- 學位論文
台灣北部2000-2004年流行性感冒病毒(A/H3N2)神經胺酸酶之基因變化
Genetic variation in neuraminidase gene of influenza A/H3N2 virus in northern Taiwan, 2000-2004
摘要
流行性感冒病毒屬於正黏液病毒科(Family Orthomyxoviridae)，老年人及小孩感染流行性感冒病毒常造成極高的罹病率及死亡率。A型流行性感冒的外套膜上具有兩種醣蛋白質，分別是血球凝集素(Hemagglutinin; HA)及神經胺酸酶(Neuraminidase; NA)，這兩個蛋白質皆會發生抗原移變和抗原飄變。HA蛋白引起的抗體可以中和流感病毒的感染力，因此是最重要的免疫決定因素，雖然抗-NA抗體不會中和病毒，但是它會緩和疾病，並且減少肺部的病毒量及降低肺部損害，因此，若要分析流行性感冒病毒的傳播和預測新興病毒，必須考慮到NA蛋白的抗原變異。然而關於流感病毒NA基因變異的資訊有限，如今又因為NA抑制物的使用使得NA基因的分析顯得更加重要。 為了瞭解北台灣地區A型流感病毒(H3N2) NA基因的變異，本研究分析了2000-2004年之間於台北分離的43支臨床病毒株。結果顯示NA基因每年的胺基酸變化率約為0.5%。和疫苗株A/Moscow/10/99比較的結果發現，有將近半數分離株(20/43)在NA蛋白的七個抗原決定位置中(I-VII)，至少改變了一個胺基酸，其中最容易發生變化的位置在332、401、431及432。在NA蛋白的酵素活化位置中，只有出現一個胺基酸改變(D151G)。所有分離株的NA基因上皆擁有七個和A/Hong kong/8/68同樣的天門冬酸-連接醣化位置(Asparagine-linked glycosylation site)，此外，大多數分離株(38/43)在胺基酸93及329位置上具有新的醣化位置。想瞭解這段時間內是否出現基因重組，我們也分析了這些分離株NA及HA基因的演化關係，結果顯示這段時間內的A型流感病毒在HA及NA基因之間沒有出現互換的現象。 在病毒溶斑實驗中觀察到大小不一的溶斑，經過病毒純化之後得到一株小溶斑病毒株(NA-)，此病毒株之NA基因出現了長度為586個核苷酸的刪去(303-888nt)。雖然NA-病毒株失去了NA酵素活性，但依舊能夠在MDCK細胞株生長，但是當病毒接種的MOI較低時(10-5)，NA-病毒株產生的病毒量較野生株低了45倍。NA-病毒株的受體結合能力降低，但是在其HA基因上並未出現補償性之胺基酸替換。克流感TM這種抗病毒藥物在試管內試驗中不會對NA-病毒株產生任何影響。綜合以上結果，我們推測NA蛋白酵素活性在A型流感病毒生長過程中不是必需的。
並列摘要
Influenza virus is a member of Orthomyxoviridae, and infection of influenza virus can cause severe morbidity and mortality in the elderly and children. Influenza A viruses are enveloped and have two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Both of HA and NA undergo antigenic shift and antigenic drift. Antibody to HA is the most important determinant of immunity because it can neutralize the infectivity of influenza virus. Although anti-NA antibodies do not neutralize virus infectivity, they appear to modify the disease and reduce both pulmonary virus titer and the extent of lung lesions. Therefore, antigenic variability of the NA protein should also be considered when analyzing the epidemic impact of influenza virus and predicting newly emerging viruses. However, limited information is available concerning the molecular change of the influenza NA genes. Analysis of NA gene is particularly important since the use of influenza NA inhibitors that target the highly conserved catalytic site of the enzyme. In order to understand the variation of NA gene of influenza A (H3N2) virus in northern Taiwan, 43 strains of clinical isolates in Taipei during 2000-2004 were collected for this study. The result indicated that the amino acid variation rate of NA was about 0.5% per year. As compared with the A/Moscow/10/99 vaccine strain, amino acid changes within at least one of the seven NA antigenic determinants (I-VII) were found in approximate half of the isolates (20/43) and the most common changes were at position 332, 401, 431 and 432. Only one amino acid change (D151G) was observed in the catalytic site of NA. All isolates contained the seven conserved asparagine-linked glycosylation sites found in the NA of the progenitor A/Hong Kong/8/68 strain. In addition, most strains (38/43) had the new glycosylation sites at positions 93 and 329. To understand whether there is gene reassortment recently, we also analyzed the evolutionary relationship of these isolates. It appears that no HA/NA reassortment was found. Variation of plaque size was observed in the plaque assay. After purification of virus, a small-plaque virus strain (NA-) was obtained and a 586 nucleotide deletion (303-888nt) of NA gene was found. Although the deficiency of NA enzyme activity, it still can grow in MDCK cell. However, the virus yield was 45-fold less than wild-type when low MOI (10-5) was used. The receptor-binding ability of the defective virus was low but no compensatory substitutions in the HA gene were found. TamifluTM, a kind of anti-influenza drug, did not influence on NA- virus in vitro. Thus, our results suggest that NA activity may not be essential for influenza A virus growth.