Premium
Detection of hantavirus RNA in tissues of experimentally infected mice using reverse transcriptase‐directed polymerase chain reaction
Author(s) -
Xiao ShuYuan,
Yanagihara Richard,
Godec Mark S.,
Eldadah Zayd A.,
Johnson Bruce K.,
Gajdusek D. Carleton,
Asher David M.
Publication year - 1991
Publication title -
journal of medical virology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.782
H-Index - 121
eISSN - 1096-9071
pISSN - 0146-6615
DOI - 10.1002/jmv.1890330413
Subject(s) - hantavirus , virology , reverse transcriptase , biology , trizol , hantaan virus , polymerase chain reaction , vero cell , reverse transcription polymerase chain reaction , virus , complementary dna , microbiology and biotechnology , rna , rna extraction , gene , messenger rna , biochemistry
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 intra‐cerebrally with 100 LD 50 of hantavirus, was initially reverse transcribed using avian myeloblas‐tosis 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 Han‐taan 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 Alul and Hpal . 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.