Antiviral Effects Of Anthraquinone Extracts From Polygonum Cuspidatum Against Koi Herpesvirus

Koi herpesvirus?KHV?is a pathogen that causes highly contagious,highly pathogenic,and high mortality of Koi herpesvirus disease?KHVD?,also called Cyprinid herpesvirus 3?CyHV-3?.Since the first report on KHV in the late 1990s,the virus has become prevalent in the world,causing major economic losses to koi and carp aquaculture.As an aquatic animal disease virus,KHV has received more and more attention.The research on the diagnostic methods and control measures of KHVD has become more and more in-depth,but no effective drugs and vaccines have been developed to treat the disease.Anthraquinone extracts from Polygonum cuspidatum have many pharmacological effects including antiviral,antibacterial,anti-inflammatory and anticancer properties.Several reports have confirmed that anthraquinone have certain inhibitory effects on HSV-1,HSV-2,HIV,SARS,hepatitis B virus,and influenza viruses.The aim of this study was to detect the antiviral activity of anthraquinone extracts from Polygonum cuspidatum against KHV infection and to find an effective pharmaceutical preparation for the treatment of Koi herpes virus disease.The contents and results of the study are as below:1.Determination of maximum safe concentration in CCB cells of anthraquinone extracts from Polygonum cuspidatum and positive controlled drug ribavirin.MTT assay and CPE microscopy were used to detect the cytotoxicity effects of anthraquinone extracts from Polygonum cuspidatum and broad-spectrum antiviral drug ribavirin on CCB cells within 48hours.8 groups of experimental groups of different concentrations of anthraquinone extract of Polygonum cuspidatum were set up,with treatment groups of 1.96?g/ml?3.91?g/ml?7.28?g/ml?15.63?g/ml?31.25?g/ml?62.5?g/ml?125?g/ml?250?g/ml,9 groups of different concentrations of ribavirin 15.63?g/ml?31.25?g/ml?62.5?g/ml?125?g/ml?250?g/ml?500?g/ml?1000?g/ml?2000?g/ml?4000?g/ml and control group without drug effect.The results showed that the survival rate of anthraquinone extracts from Polygonum cuspidatum was over80%when the concentration of anthraquinone extracts was 31.25?g/ml.Polygonum cuspidatum anthraquinone extracts and positive control drug ribavirin in CCB cells CC50 were 72.67±2.12?g/ml and 2638.7±2.36?g/ml.31.25?g/ml of anthraquinone extracts from Polygonum cuspidatum did not affect the significant changes in cell morphology or cell density of CCB,in the cell toxicity test was identified as the maximum non-toxic concentration and low concentration range??31.25?g/ml?cytotoxicity was basically zero,and there is a certain promoting the proliferation of the CCB cells.2.Study on the anti KHV virus activity of anthraquinone extracts from Polygonum cuspidatum.The virus replication inhibition test was performed after confirming the maximum nontoxic range of anthraquinone extract from Polygonum cuspidatum?1.96-31.25?g/ml?and ribavirin?15.63-500?g/ml?.The results showed that the maximum inhibition rate of anthraquinone extracts from Polygonum cuspidatum was 31.25?g/ml,IC50=13.3±0.47?g/ml,TI_?Pc?=5.48±0.49,and the positive control drug ribavirin IC50=492.15±2.13?g/ml,TI_?R?=5.36±0.03.In the virucidal Assay,anthraquinone extracts from Polygonum cuspidatum has apparent ability to directly kill KHV virus particles,and the highest virus inhibition rate is32.21%.The pre-dosed CCB cells did not produce the anti-KHV virus adsorption ability,CPE examination showed that the drug pre-incubation group had no significant difference with the negative KHV virus control group.The results of RT-PCR showed that 31.25?g/ml anthraquinone extracts from Polygonum cuspidatum in the 0.1TCID₅₀0 KHV virus-infected group showed good anti-KHV virus ability,completely inhibiting the transcription of KHV life cycle-related genes.In the animal challenge experiment,anthraquinone extracts from Polygonum cuspidatum also showed a higher anti-KHV virus activity,prolonging the number of test carp deaths,and increasing the survival rate of challenged carp.