**2. Methods**

260 Epidemiology Insights

Vaccinations started 1956 with Danish vaccine (IPV); since 1957 potentially contaminated U.S.vaccine (IPV) (Thu et al, 2006), change to OPV from 1967 to 1979, then back to IPV from 1979 onwards (Murdin et al, 1996).

Mass vaccinations (OPV) since 1958 with Koprowski strain live vaccine [94]; vaccine was claimed to be Russian made (Minor et al, 2003), but Russian vaccines were derived from Sabin's strain

(Chumakov et al, 1961)

(De Sanjose et al, 2003).

Mass vaccinations since 1959 with contaminated Russian vaccine (OPV). A small proportion of persons were vaccinated with IPV at the beginning of the mass vaccinations (Chumakov et al, 1961; Chumakov et al, 1963; Shah & Nathanson , 1976; Levine et al, 1998;Chron Wld Hlth Org 1960).

Mass vaccinations since 1963 with British vaccine (OPV) [101]; British vaccines were SV40-free since 1962 (Sangar et al, 1999); in contrast some vaccines were later claimed to have been contaminated

In 1957 potentially contaminated U.S. vaccine (IPV), from 1958 SV40-free Swedish vaccine (IPV). Sweden has never used OPV (Murdin et al, 1996)

Vaccination was not started before 1970, at a time where polio vaccines were required to be SV40-free (De Rienzo et al, 2002). The type of the vaccine is unclear. In a global poliomyelitis eradication initiative starting in 1989, OPV was used.

Vaccination started in 1956 with OPV (Chron Wld Hlth Org, 1958; Chron Wld Hlth Org, 1960). SV40-free since 1962

(Rev Sanid Hi Publica, 1965)

Positive

Unclear

Positive

Unclear

Positive

Negative

Positive

Norway

Poland

Russia (USSR)

Spain

Sweden

Turkey

United Kingdom

Table 1. (Leithner, 2005)

For PCR analysis 100 ul of blood was put into a test tube containing EDTA and kept at 4o C until it was delivered to the laboratory for DNA extraction. After extraction DNA samples were kept at -20o C.

Genome of SV40 can be present in the cell in 2 forms: episomal or integrated into genome of host cell. Because of few SV40 copies in the sample the best method allowing detecting episomal forms of SV40 DNA in RQ-PCR are methods based on DNA sorption by SiO2. That is why we used modified GuSCN protocol (Boom et al., 1992, Testa et al., 1998) for extraction of DNA from whole blood.

Three hundreds microliters of solution containing 5 M GuSCN,1% Triton X-100(v/v), 20 mM EDTA, 50 mM Tris-HCl (pH 6,4), 10 ul SiO2 was added to the blood sample (100 ul) and incubated 15 min, centrifugated and the sediment was washed once in 5 M GuSCN, 50 mM Tris-HCl (pH 6,4) and twice in 10 mM Tris-HCl (pH 7,3), 50 mM NaCl, 50% ethanol. The sample was desiccated in thermostat and DNA was eluted by TE-buffer (10 mM Tris-HCl (pH 8,0), 1Mm EDTA). For PCR amplification 5 ul were used. Rest was kept at -20o C.

As it was mentioned above SV40 detection was carried out by RQ-PCR method with internal probe directed to SV40 genome region in 4517 position, characterized by deletion of 9 nucleotides distinguishing SV40 from viruses JCV and BKV. PCR was carried out on BioRad Thermocycler according to the following program: 5 min initial denaturation, then 8 sec at 94oC, 23 sec at 60oC, 30 sec at 72oC, during 50 cycles. DNA extracted from M.mulatta blood was used as positive control. Water 5 ul from opened test tube kept together with extracted DNA samples was used as negative control.
