Establishment of SYBR green-based qPCR assay for rapid evaluation and quantification for anti-Hantaan virus compounds in vitro and in suckling mice

• Published in: Virus genes 2013-02, Vol.46 (1), p.54-62
• Main Authors: Wei, Fei, Li, Jin-lin, Ling, Jia-xin, Chen, Liang-Jun, Li, Ning, Liu, Yuan-Yuan, Luo, Fan, Xiong, Hai-Rong, Hou, Wei, Yang, Zhan-Qiu
• Format: Article
• Language: English
• Subjects: Animals; Antiviral agents; Antiviral Agents - pharmacology; Antiviral Agents - therapeutic use; Biomedical and Life Sciences; Biomedicine; Cercopithecus aethiops; Curcumin; detection limit; Disease Models, Animal; Female; fluorescent antibody technique; genes; Glyceraldehyde-3-phosphate dehydrogenase; Hantaan virus; Hantaan virus - genetics; Hantaan virus - isolation & purification; Hemorrhagic Fever with Renal Syndrome - drug therapy; Hemorrhagic Fever with Renal Syndrome - virology; Immunofluorescence; Indomethacin; Infection; Medical Microbiology; Mice; Mice, Inbred BALB C; Microbial Sensitivity Tests; Plant Sciences; Plasmids; Polymerase chain reaction; quantitative polymerase chain reaction; Real-Time Polymerase Chain Reaction - methods; Ribavirin; RNA, Viral - analysis; RNA, Viral - genetics; Sensitivity and Specificity; suckling; Suckling behavior; Treatment Outcome; Vaccines; Vero Cells; Viral Load - methods; Virology; viruses
• ISSN: 0920-8569; 1572-994X
• DOI: 10.1007/s11262-012-0834-6

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.