Wei Rongrong, Ma Xiaoqian, Wang Guihua, Guo Huijun, Liu Jianzhu, Fan Lingxiao, Cheng Ziqiang
College of Veterinary Medicine, Shandong Agricultural University, Tai'an, 271018, China.
Xiangya School of Medicine, Changsha, 410013, China.
Virol J. 2015 Mar 21;12:45. doi: 10.1186/s12985-015-0277-5.
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.
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.
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.
These data demonstrated that the miRNA-embedded siRNA interference is an efficient method for inhibition of ALV-J replication, especially double targets.
禽白血病病毒J亚群（ALV-J）引起的疾病已成为家禽业的一个严重问题。由于现有疫苗大多效果不佳，需要开发新的防控措施。RNA干扰（RNAi）已成为家禽抗病毒的一种有前景的措施。
本研究设计并使用嵌入miRNA的siRNA干扰在体外和体内抑制ALV-J复制。将源自ALV-J的gag（p15）、pol（p32）、env（gp85）和LTR（U3）基因的每个靶标siRNA序列嵌入小鼠miR-155骨架中，形成前体miRNA发夹寡核苷酸序列。退火后，分别将它们克隆到pcDNA6.2-GW/EmGFP-miR载体中。为检测干扰效果，将重组载体导入感染ALV-J的DF-1细胞和1日龄SPF鸡。
在体外，单靶标干扰显示对ALV-J mRNA的有效抑制率为74%85%。双靶标显示出更有效的抑制作用，对ALV-J mRNA的抑制率为96%98%。在体内，雏鸡在出壳当天腹腔接种各重组质粒，感染后每隔1天监测感染状态，持续4周。单靶标或双靶标miRNA的递送显著降低了体内ALV-J引起的病毒血症和致病性，尤其是双靶标。
这些数据表明，嵌入miRNA的siRNA干扰是抑制ALV-J复制的有效方法，尤其是双靶标干扰。
