Detection of yellow fever virus by polymerase chain reaction.
Parole chiave
Astratto
BACKGROUND
Yellow fever virus continues to cause major epidemics. A sensitive rapid diagnostic test is required to identify cases and contacts in order to implement emergency immunization campaigns.
OBJECTIVE
To identify YFV envelope protein gene fragments, construct a polymerase chain reaction (PCR) assay and test its utility in identifying viruses isolated from laboratory and clinical specimens.
METHODS
YFV RNA was transcribed with reverse transcriptase and the cDNA amplified by PCR using primers encoding a portion of the viral envelope protein gene. The identity of the 482 bp amplified product was confirmed by restriction enzyme analysis and by dot blot hybridization with a labelled oligonucleotide probe. The assay was tested for sensitivity and specificity on isolates from South America and Africa. Detection limits were determined using different probe labels. PCR inhibitory effects were analyzed with laboratory and clinical specimens.
RESULTS
The assay was specific for YFV and did not detect any of 15 other flaviviruses. The amplified region was conserved among all 32 South American and African isolates tested. Four strains from Africa did not hybridize with the probe, indicating sequence divergence in the envelope protein gene. Samples containing 30 pfu of virus were detected by visual inspection of the ethidium bromide stained 482 bp DNA amplimer and 10 pfu were detected with a digoxigenin labelled probe. Inhibitory effects of human serum on the PCR were overcome by diluting samples 4-fold in buffer. Viral neutralizing antibody in experimental samples did not affect the sensitivity of detection. Yellow fever virus in serum from experimentally infected Cynomolgus monkeys (10(3.7)-10(7.0) pfu/0.1 ml) was detected with signal intensities corresponding to the amount of virus in the sample. When YFV was added to normal human serum and held at 27 degrees C and 80% humidity, the RNA could be detected for up to 3 weeks in samples that had no infectious virus.
CONCLUSIONS
A PCR assay was constructed which detected YFV RNA in isolates from patients infected in South America and Africa. This assay is specific for YFV but some African strains were not detected. More clinical samples should be tested.
