Construction and applications of rabbit hemorrhagic disease virus replicon.

The study of rabbit hemorrhagic disease virus (RHDV) has long been hindered by the absence of an in vitro culture system. In this study, using RHDV as a model, a series of DNA-based reporter replicons were constructed in which the firefly luciferase (Fluc) gene was fused in-frame with the open reading frame of the replicon. In this construct, the Fluc gene was inserted where the coding region of viral structural protein was deleted and was under the control of a minimal cytomegalovirus (CMV) immediate-early promoter. Fluc activity analysis showed that these reporter replicons replicate efficiently in mammalian cells. On the basis of the replicon, 5'non-coding regions (5'NCR) and genome-linked protein (VPg) were deleted, and the effect on the expression of replicon was analyzed. The results showed that the expression level of Fluc was reduced in the absence of 5'NCR and VPg, suggesting that the 5'NCR and VPg may play an important role in replication and/or translation of RHDV. To further verify the speculation, we also constructed a replication deficient mutant (pRHDV-luc/Δ3D), and the impact of 5'NCR and VPg deletion on viral translation efficiency was analyzed, our results indicated that both VPg and 5'NCR were involved in RHDV translation

RIG-I is responsible for activation of type I interferon pathway in Seneca Valley virus-infected porcine cells to suppress viral replication

Abstract Background Retinoic acid-inducible gene I (RIG-I) is a key cytosolic receptor of the innate immune system. Seneca valley virus (SVV) is a newly emerging RNA virus that infects pigs causing significant economic losses in pig industry. RIG-I plays different roles during different viruses infections. The role of RIG-I in SVV-infected cells remains unknown. Understanding of the role of RIG-I during SVV infection will help to clarify the infection process of SVV in the infected cells. Methods In this study, we generated a RIG-I knockout (KO) porcine kidney PK-15 cell line using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) genome editing tool. The RIG-I gene sequence of RIG-I KO cells were determined by Sanger sequencing method, and the expression of RIG-I protein in the RIG-I KO cells were detected by Western bloting. The activation status of type I interferon pathway in Sendai virus (SeV)- or SVV-infected RIG-I KO cells was investigated by measuring the mRNA expression levels of interferon (IFN)-β and IFN-stimulated genes (ISGs). The replicative state of SVV in the RIG-I KO cells was evaluated by qPCR, Western bloting, TCID50 assay and indirect immunofluorescence assay. Results Gene editing of RIG-I in PK-15 cells successfully resulted in the destruction of RIG-I expression. RIG-I KO PK-15 cells had a lower expression of IFN-β and ISGs compared with wildtype (WT) PK-15 cells when stimulated by the model RNA virus SeV. The amounts of viral RNA and viral protein as well as viral yields in SVV-infected RIG-I WT and KO cells were determined and compared, which showed that knockout of RIG-I significantly increased SVV replication and propagation. Meanwhile, the expression of IFN-β and ISGs were considerably decreased in RIG-I KO cells compared with that in RIG-I WT cells during SVV infection. Conclusion Altogether, this study indicated that RIG-I showed an antiviral role against SVV and was essential for activation of type I IFN signaling during SVV infection. In addition, this study suggested that the CRISPR/Cas9 system can be used as an effective tool to modify cell lines to increase viral yields during SVV vaccine development

Additional file 1: Figure S1. of Molecular cloning of Peking duck Toll-like receptor 3 (duTLR3) gene and its responses to reovirus infection

Amino acid alignment of Peking duck, Anas platyrhynchos, Cairina moschata, Gallus, Homo sapiens, and Mus musculus TLR3. Alignment was performed using the CLUSTAW program and edited with BOXSHADE. Black boxes indicate amino acid identity; gray boxes indicates similarity (50 % threshold). LRR, leucine rich repeat. TIR, toll-interleukin 1 receptor signaling domain. The TLR3 sequences are shown for Muscovy duck (Cai), human (Hom), mouse (Mus), chicken (Gal), Jinding duck (Ana), and peking duck (Pek). (DOC 51 kb

Schematic diagrams of the organization of RHDV luciferase replicon and the derived RHDV deletion mutants.

<p>(A) In bold, a schematic representations of RHDV luciferase replicon constructs; the unique <i>Cla</i>I site was introduced at 7381 nt. Fluc replaced most of the viral structural protein coding region by fusion PCR. White boxes denote RHDV nonstructural protein coding sequence. Gray boxes indicate a structural protein coding sequence. Lines indicate the 5′ and 3′ NCRs. Arrows point to the positions of the cleavage sites at the N and C termini of the proteins. (B) In-frame deletions of RHDV plasmids were generated by fusion PCR. The deleted sequence and positions are indicated for each mutant. Arrows point to the positions of the restriction site used to construct deletion mutants.</p

Oligonucleotides used for construction of RHDV luciferase replicons, VPg deletion mutant, 3D deletion mutant, 5′NCR deletion mutant, and detection of replicon.

*<p>Underlined nucleotides were required for cloning.</p

Time kinetics of the replicon in RK 13 cells.

<p>During time kinetics with the pRHDV-luc experiments, RK13 cells (200000 per well of a 6-well plate) were transfected with 1 µg of pRHDV-luc and pGL4.75. The cells were then lysed at different times post-transfection, and the Fluc activity was measured in RLU and normalized with respect to a cotransfected plasmid encoding a Renilla luciferase. Similar results were obtained in two independent experiments. pRHDV was used as negative control. In most cases, the variations are very small and therefore the error bars are not visible.</p