| Avian leukosis(AL)caused by avian leukosis virus(ALV)of the family Retroviridae,is an infectious disease which has caused huge economic losses to the poultry industry worldwide.ALV can be divided into 11 subgroups A to K according to the different characteristics of viral envelope glycoprotein,and avian leukosis virus subgroup J(ALV-J)has been considered to be the most serious harm and more prevalent in China.Vertical and horizontal transmission are important routes for ALV transmission in chicken flocks,and it is also one of the ways of AI transmission due to the chickens were vaccinated with contaminated ALV.So far,however,there is no solution that can be used to effectively inhibit the ALV replication in vitro.Previous studies have found that the reverse transcriptase inhibitor Zidovudine(AZT)inhibit ALV replication,but the cytotoxicity is also increases with the increasing in concentration of drug,and the problem of drug resistance generation may exist under long-term AZT selection pressure.In order to establish a solution which was low toxicity and efficient to inhibit ALV-J replication in vitro,two new strategies by RNAi in reverse transcriptase gene region to inhibit ALV-J replication were proposed in this study.1.shRNA combined with AZT inhibits ALV-J replication in vitroFirst,the cytotoxicity of the in vitro culture system with different concentrations of AZT was determined by the CCK-8 assay,and the inhibitory activity against ALV-J with different concentrations of AZT was observed by the dose effect curve,the concentration of the AZT working solution should not exceed 1 ?g/m L,the cell viability was more than 95%(inhibition rate>70%).Sh RNA expression plasmids were designed and constructed against the active region of the ALV-J reverse transcriptase gene and the RNase H,it was verified that the silencing effects on target genes was about 60%.The inhibitory effects of AZT and shRNA expression plasmids alone or in combination on ALV-J in vitro were compared by ELISA assay.The relative expression of ALV-J viral RNA in DF-1 cells revealed that the relative expression of specific shRNA transfection ranged from 0.40 to 0.51;the relative expression of specific shRNA combined with AZT ranged from 0.028 to 0.037,respectively.The results indicated that the synergistic inhibitory effect of shRNA and AZT.The similar results were obtained by using Western blot to detect the expression of ALV-J capsular protein.The copy number of ALV-J in the DF-1 cell supernatant was detected by Real-time PCR assay,,it was found that the copy number was reduced to 104.216 104.313 copies / 100 ?L in specific shRNA transfection group compared to 105.126 copies/100 ?L in sh-NC group,,it was reduced to 103.443 copies/100 ?L in AZT alone group,However,the viral RNA copy number was further reduced to 102.729 102.917 copies/100 ?L in the specific shRNA combined with AZT group.The p27 antigen levels in cell supernatants revealed that the S/P values of cell supernatants were 0.62 in sh-NC group,0.32 0.37 in specific shRNA transfection group,and 0.30 in AZT alone group,whereas the ratios of specific shRNA combined with AZT group further decreased to 0.18 0.25,and the synergistic effects were observed in combination of shRNA and AZT(left shift of dose effect curves).The proviral synthesis revealed that shRNA targeting the reverse transcriptase activity domain had a better synergistic effect than shRNA targeting the RNase H activity domain after further detection.Overall,the results showed that shRNA and AZT had good inhibitory effects on ALV-J,but the inhibitory effect in vitro of shRNA alone was not as good as AZT alone in contrast,and the combination of the combination with shRNA and AZT can improve the inhibitory efficiency on ALV-J without increasing cytotoxicity,it provides a good application prospects.2.Inhibition of ALV-J replication by gold nanoparticle loaded siRNA in vitroIn order to achieve efficient delivery of siRNA,based on the designed shRNA targeting reverse transcriptase activity domain,(Cy3-)siRNA targeting RT gene was synthesized and loaded with siRNA using gold nanoparticles by using polyethylenimine modification(Au@PRP NPs).Laser confocal microscopy observation and flow cytometric analysis showed that the positive cells were 92.1% and 66.6% in the Au@PRP NPs group and RNAi Max group,respectively.The results confirmed that the Au@PRP NPs couldeffectively deliver siRNA into the DF-1 cells for shearing of ALV-J RNA,and the transfection efficiency was superior to that the commercial transfection reagent Lipofectamine RNAi Max.CCK-8 detection found that the cell viability of the gold Nanodelivery group was 100% compared to the blank control group,indicating that the delivery of siRNA using gold Nanodelivery was safe and non-toxic.Subsequently,we examined the amount of relative viral RNA expression in DF-1 cells in this assay,the relative expression decreased to 0.23 0.26 in the Au@PRP NPs delivered group and 0.45 0.52 in the RNAi MAX delivered group,indicating Au@PRP NPs could significantly inhibit the ALV-J replication on DF-1 cells with better efficacy than RNAi Max.The number of viral RNA copies in the DF-1 cell supernatant by Real-time PCR assay,it was found that the RNAi MAX delivery group reduced to 105.525 copies/100 ?L and 104.781 copies/100 ?L,respectively.The Au@PRP NPs delivery group reduced to 105.526 copies/100 ?L and 104.284 copies/100 ?L.The p27 antigen level in cell supernatant revealed that RNAi MAX delivery group reduced to 1.036 and 0.529 0.579,Au@PRP NPS group reduced to 1.044 and 0.272 0.326.Overall,the above results showed that siRNA targeting RT gene exhibit better anti-ALV-J effect in vitro after delivered using gold nanoparticles,and the transfection toxicity was lower than Lipofectamine RNAi Max.In conclusion,a new method based on RNAi to inhibit the ALV-J replication in vitro was established in this study,and innovatively proposed a new idea of combined application of RNAi and AZT to enhance the inhibition of ALV-J,and gold nanomaterials were developed as an efficient platform to achieve very efficient in delivering siRNA.The above inhibition strategy has been verified in the application scenarios of eradicating the contamination of avian attenuated vaccine seed virus ALV.These strategies provide more options to accelerate the eradicaiton of ALV and also provide references for other retroviral antiviral studies. |
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