A simple nucleic acid amplification assay for the rapid detection of Junín virus in whole blood samples.
Argentine hemorrhagic fever (AHF) is an endemoepidemic disease with cardiovascular, renal and neurologic alterations acquired in the richest farming land in Argentina. It is caused by Junín virus, one of the few human pathogenic arenaviruses. The S RNA of Junín virus has been molecularly cloned and its nucleotide sequence determined in our laboratory. This information was used to develop a rapid nucleic acid-based diagnostic test commensurate with the low viraemia detected in AHF patients. Junín virus-specific cDNA probes labeled using various methods proved insensitive in dot-hybridizations. Therefore, a RT polymerase chain reaction (PCR) was developed using a pair of oligonucleotide primers to reverse-transcribe and amplify the viral S RNA. The amplification of the target sequences was measured by ethidium bromide staining of the DNA fragments after agarose gel electrophoresis. This type of assay allowed the specific detection of Junín virus RNA sequences present in a single infected BHK21 cell over a background of 10(4) uninfected cells. Control reactions were performed on RNA samples extracted from uninfected cells or cells infected with a high multiplicity of LCMV, another arenavirus present in the AHF endemic area. The PCR was first adapted to detect viral RNA in peripheral blood mononuclear cells, described to harbor most of the virus. A simplification of this assay allows the detection of Junín virus in RNA extracted from 100 microliters of whole blood using guanidium thiocyanate disruption and acid phenol extraction. Under the conditions described in this paper, it is possible to detect up to 0.01 pfu of Junín virus in a blood sample. An early and rapid laboratory diagnostic test for AHF is important since the only effective therapy that reduces the mortality rate from 30% to less than 1% consists of early treatment with immune plasma.
Instituto de Bioquímica y Biología Molecular, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, Argentina.
M E
ME
Lozano
P D
PD
Ghiringhelli
V
V
Romanowski
O
O
Grau
D000818:Animals; D006477:Arenaviruses, New World / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D002460:Cell Line; D004279:DNA, Viral / Q000235:genetics; D005069:Evaluation Studies as Topic; D006478:Hemorrhagic Fever, American / Q000175:diagnosis; D006801:Humans; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction / Q000379:methods / Q000706:statistics & numerical data; D012367:RNA, Viral / Q000097:blood / Q000235:genetics*; D012680:Sensitivity and Specificity
D004279:DNA, Viral; D012367:RNA, Viral