Samples were Anlotinib clinical trial also tested specifically for SIVwrc with a semi-nested PCR with primers specifically designed for the detection of pol regions of SIVs from the western red colobus/olive colobus lineage (SIVwrc S1 [CATGGCAAATGGATTGTACTCA], SIVwrc R2 [GTGCCATTGCTAATGCTGTTTC], SIVwrc S3 [CCAAATTCTTGTTCT ATCCCTAACC], and SIVwrc R3 [AGCAAAAATCATATCAGCAGAAGAT]). These primers were based on SIVwrc and SIVolc sequences published by Courgnaud and colleagues [24]. We used SIVwrc S1 and SIVwrc R2 in the first round PCR,
and SIVwrc S1 and SIVwrc R3 (expected amplicon size approximately 250 bp), and SIVwrc S3 and SIVwrc R2 (expected amplicon size approximately 300 bp) in two parallel semi-nested PCRs. The cycler conditions were 94°C for 5 minutes, 30 × [94°C for 15 seconds, 55°C for 30 seconds, 72°C for 30 seconds], 72°C for 10 minutes, then cooling to 4°C. The PCRs included positive control samples from confirmed SIVwrc positive red colobus monkeys [21]. PCR products were visualised with gel electrophoresis. A subset of samples (n = 4; Loukoum, Leonardo, Lefkas, Tita) was also tested with additional primers targeting SIVwrc/SIVolc/SIVcol in the gag, env and pol regions and primers amplifying gag and env regions of SIVsmm isolated from sooty mangabeys (Table 2). Table 2 Additional PCRs for SIV testing of a subset of samples (n = 4). Primers tested Primer sequences Estimated
amplicon size Region DihydrotestosteroneDHT targeted Reference DR1-DR2/DR4-DR5 DR1 (5′-TRCAYACAGGRGCWGAYGA-3′) 800 Pol [44] DR2 (5′-AIADRTCATCCATRTAYTG -3′)
DR4 (5′-GGIATWCCICAYCCDGCAGG-3′) 200 DR5 (5′-GGIGAYCCYTTCCAYCCYTGHGG -3′) polOR-polis4/polis2uni2 polOR(5′-ACBACYGCNCCTTCHCCTTTC -3′) 800 Pol [10] polis4(5′-CCAGCNCACAAAGGNATAGGAGG-3′) polis2(5′-TGGCARATRGAYTGYACNCAYNTRGAA-3′) 650 uni2(5′-CCCCTATTCCTCCCCTTCTTTTAAAA -3′) wrcpol wrcpolF1 (5′-TAGGGACAGAAAGTATAGTAATHTGG-3′) 1100 Pol [25] wrcpolR1 (5′-GCCATWGCYAA TGCTGTTTC-3′) GNA12 wrcpolF2 (5′AGAGACAGTAAGGAAGGGAAAGCAGG-3′) 650 wrcpolR2 (5′-GTTCWATTCCTAACCACCAGCADA-3′) wrcenv wrcenvF1 (5′-TGGC AGTGGGACAAAAATATAAAC-3′) 750 Env [25] wrcenvR1 (5′-CTGGCAGTCCCTCTTCCA AGTT GT-3′) wrcenvF2 (5′TGATAGGGMTGGCTCCTGGTGATG3′) 550 wrcenvR2 (5′-AATCCCCATTTYAACCAGTTCCA-3′) wrcgag wrcgagF1 (5′-ATDGAGGATAGAGGNTTTGGAGC-3′) 600 Gag [46] wrcgagR1 (5′-GCCCTCCTACTCCTTGACATGC-3′) wrcgagF2 (5′-CCAACAGGGTCAGATATAGCAG-3′) 250 wrcgagR2 (5′-ACTTCTGGGGCTCCTTGTTCTGCTC-3′) olcpol olcpolF1(5-TAGATACAGGRGCAGATGAYACAGTAAT-3′) 700 Pol S. Locatelli, unpublished data olcpolR1 (5′TCCAYCCYTGAGGHARYACATTATA-3′) olcpolF2 (5′-CTAGAATWATWGGRGGRATAGGRGG-3′) 300 olcpolR2 (5′-ATYTTWCCTTCTKCTTCYARTCTRTCACA-3′) bwcpol bwcpolF1 (5′-TAGATACAGGAGCAGATGATACAGT-3′) 1000 pol S.