Inhibition of porcine reproductive and respiratory syndrome virus replication in vitro using DNA-based short antisense oligonucleotides.
BACKGROUND
Porcine reproductive and respiratory syndrome (PRRS) is caused by porcine reproductive and respiratory syndrome virus (PRRSV) and is an economically important disease in swine-producing areas. The objective of this study was to screen for effective antisense oligonucleotides (AS-ONs) which could inhibit PRRSV replication in MARC-145 cells and in pulmonary alveolar macrophages (PAM).


RESULTS
Nine short AS-ON sequences against the well-conserved regions of PRRSV (5'-UTR, NSP9, ORF5 and ORF7) were selected. When MARC-145 cells or PAM were infected with PRRSV followed by transfection with AS-ONs, four AS-ON sequences targeting 5'-UTR, ORF5 or NSP9 were found to be the most effective oligonucleotides in decreasing the cytopathic effect (CPE) induced by PRRSV infection. Quantitative PCR and indirect immunofluorescence staining confirmed that ORF7 levels were significantly reduced both at RNA and protein levels. The PRRSV titration data furthermore indicated that transfection with AS-ON YN8 could reduce the PRRSV titer by 1000-fold compared with controls.


CONCLUSIONS
The results presented here indicate that DNA-based antisense oligonucleotides can effectively inhibit PRRSV replication in MARC-145 cells and in PAM. Furthermore, comparing with the reported hit rates (approximately 10-30 %), we achieved a higher success rate (44 %). The strategy we took to design the antisense sequences might be applied to select AS-ONs that more efficiently reduce the expression of target genes.
Department of Veterinary Medicine, College of Animal Science and Technology, Yunnan Agricultural University, Kunming, China. 18314575103@163.com.
Longlong
L
Zheng
Present address: Wulan Institute for Animal Health, Lingyuan, Chaoyang City, Liaoning province, China. wlbjds22@126.com.
Xiang
X
Li
Department of Veterinary Medicine, College of Animal Science and Technology, Yunnan Agricultural University, Kunming, China. Zhulingyun_ZYS@163.com.
Lingyun
L
Zhu
Department of Veterinary Medicine, College of Animal Science and Technology, Yunnan Agricultural University, Kunming, China. wenguili@yeah.net.
Wengui
W
Li
Present address: Center for Animal Disease Control and Prevention of Chuxiong City, Chuxiong City, Yunnan province, China. junlongbi@aliyun.com.
Junlong
J
Bi
Department of Veterinary Medicine, College of Animal Science and Technology, Yunnan Agricultural University, Kunming, China. 2442739714@qq.com.
Guishu
G
Yang
Department of Veterinary Medicine, College of Animal Science and Technology, Yunnan Agricultural University, Kunming, China. yingefen@ynau.edu.cn.
Gefen
G
Yin
Present address: Karolinska Institute, Department of Biosciences and Nutrition, Novum, Huddinge, Sweden. Jianping.liu@ki.se.
Jianping
J
Liu
D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D002460:Cell Line; D020868:Gene Silencing; D000882:Haplorhini; D008264:Macrophages / Q000821:virology; D020320:Oligoribonucleotides, Antisense / Q000502:physiology*; D019316:Porcine respiratory and reproductive syndrome virus / Q000502:physiology*; D011650:Pulmonary Alveoli / Q000166:cytology; D012367:RNA, Viral; D013552:Swine; D014779:Virus Replication
D000998:Antiviral Agents; D020320:Oligoribonucleotides, Antisense; D012367:RNA, Viral