RESEARCH CA, USA). Briefly, 20 µ L protease and 200 µ L buffer AL were combined with 200 µ L whole blood and incubated at 56°C for 10 min. After incubation, 200 µ L ethanol was added, and the entire mixture was applied to a QIAamp spin column. The purified DNA was eluted from the col- umn with 70 µ L nuclease-free water, and concentration was determined spectrophotometrically at optical density 260 nm. The presence of SFV DNA was determined by using nested PCR. Five hundred ng purified DNA was combined with a PCR reaction mixture with a final concentration of 10 mmol/L Tris (pH 9.0), 50 mmol/L KCl, 0.1% Triton X- 100, 2 mmol/L MgCl2, 200 µ mol/L each dNTP, 0.15 mg/mL BSA, 1 µ m Taq polymerase, and 400 nmol/L of each primer in a total volume of 50 µ L. The following primer pairs were used: first round, forward 5′ CAG TGA ATT CCA GAA TCT CTT C 3′, reverse 5′ CAC TTA TCC CAC TAG ATG GTT C 3′; and second round, forward 5′ CCA GAA TCT CTT CAT ACT AAC TA 3′, reverse 5′ GAT GGT TCC CTA AGC AAG GC 3′ (29). “Touchdown PCR” was used for both rounds with reaction conditions of initial denaturation at 94°C for 2 min, followed by 7 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 45 s, with a 2°C decrease in annealing temperature per cycle to 48°C, followed by 33 cycles of 94°C for 30 s, 48°C for 30 s, and 72°C for 45 seconds with a final extension at 72°C for 2 min. Second-round conditions were the same, except 19 cycles were used instead of 33. Twenty µ L of the PCR reaction underwent electrophore- sis on a 1% low melting point agarose gel. DNA bands (365-bp product) were excised from the gel and purified by using Wizard PCR Preps DNA Purification System (Promega Corp., Madison, WI, USA). The PCR product was ligated into the pCR 2.1-TOPO vector by using the TOPO TA Cloning Kit (Invitrogen Corp., Carlsbad, CA, USA.). An SFV DNA plasmid (pSFV-1Lgp), representing long terminal repeat, gag, and pol of SFV-1, was included as a positive PCR control and for determining sensitivity of detection by serial dilution (provided by A. Mergia). TOP10 cells were transformed with the ligation reaction, plated onto Luria broth agar plates containing 50 µ g/mL kanamycin, and incubated overnight at 37°C. Miniscreen DNA was purified by using Wizard Plus Minipreps DNA Purification System (Promega). Samples were sequenced with the ABI 373 automated fluorescent sequencer using BigDye Terminator cycle sequencing chemistry (Applied Biosystems, Foster City, CA, USA). Amplification of Mitochondrial Sequences Five hundred ng purified DNA from whole blood was combined in a PCR reaction mixture with a final concen- tration of 10 mmol/L Tris (pH 9.0), 50 mmol/L KCl, 0.1% Triton X-100, 2.5 mmol/L MgCl2, 200 µ mol/L each dNTP, 0.15 mg/mL BSA, 1 U Taq polymerase, and 400 nmol/L of each primer in a total volume of 50 µ L. The following primers were used: forward, 12SA, 5´ CTG GGA TTA GAT ACC CAC TAT 3´, and reverse, 12SO, 5´ GTC GAT TAT AGG ACA GGT TCC TCT A 3´ (35). Cycling condi- tions were the following: initial denaturation at 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 5 min, with a final extension at 72°C for 5 min. The 101-bp product underwent electrophoresis and was processed for DNA sequencing essentially as described for SFV. The alignments were made in Bioedit (http://www. mbio.ncsu.edu/BioEdit/bioedit.html) and ClustalX 1.81 (ftp://ftp-igbmc.u-strasbg.fr/pub/ClustalX/). Columns in the alignment in which gaps had been inserted in regions with insertions, and deletions were stripped before the analyses. DNA trees were created with the neighbor-join- ing method by using the Phylip program (DNAdist; Neighbor), and the output was generated with Treeview (http://taxonomy. zoology.gla.ac.uk/rod/treeview.html). The GenBank accession numbers for the SFV and mito- chondrial DNA sequences reported here are AY628152-69 and AY633510-39, respectively. Results Seroprevalence of SFV among Macaques The seroprevalence of SFV among the Sangeh macaques is presented in Table 1. Thirty-eight macaques (29 males and 9 females; 4 juveniles, 6 subadults, 28 adults) were sampled. Thirty-four (89.5%) of the 38 macaques were seropositive for SFV by Western blot; 2 (50%) of 4 juveniles, all 6 (100%) subadults, and 26 (93%) of the adults were antibody positive. All 9 females were SFV seropositive. SFV seroprevalence in this free-ranging macaque population was consistent with seroprevalence studies done in captive (25) and other free-ranging macaque populations (L. Jones-Engel, unpub. data). 1030 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 7, July 2005