■重组牛干扰素λ3(bovIFN-λ3)对牛病毒性腹泻病毒(BVDV)的抗病毒活性已在Madin-Darby牛肾细胞(MDBK)中和在牛体内得到证实。然而,bovIFN-λ3的抗BVDV活性尚未在牛呼吸道上皮细胞中进行研究,据推测是通过口鼻途径进入宿主时BVDV感染的主要目标。
■在这里,我们使用BVDV感染和免疫过氧化物酶染色研究了bovIFN-λ3在牛鼻甲衍生的原代上皮细胞(BTu)中的抗BVDV活性,TCID50,RT-qPCR,DNA和转录组测序,用含有两个亚基的质粒转染,构成bovIFN-λ3受体的IL-28Rα和IL-10Rβ。
■我们的免疫过氧化物酶染色,RT-qPCR,和TCID50结果表明,尽管用bovIFN-λ3和bovIFN-α处理的MDBK细胞中BVDV被成功清除,只有后者,bovIFN-α,清除BTu细胞中的BVDV。需要在BVDV感染之前将MDBK细胞与bovIFN-λ3预孵育以诱导最佳抗病毒状态。两种细胞类型均显示完整的I型和III型IFN信号传导途径,并表达相似水平的III型IFN受体的IL-10Rβ亚基。IL-28Rα亚基的PCR扩增子的测序揭示了BTu细胞中完整的跨膜结构域和单核苷酸多态性(SNP)的缺乏。然而,RT-qPCR和转录组分析显示,与MDBK细胞相比,BTu细胞中IL-28Rα转录物的表达较低。有趣的是,用编码IL-28Rα亚基的质粒转染BTu细胞,但不是IL-10Rβ亚基,建立了bovIFN-λ3敏感性,显示出与MDBK细胞反应相似的抗BVDV活性。
■我们的结果表明,细胞对bovIFN-λ3的敏感性不仅取决于异源二聚体受体的IL-28Rα亚基的质量,而且还取决于其数量。与MDBK细胞相比,在BTu中检测到IL-28Rα转录物表达的减少,尽管没有剪接的变体或SNP。在用IL-28Rα质粒转染的BTu细胞中bovIFN-λ3诱导的抗BVDV活性的建立表明,该受体亚基的表达水平对于这些细胞中III型IFN的特异性抗病毒活性至关重要。
UNASSIGNED: The antiviral activity of recombinant bovine interferon lambda 3 (bovIFN-λ3) against bovine viral diarrhea virus (BVDV) has been demonstrated in vitro in Madin-Darby bovine kidney cells (MDBK) and in vivo in cattle. However, anti-BVDV activity of bovIFN-λ3 has not been studied in bovine respiratory tract epithelial cells, supposedly a primary target of BVDV infection when entering the host by the oronasal route.
UNASSIGNED: Here we investigated the anti-BVDV activity of bovIFN-λ3 in bovine turbinate-derived primary epithelial cells (BTu) using BVDV infection and immunoperoxidase staining, TCID50, RT-qPCR, DNA and transcriptome sequencing, and transfection with plasmids containing the two subunits, IL-28Rα and IL-10Rβ that constitute the bovIFN-λ3 receptor.
UNASSIGNED: Our immunoperoxidase staining, RT-qPCR, and TCID50 results show that while BVDV was successfully cleared in MDBK cells treated with bovIFN-λ3 and bovIFN-α, only the latter, bovIFN-α, cleared BVDV in BTu cells. Preincubation of MDBK cells with bovIFN-λ3 before BVDV infection was needed to induce optimal antiviral state. Both cell types displayed intact type I and III IFN signaling pathways and expressed similar levels of IL-10Rβ subunit of the type III IFN receptor. Sequencing of PCR amplicon of the IL-28Rα subunit revealed intact transmembrane domain and lack of single nucleotide polymorphisms (SNPs) in BTu cells. However, RT-qPCR and transcriptomic analyses showed a lower expression of IL-28Rα transcripts in BTu cells as compared to MDBK cells. Interestingly, transfection of BTu cells with a plasmid encoding IL-28Rα subunit, but not IL-10Rβ subunit, established the bovIFN-λ3 sensitivity showing similar anti-BVDV activity to the response in MDBK cells.
UNASSIGNED: Our results demonstrate that the sensitivity of cells to bovIFN-λ3 depends not only on the quality but also of the quantity of the IL-28Rα subunit of the heterodimeric receptor. A reduction in IL-28Rα transcript expression was detected in BTu as compared to MDBK cells, despite the absence of spliced variants or SNPs. The establishment of bovIFN-λ3 induced anti-BVDV activity in BTu cells transfected with an IL-28Rα plasmid suggests that the level of expression of this receptor subunit is crucial for the specific antiviral activity of type III IFN in these cells.