新興流感病毒之神經胺酸酶功能性探討

Abstract

流行性感冒是由流感病毒(Influenza virus)所引起的呼吸道疾病，典型症狀包含發燒、頭痛、咳嗽、四肢無力、咽喉炎及鼻炎等，嚴重者甚至死亡；近來因為疫苗以及藥物的使用，對於疾病的預防及治療有著顯著功效。 2007 年一月到十月由台大檢驗醫學部所分離出的H3N2流感病毒，經過高全良老師實驗室針對這些病毒的神經胺酸酶(Neuraminidase，NA)基因定序後，發現這些檢體大致上可依核酸序列分為兩個群體：Group I (2007 年一、二月份所分離出的檢體)及GroupII (其他月份所分離出的檢體)，這兩個群體在胺基酸位點 86、93、194、296、307、310、335、370、372、387有差異，並且這些差異是群體特異(Group specific)的。經過結構分析，我們推測335/387、370/372 這兩組胺基酸位點的改變可能會影響神經胺酸酶的構形。我們發現原始病毒(parental virus) Grou I神經胺酸酶的活性較Group II低，且在溶斑實驗中，Group I形成小溶斑的比例較高。為了解前後期檢體神經胺酸酶胺基酸序列改變是否影響病毒的表現型，我們利用反遺傳學(reverse genetics)製備僅在神經胺酸酶基因有差異的兩重組病毒(Group I：754；Group II：4517)。進一步測量重組病毒(recombinant virus)神經胺酸酶活性、生長曲線、以及對於藥物的感受性，我們發現：兩重組病毒神經胺酸酶活性差距較原始病毒為小；雖然病毒生長速率均小於原始病毒十至一百倍，但兩者在低MOI下的生長速率沒差別；在藥物的感受性實驗中，754 重組病毒較4517 重組病毒對於NHRI藥物(BPR2P000150)為敏感；2-deoxy-2,3-didehydro-D-N- acetylneuraminic acid (DANA) 抑制754病毒50%溶斑所需濃度(EC50)在原始或重組病毒中均大於4517 檢體3~5 倍。 綜合以上實驗，這些變異可能導致神經胺酸酶的結構產生差異並影響到病毒的生長速率以及對藥物的感受性。我們將更進一步探討造成神經胺酸酶活性以及藥物敏感性差異的位點，希望藉由這些資訊可以對神經胺酸酶有更深層的認識。

Influenza virus has caused worldwide pandemic every year. Although influenza vaccine and therapeutic compounds have effectively prevented people from being infected, the appearance of natural variants and drug-resistant viruses have become an important issue. Therefore, in this study, we aimed to characterize the natural variants of neuraminidase (NA) diverse in H3N2 collected at National Taiwan University Hospital in 2007. From January 2007 to October, the NA genes of H3N2 influenza virus strains isolated from Department of Laboratory Medicine, National Taiwan University were sequenced by Dr. Kao’s laboratory. By phylogenetic analysis, these genesequence were separated into two groups – Group I (mainly composed of the virus strains isolated in January and February) and Group II (composed of the virus strains isolated in later time period). Some group-specific amino acid changes were identified at residues 86, 93, 194, 296, 307, 310, 335, 370, 372 and 387. Among these, residue changes at 335/387 and 370/372 are speculated to affect the protein conformation by protein structural prediction. The NA activity of Group I strains is lower than that of Group II strains and small plaque percentage in Group I is higher than that of Group II. To understand whether these genotype variations did correlate with the phenotype changes, reverse genetics was performed to analyze the differences of NA gene from Group I (754) and Group II (4517). Comparison of enzyme activity, and virus growth curve between these two recombinant viruses, we found that Group I virus had lower NA activity, yet similar growth kinetics as compared to Group II virus while both viruses exhibited comparable sensitivity to a compound from NHRI (BPR2P000150) which is not targeted to NA, Group I virus are 3~5 times more resistant to one NA inhibitor, 2-deoxy-2,3-didehydroD-N-acetylneuraminic acid(DANA). Taken together, we speculate that natural variations in NA do exist and some of them may result in phenotypic changes. Further analysis is required to determine the role of specific amino acid change in NA structure and enzyme activity.