SPADA

Maximum likelihood analysis of single nucleotide matrices ( Variola virus )

48 isolates passaged 2-3 times to high titer master seed stocks

African minor AND African major

NON- WEST AFRICA

DNA extracted Sequenced using primer walking methodology

BANGLADESH NEPAL INDIA SUMATRA MIDDLE EAST

Asian Variola major

EAST ASIA ORIENT WAFRICA BRAZIL

Alastrimminor

Li Y.et.al.PNAS 2007;104:15787-15792 Esposito et. al Science 2006

Diversity of Variola virus ~200 kb dsDNA, ~200 ORFs

• Diversity of Variola virus strains is associated with geographic distance • Alastrimminor (South America) / Variola major (Asia): ~600 SNPs, ~80 Indels • Alastrimminor / Variola intermediate (West Africa): ~350 SNPs, ~45 Indels • Variola African minor/major / Variola major (Asia): ~150 SNPs, ~30 Indels

• Central region: virion structural proteins, enzymes - 30 gene sequences are perfectly conserved or have only synonymous SNPs, highly conserved, essential function. • Left and Right end regions: Host range and immunomodulatory genes -

majority of Indels/frameshift mutations, fragmented sequences, additional/absent of ORFs, - likely reflecting selection pressures.

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Versus other Orthopoxviruses : Variola / Camelpox-Taterapox viruses :

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~3200 SNPs, ~380 Indels ~7500 SNPs, ~600 Indels

Variola / Monkeypox virus :