2012高血壓回顧整理（精簡版）

2012再次複習高血壓！13,July,2013 人血壓的分佈跟身高是一樣呈非線性分佈！ 從統計學角度，找出140/90mmHg以上，心血管疾病增加的角度陡！ 每增加20/10mmHg,心血管病機率增2倍！ 現今高血壓定義，140/90以上！JNC,ESH,WHO在這點上有共識！但對其分類，就不同！台灣採JNC美式標準，因較簡潔！ 正常血壓：120/80, 高血壓：140/90, 2級高血壓：160/100 (也就是差一級,S多20，D多10，心血管病多2倍！） 造成高血壓的機轉：複雜！多重因子！ 最重要的為：鹽過多！RAAS系統活化！壓力！血管內皮受損！ 血壓=心輸出X周邊血管阻力 高血壓會造成那些不良後果？ 1：心：左室肥大，心衰竭，冠心症(CAD) 2：腦：一過性腦缺血發作（TIA),中風(stroke),高血壓腦 病變

肝癌

從友人配偶急性腹痛而診斷出肝癌説起 　　我國因慢性肝病及肝癌死亡的病例，約佔整體死因的近一成。其中大部份是由B及C型肝炎引起，也就是眾所周知的肝炎、肝硬化、肝癌三部曲。也因此B及C型肝炎，一直都是我國重大的公共衛生問題，近四十年來，從澳洲抗原的發現、血源篩檢、拋棄式針筒及針頭的使用、B型肝炎疫苗的全面施打、C型肝炎抗體的發現、抗病毒藥物的發明及分子生物學的突飛猛進，對於B及C型肝炎的流行病學有重大的影響。 　　台灣是B型肝炎的高盛行地區，一般25歲以上居民的帶原率約在15~20% ，不論是感染率、帶原率、 HBeAg的清除率及肝硬化、肝癌的發生率，男性皆明顯高於女性。B型肝炎帶原率的地理分佈差異不大，但仍稍有差距。民國七十三年七月開始對HBsAg陽性的媽媽所生的嬰兒施打B型肝炎疫苗，兩年後推廣為新生兒全面施打，但如今25歲以下族群帶原率皆已降至1%以下。疫苗施打後，避免掉大部份B型肝肝炎的垂直感染，也明顯降低小兒肝癌的發生。疫苗全面施打後，產生一些新的議題，如：疫苗是否引起B型肝炎表面抗原的突變、HBeAge陽性孕婦是否應該在懷孕末期接受抗病毒治療以避免子官內感染的保護及B型肝炎疫苗在青少年之後是否需要追加以延長保護效果，等。解決這些問題可以將B型肝炎的防治更臻完美。 　　在接種疫苗的世代中，仍有少部份因子宮內感染、未接種疫苗或疫苗免疫失敗而感染B型肝炎。來自中國的論文顯出子宮內感染的發生率高於其他國家，因此有學者建議HBeAge陽性孕婦應該在懷孕末期接受抗病毒治療以避免子官內感染的保護，國內亦有相關的研究，目前國內正在進行隨機對照試驗之中。這個世代已感染B型的個案，在國中時期，HBeAg的清除率已達七成五，而這些早期清除的個案，九成以上HBV DNA<10的四次方copies/ml ，這是長期有良好預後的表徵。也就是說這個世代的B肝個案，會演變到肝炎、肝硬化、肝癌的機會較低。這個世代些在國中時期，已接種疫苗者，雖然有很高的比率會在血中測不到anti-HBs但在追加一劑疫苗之後都會提升anti-HBs至有效的保護濃度，接受基因工程疫苗與血清疫苗者anti-HBs的雖消失率較高，但追加接種後，有保護力的比率相當。有極少部份保護力減弱的個案，確實有感染B型肝炎，但有可能在感染後誘發免疫記憶來清除病毒而型成anti-HBc單獨陽性(HBsAg(-), anti-HBs(-), anti-HBc(+))的狀態，而未變成帶原者。因此認為這個接種過B肝疫苗的世代無需再追加接種。但年齡更大之後，是否要再追加，或重新接種，則還未定論。 　　分子生物學的突飛猛進，應用分子生物學檢驗來進行流行病學研究稱為分子流行病學，HBV DNA過去用偶合法(hybridization)來檢測，後來應用PCR聚合鏈反應(polymerase chain reaction, PCR)可測得更微量的病毒，後來的realtime PCR使其定量的檢測更為方便，隨著國際單位(international unit, IU)的訂定，使各種檢驗試劑或方法有了一致的標準。