台灣第一例禽流感H7N9病毒基因組之探討

Abstract

流感病毒(Influenza virus)主要藉由飛沫傳播感染宿主呼吸道。患者通常會有咳嗽、發燒、倦怠、四肢無力及鼻炎，嚴重者可能引起呼吸衰竭甚至死亡。 西元2013年春季在中國大陸東南省份爆發的H7N9新型禽流感病毒，截至2013年六月為止共造成了131例的感染及36例的死亡。H7N9感染所造成的高死亡率使得H7N9的致病能力及其是否會發生人傳人感染成為重要的研究課題。台灣在四月下旬確診一名重症住院病患感染H7N9，我們從採集的檢體中分離病毒核酸並進行質體構築，同時比較GISAID流感數據庫中所有不同宿主來源的H7N9參考株序列，釐清是否存在特異性胺基酸位點取代出現於跨宿主感染，與潛在抗藥性位點的有無。H7N9的質體與參考株經過序列分析，PB2的第627位點有屬於禽鳥病毒特異性胺基酸位點穀胺酸(glutamic acid)相異於人類的精胺酸(arginine)；神經胺酸酶則在第289位點發現抗藥型的離胺酸(lysine) 存在。此外，質體株的神經胺酸酶在第219骨架性位點由異亮胺酸(isoleucine)突變為精胺酸。 我們以構築之質體進行反轉錄系統製造重組病毒，重組病毒在經過培養與放大後，在MDCK細胞中觀察到細胞病變的情形，確認重組病毒的確成功製作。

Influenza virus is mainly spread by droplet to infect the respiratory tract and lungs. Patients usually have cough, fever, fatigue, weakness and rhinitis, while severe cases can cause respiratory failure and even death. Novel H7N9 avian-origin influenza outbreak occurred in the coastal provinces of China in AD 2013 spring. As of June, raised great concern about a total of 131 cases of infection and 36 cases of death were reported. High mortality rates of H7N9 infection make the pathogenicity of H7N9 and the potential of human to human transmission. A severe case of H7N9 infection was diagnosed in Taiwan in late April. Viral nucleic acid was extracted from collected samples and subjected to PCR and sequencing. The H7N9 sequences from GISIAD influenza database were used to compare to specific amino acid site substitutions appear in the viral population from cross-hosts and the presence of potential drug resistance. Total H7N9 plasmids and reference strains were analyzed to release some information. At the 627 amino acid site of PB2 protein, there are glutamic acid from avian-origin virus different in the arginine from human-origin. The 289 amino acid site of neuraminidase presents lysine for resistance strain and arginine for wild type strain. In addition, as a neuraminidase skeleton site, the 219 site from plasmid is mutated from isoleucine to arginie. And the correlation between point mutation and resistance needs to be further evaluated. We constructed the plasmid for reverse genetic system manufacturing recombinant viruses. Cytopathic effects caused by recombinant viruses observed after culture and amplification. Confirmed the recombinant virus is indeed successful production.