Abstract
BackgroundThe 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.MethodsIn 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.ResultsIn 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.ConclusionsThese data demonstrated that the miRNA-embedded siRNA interference is an efficient method for inhibition of ALV-J replication, especially double targets.
Highlights
The diseases caused by avian leukosis virus subgroup J (ALV-J) has become a serious problem in the poultry
Construction of expression vectors and its transfection efficiency The synthesis of four pre-miRNA 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
The combined plasmids were transfected into DF-1 cells that infected with ALV-J
Summary
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. Avian leukosis virus subgroup J (ALV-J), an oncogenic retrovirus, isolated in the United Kingdom in 1991, has been spread primarily in meat-type chickens and later in egg-type chickens [1,2,3,4,5]. Due to vaccines are largely ineffective, new measures against ALV-J are needed to be developed. RNA interference (RNAi) has shown a promising antiviral strategy in poultry [7,8,9,10]
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