Screening Of Chinese Herbal Medicine Against Largemouth Bass Virus And Study On The Activity Of Berberine

Largemouth bass（Micropterus salmoides）,an important freshwater aquaculture fish species in China,has been increasing in production by an average of 9%per year.However,the deteriorating water environment caused by the increasing scale of largemouth bass aquaculture has led to frequent outbreaks of diseases,resulting in significant economic losses.Among them,largemouth bass virus（LMBV）is one of the most serious pathogens in cultivation and commercial fish production.Once it occurs,the mortality rate of largemouth bass can reach over 60%.It is well-known that immunological control,drug control,and ecological control are the three major methods for preventing aquatic diseases.Currently,vaccine research and application are developing slowly in China,especially for LMBV in largemouth bass.Therefore,drug control has become the most direct and effective method for preventing this disease.Herbal medicine,as a natural product in nature,is mostly biodegradable,has low toxicity to non-target organisms,and has anti-viral activity against various viruses.It is an important source of innovation for anti-viral drugs.Based on the screening of anti-LMBV activity among 88 herbal medicines,this study evaluated the anti-LMBV effect of Phellodendron chinense Schneid.and its active ingredient,Berberine（BBR）,at the cellular and individual levels.Further investigations were conducted to explore the anti-viral mechanism of BBR using flow cytometry,transmission electron microscopy,and Western blot.The results of this study are as follows:1.Antiviral activity of Chinese medicinal herbs and active components against LMBV.88 Chinese herbal medicines were investigated for their ability to inhibit LMBV using epithelioma papulosum cyprinid cells（EPC）as a cell screening model.The inhibitory effects of the herbs on LMBV were evaluated by RT-q PCR.Among the herbs screened,eight,namely Phellodendron chinense Schneid.,Acanthopanax gracilistylus W.W.Smith,Anemarrhena asphodeloides Bge.,Atractylodes japonica Koidz.ex Kitam.,Pharbitis purpurea（L.）Voigt,Plantago asiatica L.,Eupatorium fortunei Turcz.,and Ruta graveolens L.,were found to have inhibitory rates exceeding 90%,with Phellodendron chinense Schneid.showing the highest anti-LMBV activity with an inhibition rate of 99.9%.The anti-viral activity of the main component of Phellodendron chinense Schneid.,BBR,was also investigated,and the results showed that BBR significantly inhibited LMBV proliferation,with a half maximal inhibitory concentration(IC₅₀)of 11.29μM,indicating that BBR may be the main active ingredient of Phellodendron chinense Schneid.Virus titration and cell structure observation demonstrated that BBR could significantly reduce the viral titer and inhibit LMBV-induced cell damage,indicating that BBR has in vitro anti-LMBV activity.2.Berberine（BBR）inhibits LMBV replication through the mitochondrial apoptosis pathway.Flow cytometry was used to detect the degree of cell apoptosis in BBR-treated cells,and it was found that the apoptotic rate of the BBR-treated group was increased by 13.73%compared to the virus group after 6 h of LMBV infection,indicating that BBR can effectively induce apoptosis in LMBV-infected cells.Transmission electron microscopy observation of the ultrastructure inside EPC cells revealed that after 6 h of LMBV infection,BBR-treated EPC cells showed obvious apoptotic features compared to the virus group,such as chromatin condensation,cytoskeleton breakdown,and formation of intracellular vesicles.This suggests that BBR can effectively induce infected cells to destroy the site of LMBV replication through apoptosis.RT-q PCR,CPE,and titer detection methods were used to verify the effects of activators and inhibitors of cell apoptosis on LMBV proliferation.Staurosporine,Etoposide,and Z-VAD-FMK were found to effectively inhibit LMBV proliferation.The mitochondrial membrane potential detection results showed that the mitochondrial membrane potential of BBR-treated EPC cells was significantly decreased compared to the virus group after 6 h of LMBV infection.Further Western blot detection of the key proteins in the mitochondrial apoptosis pathway,caspase-9,and caspase-3,showed that the expression levels of cleaved caspase-9 and cleaved caspase-3 in EPC cells infected with LMBV for 6 h were not significantly different from the control group,while the expression levels of cleaved caspase-9 and cleaved caspase-3 in BBR-treated infected cells were significantly increased,indicating that BBR can effectively reduce the mitochondrial membrane potential of LMBV-infected cells and induce the mitochondrial apoptosis pathway.3.In vivo antiviral activity study of berberine（BBR）against LMBVThe correlation between the viral load of LMBV and the mortality rate of largemouth bass was investigated by injection,and the viral dose of 10³ TCID₅₀ was selected.BBR was used as the research object,and its in vivo anti-LMBV activity was verified by injection.The results showed that after treatment with 3.93 mg/kg BBR,the cumulative mortality rate of largemouth bass relative to the virus group decreased by 15%,indicating that BBR can effectively improve the survival rate of largemouth bass after LMBV infection.The proliferation of LMBV in largemouth bass liver,spleen,and kidney was detected by RT-q PCR,and it was found that BBR significantly reduced virus expression in liver,spleen,and kidney tissues of largemouth bass at 1 and 4 d post-infection,indicating that BBR can effectively inhibit the proliferation of LMBV in largemouth bass liver,spleen,and kidney tissues.Additionally,the viral titer in largemouth bass was also significantly reduced after BBR treatment.In conclusion,BBR was found to effectively inhibit effectively inhibit LMBV replication both in vitro and in vivo,reducing the mortality rate of LMBV-infected largemouth bass.Furthermore,BBR suppresses virus replication via the mitochondrial apoptosis pathway.Therefore,these findings have important implications for the research on anti-LMBV activity of Chinese medicinal herbs and provides a theoretical foundation for the development of novel anti-LMBV drugs.