2018年4月4日，Journal of Virology杂志在线发表浙江大学动物预防医学研究所、农业部动物病毒学重点实验室黄耀伟教授课题组文章《Porcine deltacoronavirus engages the transmissible gastroenteritis virus functional receptor porcine aminopeptidase N for infectious cellular entry》,首次报道发现动物丁型冠状病毒属成员PDCoV的细胞入侵受体。
2010年下半年起，我国各地猪场频发新生仔猪腹泻疫情，造成重大经济损失。目前认为四种新发与再现猪肠道冠状病毒（Swine Enteric Coronaviruses, SECoVs）是主要致病病原，包括：猪流行性腹泻病毒（PEDV）、传染性胃肠炎病毒（TGEV）、猪丁型冠状病毒（porcine deltacoronavirus，PDCoV）、以及黄耀伟课题组2017年在广东省首次分离发现的新型猪肠道甲型冠状病毒（SeACoV）。除SeACoV之外，2012年在香港报道的另一种新突发病毒PDCoV则属于丁型冠状病毒属(Deltacoronavirus)，是被国际病毒分类委员会于2014年明确划分的新病毒属。冠状病毒亚科现分为甲(Alpha-)、乙(Beta-)、丙(Gamma-)、丁(Delta-)4个病毒属。丙型和丁型冠状病毒属的细胞入侵受体此前还未发现。
本研究主要从三方面证实猪氨基肽酶N（pAPN）作为PDCoV入侵宿主细胞受体：
1.介导PDCoV入侵宿主细胞的结构蛋白——纤突蛋白（Spike）亚基S1可以在细胞表面结合PDCoV猪源易感细胞与稳定表达pAPN的非易感细胞；
2.PDCoV-S1与pAPN在体外直接相互作用；
3.稳定表达pAPN的非易感细胞可被PDCoV感染并产生子代病毒。由于pAPN同时也是甲型冠状病毒TGEV的受体，因此pAPN是一种跨不同冠状病毒属（cross-genus）的细胞受体，类似于乙型的SARS冠状病毒与甲型人类冠状病毒NL63共用血管紧张素转换酶2 （ACE2）作为cross-genus入侵受体。
文章还对比分析了其它两种SECoVs是否使用pAPN作为细胞受体：特别是对仔猪腹泻临床危害性最大的PEDV，此前长达11年一直被认为也使用APN入侵宿主细胞。但最近已有来自日本和荷兰的两项研究质疑了这个定论。本研究表明，与PDCoV相反，PEDV并不能感染稳定表达pAPN的非易感细胞。因此，结合之前日本和荷兰的两篇研究论文与黄耀伟课题组今年在Virology杂志发表的另一篇文章《Aminopeptidase-N-independent entry of porcine epidemic diarrhea virus into Vero or porcine small intestine epithelial cells》，进一步确认APN很可能不是PEDV受体。
至于去年发现的第四种猪肠道冠状病毒新突发SeACoV，其S蛋白接近于乙型冠状病毒，并且感染不表达APN的Vero细胞，因此其受体也不可能是pAPN。
鉴定病毒细胞入侵受体是了解病毒跨种传播、致病机制和制定干预策略的关键。PDCoV功能性受体的发现为研究丁型冠状病毒的自然界动物起源及入侵细胞的分子机制开辟了道路，同时有助于针对APN靶标研发抗PDCoV及潜在的危害人畜的其它丁型冠状病毒疫苗、药物和诊断试剂，从而有效防控疾病（如由PDCoV引发的仔猪腹泻），兼具重要的科学意义和应用前景。另一方面，提供PEDV不使用APN作为功能性细胞受体的实验证据也是猪肠道冠状病毒研究领域的重要转折性进展，或可避免相关研究方向、经费与时间的继续无效投入。
本研究受国家重点研发计划“畜禽重大疫病防控与高效安全养殖综合技术研发”重点专项(2016YFD0500102)、国家自然科学基金(31572518)、浙江省重大科技专项重点农业项目(2015C02021)、浙江省杰出青年基金(LR14C180001) 及中组部青年千人计划资助。
ABSTRACT
Identification of cellular receptors used by coronavirus (CoV) entry into the host cells is critical to understand pathogenesis and to develop intervention strategies. The fourth CoV genus, Deltacoronavirus, evolutionally related to theGammacoronavirus, has just been defined recently. In the current study, we demonstrate that porcine aminopeptidase N (pAPN) acts as a cross-genus CoV functional receptor for both enteropathogenic porcine DeltaCoV (PDCoV) and AlphaCoV (transmissible gastroenteritis virus, TGEV) based upon three lines of evidences. First, the soluble S1 protein of PDCoV efficiently bound to surface of target porcine cell lines known to express pAPN as TGEV-S1 did, which could be blocked by soluble pAPN pre-treatment. Second, either PDCoV-S1 or TGEV-S1 physically recognized and interacted with pAPN by co-immunoprecipitation in pAPN-cDNA-transfected cells and by dot blot hybridization assay. Finally, exogenous expression of pAPN in refractory cells conferred susceptibility to PDCoV-S1 binding and for PDCoV entry and productive infection. PDCoV-S1 appeared to have a lower pAPN-binding affinity and likely consequent lower infection efficiency in pAPN-expressing refractory cells as compared to TGEV-S1, suggesting that there may be difference in virus-binding regions in pAPN between these two viruses. This study paves the way for dissecting the molecular mechanisms of PDCoV-host interactions and pathogenesis as well as facilitates future vaccine development and intervention strategies against PDCoV infection.
IMPORTANCE
The emergence of new human and animal coronaviruses is believed to have occurred through interspecies transmission that is mainly mediated by species-specific receptor of the host. Among the four genera of theCoronavirinae, a couple functional receptors for the representative members in the genera Alphacoronavirus and Betacoronavirus have been identified, whereas receptorsfor Gammacoronavirus and Deltacoronavirus , which are believed to originate from birds, are still unknown. Porcine coronaviruses including the newly discovered porcine deltacoronavirus (PDCoV) associated with diarrhea in newborn piglets have posed a serious threat to the pork industry in Asia and North America. Here we report that PDCoV employs alphacoronavirus TGEV functional receptor porcine aminopeptidase N (pAPN) for cellular entry, demonstrating the usage of pAPN as a cross-genus CoV functional receptor. The identification of PDCoV receptor provides another example of the expanded host range of CoV, and paves the way for further investigation of PDCoV-host interaction and pathogenesis.
