Abstract
BackgroundPorcine reproductive and respiratory syndrome virus (PRRSV) causes porcine reproductive and respiratory syndrome (PRRS), which is currently insufficiently controlled. From a previous small-scale screen we identified an effective DNA-based short antisense oligonucleotide (AS-ON) targeting viral NSP9, which could inhibit PRRSV replication in both Marc-145 cells and pulmonary alveolar macrophages (PAMs). The objective of this study was to explore the strategy of incorporating locked nucleic acids (LNAs) to achieve better inhibition of PRRSV replication in vitro.MethodsThe effective DNA-based AS-ON (YN8) was modified with LNAs at both ends as gap-mer (LNA-YN8-A) or as mix-mer (LNA-YN8-B). Marc-145 cells or PAMs were infected with PRRSV and subsequently transfected.ResultsCompared with the DNA-based YN8 control, the two AS-ONs modified with LNAs were found to be significantly more effective in decreasing the cytopathic effect (CPE) induced by PRRSV and thus in maintaining cell viability. LNA modifications conferred longer lifetimes to the AS-ON in the cell culture model. Viral ORF7 levels were more significantly reduced at both RNA and protein levels as shown by quantitative PCR, western blot and indirect immunofluorescence staining. Moreover, transfection with LNA modified AS-ON reduced the PRRSV titer by 10-fold compared with the YN8 control.ConclusionTaken together, incorporation of LNA into AS-ON technology holds higher therapeutic promise for PRRS control.
Highlights
Porcine reproductive and respiratory syndrome virus (PRRSV) causes porcine reproductive and respiratory syndrome (PRRS), which is currently insufficiently controlled
Both pulmonary alveolar macrophages (PAMs) and Marc-145 cells were applied in this study, as PRRSV can replicate in these two culture systems
Locked Nucleic Acid (LNA) modification conferred longer lifetime to antisense oligonucleotides To investigate how long AS-ONs with LNA modifications are present in the cell culture system, 32 μM cy-3 labelled LNA modified antisense oligonucleotides LNA-YN8-A and LNA-YN8-B were transfected into Marc-145 cells
Summary
Porcine reproductive and respiratory syndrome virus (PRRSV) causes porcine reproductive and respiratory syndrome (PRRS), which is currently insufficiently controlled. From a previous small-scale screen we identified an effective DNA-based short antisense oligonucleotide (AS-ON) targeting viral NSP9, which could inhibit PRRSV replication in both Marc-145 cells and pulmonary alveolar macrophages (PAMs). In order to develop improved methods to manage PRRS, we selected the best antisense sequence YN8 from our previous small-scale screening [5] for LNA modifications and applied the two modified sequences to in vitro studies (lifetime of the antisense oligonucleotides, cytotoxicity, cytopathic effect observation, qPCR, virus titer assessment, western blot and indirect immunofluorescence) to evaluate the inhibitory effects on PRRSV replication in Marc-145 cells and in PAMs between the DNA- and LNA-based AS-ONs. Our data showed that incorporation of LNA into AS-ON technology holds higher therapeutic promise for PRRS control
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