Study On The Effect Of Rhodomyrtus Tomentosa Against Largemouth Bass Ranavirus

Largemouth bass ranavirus（LMBV）is one of the most damaging pathogens in the culture of Largemouth bass and is characterized by high lethality and high infectivity.It causes serious economic losses every year,but there are no effective prevention and control measures.Rhodomyrtus tomentosa Aiton Hassk is a plant of the Myrtle family with antibacterial,antiviral,and anti-inflammatory functions.In this study,we investigat the anti-LMBV efficacy of different extracted parts of Rhodomyrtus tomentosa to provide basic data for its application in disease prevention and control of aquatic animals in vivo and in vitro.The main results are as follows:1.Study on the anti-LMBV effect of Rhodomyrtus tomentosa aqueous extract In vitro experiments were first conducted to study the safe concentration of each aqueous extract of Rhodomyrtus tomentosa rhizome on EPC cells using the MTT method and to study the effect of Rhodomyrtus tomentosa aqueous extract against LMBV in vitro by using the MTT method,cell nuclear damage,and mitochondrial transmembrane potential assays.In vivo experiments were conducted at five concentrations of 3 g/kg,6 g/kg,10 g/kg,15 g/kg,and 20 g/kg,and were fed at three days to attack the toxicity and determine the mortality rate.The viral load in vivo was determined by RT-PCR,and the changes of immune-related indexes such as catalase（CAT）,acid phosphatase（AKP）,alkaline phosphatase（ACP）,and superoxide dismutase（SOD）in liver tissues after the administration of the drug were determined.The results showed that:the safe concentration of Rhodomyrtus tomentosa aqueous extract for EPC cells was≤1mg/m L,and its concentration of 1 mg/m L achieved 45%inhibition of LMBV;Rhodomyrtus tomentosa aqueous extract had a certain protective effect on the nucleus damage and the decrease of mitochondrial membrane potential caused by LMBV;feeding different concentrations of Rhodomyrtus tomentosa aqueous extract had a certain protective effect on the bass after the attack,the highest protection rate of largemouth bass was achieved at the feeding concentration of 10 g/kg,which was33%;compared with the control group,the viral load of the experimental group decreased significantly,and the viral load of the 10 g/kg group decreased by 93%on day 14;the results of immune-related enzyme activities showed that the activities of ACP,CAT,AKP,and SOD were significantly higher than those of the control group on day 7（P<0.05）.2.Study on the anti-LMBV effect of Rhodomyrtus tomentosa solvent extracts.In vitro experiments were first conducted to study the safe concentration of each organic solvent extract of Rhodomyrtus tomentosa rhizome on EPC cells using the MTT method and to study the effect of Rhodomyrtus tomentosa aqueous extract against LMBV in vitro using the MTT method.In vivo experiments were conducted by feeding the extracts at a concentration of 10 g/kg、15 g/kg and 20 g/kg,and the attack was performed after 3 days of feeding,and mortality was determined.The viral load in vivo was determined using the RT-PCR method.The results of in vitro experiments showed that:At 10 g/kg addition,the acetone extract and 95%methanol extract of Rhodomyrtus tomentosa showed some killing effect on LMBV with inhibitory rates of33%and 35%correspondingly;75%ethanol extract had no protective effect on LMBV-infected bass,and the protection rate for largemouth bass was 25%and10.25%when the content of 95%methanol extract and acetone extract in the feed was10 g/kg.On day 14,the viral load was significantly reduced in the methanol extract and acetone extract groups by 76%and 46%respectively,compared to the control group.3.Study of the main active components of Rhodomyrtus tomentosa against LMBV.In vivo and in vitro antiviral efficacy studies were carried out on three important active substances of myrtenol,myrtenal,and rhodomyrtone.The three active substances were fed at 500 mg/kg,250 mg/kg,and 100 mg/kg,and 0.1 ml of 10×TCID₅₀/0.1 m L LMBV was injected intraperitoneally after 3 days of feeding to study the protection rate of the three active substances against largemouth bass.The effects of the three active substances on the expression of three genes,IL-6,IL-1β,and TNF,related to inflammation in liver tissues of largemouth bass were determined by RT-PCR,and the anti-LMBV effects of the three active substances were investigated in vitro using tests such as cell nuclear damage and mitochondrial transmembrane potential.The results showed that all three active substances of Rhodomyrtus tomentosa had certain anti-LMBV effects,among which rhodomyrtone had the best effect,with a protection rate of 55.5%at 500 mg/kg addition,followed by myrtenol,with a maximum protection rate of 44.4%;compared with the control group,the viral load of rhodomyrtone group was significantly reduced.The three concentrations of100 mg/kg,250 mg/kg,and 500 mg/kg at day 14 decreased by 67.9%,87.84%,and91.52%,respectively.The results also suggested that rhodomyrtone significantly reduced the expression of inflammation-related factors induced by LMBV.The results of nucleus damage and mitochondrial transmembrane potential assay showed that the three substances had a protective effect on the nucleus damage and the decrease of mitochondrial membrane potential induced by LMBV.In conclusion,the main site of Rhodomyrtus tomentosa against LMBV is its aqueous extract,and its main active ingredient against the virus may be rhodomyrtone.