Detection of hantavirus RNA in tissues of experimentally infected mice using reverse transcriptase-directed polymerase chain reaction.
Detection of hantaviruses, the etiological agents of hemorrhagic fever with renal syndrome (HFRS), by virus isolation using experimental animals or cell culture is time-consuming. A more rapid but equally specific method is needed. We used a reverse transcriptase-directed polymerase chain reaction (RT-PCR) to detect hantavirus genomic sequences and compared its sensitivity with conventional virus isolation. RNA, extracted by the guanidinium isothiocyanate-cesium chloride method from hantavirus-infected Vero E6 cells and from tissues of infant mice inoculated intracerebrally with 100 LD50 of hantavirus, was initially reverse transcribed using avian myeloblastosis virus reverse transcriptase. The resulting complementary DNA (cDNA) was used as template to amplify the glycoprotein 2-encoding region of the hantavirus M segment. With this method, Vero E6 cell cultures infected with Hantaan virus strains 76-118 (prototype) and HV114 (an isolate from the urine of an HFRS patient in China) were positive, while control cultures were negative. Brain, lung, and heart tissues from hantavirus-infected mice were positive by RT-PCR at 5, 8, and 11 days after intracerebral inoculation. The specificity of the positive results was confirmed by restriction endonuclease digestion of the amplified fragments with AluI and HpaI. The sensitivity of the RT-PCR was equal to cell culture amplification but required less time. This method is being adapted for detection of hantavirus genomic sequences in clinical specimens and postmortem tissues from patients with HFRS.
Xiao
S Y
SY
Laboratory of Central Nervous System Studies, National Institute of Neurological Disorders and Stroke, Bethesda, Maryland 20892.
Yanagihara
R
R
Godec
M S
MS
Eldadah
Z A
ZA
Johnson
B K
BK
Gajdusek
D C
DC
Asher
D M
DM
D000818:Animals; D000831:Animals, Newborn; D001921:Brain / Q000382:microbiology; D004279:DNA, Viral / Q000737:chemistry; D006476:Orthohantavirus / Q000235:genetics* / Q000472:pathogenicity; D006321:Heart / Q000382:microbiology; D006480:Hemorrhagic Fever with Renal Syndrome / Q000175:diagnosis* / Q000382:microbiology; D007928:Lethal Dose 50; D008168:Lung / Q000382:microbiology; D051379:Mice; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000032:analysis*; D012194:RNA-Directed DNA Polymerase; D012680:Sensitivity and Specificity; D014709:Vero Cells
D004279:DNA, Viral; D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase