-Time PCR 14-kDa protein genef Y Variola minor-GAR (Garcia) Real-Time PCR VAR HA genek N Variola major-HAR Real-Time PCR VAR HA genek N Harper (Japan) Real-Time PCR VAR HA genee N Real-Time PCR 14-kDa protein genef Y Harvey (United Kingdom) Real-Time PCR VAR HA genee N Real-Time PCR 14-kDa protein genef Y PCR, RFLP CrmB genei Y Heidelberg, GER_hdlg (Germany, Asia Real-Time PCR VAR HA genee N import) Sequencing GeneChips whole-genome sequences of 24 VAR strainsg N Real-Time PCR 14-kDa protein geneh Y Real-Time PCR 14-kDa protein genef Y

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Herrlich (India) Real-Time PCR 14-kDa protein genef Y Hembula (Tanzania) Real-Time PCR 14-kDa protein genef Y Higgins, UNK47_hig (United Kingdom) Real-Time PCR VAR HA genee N Sequencing GeneChips whole-genome sequences of 24 VAR strainsg N Real-Time PCR 14-kDa protein geneh Y Real-Time PCR 14-kDa protein geneh Y Helder, S/HEL Microarray VAR C33L/B29R, B19R genesa Y Hinden (United Kingdom) Real-Time PCR 14-kDa protein genef Y Hinton Real-Time PCR VAR HA genee N Horn (China) Real-Time PCR VAR HA genee N Real-Time PCR 14-kDa protein genef Y Ind-3a Microarray VAR C33L/B29R, B19R genesa Y MPCRc five pairs of primersd Y Microarray CrmB geneb Y Microarray C23L/B29R genel Y Variola major-BOM (India) Real-Time PCR VAR HA genek N Variola major-MAD (India) Real-Time PCR VAR HA genek N India, S/IND (India) Microarray VAR C33L/B29R, B19R genesa Y  continued

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Method for Detection of VAR from Other  VAR Strain (Origin) Viral DNA Probes/Targets OPVs? (Y/N) India 164, S/I164 (India) Microarray VAR C33L/B29R, B19Ra Y MPCRc five pairs of primersd Y India 378 (India) Microarray VAR C33L/B29R, B19Ra Y Microarray CrmB geneb Y India-1967, S/IND67 (India) Microarray VAR C33L/B29R, B19Ra Y India 7124 (India) Real-Time PCR VAR HA, B9R, B10R genesj Y India 7125 (India) Real-Time PCR 14-kDa protein geneh Y Iran (Iran) Real-Time PCR VAR HA genee N IRN72_tbrz, Iran 2602, Tabrez (Iran) Sequencing GeneChips whole-genome sequences of 24 VAR strainsg N Real-Time PCR 14-kDa protein genef Y Juba (Sudan) Real-Time PCR VAR HA genee N Real-Time PCR 14-kDa protein genef Y K1629 (Kuwait) Real-Time PCR VAR HA genee N Real-Time PCR 14-kDa protein genef Y Kali Mathu (India) Real-Time PCR VAR HA genee N Real-Time PCR 14-kDa protein genef Y Kembula Real-Time PCR VAR HA genee N Real-Time PCR 14-kDa protein geneh Y

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Khateen, S/WSI (Pakistan) Microarray VAR C33L/B29R, B19R genesa Y MPCRc five pairs of primersd Y Kudano (Nigeria) Real-Time PCR 14-kDa protein genef Y Kuw-5, S/DUW5 (Kuwait) Microarray VAR C33L/B29R, B19R genesa Y MPCRc five pairs of primersd Y Microarray CrmB geneb Y Microarray C23L/B29R genel Y Lee (Korea) Real-Time PCR 14-kDa protein genef Y M-Abr-60, S/ABR (Russia) Microarray VAR C33L/B29R, B19R genesa Y MPCRc five pairs of primersd Y Microarray CrmB geneb Y M-Sok-60, S/SOK (Russia) Microarray VAR C33L/B29R, B19R genesa Y MPCRc five pairs of primersd Y Microarray CrmB geneb Y M-Sur-60, S/SUR (Russia) Microarray VAR C33L/B29R, B19R genesa Y MPCRc five pairs of primersd Y Microarray CrmB geneb Y M-N-60 Microarray VAR C33L/B29R, B19R genesa Y Mannan (Bangladesh) Real-Time PCR 14-kDa protein genef Y Minnesota124 (United States) Real-Time PCR VAR HA genee N Real-Time PCR 14-kDa protein genef Y MS Lee Real-Time PCR VAR HA genee N Real-Time PCR 14-kDa protein geneh Y  continued

