College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, PR China.
College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, PR China.
Virus Res. 2014 Jan 22;179:85-92. doi: 10.1016/j.virusres.2013.11.008. Epub 2013 Nov 15.
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 ∼3.5 log) reduction in the viral titers (TCID50) than that of co-cultivation with the single vector-transduced cells (by ∼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.
猪繁殖与呼吸综合征（PRRS）是一种对全球养猪业具有重要经济意义的猪病。目前的 PRRS 疫苗仅部分有效，新疫苗的开发面临巨大挑战。唾液酸结合蛋白（Sn）和 CD163 是 PRRSV 感染猪肺泡巨噬细胞（PAM）的两个必需受体。为了研究可溶性病毒受体在 PRRS 控制中的可行性，本研究构建了表达猪 Sn 的四个 N 端 Ig 样结构域（Sn4D）、第五个 SRCR 结构域（SRCR5）或猪 CD163 的结构域 5-9（SRCR59）的重组腺病毒（rAd），作为猪 Fc（pFc）融合蛋白。通过 RT-PCR 和 Western blot 证实了可溶性病毒受体在 rAd 转导细胞中的有效表达。为了检测它们的抗病毒活性，从 rAd 转导细胞的培养基中纯化了可溶性病毒受体，并通过 Western blot 进行鉴定。病毒结合实验表明，可溶性受体 Sn4D-Fc 和 SRCR59-Fc 能够结合 PRRSV 颗粒，但 SRCR5-Fc 和对照 pFc 不能。病毒感染阻断实验表明，与不同浓度的 Sn4D-Fc 和 SRCR59-Fc 蛋白共同处理 PRRSV 导致 PRRSV 阳性细胞数量的减少明显高于单一蛋白处理（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 培养系统将两种猪细胞系用 rAd-Sn4D-Fc 和/或 rAd-SRCR59-Fc 转导。用 PRRSV 感染 PAM 细胞，然后与 rAd 转导的细胞共培养。病毒滴定实验表明，与单独使用载体转导的细胞共培养相比，与 rAd-Sn4D-Fc 和 rAd-SRCR59-Fc 转导的细胞共培养可使病毒滴度（TCID50）降低约 3.5 log（∼3.5 log）。进一步的研究表明，rAd 共递送的可溶性受体 Sn4D-Fc 和 SRCR59-Fc 对三种不同的 PRRSV 株具有剂量依赖性和时间依赖性的抗病毒作用。由于所呈现的数据表明可溶性受体 Sn4D-Fc 和 SRCR59-Fc 之间具有相加的抗 PRRSV 活性，因此我们得出结论，所产生的两种 rAd 载体将有助于开发 PRRS 控制的新型试剂。
