Research Of Moroxydine Hydrochloride Inhibits Grass Carp Reovirus

Grass carp farming is the largest fishery industry in China and is of significance in providing enough animal protein as food for human beings.However,this species is suffering from the infection of reovirus for many years and cause huge economic losses.Currently,it is still difficult to efficiently control grass carp reovirus(GCRV)with vaccine,due to the frequent variation of GCRV strains and the application bottleneck.Therefore,the medicinal prevention and treatment of grass carp hemorrhagic disease is still the most effective way.A biguanide medicine,Moroxydine hydrochloride(Mor)was forbidding to use since 1999,because lacking of evidence of pharmacology and toxicology research.The present study was early found to have potential anti-GCRV activity.Based on the safety in vitro and in vivo results of Mor,Ctenopharyngodon idella kidney(CIK)cells and grass carp were introduced into the present study to explore the antiviral and anti-apoptosis mechanism of Mor.The results obtained in this study were as follows: 1.In vitro antiviral activity of Mor against GCRVIn the in vitro drug screening tests,Mor showed different degree of positive protective efficiency to four aquatic virus-infected host cells.GCRV104 was found to have higher virulence than GCRV873 and selected in further antiviral tests.Especially,the 100 μg mL-1 of Mor achieved 100% protection to GCRV-infected CIK cells.The 96 h safe concentration(SC)of Mor to CIK and GCO cells were calculated as 218.5 ± 9.5 μg mL-1 and 140.9 ± 25.7 μg mL-1,respectively.Moreover,the Mor showed a good metabolic features with the maximum content of medicine in CIK cells reaching 16.7 ± 0.6 mg per 106 cells within 4 h.Therefore,Mor shows favourable safety and metabolism characteristic.Under the condition of GCRV infections,CIK cells and GCO cells exposed to Mor showed concentration-and time-dependent induction of cell viability.And the CIK and GCO cells maintained more than 90% of viability when treated with 39.8 μg mL-1 Mor for 96 h,which is slightly greater than the same concentration designed of Rib with 80% cell viability.The cytopathic effect was also avoided according to microscope observation.Moreover,the virulence showed a respective 7.7 and 5.7 natural exponential reduction when expose GCRV infected CIK and GCO cells in 20 μg mL-1 of Mor in the further tests.And the expressions of viral protein genes were significantly inhibited with time-dependent induction.Western blot test also showed a inhibition of GCRV viral protein expression in host cells.2.In vivo antiviral research of Mor against GCRVThere is no significant acute toxicity of Mor to the grass carp within 1000 μg mL-1.The anti-GCRV activity of Mor in grass carp was detected after performing inhalation and injection.And a series of pathological changes were appeared in grass carp after virus infection,including the virus proliferation and oxidation damage,and resulting a 56.7% of cumulative mortality post 7 d infection.Both the VP3 and VP6 relative expressions were less than 0.07 in the kidneys,liver,gill and muscle after Mor 7 d injection,which is significantly lower than the Mor inhalation group.Meanwhile,the GCRV-induced reduction of serum superoxide dismutase(SOD),total antioxidant capacity(T-AOC)and catalase(CAT)activity could avoided post Mor injection.There is no significant difference in survival rate in the Mor injection group within 7 d,as compared with control group.3.Inhibition of Mor on virus protein expressionThe recombinant plasmid pIRES2-AcGFP1-VP6 was successfully constructed.And the recombinant VP6 protein was expressed in CIK cells post plasmid transfection.Cell viability showed no significant influence within 48 h post recombinant plasmid transfection.Flow cytometry results also showed no significant apoptosis at 48 h post pIRES2-AcGFP1-VP6 transfection.However,the VP6 protein expression was significantly suppressed post Mor treatment.There are only a few aims fluorescent appeared in local position inside the Mor-treated cell.The VP6 gene expression was up-regulated in a time dependent manner after plasmid transfection.And the relative expression was reached to 0.59 as comparing with GCRV infection group.But 20 μg mL-1 of Mor showed a significant inhibition for VP6 expression with 72 h relative expression decreasing to 0.14.Taken together,Mor showed a direct inhibitory effect for viral protein expression.4.The research of anti-apoptosis mechanism of MorThe detection of apoptosis was executed by a variety of detection technology including fluorescence imaging,DNA electrophoresis,flow cytometry,scanning and transmission electron microscope.And a series of characteristics of cell apoptosis were detected post 48 h of GCRV infection,including karyolysis,DNA fragmentation,cell shrinkage and fracture,mass proliferation of GCRV particles,inclusion body appearance,virus releases,etc.The preliminary results of inhibitors test was confirmed that the activation of GCRV-induced apoptosis in connection with caspase pathway.Western blot analysis showed a dose-dependent increase of activated procaspase-9 and-3 at 48 h,and their full-length procaspases decreased with the increased infection doses.In GCRV infected cells,protein levels of Bcl-2 exhibited an apparent down-regulated in a time dependent manner,whereas the protein levels of Bax were up-regulated in a time dependent manner.Meanwhile,all the characteristics of apoptosis and the activation of mitochondrial apoptosis pathway were avoided post Mor treatment,contributing to the protection of host cells.5.Regulation of Mor on the host immune responseThe immune regulation of Mor on the CIK cell and grass carp were detected post GCRV infection and medication.However,the Mor significant decreases the virus-induced gene overexpression of Myd88、Mx1、IL-1β、IL-8、I-IFN and TNFα in CIK cells.And Mor effective blocks the GCRV-induced immune response.Furthermore,the grass carp immune stimulation reaction was also detected in grass carp post GCRV infection.And the GCRV-induced up-regulation of IFN-I、IL-1β and TNF-α gene expressions in kidneys,liver,gill and muscle also inhibited post medication.In summary,the GCRV proliferation could effectively controlled by direct antiviral effect of Mor.And the activation of apoptosis pathway was inhibited post Mor treatment,contributing to the protection of host cells and suppression of GCRV virus particle.At the same time,Mor showed the high safety index exhibiting it’s great application prospects in the control of grass carp hemorrhagic disease.