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Synergistic inhibition of avian leukosis virus subgroup J replication by miRNA-embedded siRNA interference of double-target.

Wei R, Ma X, Wang G, Guo H, Liu J, Fan L, Cheng Z - Virol. J. (2015)

Bottom Line: Due to largely ineffective vaccines, new control measures are needed to be developed.For detecting the interference effect, recombinant vectors were introduced into DF-1 cells and day-old SPF chickens that infected with ALV-J.In vivo, chicks were inoculated with each recombinant plasmid in peritoneal cavity at day of hatch, and monitored infection status at interval 1 day postinfection for 4 weeks.

View Article: PubMed Central - PubMed

Affiliation: College of Veterinary Medicine, Shandong Agricultural University, Tai'an, 271018, China. leng.lengaaa@163.com.

ABSTRACT

Background: The diseases caused by avian leukosis virus subgroup J (ALV-J) has become a serious problem in the poultry. Due to largely ineffective vaccines, new control measures are needed to be developed. RNA interference (RNAi) has been developed a promising measure for antivirus in poultry.

Methods: In this study, miRNA-embedded siRNA interference was designed and used to inhibit ALV-J replication in vitro and in vivo. Each sequence of target siRNA derived from the gag (p15), pol (p32), env (gp85) and LTR (U3) gene of ALV-J was embedded into mouse miR-155 backbone as a pre-miRNA hairpin oligonucleotide sequence. After annealing, they were cloned into pcDNA6.2-GW/EmGFP-miR vector, respectively. For detecting the interference effect, recombinant vectors were introduced into DF-1 cells and day-old SPF chickens that infected with ALV-J.

Results: In vitro, single target interference showed effective inhibition of reducing 74% ~ 85% mRNA of ALV-J. Double targets showed more efficient inhibition of reducing 96% ~ 98% mRNA of ALV-J. In vivo, chicks were inoculated with each recombinant plasmid in peritoneal cavity at day of hatch, and monitored infection status at interval 1 day postinfection for 4 weeks. Delivery of single target or double targets miRNA significantly reduced viremia and pathogenicity caused by ALV-J in vivo, especially the double targets.

Conclusions: These data demonstrated that the miRNA-embedded siRNA interference is an efficient method for inhibition of ALV-J replication, especially double targets.

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Related in: MedlinePlus

Transfection effect of recombinant expression vectors observed by fluorescence microscope. The miRNA-embedded siRNA expression plasmids were transfected into DF-1 cells and GFP expression was monitored at 48 h post-transfection by fluorescence microscopy (magnification 40×).
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Fig2: Transfection effect of recombinant expression vectors observed by fluorescence microscope. The miRNA-embedded siRNA expression plasmids were transfected into DF-1 cells and GFP expression was monitored at 48 h post-transfection by fluorescence microscopy (magnification 40×).

Mentions: The synthesis of four pre-miRNA (schematic diagram was shown in Figure 1) and a control sequence was cloned into pcDNA6.2-GW/EmGFP-miR, respectively, and named p-miR-gag, p-miR-pol, p-miR-env, p-miR-LTR and vector control. Sequence analysis and recombinant plasmid electrophoresis results confirmed that miRNA-embedded siRNA expression vectors were successfully constructed. At 48 h post transfection, typical green fluorescence-positive cells were observed by fluorescence microscopy. As shown in Figure 2, about 70% of the DF-1 cells are positive, indicating the transfection efficiencies is suitable for RNA interference experiment.Figure 1


Synergistic inhibition of avian leukosis virus subgroup J replication by miRNA-embedded siRNA interference of double-target.

Wei R, Ma X, Wang G, Guo H, Liu J, Fan L, Cheng Z - Virol. J. (2015)

Transfection effect of recombinant expression vectors observed by fluorescence microscope. The miRNA-embedded siRNA expression plasmids were transfected into DF-1 cells and GFP expression was monitored at 48 h post-transfection by fluorescence microscopy (magnification 40×).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4376366&req=5

Fig2: Transfection effect of recombinant expression vectors observed by fluorescence microscope. The miRNA-embedded siRNA expression plasmids were transfected into DF-1 cells and GFP expression was monitored at 48 h post-transfection by fluorescence microscopy (magnification 40×).
Mentions: The synthesis of four pre-miRNA (schematic diagram was shown in Figure 1) and a control sequence was cloned into pcDNA6.2-GW/EmGFP-miR, respectively, and named p-miR-gag, p-miR-pol, p-miR-env, p-miR-LTR and vector control. Sequence analysis and recombinant plasmid electrophoresis results confirmed that miRNA-embedded siRNA expression vectors were successfully constructed. At 48 h post transfection, typical green fluorescence-positive cells were observed by fluorescence microscopy. As shown in Figure 2, about 70% of the DF-1 cells are positive, indicating the transfection efficiencies is suitable for RNA interference experiment.Figure 1

Bottom Line: Due to largely ineffective vaccines, new control measures are needed to be developed.For detecting the interference effect, recombinant vectors were introduced into DF-1 cells and day-old SPF chickens that infected with ALV-J.In vivo, chicks were inoculated with each recombinant plasmid in peritoneal cavity at day of hatch, and monitored infection status at interval 1 day postinfection for 4 weeks.

View Article: PubMed Central - PubMed

Affiliation: College of Veterinary Medicine, Shandong Agricultural University, Tai'an, 271018, China. leng.lengaaa@163.com.

ABSTRACT

Background: The diseases caused by avian leukosis virus subgroup J (ALV-J) has become a serious problem in the poultry. Due to largely ineffective vaccines, new control measures are needed to be developed. RNA interference (RNAi) has been developed a promising measure for antivirus in poultry.

Methods: In this study, miRNA-embedded siRNA interference was designed and used to inhibit ALV-J replication in vitro and in vivo. Each sequence of target siRNA derived from the gag (p15), pol (p32), env (gp85) and LTR (U3) gene of ALV-J was embedded into mouse miR-155 backbone as a pre-miRNA hairpin oligonucleotide sequence. After annealing, they were cloned into pcDNA6.2-GW/EmGFP-miR vector, respectively. For detecting the interference effect, recombinant vectors were introduced into DF-1 cells and day-old SPF chickens that infected with ALV-J.

Results: In vitro, single target interference showed effective inhibition of reducing 74% ~ 85% mRNA of ALV-J. Double targets showed more efficient inhibition of reducing 96% ~ 98% mRNA of ALV-J. In vivo, chicks were inoculated with each recombinant plasmid in peritoneal cavity at day of hatch, and monitored infection status at interval 1 day postinfection for 4 weeks. Delivery of single target or double targets miRNA significantly reduced viremia and pathogenicity caused by ALV-J in vivo, especially the double targets.

Conclusions: These data demonstrated that the miRNA-embedded siRNA interference is an efficient method for inhibition of ALV-J replication, especially double targets.

Show MeSH
Related in: MedlinePlus