浙江大學黃耀偉組發現新突發豬丁型冠狀病毒細胞入侵受體

2018年4月4日，Journal of Virology雜誌在線發表浙江大學動物預防醫學研究所、農業部動物病毒學重點實驗室黃耀偉教授課題組文章《Porcine deltacoronavirus engages the transmissible gastroenteritis virus functional receptor porcine aminopeptidase N for infectious cellular entry》,首次報道發現動物丁型冠狀病毒屬成員PDCoV的細胞入侵受體。

2010年下半年起，我國各地豬場頻發新生仔豬腹瀉疫情，造成重大經濟損失。目前認為四種新發與再現豬腸道冠狀病毒（Swine Enteric Coronaviruses, SECoVs）是主要致病病原，包括：豬流行性腹瀉病毒（PEDV）、傳染性胃腸炎病毒（TGEV）、豬丁型冠狀病毒（porcine deltacoronavirus，PDCoV）、以及黃耀偉課題組2017年在廣東省首次分離發現的新型豬腸道甲型冠狀病毒（SeACoV）。除SeACoV之外，2012年在香港報道的另一種新突發病毒PDCoV則屬於丁型冠狀病毒屬(Deltacoronavirus)，是被國際病毒分類委員會於2014年明確劃分的新病毒屬。冠狀病毒亞科現分為甲(Alpha-)、乙(Beta-)、丙(Gamma-)、丁(Delta-)4個病毒屬。丙型和丁型冠狀病毒屬的細胞入侵受體此前還未發現。

本研究主要從三方面證實豬氨基肽酶N（pAPN）作為PDCoV入侵宿主細胞受體：

1.介導PDCoV入侵宿主細胞的結構蛋白——纖突蛋白（Spike）亞基S1可以在細胞表面結合PDCoV豬源易感細胞與穩定表達pAPN的非易感細胞；

2.PDCoV-S1與pAPN在體外直接相互作用；

3.穩定表達pAPN的非易感細胞可被PDCoV感染併產生子代病毒。由於pAPN同時也是甲型冠狀病毒TGEV的受體，因此pAPN是一種跨不同冠狀病毒屬（cross-genus）的細胞受體，類似於乙型的SARS冠狀病毒與甲型人類冠狀病毒NL63共用血管緊張素轉換酶2 （ACE2）作為cross-genus入侵受體。

文章還對比分析了其它兩種SECoVs是否使用pAPN作為細胞受體：特別是對仔豬腹瀉臨床危害性最大的PEDV，此前長達11年一直被認為也使用APN入侵宿主細胞。但最近已有來自日本和荷蘭的兩項研究質疑了這個定論。本研究表明，與PDCoV相反，PEDV並不能感染穩定表達pAPN的非易感細胞。因此，結合之前日本和荷蘭的兩篇研究論文與黃耀偉課題組今年在Virology雜誌發表的另一篇文章《Aminopeptidase-N-independent entry of porcine epidemic diarrhea virus into Vero or porcine small intestine epithelial cells》，進一步確認APN很可能不是PEDV受體。

至於去年發現的第四種豬腸道冠狀病毒新突發SeACoV，其S蛋白接近於乙型冠狀病毒，並且感染不表達APN的Vero細胞，因此其受體也不可能是pAPN。

鑒定病毒細胞入侵受體是了解病毒跨種傳播、致病機制和制定干預策略的關鍵。PDCoV功能性受體的發現為研究丁型冠狀病毒的自然界動物起源及入侵細胞的分子機制開闢了道路，同時有助於針對APN靶標研發抗PDCoV及潛在的危害人畜的其它丁型冠狀病毒疫苗、藥物和診斷試劑，從而有效防控疾病（如由PDCoV引發的仔豬腹瀉），兼具重要的科學意義和應用前景。另一方面，提供PEDV不使用APN作為功能性細胞受體的實驗證據也是豬腸道冠狀病毒研究領域的重要轉折性進展，或可避免相關研究方向、經費與時間的繼續無效投入。

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.

來源：綜合浙江大學

本期編輯：Tony

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