Figure. Phylogenetic tree generated from 309 nucleotides of the 3' noncoding region of the strain of yellow fever (YF) isolated in French Guiana in 1998 (in bold) and of 14 other YF strains by using the DNAPARS program. Numbers indicate bootstrap values for groups to the right. One µl (30 ng) of primer VD8 (5'-GGGTCTCCTCTAACCTCTAG-3') was mixed with RNA resuspended in 10 µl of distilled water; the mixture was heated at 95°C for 2 minutes and placed on ice. cDNA was synthesized in a mixture containing reverse transcriptase (RT) incubation buffer (provided by the manufacturer), 0.2 mM of each of the four dNTPs, 20 units RNasine, and five units of AMV RT (Promega, Charbonnières, France) by incubation for 1 hour at 42°C. cDNA was amplified by PCR. Four µl of the cDNA sample was added to 46 µl of a mixture containing Taq polymerase buffer (provided by the manufacturer), 2 mM MgCl2, 0.5 mM of each of the 4 dNTPs, 300 ng of primer VD8 and of degenerate primer EMF1 (5'-TGGATGACSACKGARGAYATG-3') (S = C, G; K = G, T; R = A, G; Y = C, T), and 0.5 unit of Taq polymerase (Promega, Charbonnières, France). After 5 minutes of denaturation at 95°C, the mixture was subjected to 30 polymerase chain reaction (PCR) cycles: 95°C for 30 seconds, 53°C for 90 seconds, and 72°C for 60 seconds, followed by a final 10-minute polymerization step at 72°C. Four µl of a 1 in 100 dilution of the PCR products was used for seminested PCR using primers VD8 and NS5YF (5'-ATGCAGGACAAGACAATGGT-3'). After the denaturation step, DNA was amplified by 25 cycles of PCR: 94°C for 30 seconds, 55°C for 90 seconds, and 72°C for 120 seconds, followed by a final extension step at 72°C. Negative controls (serum from healthy persons) were included in the series. The positive control (supernatant of infected mosquito cells) was tested separately to avoid any contamination. The phylogenetic analysis was conducted by the Pasteur Institute in Paris.
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