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Method for Detection of VAR from Other  VAR Strain (Origin) Viral DNA Probes/Targets OPVs? (Y/N) Nepal (Nepal) Real-Time PCR VAR HA genee N Nepal 73 (Nepal) Real-Time PCR 14-kDa protein genef Y New Delhi, IND53_ndel (India) Real-Time PCR VAR HA genee N Sequencing GeneChips whole-genome sequences of 24 VAR strainsg N Real-Time PCR 14-kDa protein genef Y Ngami, S/NGA (Tanzania) Microarray VAR C33L/B29R, B19R genesa Y MPCRc five pairs of primersd Y Microarray CrmB geneb Y Variola major-NIG (Nigeria) Real-Time PCR VAR HA genek N Nigeria Hudano (Nigeria) Real-Time PCR VAR HA genee N Nur Islam, BSH74_nur (Bangladesh) Real-Time PCR VAR HA genee N Sequencing GeneChips whole-genome sequences of 24 VAR strainsg N Real-Time PCR 14-kDa protein genef Y Parvin (Bangladesh) Real-Time PCR 14-kDa protein genef Y Variola major-RAT (Pakistan) Real-Time PCR VAR HA genek N Rafiq, Lahore, PAK69_lah (Pakistan) Real-Time PCR VAR HA genee N Sequencing GeneChips whole-genome sequences of 24 VAR strainsg N Real-Time PCR 14-kDa protein genef Y Rumbec (Sudan) Real-Time PCR VAR HA genee N Real-Time PCR 14-kDa protein genef Y

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Rw18, S/RW18 Microarray VAR C33L/B29R, B19R genesa Y Semat, S/SEM (Tanzania) Microarray VAR C33L/B29R, B19R genesa Y MPCRc five pairs of primersd Y Microarray CrmB geneb Y Shahzaman (Bangladesh) Real-Time PCR VAR HA genee N Real-Time PCR 14-kDa protein geneh Y Real-Time PCR 14-kDa protein genef Y Variola major-SLN (Sierra Leone) Real-Time PCR VAR HA genek N Solaiman (Bangladesh) Real-Time PCR VAR HA genee N Real-Time PCR 14-kDa protein genef Y SOM77_ali (Somalia-1977, Sequencing GeneChips whole-genome sequences of 24 VAR strainsg N CDC V77-2479 [last case]) Microarray VAR C33L/B29R, B19R genesa Y Real-Time PCR VAR HA genek N PCR, RFLP CrmB genei Y Stillwell (Japan) Real-Time PCR VAR HA genee N Real-Time PCR 14-kDa protein genef Y Taj Barin, S/TAJ Microarray VAR C33L/B29R, B19R genesa Y Variola minr-BUT Real-Time PCR VAR HA genek N (United Kingdom) Yamada (Japan) Real-Time PCR VAR HA genee N Real-Time PCR 14-kDa protein genef Y  continued (notes on following page)