B型肝炎基因型的測定及由核酸系列來釐清各種突變，不只對於臨床治療，對於流行病學也有莫大的幫助。大規模的社區長期追蹤研究 (the Risk Evaluation of Viral Load Elevation and Associated Liver Disease/Cancer-Hepatitis B Virus, REVEAL-HBV study)，提供了相當多B型肝炎自然史的資訊。慢性B型肝炎的帶原者在30歲以後仍然為HBeAg陽性或HBV DNA濃度在10的五次方copies/ml有較高的風險會演變成肝硬化、肝癌，若血中病毒濃度高則風險更大。若血中病毒濃度較低則發生肝硬化、肝癌的風險相對減少，但仍高於非帶原者、血中病毒濃度較低發生HBsAg陰轉的機會較大。感染基因型C的個案、無核前區突變或核心驅動子突變都可增加發生肝癌的風險。在B型肝炎的抗病毒治療發明及普及之後，若要再進行自然史的觀察研究就需要有更合理倫理顧慮。 　　慢性B型肝炎的帶原者約四分之三會在四十歲以前轉變成穩定的狀態，甚至逐漸清除病毒。而在四十歲之後B型肝炎仍有活動性者，就是日後有可能發展成肝硬化及肝癌的高危險群。這些人需要臨床考量其治療的必要性。治療的目標由原本的病毒的根除，因藥物療效不甚理想，退而變成病毒活動性的長期壓制。如今疫苗接種世代已近三十歲，他們之中僅有少數的帶原者，而未接種疫苗世代在十幾年就都到達四十歲，到時候哪些人需要積極治療就會更清楚了。但如今，尚未滿四十歲的未接種疫苗的世代，臨床上的處理方式仍會有些討論的空間。至於四十歲以上血病毒濃度血中濃度在10的四次方copies/ml (or 2000 IU)以上的處理原則，已有各家臨床指引的建議。高危險群肝癌篩檢的必要性，會在肝癌的部份討論。 　　台灣的肝病一直都被認為以B型肝炎為大宗（6成），在台灣最早發表的論文C型肝炎抗體的盛行率並不高，直到C型肝炎盛行地區一一被發現以後，才逐漸開始注意到其重要性。C型肝炎盛行地區主要集中在台灣的中南部，以雲嘉南地區為多，但其他地區也有小地方聚集。盛行地區可小到一個村的一部份也可以大到好幾個鄉鎮。C型肝炎抗體陽性率最高的村可高達90%，一般認為與打針習慣有關。這些社區聚集也同時有基因型的聚集。在約二十年前的研究皆顯示年發生率甚高，可達近5% ，但近來的研究發現盛行地區的發生率有明顯下降。在這些地區的C型肝炎抗體陽性率在民國35年以後出生的世代隨年齡減少而逐漸下降。對於新的一代，除靜脈毒癮者來，C型肝炎的罹患率將會降到很低。而較年輕的病人感染第二基因型的比率較高，而靜脈毒癮者則出現一些台灣較少見的基因型。由我們肝癌病因中B或C型肝炎的消長，可以推論這個病毒危害台灣的期間前後僅數十年。雖然C型肝炎是報告傳染病，但由於急性C型肝炎的診斷標準是「抗體陽轉」，因此診斷上有所限制，在疾病管制局的報告病例中，應該僅不到四成是真正的急性C型肝炎。而醫院的研究也顯示僅極少數的急性C肝的病人有完成通報。所謂「通報的不一定是真正病例，而真正的病例不一定有通報」，也就是說由官方資料並無法反應出真正的急性病例。 　　這些C型肝炎盛行地區，可由其肝癌死亡率高、血小板低下的盛行率高及ALT異常率高可推知。換句話說，這些地區有較高肝癌、肝硬化及肝炎的發生率。因此對這些地區應該有獨特且積極的防治，包括：找出高盛行區、盛行區的肝炎篩檢及轉介治療、低血小板C肝個案之肝癌篩檢等。但種種因素使這些策略未被及早積極推行。隨著時間過去高感染世代的凋零，C型肝炎盛行區的問題就自然解決，公共衛生的介入有多少貢獻，則留待後世的評價。 　　C型肝炎雖無疫苗的發明，由於目前傳染並不活絡，對於防治的影響不大。