H6N1流感病毒在細胞的感染性分析

Cellular Infectivity Analysis of the H6N1 Influenza Viruses

10.6342/NTU201700730

許偉恩

H6N1流感病毒 ； 親緣樹狀圖 ； 病毒複製 ； 細胞傷害性 ； 台灣 ； H6N1 Influenza Virus ； Phylogenetic tree ； Viral Replication ； Cell Injury ； Taiwan

臺灣大學獸醫學研究所學位論文

2017年

碩士

王金和

繁體中文

A型流感病毒為威脅人類健康的重要病原。一般情形下，A型流感病毒具有宿主專一性。如H6N1亞型會感染禽鳥。在台灣於2014年發現H6N1感染犬隻的案例。該病毒由犬隻的鼻腔拭子經胚胎蛋增殖2代所分離，命名為A/canine/Taiwan/E01/2014 (H6N1)。此犬H6N1於PB2蛋白質中擁有已知能增加病毒在哺乳類宿主複製的E627K突變。然而，發現將此病毒在胚胎蛋增殖1代後於PB2基因中見K627E的胺基酸突變。將該病毒以MDCK細胞增殖2代後恢復PB2 627K。顯示於不同系統的增殖導致了該病毒基因的突變。本研究目的為對H6N1在細胞的感染性進行分析。包含 (1)犬H6N1以胚胎蛋或MDCK細胞增殖後胺基酸的突變。(2)病毒對不同物種來源細胞的複製能力及細胞傷害性。 (3) 於台灣所分離到的H6N1在HA、PB2、PB1及PA基因中自1972年至2014年的演化趨勢。 (4)演化趨勢與病毒在細胞中複製動力學與傷害性的關係。透過病毒溶斑純化試驗，純化確認PB2的627個別為E (病毒93)和K (病毒99)的2株H6N1病毒，並重新做8段基因的定序。此外，挑選5株實驗室中於不同年份分離之家禽來源H6N1 (病毒04、11、20、37及72)，以RT-PCR增幅HA、PB2及PA基因片段，與病毒99及基因資料庫中其他台灣H6N1比對，繪製親緣樹狀圖。依據親緣樹狀圖將病毒分作2組，是為2012年前分離組 (病毒04、37及72)與2012年後分離組 (病毒11、20及99)。以MOI=0.01的病毒感染MDCK、A549及DF-1此3種不同物種來源細胞檢視病毒在細胞中的複製動力學。此外，經由MTT assay檢視感染後病毒對細胞的傷害性。結果顯示，病毒93與病毒99有3個胺基酸位點的胺基酸改變。93的HA基因有142I、PB2基因有627E，及PA基因有739E。99的HA基因有142N、PB2基因有627K，及PA具有739G。病毒99比病毒93可在MDCK細胞上產生較大的病毒溶斑。病毒99在MDCK、A549及DF-1中都有比93更高的病毒複製力價，並在感染後能對MDCK細胞造成比病毒93更顯著的傷害。親緣樹狀圖顯示，2012年後於台灣所分離的H6N1於HA、PB2及PA都有相近的親緣。在HA胺基酸中，2005年後H6N1普遍具有E190V和G228S此2會可能病毒在H6亞型增加與哺乳類受體結合能力的突變點。在PB2胺基酸中只有於2014年分離的犬H6N1和病毒99具有627K的突變。病毒99比病毒04、11、20、37及72在MDCK、A549及DF-1細胞都能更有效複製。在MDCK細胞中病毒99可造成嚴重的細胞傷害。然而，對2012年前分離的病毒與2012年後分離的病毒做組間比較，發現2012年後分離的病毒並沒有都比2012年前分離的病毒有更好的複製能力。即使組內基因具有較高的相似度，2012年後分離的病毒並沒有都比2012年前分離的病毒有更好的複製能力。而在A549及DF-1細胞中，病毒複製能力和病毒的細胞傷害性沒有絕對的相關性。結論，擁有PB2 E627K此一位點突變，可能是導致病毒99比93和其他禽來源H6N1在哺乳類細胞有更好複製效率的原因。

Influenza A viruses are important life threatening pathogen to human. Generally, influenza A viruses have host restriction. H6N1 subtype viruses infect avian species. A canine H6N1influenza virus was first noted in Taiwan in 2014. The virus on nasal swab was isolated from 2 passages in chicken embryo eggs and designated as A/canine/Taiwan/E01/2014 (H6N1). PB2 protein of the virus possessed E627K mutation, which was already known to enhance virus replication in mammalian hosts. However, the amino acid had mutated to PB2 627E after a passage in chicken embryo eggs. The virus recovered PB2 E627K after being cultivated in MDCK (Madin-Darby canine kidney) cell for 2 passages. It showed that passaging in different systems may lead to gene mutation. The aims of this study were cellular infectivity analysis of the H6N1 influenza viruses: (1) The amino acid mutations of the dog virus after passaging in chicken embryo eggs and MDCK cells. (2) Viral replication efficiency and injury of cells from different hosts. (3) The evolutionary trend of HA, PB2, PB1 and PA genes in H6N1 in Taiwan from 1972 to 2014. (4) The relationship between evolutionary trend, viral replication efficiency and injury of cells from different hosts. Plaque purification was used for purifying both the canine H6N1 viruses which possessed PB2 627E (virus 93) and PB2 627K (virus 99). Virus 93 and virus 99 were sequenced. In addition, five H6N1 viruses (virus 04, 11, 20, 37 and 72) were selected. HA, PB2, PB1 and PA segments of the viruses were amplified by RT-PCR. Phylogenetic tree was built up in order to analyze evolutionary trend. Viruses were grouped into the viruses isolated before 2012 (included 04, 37 and 72) and the viruses isolated after 2012 (included 11, 20 and 99) based on phylogenetic tree. Using MOI=0.01 viruses to infect MDCK, A549 and DF-1 cells to investigate viral replication efficiency. Moreover, MTT assay was used for detecting cell injury. Results showed that virus 93 had three amino acids different from virus 99. Virus 93 possessed 142I in HA gene, 627E in PB2 gene and 739E in PA gene. Virus 99 possessed 142N in HA gene, 627K in PB2 gene and 739G in PA gene. Plaques generated by virus 99 were larger than virus 93. Higher virus titers were observed in MDCK, A549 and DF-1 cells infected with virus 99 than infected with virus 93. Infected with virus 99 caused distinct cytopathic effect in MDCK cells than virus 93. Phylogenetic tree showed that the HA, PB2, PB1 and PA genes of H6N1 which were isolated after 2012 shared close relationship. Most of H6N1 possessed E190V and G228S in HA gene, which may enhance the binding between viruses and receptor of mammalian hosts. Only the canine virus isolated in 2014 and virus 99 possessed E627K in PB2 gene. Virus 99 could replicate better in MDCK, A549 and DF-1 cells than virus 04, 11, 20, 37 and 72. Infected with virus 99 could couse severe cell injury in MDCK cells. However, the viruses isolated after 2012 did not show better viral replication efficiency than the viruses isolated before 2012. Although having similar amino acid compositions, not every virus isolated after 2012 could have better viral replication efficiency than the viruses isolated before 2012. The correlation between viral replication efficiency and cell injury in A549 and DF-1 cells was poor. In concusion, possessed E627K in PB2 gene may lead virus 99 to have better viral replication efficiency in mammalian hosts than virus 93 and other H6N1 viruses in this study.