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

Abstract

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.

Migratory birds in southern Brazil are a source of multiple avian influenza virus subtypes

“…Despite Brazil being considered free of HPAI, surveillance of AIV in migratory birds remains incomplete in that country, and AIV data remain scarce. Although H6N1 viruses are considered low‐pathogenic, an isolate of this subtype, albeit genetically distinct from our isolates, has been isolated from a human infection in Taiwan 46. Regardless of the zoonotic potential of the viruses found in Lagoa do Peixe National Park, increased surveillance in all animal species in the country is prudent.…”

Arbidol as a broad-spectrum antiviral: An update

“…Since these studies were performed by only one research group, with a limited number of animals, they should be reproduced by others before concluding to a beneficial effect of ARB against hantavirus infection. However ARB efficacy compared well in vivo with that of ribavirin, the reference treatment for such a disease (Wei et al, 2013).…”

“…Recently, Chinese studies demonstrated antiviral activity of ARB against the Hantaan virus, an enveloped virus from the Bunyaviridae family Wei et al, 2013), causing an often lethal hemorrhagic fever with renal syndrome (HFRS). In vitro, ARB was more efficient when added before infection, with an IC50 in the 2 lM range.…”

In Vitro and in Vivo Studies of the Inhibitory Effects of Emodin Isolated from Polygonum cuspidatum on Coxsakievirus B4

“…All reactions were performed in at least duplicate for each sample. The relative mRNA expressions were normalized to the level of GAPDH and were calculated using the formula as described elsewhere [25].…”