因目前抗病毒藥物在台灣的治療成效良好，完全病毒反應率高達七成，理當是防治的主力，但諸多C肝患者為65歲以上的老者，較難承受治療的副作用。期待有較溫和的療法可以使用。由於早期診斷及適當治療，肝癌篩檢的必要性逐漸確立，唯對這群年長的族群肝癌篩出後存活時間的延長僅局限在小於兩公分且接受治癒性療法個案，因此建議年長者的肝癌篩檢要積極執行。總之，目前需要從老年醫學的角度來考慮C型肝炎的防治。 　　隨著治療達成完全病毒反應的比例高，治療成功的諸多病人的後續長期變化為何，也是臨床醫師所應該知道的。他們的C型肝炎抗體效價大多會明顯下降，但鮮少會陰轉。大部份的病人，以病理切片、肝纖維化測定儀及血小板計數做為指標，顯示其肝纖維程度都會有所改善。發生肝癌的風險也會隨時間而減少，但偶爾會有八至十年後才發生的病例。至於肝外表徵的發生率也有些研究顯示會下降。達到完全病毒反應的病人僅有極少數會延遲復發。 　　C型肝炎有諸多的肝外表徵，其中最重要的就是糖尿病，大部份的研究認為慢性C型肝炎的患者容易罹患糖尿病，南台灣的三個社區研究，分別指出糖尿病的發生率增高、 HCV RNA陽性而非anti-HCV陽性才是真正的危險因子、C型肝炎的引起的糖尿病在無高血脂症者較為顯著。其中兩個社區亦報告，蛋白尿及腎功能亦與C肝相關。其中一個社區報告慢性C型肝炎的患者的血脂肪較低，而另一個社區研究指出慢性B或C型肝炎患者都有血脂肪較低的現象，REVEAL - HBV study 也有報告慢性B型肝炎有血脂肪較低的現象。這些慢性肝炎與代謝症候群領域的重疊，將是接下來的焦點所在。至於糖尿病是否會增加肝癌的機會，則有相當不一致的結果，僅國內研究就有贊成、反對及僅影響某些族群的說法，這個議題將有待釐清。 　　慢性B型及C型肝炎對台灣公共衛生的重要性，在十餘年後將逐漸減少，為減少其罹病率及死亡率的努力還是要持續到疾病幾乎根除的那一刻。 　　癌症是我國第一大死因，而肝癌高居於前兩位，是我國重要的癌症。在我國B型肝炎為主要原因，而C型肝炎次之，有九成的肝癌病患在診斷時呈現HBsAg或anti-HCV陽性。 在這三十餘年來肝癌有逐漸增加的趨勢，死亡率從民國70年以來逐年增加。在開始實行癌症登記以後，也顯示發生率逐年增加。在死亡率增加的同時，男女性比逐漸下降，以上兩個現象都可由C型肝炎相關肝癌的增加所致。肝癌的死亡發生比(mortality to incidence ratio)有逐漸減少的趨勢。此一趨勢可由部份死亡診斷書無法載明真正病因、早期癌症登記不完整、慢性B或C型肝炎患者早期篩檢及肝癌治療技術及概念進步所致。 　　在70年代就有臨床及流行病學研究顯示慢性B型感染可導致肝硬化及肝癌，之後也發現HBeAg的延遲消失亦為重要因素，後續的基礎研究的結果都支持這樣的看法。最近由大規模的社區長期追蹤研究 REAVEAL-HBV study 提供慢性B型肝炎演變成肝癌的自然史的資訊，指出不論以HBV DNA, HBeAg或HBsAg為標記，B型肝炎病毒的延遲消失都會增加發生肝癌的風險，同時已指出病毒基因型與非抗藥性引起的突變，亦與發生肝癌的風險有關。 　　慢性非A非B型肝炎會演變成肝癌早在anti-HCV還不能檢測的時代已有臨床觀察，在anti-HCV的試劑問市後證實為C型肝炎所致，不論是回溯或前瞻的流行病學研究都提供了相同的結果。C肝相關肝癌與B肝相關肝癌的差異大致為男女性比較低、年齡較長及ALT異常率高，對於年齡較長的解釋也許是因感染世代逐漸老化所致。 C型肝炎感染有地區聚集的現象，在台灣相當明顯，在C型肝炎高盛行區C型肝炎相關的肝癌也有明顯較多，以整個台灣來說，以雲嘉南的C肝最為盛行，但在此地區之外也有些鄉鎮市區級或村里級的盛行區，有些學者認為基因型有關，但如無一致的結論。 　　