Inhibitory Activity of Honeysuckle Extracts against Influenza A Virus In Vitro and In Vivo

Honeysuckle has been used in the treatment of influenza virus infection for thousands of years in China.However,its main active components and the functional mechanisms remain to be elucidated.Here,four honeysuckle extracts,including acids extract,flavonoids extract,total extract and acids-flavonoids mixture,were prepared to clarify the main active antiviral components.The cytopathic effect reduction assay showed that all the four extracts inhibited the replication of influenza viruses H1N1,H3N2 and the oseltamivir-resistant mutant strain H1N1-H275Y.The acids-flavonoids mixture had the strongest inhibitory effects in vitro with EC_(50) values of 3.8,4.1,and >20 lg/m L against H1N1,H3N2 and H1N1-H275Y,respectively,showing competitive antiviral activity with oseltamivir and ribavirin.Honeysuckle acids extract also showed the most significant antiviral activity in vivo.Oral administration of the acids extract at a dosage of 600 mg/kg/d effectively alleviated viral pneumonia,maintained body weight and improved the survival rate to 30% of the mice infected with a lethal dose of H1N1.The results of time-of-drug addition experiment and neuraminidase (NA) inhibition assay showed that honeysuckle extracts had a broad-spectrum inhibitory effect against influenza virus NAs.The flavonoid extract showed the strongest inhibitory effect on the NA of influenza virus H7N9 with an IC_(50) of 24.7 lg/m L.These results suggested that these extracts might exert their antiviral activity by suppressing the release of influenza viruses.Briefly,our findings demonstrate that acids and flavonoids extracts of honeysuckle are the major antiviral active components,and the acids extract has the potential to be developed into an antiviral agent against influenza virus,especially for oseltamivirresistant viruses.

Construction and characterization of a full-length infectious clone of Getah virus in vivo

Getah virus(GETV)is a mosquito-borne virus of the genus Alphavirus in the family Togaviridae and,in recent years,it has caused several outbreaks in animals.The molecular basis for GETV pathogenicity is not well understood.Therefore,a reverse genetic system of GETV is needed to produce genetically modified viruses for the study of the viral replication and its pathogenic mechanism.Here,we generated a CMV-driven infectious cDNA clone based on a previously isolated GETV strain,GX201808(pGETV-GX).Transfection of pGETV-GX into BHK-21 cells resulted in the recovery of a recombinant virus(rGETV-GX)which showed similar growth characteristics to its parental virus.Then three-day-old mice were experimentally infected with either the parental or recombinant virus.The recombinant virus showed milder pathogenicity than the parental virus in the mice.Based on the established CMV-driven cDNA clone,subgenomic promoter and two restriction enzyme sites(BamHI and EcoRI)were introduced into the region between E1 protein and 3’UTR.Then the green fluorescent protein(GFP),red fluorescent protein(RFP)and improved light-oxygen-voltage(iLOV)genes were inserted into the restriction enzyme sites.Transfection of the constructs carrying the reporter genes into BHK-21 cells proved the rescue of the recombinant reporter viruses.Taken together,the establishment of a reverse genetic system for GETV provides a valuable tool for the study of the virus life cycle,and to aid the development of genetically engineered GETVs as vectors for foreign gene expression.