四種不同型別冠狀病毒核殼蛋白的交叉抗原性探討

Abstract

冠狀病毒是一群有外套膜的單股正向 RNA 病毒，可感染人類和動物。冠狀病毒的核殼蛋白 (N) 是一種大量表現的多功能結構蛋白，對病毒的複製、組裝與釋放至關重要，N蛋白也具有高度免疫原性，因此常用於血清學診斷的目標抗原。然而，人與動物血清對不同冠狀病毒蛋白質是否存在著交叉反應，目前並不清楚。在這項研究中，我們旨在研究四種冠狀病毒 N蛋白之間的抗原交叉反應性。我們成功選殖並表現了group 1冠狀病毒貓冠狀病毒 (FCoV)、group 2冠狀病毒嚴重特殊傳染性肺炎冠狀病毒 (SARS-CoV-2)、group 3冠狀病毒傳染性支氣管炎病毒 (IBV)、和group 4冠狀病毒豬冠狀病毒 (PDCoV) 的重組N蛋白，同時使用小鼠製備這些重組 N蛋白的抗血清，另一方面，我們也分別收集了針對這些冠狀病毒感染呈現陽性或陰性的人類或動物臨床血清，在齊備各式抗原抗體之後，我們進行西方墨點法與酵素連結免疫吸附法進行交叉反應分析。結果顯示，我們在 FCoV與SARS-CoV-2 之臨床血清中觀察到雙向交叉反應，其中貓血清的FCoV N 蛋白抗體力價與 SARS-CoV-2 N 蛋白抗體力價之間存在顯著相關性；IBV與其他的冠狀病毒在N蛋白的抗原性未觀察到交叉反應，唯SARS-CoV-2 N 之兔抗血清能夠清楚辨識到 IBV 重組N 蛋白；另外，雖然我們在PDCoV N與 FCoV N 之老鼠抗血清之間觀察到雙向交叉反應，但因收集到PDCoV之臨床血清不論陰陽性對於PDCoV N蛋白結合力價皆不高，因此PDCoV與其他冠狀病毒之間的關係尚無法下定論；最後，我們以序列分析和蛋白質結構模擬呈現了四種冠狀病毒N蛋白的保守區段，顯示冠狀病毒之間可能發生交叉反應的抗原位點。藉由這項研究，我們指出人與動物的血清對於跨型別的冠狀病毒N蛋白存在著交叉反應，這些資訊可作為發展或進行冠狀病毒感染血清學診斷時的參考。

Coronavirus is a group of enveloped single-stranded positive-sense RNA viruses which infect humans and animals. The nucleocapsid (N) protein of coronavirus is a multifunctional structure protein that expresses abundantly and is essential for viral replication, assembly and release. N protein is frequently applied in serological assays for coronavirus detection due to its high immunogenicity. However, potential cross-reactivity among coronaviruses N proteins can complicate diagnosis and epidemiological analysis. In this study, we aim to investigate the antigenic cross-reactivity among four types of coronavirus N proteins. In this study, we have successfully cloned and expressed recombinant N proteins of feline coronavirus (FCoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), infectious bronchitis virus (IBV) and porcine deltacoronavirus (PDCoV), which belong to alpha, beta, gamma, and delta coronavirus, respectively. Polyclonal antisera against these recombinant N proteins were prepared in mice. Meanwhile, positive or negative human/animal clinical sera against these coronaviruses were also collected. Western blot assays and ELISAs were performed to assess the antigenic cross-reactivity among coronavirus N proteins. Results showed that two-way cross-reactive responses were observed between the clinical sera of FCoV and SARS-CoV-2, and there exists a significant correlation between the FCoV N antibody titer and SARS-CoV-2 N antibody titer in feline clinical sera. No cross-reaction was found among IBV and other three coronaviruses, however, the SARS-CoV-2 N rabbit antiserum clearly detected IBV recombinant N protein. Although a two-way cross-reaction between mouse antiserum of PDCoV N and FCoV N was observed, the relationships among PDCoV and other coronaviruses remain inclusive as all the pig clinical sera showed a weak response with the PDCoV N protein. Furthermore, sequence analysis and protein structure prediction showed universal residues in conserved regions of the coronavirus N protein, highlighting potential common epitopes for anti-N antibody induction. Through this study, we address the antigenic cross-reactivity among coronavirus N proteins and provide essential information toward the development of serological diagnostics and surveillance for coronavirus infections.