Improved detection of Zika virus RNA in human and animal specimens by a novel, highly sensitive and specific real-time RT-PCR assay targeting the 5'-untranslated region of Zika virus.

OBJECTIVE AND METHOD: We developed and evaluated five novel real-time RT-PCR assays targeting conserved regions in the 5'-untranslated region (5'-UTR), envelope (E'), non-structural protein 2A (NS2A), NS5 and 3'-UTR of the ZIKV genome. RESULTS: The ZIKV-5'-UTR assay exhibited the lowest in vitro limit of detection (5-10 RNA copies/reaction and 3.0 × 10-1 plaque-forming units/ml). Compared to the modified version of a widely adopted RT-PCR assay targeting the ZIKV-E gene, the ZIKV-5'-UTR assay showed better sensitivity in human clinical specimens, and representative mouse specimens, including many organs which are known to be involved in human ZIKV infection but difficult to obtain in clinical settings. The ZIKV-5'-UTR assay detected ZIKV RNA in 84/84 (100.0%) ZIKV-E'-positive and an additional 30/296 (10.1%, P < 0.01) ZIKV-E'-negative mouse specimens. The higher sensitivity of the ZIKV-5'-UTR assay was most significant in kidney and testis/epididymis specimens (P < 0.01). No in vitro or in vivo cross-reactivity was found between the ZIKV-5'-UTR assay and dengue virus, yellow fever virus, Japanese encephalitis virus, West Nile virus, hepatitis C virus and Chikungunya virus. CONCLUSIONS: The highly sensitive and specific ZIKV-5'-UTR assay may help to improve the laboratory diagnosis of ZIKV infection.

Screening of an FDA-Approved Drug Library with a Two-Tier System Identifies an Entry Inhibitor of Severe Fever with Thrombocytopenia Syndrome Virus.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne bunyavirus that causes severe disease in humans with case-fatality rates of up to 30%. There are currently very limited treatment options for SFTSV infection. We conducted a drug repurposing program by establishing a two-tier test system to rapidly screen a Food and Drug Administration- (FDA)-approved drug library for drug compounds with anti-SFTSV activity in vitro. We identified five drug compounds that inhibited SFTSV replication at low micromolar concentrations, including hexachlorophene, triclosan, regorafenib, eltrombopag, and broxyquinoline. Among them, hexachlorophene was the most potent with an IC50 of 1.3 ± 0.3 µM and a selectivity index of 18.7. Mechanistic studies suggested that hexachlorophene was a virus entry inhibitor, which impaired SFTSV entry into host cells by interfering with cell membrane fusion. Molecular docking analysis predicted that the binding of hexachlorophene with the hydrophobic pocket between domain I and domain III of the SFTSV Gc glycoprotein was highly stable. The novel antiviral activity and mechanism of hexachlorophene in this study would facilitate the use of hexachlorophene as a lead compound to develop more entry inhibitors with higher anti-SFTSV potency and lower toxicity.