人類第三型副流感病毒在北臺灣地區之分子流行病學研究

Abstract

人類第三型副流感病毒(hPIV-3)是造成小孩及免疫缺乏病人下呼吸道疾病的主要病原之一，它在每年春夏之際造成流行。在急性嚴重呼吸道感染中是第二個最常見的病毒因子，其重要性僅次於呼吸道融合病毒。目前尚無有效的藥物和疫苗上市。hPIV-3屬於套膜病毒，基因特性為單股負向的RNA，總長約為15 kb。其基因組合為3’-NP-P/C/D-M-F-HN-L-5’。其中，HN基因和F基因被認為具有較高的變異性，所以常被用來做為基因分型及研究其分子流行病學。它們製造出兩種病毒表面醣蛋白質-血球凝集素-神經胺酸脢蛋白質(HN protein)及融合蛋白質(F protein)，此兩種蛋白質在病毒接觸、進入及釋出細胞扮演重要角色。另外，NP基因和L基因相對於HN基因和F基因保守性較高，而較少用來做基因型分析。種系分析將hPIV-3分成三群，cluster A、B及C。Cluster A及B多流行於上世紀的西方國家、而cluster C則於2000年後才陸續在各國分離出。 本研究針對1988年到1996年、2002年到2004年及2006年 到2013年台大醫院所分離的hPIV-3病毒株進行分析，共計404株。首先，以反轉錄-聚合酶連鎖反應增幅HN基因全長，並定序HN基因序列。在404株 hPIV-3病毒株中，有387株定序成功。利用種系分析此387株hPIV-3，發現cluster C 早於1988年即出現在台灣，仍持續存在直至2013年。根據核酸序列差異及種系分析，我們將cluster C分成2個亞群，其中cluster C1 又再細分成6個分支 (C1.1-C1.6)。種系分析結果顯示，1988年到1996年所分離的hPIV-3多屬於C1.1、C1.2及C1.3分支(28/30)，另外有1株1996年的hPIV-3 屬於cluster A (96TW793)及1株1990年的屬於cluster B (90TW308)。2002年到2004年所分離的hPIV-3多屬於sub-cluster C2 (30/50)。然而2006年到2013年所分離的hPIV-3則多屬C1.4 (241/320)，這些結果顯示C1.4可能與2006年後hPIV-3分離株的增加有關。 比較HN、F及NP等基因之核酸及胺基酸序列相似性其結果顯示無論是cluster A、B或 C 之hPIV-3 彼此相似性極高。另外HN蛋白質及F蛋白質胺基酸變異分析顯示，只有少數胺基酸變異會出現在其抗原決定位上。最後，由於cluster C hPIV-3 已於1988年被分離出，所以我們認為cluster C hPIV-3在上世紀可能已流行於亞洲。

Human parainfluenza virus type 3 (hPIV-3) is one of the major pathogens to cause lower respiratory tract illness among children and immunocompromised individuals. HPIV-3 is second to the most common viruses that causes acute respiratory infection in children and mainly appears in spring and summer every year. So far, there are no efficient drugs or vaccines available against HPIV-3. HPIV-3 is an enveloped and negative-sense, single-stranded RNA virus with a genome size of about 15 kb. Among six structural proteins encoded (NP, P, M, F, HN and L), hemagglutinin-neuraminidase (HN) protein and fusion (F) protein, which play important roles in viral attachment, entry, and releasing, are more diverse in their sequences and are frequently used in genotyping to investigate molecular epidemiology of hPIV-3. Phylogenetic analysis of complete HN gene reveals that there are three major clusters of hPIV-3, which are cluster A, B and C. Previous studies indicated that both clusters A and B were the major hPIV-3 circulating in most western countries in the last century, while cluster C became the major hPIV-3 in this century in Asia countries. In this study, a total of 404 hPIV-3 strains isolated during the periods of 1988~1996, 2002~2004, and 2006~2013 in NTUH were analyzed by using RT-PCR and sequencing. The full length HN gene was sequenced successfully for 387 strains. Phylogenetic analysis reveals that cluster C could be divided into 2 sub-clusters, C1 and C2. Sub-cluster C1 could be further divided into 6 lineages (C1.1 to C1.6). Most of the hPIV-3 strains isolated in NTUH from 1988 to 1996 belonged to C1.1, C1.2, and C1.3, only one strain belonged to cluster A (96TW793) and one strain belonged to cluster B (90TW308). In the period of 2002 to 2004, most of the hPIV-3 strains belonged to sub-cluster C2 (30/50) and one strain belonged to cluster B (02TW1901) while in the period of 2007 to 2013, most of the hPIV-3s belonged to C1.4 (241/320). These results suggested that C1.4 replaced sub-cluster C2 and was responsible for the increasing hPIV-3 isolates after 2007. Comparison of the nucleotide and amino acid sequences of the HN, F, and NP genes reveals that the hPIV-3 strains shared similarity more than 90% among clusters. Based on amino acid sequence alignment, we found that 27 of 134 amino acid changes occurred on epitopes of HN protein and 1 of 73 amino acid changes occurred on epitopes of F protein. Because the cluster C strains have been isolated in Taiwan since 1988, we suggest that cluster C hPIV-3 has been circulated among Asia countries in last century.