| Background and purpose of research Dengue fever is an acute insect-borne infectious disease caused by dengue virus(DENV),which is transmitted by mosquitoes.There are many clinical manifestations after dengue virus infection,such as latent infection,dengue fever,dengue hemorrhagic fever and so on.The typical clinical manifestations of dengue fever include sudden onset,high fever,headache,and severe muscle and joint soreness.Some patients may present with rash,bleeding tendency,lymph node enlargement,decreased white blood cell count,and thrombocytopenia.This disease is mainly endemic in tropical and subtropical areas.In our country Yunnan,Guangdong,Hong Kong,and Macao are dengue endemic areas with many local small outbreaks of dengue fever.At present,the clinical research and development of antiviral drugs is difficult,and the effect is generally poor.Different from common infectious diseases,dengue virus is spread in the population through insect-borne transmission.At present,most patients infected with dengue virus can only take symptomatic treatment.However,symptomatic treatment cannot achieve the effect of inhibiting virus replication and antiviral,and dengue patients have serious clinical symptoms and long course of disease,affecting their normal life.Therefore,there is no specific antiviral treatment for dengue.Therefore,a specific anti-dengue drug is urgently needed to provide the technical means to prevent and control such outbreaks.Antisense oligonucleotides refers to a single stranded DNA or RNA sequence that is complementary to the target gene or m RNA and produces specific gene allosteric effects.A single stranded DNA or RNA sequence,usually composed of 15 to 30 nucleotides paired with a target gene,produced by means of chemical synthesis and biological modification.Its effect is to silence or degrade genes and thus inhibit gene expression.Due to its advantages of high specificity,high efficiency,safety and low toxicity,ASO has shown a broad application prospect in gene therapy and other fields.For example,nusinersen was the first drug approved for spinal muscular atrophy and the first drug to demonstrate that increased expression of a specific protein in spinal muscular atrophy patients can improve motor function.Inotersen has been shown to be useful in the treatment of transthyretin amyloidosis cardiomyopathy.Eteplirsen has been shown to treat Duchenne muscular dystrophy by increasing the amount of dystrophin fibers in the human body and correspondingly increasing the average staining intensity of dystrophin.This study aims to screen ASO targets that can effectively inhibit dengue virus,further evaluate and optimize its antiviral efficiency,and finally obtain anti-dengue virus antisense oligonucleotide candidate targets,so as to provide target suggestions and technical support for subsequent research and development of related drugs.Research content(1)Design and screening of ASO targets against Dengue virus.By sequence alignment,10 ASO candidate targets with a length of 28 bp were selected from relatively conserved regions of the dengue virus genome,and specific sequences targeting mutations in the genome of thalassemia patients were used as irrelevant target controls.The above targets were synthesized into ASO targets,and the morpholine ring and diaminophosphate groups were modified at both ends of ASO target sequence.Subsequently,quantitative fluorescent PCR was used to evaluate the inhibitory efficiency of ASO containing different targets on dengue viral load in Vero cell models infected with dengue type II virus.The antisense oligonucleotides with the highest viral load inhibition efficiency were screened.(2)Condition optimization and effect evaluation of ASO against Dengue virus.In order to obtain the optimal anti-dengue virus conditions of antisense oligonucleotides,the optimal dose and the optimal administration time of ASO against Dengue virus were evaluated.Meanwhile,we further verified the inhibition of ASO on Dengue virus activity by viral plaque assay.In addition,to verify its antiviral effect in different cell lines,we evaluated the antiviral efficiency of ASO in Vero,Hep G2 and other cell lines respectively.(3)Inhibitory effect of antisense oligonucleotides on subtypes of Dengue virus.In order to further evaluate the inhibitory effect of anti-Dengue virus ASO on the replication of different types of Dengue virus,we respectively established cell models infected with Dengue virus types I,III and IV,and then used ASO screened in the above studies to detect the inhibitory efficiency of viral load of different types of Dengue virus.To evaluate its inhibitory effect on different serotypes of dengue virus.Research results(1)The antisense oligonucleotide targeting UTR region with the best anti-dengue virus effect was screened(hereafter referred to as ASO-UTR).ASO-UTR showed the best antiviral effect by using antisense oligonucleotides targeting 10 gene targets of Dengue virus at the same concentration.The cell growth of the ASO-UTR group was similar to that of the control group,suggesting less infection and a decrease of viral load of 99.8%in 72 hours.(2)The optimal concentration of antisense oligonucleotides anti-dengue virus was selected.The results showed that the inhibitory efficiency of ASO-UTR at 0.01μM,0.1μM,1μM and 10μM concentrations was 0.01μM,84.46%±4.23%.0.1μM,97.48%±3.96%;1μM,99.58%±3.48%,10μM,99.97%±3.46%.It can be seen that ASO-UTR at 10μM concentration had the highest viral inhibition efficiency,reaching99.97%±3.46%.(3)The difference of antiviral effect was analyzed according to the time of administration.After 2 hours,6 hours and 12 hours of administration,the 24-hour viral inhibition efficiency was 99.88%±20.84%,respectively.6 hours: 99.89%±20.84%;12 hours: 98.99%±20.84%.The 48-hour virus inhibition efficiency was as follows: 2 hours:99.99%±26.23%;6 hours: 99.98%±26.23%;12 hours: 99.63%±26.23%.72-hour virus inhibition efficiency was 99.94%±10.98% for 2 hours,99.82%±10.98% for 6 hours.12 hours: 98.78%±10.98%.It is concluded that the earlier the actual time of medication,the better the effect.(4)The inhibitory effect of ASO on dengue virus in different cell lines was verified.In Vero cells,the inhibitory efficiency of ASO-UTR against Dengue virus decreased by99.99%±10.66% at 72 h.In Hep G2 cells,the inhibition efficiency of ASO-UTR against Dengue virus decreased by 99.99%±29.89% at 72 hours.(5)It was verified that the target ASO-UTR had inhibitory effect on all subtypes of dengue virus.The inhibition efficiency of ASO-UTR against Dengue I virus decreased by 98.69%±15.22% at 24 hours,99.83±12.03% at 48 hours and 99.99%±11.82% at 72 hours.The inhibition efficiency of ASO-UTR against dengue III virus decreased by99.71%±21.14% at 24 h,99.96%±29.92% at 48 h and 99.99%±8.26% at 72 h.The inhibition efficiency of ASO-UTR against dengue IV virus decreased by 99.75%±4.89%at 24 hours,99.99%±13.45% at 48 hours and 99.99%±18.94% at 72 hours.Research conclusionsIn this study,ASO targeting the UTR region of the dengue virus genome was found to be highly effective in inhibiting the replication of Dengue virus.ASO reduced the viral load of Dengue virus by 99.99%±29.89% in the cell model,and inhibited all four serotypes of Dengue virus.In addition,this study also explored the optimal dose and administration time of ASO,which will provide candidate targets and technical basis for the subsequent development of ASO based anti-dengue virus drugs. |
