Inhibition of porcine reproductive and respiratory syndrome virus replication in vitro using DNA-based short antisense oligonucleotides.

Longlong Zheng,Xiang Li,Junlong Bi,Gefen Yin,Guishu Yang,Jianping Liu,Wengui Li,Lingyun Zhu

doi:10.1186/s12917-015-0518-2

Abstract

BackgroundPorcine 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).ResultsNine 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.ConclusionThe 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.

Highlights

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
It is known that the PRRSV can replicate in pulmonary alveolar macrophages (PAM) or in MARC-145 cells, both culture systems were applied in this study
Antisense oligonucleotide design An effective antisense oligonucleotides (AS-ONs) should be selected at the RNA regions which are accessible for hybridization [17, 18]

Summary

Introduction

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. Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically significant viral diseases in the swine industry, which is characterized by respiratory disorders in piglets and reproductive failure in sows [1]. This disease is due to the infection by porcine reproductive and respiratory syndrome virus (PRRSV), which belongs to the family Arteriviridae, genus Arterivirus [2]. It is reasonable to speculate that antisense oligonucleotides targeting NSP9, ORF5 and ORF7, as well as 5′UTR, will result in degradation of the viral genome and inhibition of viral production

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