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0 LIVE VARIOLA VIRUS NOTES: aRyabinin et al., 2006. bLapa et al., 2002. The microarray contained 15 oligonucleotide probes directed toward five species-specific segments of the CrmB gene. cMPCR = Multiplex PCR. dSchelkunov et al., 2005. Five pairs of oligonucleotide primers—one genus-specific, and the rest species-specific for variola, monkeypox, cowpox, and vaccinia viruses. eIbrahim et al., 2003. fOlson et al., 2004. gSulaiman et al., 2007. Seven GeneChips, containing ~240,000 different types of 25-mer oli- oli- gonucleotides, each designed to analyze a divergent segment of approximately 30,000 bases of VAR genome. hScaramozzino et al., 2007. One probe to recognize human pathogenic orthopoxviruses (OPV) and one specific for VAR. iLoparev et al., 2001. jKulesh et al., 2004. Assays used cloned genes. kSchoepp et al., 2004. lLaassri et al., 2003. REFERENCES Ibrahim, M. S., D. A. Kulesh, S. S. Saleh, I. K. Damon, J. J. Esposito, A. L. Schmaljohn, and P. B. Jahrling. 2003. Real-time PCR assay to detect smallpox virus. Journal of Clinical Microbiology 41(8):3835–3839. Kulesh, D. A., R. O Baker, B. M. Loveless., D. Norwood, S. H., Zwiers, E. Mucker, C. Hartmann, R. Herrera, D. Miller, D. Christensen, L.P., Wasieloski, J. Huggins, and P. B. Jahrling. 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. Journal of Clinical Microbiology 42(2):601–609. Laassri, M., V. Chizhikov, M. Mikheev, S. Shchelkunov, and K. Chumakov. 2003. Detection and discrimination of orthopoxviruses using microarrays of immobilized oligonucle- otides. Journal of Virological Methods 112(1–2):67–78. Lapa, S., M. Mikheev, S. Shchelkunov, V. Mikhailovich, A. Sobolev, V. Blinov, L. Babkin, A. Guskov, E. Sokunova, A. Zasedatelev, L. Sandakhchiev, and A. Mirzabekov. 2002. Spe- cies-level identification of orthopoxviruses with an oligonucleotide microchip. Journal of Clinical Microbiology 40(3):753–757. Loparev, V. N., R. F. Massung, J. J. Esposito, and H. Meyer. 2001. Detection and differentia- tion of Old World orthopoxviruses: Restriction fragment length polymorphism of the crmB gene region. Journal of Clinical Microbiology 39(1):94–100. Olson, V. A., T. Laue, M. T. Laker, I. V. Babkin, C. Drosten, S. N. Shchelkunov, M. Niedrig, I. K. Damon, and H. Meyer. 2004. Real-Time PCR System for Detection of Orthopoxvi- ruses and Simultaneous Identification of Smallpox Virus. Journal of Clinical Microbiol- ogy 42(5):1940–1946. Ryabinin, V. A., Shundrin, L.A., Kostina, E.B., Laassri, M., Chizhikov, V., Shchelkunov, S.N., Chumakov, K., and A. N. Sinyakov. 2006. Microarray assay for detection and discrimi- nation of Orthopoxvirus species. Journal of Medical Virology 78(10):1325–1340. Scaramozzino, N., A. Ferrier-Rembert, A.L. Favier, C. Rothlisberger, S. Richard, J.M. Crance, H. Meyer, and D. Garin. 2007. Real-time PCR to identify variola virus or other human pathogenic orthopox viruses. Clinical Chemistry 53(4):606–613.

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 APPENDIX Schoepp, R. J., M.D. Morin, M.J., Martinez, D. A. Kulesh, L. Hensley, T. W. Geisbert, D. R. Brady, and P. B. Jahrling. 2004. Detection and identification of Variola virus in fixed human tissue after prolonged archival storage. Laboratory Investigation 84(1):41–48. Shchelkunov, S. N., E. V. Gavrilova, and I. V. Babkin. 2005. Multiplex PCR detection and species differentiation of orthopoxviruses pathogenic to humans. Molecular & Cellular Probes 19(1):1–8. Sulaiman, I.M., T. Tang, J. Osborne, S. Sammons, and R. M. Wohlhueter. 2007. GeneChip resequencing of the smallpox virus genome can identify novel strains: A biodefense ap- plication. Journal of Clinical Microbiology 45(2):358–363.

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