在感染中HBV RNA合成的時間動力學

Abstract

B型肝炎病毒（HBV）是全球範圍內導致慢性肝病和肝細胞癌的主要原因。理解HBV感染的早期階段，特別是HBV RNA的合成和出口，對於開發有效的診斷和治療策略至關重要。病毒通常擁有獨特的RNA出口機制，使它們能夠成功地利用宿主細胞的機制進行RNA的轉運和表達。例如，HIV通過Rev-RRE系統促進未剪接RNA的出口，Mason-Pfizer猴病毒（MPMV）則利用CTE元素進行RNA的核輸出。本研究旨在探討HBV RNA在感染初期的合成動力學，並希望發現HBV RNA出口的可能機制。利用Northern blot偵測HBV mRNAs能夠在感染後48小時看見3.5 kb preC/pgRNA, 2.4 kb PreS1 RNA, and 2.1 kb PreS2/S RNA 的存在，更進一步以RT-PCR發現在感染12和24小時後只出現剪接型RNA的訊號，並沒有未剪接RNA的存在，但還需要加以證明。另外，我們希望開發一種利用特殊的引子設計的RT-qPCR方法試圖在不處理DNaseI的狀況下偵測HBV RNAs，然而並無法找出合適的引子條件能夠完全避免HBV DNA干擾，所以我們進一步對RNA進行mRNA純化。最後以20 nM 3’Poly(dT)-Adaptor的引子濃度和純化mRNA的方式進行RT-qPCR能夠避免HBV DNA的干擾，然而此方法在偵測不同感染時間點的HBV mRNA靈敏度過低，而且測得的不同感染時間點的Ct值趨勢並不符合Northerm blot的結果。

Hepatitis B virus (HBV) is a major global cause of chronic liver disease and hepatocellular carcinoma. Understanding the early stages of HBV infection, particularly the synthesis and export of HBV RNA, is crucial for developing effective diagnostic and therapeutic strategies. Viruses often possess unique RNA export mechanisms that allow them to successfully utilize host cell machinery for RNA transport and expression. For example, HIV promotes the export of unspliced RNA through the Rev-RRE system, while Mason-Pfizer monkey virus (MPMV) uses the CTE element for RNA nuclear export. This study aims to investigate the kinetics of HBV RNA synthesis during the early stages of infection and to identify potential mechanisms for HBV RNA export. Northern blot analysis revealed the presence of 3.5 kb preC/pgRNA, 2.4 kb PreS1 RNA, and 2.1 kb PreS2/S RNA at 48 hours post-infection. Further RT-PCR analysis showed that only spliced RNA signals were detected at 12 and 24 hours post-infection, with no evidence of unspliced RNA, though further confirmation is needed. Additionally, we aimed to develop an RT-qPCR method using specially designed primers to detect HBV RNAs without DNase I treatment. However, suitable primer conditions to completely avoid HBV DNA interference could not be found. Therefore, we proceeded with mRNA purification. Finally, using a 20 nM 3’Poly(dT)-Adaptor primer concentration and purified mRNA for RT-qPCR successfully avoided HBV DNA interference. Nonetheless, this method had insufficient sensitivity for detecting HBV mRNA at different infection time points, and the Ct value trends did not align with Northern blot results.