- 學位論文
分析傳染性腹膜炎貓隻積液中之貓冠狀病毒及抗體及其與第二型嚴重急性呼吸綜合症冠狀病毒之間的關聯性
Feline coronavirus and antibodies in effusions of cats with feline infectious peritonitis and their potential cross-reactivity with SARS-CoV-2
摘要
貓傳染性腹膜炎 (FIP) 是由貓冠狀病毒 (FCoV) 其中一種生物型，貓傳染性腹膜炎病毒 (FIPV) ，引起之貓致命性疾病。而對於FIPV感染期間的病理免疫機制，尤其是抗體依賴增強作用 (ADE) 缺乏研究，這導致該病毒疫苗開發方面存在不確定因素。此外，貓對於近年造成全球威脅的第二型嚴重急性呼吸道綜合症冠狀病毒 (SARS-CoV-2) 也具有感受性，當貓同時感染這兩種冠狀病毒，FIPV和SARS-CoV-2之間的潛在交叉反應便成了一大議題。本研究旨在通過對FIP患貓的積液樣本中的病毒和抗體進行分析，以增加我們對FIP病理發生機制和冠狀病毒交叉反應的了解。從2022年1月開始，46個疑似FIP貓腹水樣本經PCR和免疫螢光試驗 (IFA) 的重複驗證確診為陽性。而首先，我們對這些陽性案例的特徵進行分析以揭示FIP的危險因子，發現了2歲以下以及白蛋白與球蛋白比值 (A/G ratio) 小於等於0.4的貓隻與FIP陽性有著顯著關聯。隨後，我們成功地在36個陽性FIP積液樣本中進行了FCoV片段的序列分析，其中36個屬於基因一型，僅有一個檢體同時存在基因一型和二型FCoV，相對於基因二型FCoV，基因一型FCoV親緣樹狀圖顯示出高度多樣性。再者，積液當中的細胞激素TNF-α及IL-6濃度，雖在FIP陽性與陰性病例之間並無顯著差異，但仍使我們更進一步瞭解該疾病的免疫機制。此外，通過從FIP患貓積液中純化之免疫球蛋白G (IgG) 和建立的體外FIPV ADE測試模型，檢測到了FIP陽性積液樣本中的ADE現象。另一項研究探討了FCoV和SARS-CoV-2之間的潛在交叉反應，結果顯示，來自兔隻抗SARS-CoV-2核殼蛋白 (Nucleocapsid) 的多株抗體 (pAb) 通過西方墨點法 (Western blot) 和酵素連結免疫吸附試驗（ELISA）與FCoV產生反應，但在FIPV ADE測試模型沒有觀察到ADE現象。為進一步研究這兩種病毒之間的關係，將人類COVID-19康復血清引入測試，發現兩名患者的血清與FCoV的刺突蛋白 (Spike) 亞單位1 (subunit 1) 和核殼蛋白產生交叉反應。同時，一人的血清樣本甚至略微增強FIPV的感染狀態，是為ADE現象，也初步證實了兩種病毒的交叉反應。此外，將FIP貓積液中的IgG與SARS-CoV-2的刺突蛋白、核殼蛋白及其片段進行測試，發現IgG能夠與SARS-CoV-2的刺突蛋白亞單位2 (subunit 2) 和核膜蛋白的所有片段結合，這再次證明了FCoV和SARS-CoV-2之間的雙向交叉反應。本研究首次報告了FIP貓積液中IgG引發的ADE現象，並發現FCoV和SARS-CoV-2之間的潛在交叉反應，為未來FIPV之感染研究提供了更近一步的見解。
並列摘要
Feline infectious peritonitis (FIP) is a fatal disease in cats caused by feline infectious peritonitis virus (FIPV), a biotype of feline coronavirus (FCoV). The lack of research on antibody-dependent enhancement (ADE) during FIPV pathogenesis has resulted in uncertainties in vaccine development. Moreover, cats are susceptible to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), another coronavirus posing a pandemic threat, and the potential cross-reactivity between these two coronaviruses remains unclear. This study aimed to increase our knowledge of FIP pathogenesis and coronavirus cross-reactivity by characterizing the virus and antibodies in FIP-diseased effusion samples. From January 2022, 46 FIP-confirmed samples were collected using PCR and IFA simultaneously. The signalments of these cases were firstly analyzed to reveal the FIP risk factors, which discovered that the cats with age equal to or lower than two y/o and A/G ratio equal to or lower than 0.4 were significantly correlated to FIP-positive. Subsequently, materials related to FIP pathogenesis in FIP effusions were investigated. FCoV fragments were successfully sequenced in 36 positive samples, with 35 cases belonging to genotype I and only one co-infected with genotype I and II FCoV. The phylogenetic analysis of the type I FCoV sequences revealed high diversity. Although there was no significant difference in the level of TNF-α and IL-6 in the effusions between FIP-positive and FIP-negative cases, this finding contributes to a better understanding of the immune mechanisms underlying FIP. The ADE phenomenon was also detected in FIP-positive effusion samples through IgG purified from FIP cat’s effusions and the established in vitro FIPV ADE test model. Another investigation explored the potential cross-reactivity between FCoV and SARS-CoV-2. Results revealed that only rabbit-derived anti-SARS-CoV-2 nucleocapsid pAb reacted with FCoV through western blot and ELISA, but no ADE phenomenon was observed. To further study the relationship between these two viruses, human COVID-19 convalescent serum was introduced into the test, which surprisingly demonstrated that serum from two patients cross-reacted with FCoV spike subunit 1 (S1) and nucleocapsid proteins—meanwhile, one human serum sample even slightly enhanced FIPV infection. Also, the IgG from FIP cat effusions was incubated with SARS-CoV-2 spike, nucleocapsid proteins, and their truncated fragments. The IgG was found capable of binding to the spike subunit 2 (S2) and all fragments of nucleocapsid protein of SARS-CoV-2, which proved the two-way cross-reactivity between FCoV and SARS-CoV-2. This study is the first to report the in vitro ADE phenomenon elicited from IgG in FIP effusions and discovered potential cross-reactivity between FCoV and SARS-CoV-2, providing valuable insights for future FCoV studies.