Study On The Activity Of Luteolin Against Micropterus Salmoides Rhabdovirus

Micropterus salmoides rhabdovirus disease is a kind of fish slug virus disease caused by Micropterus salmoides rhabdovirus（MSRV）.Considering the limitation of application of vaccine in aquaculture,the virus is now mainly prevented and controlled with drugs at present.Since drugs such as ribavirin are banned,there is an urgent need for new antiviral drugs in aquaculture.Tradictional Chinese herbal medicine（TCHM）has the advantages of multi-biological activities（anti-inflammatory,antioxidant,antiviral and other activities）,environmental protection and large screening library,which makes it an ideal research direction of anti-MSRV drugs.In this study,126 kinds of TCHMs were screened for their anti-MSRV activity,and Houttuynia cordata has the best activity.Next,the anti-MSRV activity of the active components of Houttuynia cordata was determined and luteolin（LUT）was identified as the anti-MSRV active ingredient.Further,the anti-MSRV activity of LUT was verified in vitro.The antiviral mechanism of LUT was preliminarily determined and its antiviral activity was studied in vivo,which provided a theoretical basis for the application of LUT in the prevention and control of Micropterus salmoides rhabdovirus disease.The results of this study are as follows:1.Screening of anti-MSRV activity of TCHMs and its active ingredientsFirstly,CPE observation method was used to screen the anti-MSRV activity of TCHMs in the safe concentration range,and four TCHMs with significant anti-MSRV activity were obtained.Secondly,in order to determine the anti-MSRV activity of 4 kinds of TCHMs,the maximum safe concentration(CC₂₀)of these 4 TCHMs was determined by MTT method,and the relative expression level of MSRV G protein gene of 4 TCHMs at CC₂₀ was detected by real-time quantitative polymerase chain reaction（RT-q PCR）.Houttuynia cordata was confirmed as the best anti-MSRV herb（inhibition rate was 98.94%）.Then crystal violet test and CPE observation were used to further verify the anti-MSRV activity of Houttuynia cordata.The results showed that Houttuynia cordata had protective effect on the cell morphology after MSRV infection.Finally,CPE observation method and RT-QPCR method were used to study the anti-MSRV activity of four active ingredients（quercetin,kaneferol,luteolin and chlorogenic acid）of Houttuynia cordata.It was found that LUT had the best anti-MSRV activity（inhibition rate was 99.17%at 10 mg/L）.These results indicated that Houttuynia cordata and LUT had good anti-MSRV activity in vitro,and LUT could be used as the object of follow-up study.2.Study on anti-MSRV activity of Luteolin in vitroIn further to study better the anti-MSRV activity of LUT in vitro,the CC₂₀ of LUT was15.304±3.320 mg/L by MTT method.Secondly,crystal violet staining test and CPE observation showed that LUT could reduce CPE induced by MSRV.Rt-q PCR and titer assay were used to detect the effects of LUT on the expression and titer of MSRV G protein.The results showed that LUT could significantly inhibit the expression of MSRV G protein(IC₅₀was 2.13 mg/L)and significantly reduce the virus titer within 72 h,indicating that LUT could inhibit the proliferation of MSRV.In order to further study the mechanism of LUT against MSRV,the direct anti-MSRV activity of LUT and the influence of LUT on MSRV adsorption process,different stages of virus replication and particle release process were studied by ultra-speed centrifugation,RT-q PCR and virus titer assay.It was found that LUT had certain direct anti-MSRV activity.In addition,LUT exerted antiviral effect mainly by blocking MSRV particles,inhibiting MSRV G protein gene replication and virion release.These results indicated that LUT has a good anti-MSRV activity in vitro,.3.Study on anti-MSRV activity of Luteolin in vivoIn order to further study the actual anti-LUT effect of LUT,the anti-MSRV activity of LUT in vivo was evaluated by injection and oral administration.Firstly,the safe concentrations of LUT injection and oral administration as well as the challenge concentration of MSRV were determined.Then,challenge test of injection and oral of LUT were conducted under the safe concentrations.The survival rate of juvenile Micropterus salmoides and viral load in liver,spleen and kidney were measured by RT-q PCR.It was found that both injection and oral administration of LUT improved the survival rate of juvenile fish by 40%and 30%respectively,and LUT significantly reduced the MSRV content in liver,spleen and kidney in the early stage of MSRV infection.These results indicated that LUT had good anti-MSRV activity in vivo.In conclusion,Houttuynia cordata had excellent anti-MSRV activity,and its main anti-MSRV active ingredient was LUT.LUT inhibits MSRV proliferation by blocking MSRV adsorption,acting on virus replication stage and inhibiting the release of virus particles,and has certain in vivo anti-MSRV activity.