SPADA

Variola virus signature (eroded specificity): assay cross-reacts with Cowpox virus Exclusivity panel Species Strain Name Assay 1 ct for 5 ng DNA Assay 2 ct for 5 ng DNA Vaccinia Copenhagen Negative Negative Vaccinia WR Negative Negative Vaccinia ACAM2000 Negative Negative Vaccinia BRZ SERRO Negative Negative Cowpox CPXV-NOR1995-MAN Negative Negative Cowpox CPXV GER1980-EP4 19 Negative Cowpox CPXV GER1991-3 18 Negative Cowpox CPXV_GER1998_2 17 Negative Cowpox CPXV FIN 2000 Negative Ectromelia ECTV Moscow Negative Negative Monkeypox MPXV RCG 2003 358 Negative Negative Monkeypox MPXV USA 2003 044 Negative Negative Raccoonpox RACVV71-I-84 Negative Negative Skunkpox SKPV 1991 Negative Negative Volepox VPXV 2004-CA-007 Negative Negative Camelpox CMLV-78-I-2379 17 Negative Taterapox (gerbilpox) TATV-71-I-016 16 Negative Parapoxvirus Orf Vaccine for sheep Negative Negative

What we have learned about Variola virus diagnostic assay development/validation

Bioinformatic analysis should lead design of validation panels

Inclusivity panel include all Variola virus strains with differences in assay target region

Exclusivity panel (near neighbor Orthopoxvirus ) contain viruses with assay target regions most similar to Variola virus

Exceedingly difficult to construct uniform panels for all assays due to high similarity between Orthopoxviruse s

Simultaneous identification of multiple Variola virus signatures will increase confidence in initial identification/ verification of the pathogen with real-time PCR