Detection of Hantaan and Seoul Viruses by Reverse Transcriptase-Polymerase Chain Reaction(RT-PCR) and Restriction Fragment Length Polymorphism(RFLP) in Korean Hemorrhagic Fever Patients

Ahn, Cu Rie; Cho, Jong Tae; Lim, Chun Soo; Chin, Ho Jun; Kim, Young Yim; Kim, Yon Su; Han, Jin Suk; Kim, Suhng Gwon; Lee, Jung Sang

Abstract

BACKGROUND: Korean hemorrhagic fever(KHF), a severe from of hemorrhagic fever with renal syndrome(HFRS), is the most common cause of acute renal failure in far east. Two serotypes of hantavirus, Hantaan and Seoul viruses, were identified as pathogens for KHF in Korea. To elucidate the diagnostic applicability for the serotype diagnosis in KHF patients, using a nested reverse transcriptase-PCR and restriction fragment length polymorphism(nRT-PCR /RFLP), we screened 4 prototype viruses, 11 virus isolates from KHF patients, and 69 specimens obtained from 30 KHF patients.
METHODS
The nRT-PCR was performed using serotype specific primers for G1 segments for Hantaan(HF3 1140-1163, HB14 1363-1342) and Seoul(SF2 809-832, SB3 1200-1177) viruses. The PCR product was further amplified using nested primers for Hantaan(HF4, 1141-1164, HB13, 1360-1339) and Seoul(SF7 863-884, SB1 1165-1142) viruses. Amplified segments were digested with restriction enzymes specific for either Hantaan(Cla I) or Seoul(Sac I) virus sequences. In all cultured viruses, serotypes identified by nRT-PCR/RFLP were consistent with those of PRNT.
RESULTS
In KHF patients, nRT-PCR/RFLP results were compatible with Hantaan virus in 10 patients and with Seoul virus in 13 patients. In 3 patients both Hantaan and Seoul specific amplified bands were visualized in serially collected samples, and in 4 patients no detectable amplicons were detected. Among 69 specimens, 55 specimens obtained from 3 to 33 day of illness were positive. The positive rate was affected by the hospital where specimens were collected, but not by clinical phases, the day of illness, or severity of HFRS.
CONCLUSIONS
In general, nRT-PCR/RFLP was a rapid and convenient method for serotype diagnosis in most of the KHF patients. The presented method also make it possible to detect genetic variation of hantavirus within the same serotype. But unlike the viruses in culture, in testing patients' sera, the sensitivity of this methods needed to be improved especially by adequate sample handling.