Molecular detection of yellow fever virus in human sera and mice brains.

Abstract

A molecular method for the diagnosis of yellow fever virus infection was developed based on reverse transcription (RT) followed by polymerase chain reaction (PCR) amplification. Examinations were conducted on lyophilized sera from 3 fatal yellow fever cases and 4 fresh sera from 3 fatal cases and one from a symptomatic patient (positive IgM against yellow fever virus). Sera were extracted with TRIZOL-LS to isolate viral RNA for RT treatment and the PCR reaction included 2 primers sets designed specifically for yellow fever virus: sense, JM2104 (5'-CGTTGGGAGAGGAGATTC-3') y JM2249 (5'-TTCTTCACTTCGGTTGGG-3'), and antisense, JM2673 (5'-TCATCTGCCCTGCTTCTC-3') y JM2751 (5'-CCTCTCTGGTAAACATTCT-3'). The technique for demonstrating the yellow fever virus in tissue samples was used in infected mice brains treated with lysis buffer before RNA extraction. PCR reactions were evaluated in agarose gels where single bands of the expected size for each primers pair (569 bp and 502 bp) were observed for all serum samples. In addition, the results for 2 fresh positive sera were supported by histopathologic finding of yellow fever virus. The RT-PCR method permits a rapid and specific demonstration of the presence of yellow fever virus.