Preliminary Study On Antiviral Activity Of Duck Viperin Protein Against Newcastle Disease Virus

Viperin has been indentified as one of interferon-stimulated genes （ISGs） that induced by interferon and virus in 2001. Recently, Viperin was also known to be capable of inhibiting viral and bacterial infection, and even involving cell signaling pathway, thus it plays a crucial role in innate immunity. As more in-depth and comprehensive study on Viperin, the mechanism of Viperin resistence to a variety of virus and bacteria is gradually being reported. In this study, we cloned the complete duck Viperin gene from Changbai duck and carried on analysis for the sequences, focusing on the interaction between duck Viperin and Newcastle disease virus （NDV） to reveal the molecular mechanisms of duck Viperin to anti-NDV.At the beginning, we collected duck anticoagulation and then separated peripheral blood lymphocytes, and the total RNA of peripheral lymphocytes was extracted and reverse transcribed into cDNA. Based on the sequence of RSAD2 （NW₀04676922.1 and XM₀05018580.1）, we amplified the complete ORF sequence of duck Viperin, and analyzed duck Viperin sequence and predicted its function with bioinformatics tools. The results indicate that the structure of duck Viperin, including a variable N-terminal domain, a radical SAM domain, and a conserved C-terminal domain, is very similar with other species published. Based on Viperin sequence, we cloned the recombinated plasmid by inserting Viperin into pGEX 4T-1 to generate Viperin protein through prokaryotic expression and purification. The fusion protein was used to immune rabbits, and anti-Viperin serum was successful produced. Previous studies have confirmed that Viperin expression can be induced by a variety of viruses, we raised the question that whether duck Viperin could be stimulated and induced by Polyinosinic-polycytidylic （poly（I:C）） and NDV. DEF （duck embryo fibroblast） cells were prepared and cultivated, and then poly（I:C） and NDV with dose-point were choosed to infect the DEF and cells were collected for detecting Viperin expression through qRT-PCR and Western Blot analysis. Meanwhile, animal experiment was carried on for analysis the tissues distribution of duck Viperin, experimental group was infected with NDV and the control goup was treated with PBS. The different tissues, including spleen, kidneys, liver and blood, were collected for analysis the Viperin expression. The results demonstrated that Viperin, in vivo or in vitro, can be strongly induced by NDV at different levels.To further study the role of duck Viperin in anti-NDV, we cloned the full and truncation of duck Viperin （designated as Vipâ–³N and Vipâ–³C, respectively）. All the fragments, including the full and truncated Viperin, were inserted into the pCAGGS vector and transfected DEF cells. B, siRNA plasmids designed to suppress in expression of duck Viperin were also transfected DEF celss. NDV was then used to infect the cells and supernatant was harvested for analysis of NDV titer while the cells were collected for qRT-PCR. The experimental data showed that overexpresssin Viperin is able to limit the infection of NDV and the NDV titer is significant lower than that of the control group, while transfection with siRNA plasmid can decrease the expression of duck Viperin and the NDV titer is much higher. We also investigated which domains of duck Viperin is critical for its antiviral ability, the results showed that duck Viperin N-termini deletions of Viperin can retain its partial anti-NDV activity, while C-terminal truncations abrogate its antiviral activity totally.In summary, our study on structure and fuction of Viperin was performed, the results showed that duck Viperin can be strongly induced by poly（I:C） and NDV, suggesting that duck Viperin is similar with that of other specieses, also indicating that Viperin is capable of limiting the infection of NDV and C terminal domain is critical for Viperin involving antiviral activity.