Additive inhibition of porcine reproductive and respiratory syndrome virus infection with the soluble sialoadhesin and CD163 receptors
Additive inhibition of porcine reproductive and respiratory syndrome virus infection with the soluble sialoadhesin and CD163 receptors
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DOI：
10.1016/j.virusres.2013.11.008
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发表时间：
2014-01-22
期刊：
影响因子：
5
通讯作者：
Sun, Huaichang
中科院分区：
文献类型：
--
作者：
Chen, Yang;Guo, Rui;Sun, Huaichang
猪繁殖与呼吸综合征（PRRS）是一种严重危害养猪业的经济疾病。目前的PRRS疫苗仅部分有效，新疫苗开发面临巨大挑战。唾液酸粘附素（Sn）和CD 163是猪肺泡巨噬细胞（PAM）感染PRRSV的两种重要受体。为了研究可溶性病毒受体用于PRRS控制的可行性，在本研究中，我们制备了表达猪Sn的四个N-末端Ig样结构域（Sn 4D）、猪CD 163的第五个SRCR结构域（SRCR 5）或结构域5-9（SRCR 59）的重组腺病毒（rAd）作为猪Fc（pFc）融合蛋白。用RT-PCR和Western blotting方法证实可溶性病毒受体在rAd转导的细胞中得到了有效表达，并从rAd转导的细胞培养基中纯化可溶性病毒受体，用Western blotting方法鉴定其抗病毒活性。病毒结合试验显示可溶性受体Sn 4D-Fc和SRCR 59-Fc能够与PRRSV颗粒结合，但SRCR 5-Fc和对照pFc不能。病毒感染阻断试验显示，用不同浓度的Sn 4D-Fc和SRCR 59-Fc蛋白共处理PRRSV导致高得多的病毒感染阻断率。与单一蛋白质处理相比，PRRSV阳性细胞数量减少（72.1%-77.6%）（45.1%-60.0%或44.0%-56.2%）。为了研究将可溶性病毒受体递送至PAM的可行性，使用transwell培养系统用rAd-Sn 4D-Fc和/或rAd-SRCR 59-Fc转导两个猪细胞系。用PRRSV感染PAM细胞，然后与rAd转导的细胞共培养。病毒滴度测定显示，感染的PAM与rAd-Sn 4D-Fc-和rAd-SRCR 59-Fc-转导的细胞共培养导致病毒滴度（TCID 50）的降低（接近3.5 log）比与单一载体转导的细胞共培养（接近1.0 log）高得多。进一步的研究表明，rAd与可溶性受体Sn 4D-Fc和SRCR 59-Fc共转染对3种不同的PRRSV毒株具有剂量依赖性和时间依赖性的抗病毒作用，且可溶性受体Sn 4D-Fc和SRCR 59-Fc对PRRSV的抗病毒活性具有相加作用，因此，所构建的两种rAd载体可用于开发新型的PRRS控制试剂。(C)2013爱思唯尔有限公司版权所有。
Porcine reproductive and respiratory syndrome (PRRS) is an economically important swine disease to the swine industry worldwide. Current PRRS vaccines are only partially effective and new vaccine development faces great challenges. Sialoadhesin (Sn) and CD163 are the two essential receptors for PRRSV infection of porcine alveolar macrophage (PAM). To investigate the feasibility of the soluble viral receptors for PRRS control, in the present study we generated recombinant adenovirus (rAd) expressing the four N-terminal Ig-like domains of porcine Sn (Sn4D), the fifth SRCR domain (SRCR5) or domains 5-9 (SRCR59) of porcine CD163 as porcine Fc (pFc) fusion proteins. Efficient expression of the soluble viral receptors in the rAd-transduced cells was confirmed by RT-PCR and Western blotting.To detect their antiviral activities, the soluble viral receptors were purified from the media of rAd-transduced cells and identified by Western blotting. The viral binding assay showed that the soluble receptors Sn4D-Fc and SRCR59-Fc, but not SRCR5-Fc and the control pFc, were able to bind to PRRSV particles. The viral infection blocking assays showed that co-treatment of PRRSV with different concentrations of Sn4D-Fc and SRCR59-Fc proteins resulted in a much higher (72.1%-77.6%) reduction in PRRSV-positive cell number than the single protein treatment (45.1%-60.0% or 44.0%-56.2%).To investigate the feasibility of delivering the soluble viral receptors to PAM, two pig cell lines were transduced with rAd-Sn4D-Fc and/or rAd-SRCR59-Fc using a transwell culture system. PAM cells were infected with PRRSV and then co-cultured with the rAd-transduced cells. Viral titration assay showed that co-cultivation of the infected PAM with rAd-Sn4D-Fc- and rAd-SRCR59-Fc-transduced cells resulted in much higher (by similar to 3.5 log) reduction in the viral titers (TCID50) than that of co-cultivation with the single vector-transduced cells (by similar to 1.0 log). Further studies showed that the rAd co-delivered soluble receptors Sn4D-Fc and SRCR59-Fc had dose-dependent and temporal antiviral effect against three different PRRSV strains.Since the data presented indicate an additive anti-PRRSV activity between the soluble receptors Sn4D-Fc and SRCR59-Fc, we conclude that the two rAd vectors generated will be useful for development a novel reagent for PRRS control. (C) 2013 Elsevier B.V. All rights reserved.