Establishment of SYBR green-based qPCR assay for rapid evaluation and quantification for anti-Hantaan virus compounds in vitro and in suckling mice.
Hantaan viruses cause two severe diseases lacking efficient treatment, yet no effective prophylactic vaccines are available. Continued exploration of alternative antiviral agents to treat hantavirus-related syndromes remains compulsory. The fluorescence-based quantitative real-time PCR (qPCR) has become the touchstone for target gene quantification. In the present study, standard curves for Hantaan virus (HTNV), mouse, and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were generated by serial 10-fold dilutions of the constructed recombinant plasmid pGEM-T/HTNV, pGEM-T/mouse-GAPDH, and pGEM-T/human-GAPDH, respectively. Comparisons between the indirect immunofluorescence assay and qPCR assay in the detection of HTNV-infected Vero E6 cells showed improved detection limit and sensitivity of latter method. To characterize the inhibitory effect of several conventional antivirals (arbidol and ribavirin) and unconventional antivirals (indomethacin and curcumin) on HTNV, the levels of viral RNAs were measured for 4 days post-treatment of HTNV-infected Vero E6 cells and 18 days post-inoculation of HTNV-infected suckling mice. Our results validated that HTNV was sensitive to ribavirin and arbidol treatment, while indomethacin and curcumin may also be therapeutically effective in treating HTNV infection. As a result, the establishment and application of qPCR may be a useful tool for the evaluation of potential antivirals for Hantaan virus infection in vitro and in vivo.
State Key Laboratory of Virology/Institute of Medical Virology/Research Center of Food and Drug Evaluation/State Laboratory of Antiviral and Tumor of Traditional Chinese Medicine, School of Medicine, Wuhan University, Wuhan, China.
Fei
F
Wei
Jin-lin
JL
Li
Jia-xin
JX
Ling
Liang-Jun
LJ
Chen
Ning
N
Li
Yuan-Yuan
YY
Liu
Fan
F
Luo
Hai-Rong
HR
Xiong
Wei
W
Hou
Zhan-Qiu
ZQ
Yang
D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology / Q000627:therapeutic use; D002522:Chlorocebus aethiops; D004195:Disease Models, Animal; D005260:Female; D018063:Hantaan virus / Q000235:genetics / Q000302:isolation & purification*; D006480:Hemorrhagic Fever with Renal Syndrome / Q000188:drug therapy / Q000821:virology; D051379:Mice; D008807:Mice, Inbred BALB C; D008826:Microbial Sensitivity Tests; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D016896:Treatment Outcome; D014709:Vero Cells; D019562:Viral Load / Q000379:methods*
D000998:Antiviral Agents; D012367:RNA, Viral