Antiviral Mechanism Of LJ002 Based On Singlet Oxygen Mediated Lipid Oxidation

The emerging viral pathogens have caused a large number of diseases and caused huge economic losses to the world.In addition,animals carrying viruses are the main source of infection for zoonotic diseases,and about 75%of new infectious diseases are caused by animal viruses.Immunization is an effective method to prevent the infection of new-onset virulent diseases.Therefore,the development of new inactivating agents will be an effective means to resist the spread of viral diseases.The new type of inactivated vaccine can not only stimulate a specific immune response,but also overcome the adverse immune response induced by formalin,ultraviolet light,gamma radiation and heat inactivated vaccine,as well as the highly toxic and non-toxicity of aziridine derivatives.Stability and other adverse reactions.Singlet oxygen(1O2)is a kind of reactive oxygen species,which plays a very important role in the process of programmed cell death of plants and microorganisms.1O2 can also be used as a second messenger to mediate signal transduction and activate redox-sensitive transcription factors.The chemical production of 1O2 requires the light activation of photosensitizers(such as porphyrin,chlorine and thiazolidine)to enter the triplet state and transfer energy to 1O2 to form 1O2,which can be used in blood sterilization,herbicides,pesticides,wastewater treatment,Fine chemical synthesis and photodynamic therapy.In this study,the ability of ethylthiazolidine derivative LJ002 to produce 1O2 and the role of 1O2 in the process of viral infection were explored,in order to clarify that LJ002 inactivates the virus through 1O2,and provides methods for the prevention of viral diseases and the development of new inactivated vaccines.1.LJ002 produces 1O2 in solution and living cells.This study firstly used 9,10 dimethylanthracene(DMA)and Si-DMA to detect the production of 1O2 by LJ002 in solution and living cells,which proved that LJ002 produced 1O2 in solution and living cells,and added tocopherol And lycopene can quench 1O2.Subsequently,the singlet oxygen fluorescent probe was used to detect the quantum yield of LJ002 and compare the 1O2 production capacity of LJ002 and LJ001,which proved that the quantum yield of LJ002 was higher than that of LJ001.This feature confirms that LJ002 has the basis of oxidizing lipid film.2.LJ002 inhibits PRV proliferation in vitro.In order to study the role of LJ002 in the virus invading the host,this paper uses PRV as the model virus for research.Firstly,it was determined that LJ002 did not affect cell proliferation by CCK-8 method,cell counting and cell morphology observation under microscope.Subsequently,through immunofluorescence,flow cytometry,immunoblotting,and viral infection titer tests,it was found that LJ002 inhibited the number of fluorescent cells of PRV-GFP,the expression of PRV HN-1201 and PRV-QXX gE protein,and the PRV progeny virus The titer indicates that LJ002 has an inhibitory effect on PRV infection.3.LJ002 destroys the structure of the virus membrane by producing 1O2 to oxidize the lipids in the viral envelope,thereby inhibiting the fusion of the virus with the cell membrane.In this study,LJ002 addition time experiment was carried out by virus titer,flow cytometry,RT-qPCR and other methods.It was found that LJ002 inactivated PRV virus particles and impaired early entry,thereby inhibiting virus infection.Secondly,the EDU-labeled virus,immunofluorescence,immunoblotting and electron microscopy experiments showed that the LJ002 inactivated virus was non-infectious,did not attach/fused to the cell surface,and some distortions appeared on the virus particle membrane.Finally,through electron microscopy,atomic force microscopy,malondialdehyde,LC-MS,virus titer,PRV-GFP fluorescence and other tests,it was confirmed that the addition of 1O2 scavenger reversed the antiviral activity of LJ002 against PRV,and 1O2 participated in LJ002's antiviral activity.Destruction of the envelope and its antiviral activity.4.Mice immunized with LJ002 inactivated PRV vaccine can induce more neutralizing antibodies and can effectively protect mice from PRV infection.First of all,the potential toxicity of LJ002 was evaluated.According to body weight,appearance,serum alanine aminotransferase and aspartate aminotransferase levels,and HE staining to observe its toxicity to the main organs,the results proved that the dose of LJ002 has no toxic side effects on animals.Secondly,animal challenge survival experiments and immunofluorescence staining confirmed that pre-incubating PRV with LJ002 can inhibit PRV infection in vivo.Finally,the potential application of LJ002 and its PRV inactivated vaccine's ability to generate immunity were explored.Dot blot was used to analyze the changes of essential protein content of PRV virus such as viral antigen gB and virulence factor gE,and found that LJ002 had no effect on the content of gB and gE;LC-MS spectrum showed that the oxidative modification of viral polypeptides increased after LJ002 treatment.The Dot blot test showed that the antibody titer in the serum of mice vaccinated with the LJ002 inactivated vaccine was significantly higher than that of the conventional formalin inactivated vaccine.Serum neutralization experiments and animal survival challenge experiments show that compared with conventional formalin inactivated vaccines,mice immunized with the same antigen-containing LJ002 inactivated vaccine can induce more neutralizing antibodies and can effectively protect the mice Protection from PRV infection.5.LJ002 inhibits the proliferation of enveloped viruses.Studies have shown that LJ002 mediates 1O2 to oxidize the viral membrane,thereby blocking virus-cell membrane fusion and exerting antiviral activity.The resistance of LJ002 to enveloped viruses of different families was further tested.After LJ002 inactivated PRRSV,ND V,VSV,Sev and H1N1 infected cells,the virus replication was detected by fluorescence,flow cytometry,TCID50 and HA.The results showed that LJ002 has a broad-spectrum antiviral effect on enveloped viruses.In summary,LJ002 inhibits the fusion and infection between the virus and the cell membrane by producing 1O2 to oxidize the lipids in the viral envelope,and the LJ002 inactivated pseudorabies virus produces more in mice than the traditional formalin inactivated virus.Strong neutralizing antibody level and immune protection effect.This article suggests that 1O2 can be used as a new inactivator in the production of inactivated vaccines,providing a new strategy for the development of inactivated vaccines.