Abstract
Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome, which leads to serious economic losses in the pig industry worldwide. While the molecular basis of PCV2 replication and pathogenicity remains elusive, it is increasingly apparent that the microRNA (miRNA) pathway plays a key role in controlling virus-host interactions, in addition to a wide range of cellular processes. Here, we employed Solexa deep sequencing technology to determine which cellular miRNAs were differentially regulated after expression of each of three PCV2-encoded open reading frames (ORFs) in porcine kidney epithelial (PK15) cells. We identified 51 ORF1-regulated miRNAs, 74 ORF2-regulated miRNAs, and 32 ORF3-regulated miRNAs that differed in abundance compared to the control. Gene ontology analysis of the putative targets of these miRNAs identified transcriptional regulation as the most significantly enriched biological process, while KEGG pathway analysis revealed significant enrichment for several pathways including MAPK signaling, which is activated during PCV2 infection. Among the potential target genes of ORF-regulated miRNAs, two genes encoding proteins that are known to interact with PCV2-encoded proteins, zinc finger protein 265 (ZNF265) and regulator of G protein signaling 16 (RGS16), were selected for further analysis. We provide evidence that ZNF265 and RGS16 are direct targets of miR-139-5p and let-7e, respectively, which are both down-regulated by ORF2. Our data will initiate further studies to elucidate the roles of ORF-regulated cellular miRNAs in PCV2-host interactions.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-015-0172-5) contains supplementary material, which is available to authorized users.
Highlights
Porcine circoviruses (PCVs) are small, non-enveloped viruses with a circular single-stranded DNA genome of approximately 1.7 kb [1]
Solexa sequencing of small RNA cDNA libraries and data analysis Since other viruses use cellular miRNA to modulate host-cell infection, we hypothesized that PCV type 2 (PCV2) alters the miRNA expression profile of host cells during infection
We expressed individual PCV2-encoded open reading frames (ORFs) (ORF1, ORF2, and ORF3) in PK15 cells, which have been widely used to propagate PCV2 and study its replication in vitro [45,46]. This method was chosen instead of infecting these cells with PCV2 because of the low infectivity of the virus strain used in this study. This approach might enable the analysis of the potential effect of each ORF protein on cellular miRNA expression during the course of PCV2 infection
Summary
Porcine circoviruses (PCVs) are small, non-enveloped viruses with a circular single-stranded DNA genome of approximately 1.7 kb [1]. The original virus, designated PCV type 1 (PCV1), is non-pathogenic to pigs [2], while a variant strain of PCV, designated PCV type 2 (PCV2), is the principal etiological agent of postweaning multisystemic wasting syndrome (PMWS), a multifactorial disease in swine that leads to severe losses in pig production worldwide [3]. Prominent PMWS symptoms include severe progressive weight loss, dyspnea, tachypnea, anemia, diarrhea, and lymphocyte depletion in pigs between 5 and 15 weeks of age [4,5]. PCV2 infections are associated with other porcine diseases, such as porcine dermatitis and nephropathy syndrome (PDNS) and porcine respiratory disease complex (PRDC) [6].
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