感染肝炎病毒經慢性化而演變成肝癌，防治的策略不外有下略三點：阻斷傳染途徑、治療慢性肝炎及高危險群的篩檢。B型肝炎疫苗在我國已自民國73年後開始接種，已近三十年。由接種世代的帶原率下降、急性B型肝炎發生率減少及小兒肝癌的發生率減少，可以預期B型肝炎相關肝癌的減少已為期不遠。B型肝炎抗病毒治療雖然對病毒的根除率不高，但在肝衰竭死亡及肝癌發生的避免已有成效。C型肝炎雖無疫苗的發明，流行病學調查顯示地區聚集的慢性C型肝炎患者大多在民國35年以前出生的年長者，年輕族群的盛行率已明顯降低。抗病毒藥物治療有相當的成效，在台灣各地的報告完全病毒反應的比率皆在七成或以上。由上述的資料可以預期，不論是B或C型引起的肝癌的發生率，在隨著B型肝炎疫苗世代及C型肝炎高感染世代的年齡增加而減少，對於已經感染者也能藉由抗病毒治療而減少發生肝癌的風險。 　　對於已發展成肝癌高危險群的患者接受肝癌篩檢的必要性，過去的學者基本成本效益的考量，大多持負面的態度。診斷出來的病患不夠早期及治療成效不佳是主要的原因，其實肝癌有諸多值得篩檢的特點。肝癌的高危險群相當清楚，有九成為慢性B或C型肝炎的患者，這是其他癌症所少見的。對高危險施行超音波及甲型胎兒蛋白的定期篩檢，能夠找出相當早期的病例，可早年及近年初診病例的期別，顯示出篩檢的成效。治療技術的進步、臨床指引的推廣及抗病毒藥物的使用，明顯改善肝癌的存活期。至於肝癌篩檢是否要推行至盛行社區，若有更合理的篩檢設計與規劃，也有推行的空間。 　　病毒性肝炎與肝癌的關係已相關的清楚，依其自然史所推行的防治策略也有成效，但仍有些議題需要解決，如：B型肝炎子官內感染的保護、B型肝炎表面抗原的突變、B型肝炎長期保護效果、C型肝炎在靜脈毒癮者的傳播等。此外，非B非C的肝癌的比率逐漸增加，可能是日後肝癌防治的新課題，特別是與生活型態相關，可經由改變生活型態而減少風險的肝癌。酒精性肝病、糖尿病及代謝症候群，在即將由病毒性肝炎高盛行地區轉變成低盛行地區，在肝癌發生所扮演的角色及防治，也將是逐漸被重視的課題。        

到底禽流感和人流感有何關係？

到底禽流感和人流感有和關係？ 流感 流感病毒屬正黏液病毒科（Orthomyxoviridae），A型流感病毒，含8段負向單股RNA病毒，依核蛋白（nucleoprotein）或基質蛋白（matrix protein），將本病毒分為A、B、C等3型。B、C只在人發現；A型廣佈於人及各種動物（哺乳，鳥）。A型病毒又依HA及NA抗原的不同分為許多亞型，目前有17種HA抗原，9種NA抗原。二抗原組合在不同的病毒株中，造成本病毒的多樣性。此病毒與一般RNA病毒不同，其基因進入細胞核中複製RNA。此病毒的病原性變異甚大，大部份的禽流感病毒只引起輕微的消化道及呼吸道的感染。對家禽而言只有H5, H7為高病原性家禽流行性感冒（Highly pathogenic avian influenza, HPAI），高病原性毒株引起全身感染。然而很多具有H5,H7毒株為非HPAI。高病原性毒株引起嚴重損失的亞型計有H7N1、H7N7、H5N1、H5N3，歷史上稱為雞瘟（Fowl plague）。但很多H5、H7分離株屬無病原性毒株。 一、世界動物衛生組織（World Organization for Animal Health, OIE） 定義高病原性家禽流行性感冒如下: （一）任何亞型病毒由靜脈接種0.2ml 10倍稀釋含病毒的尿囊液至8隻4-6週齡SPF雞，10天內死亡6隻以上。 （二）上述接種後死亡1-5隻任何非H5或H7 AI病毒，進行細胞培養，若病毒在不加trypsin可以生長的毒株。 （三）在不加trypsin可以生長的任何亞型毒株，其HA裂解序列與其他已知高病原性的序列相似。 只要合乎上述3者的任1，即為高病原性毒株（OIE, 2004）。 二、歐盟對高病原性家禽流行性感冒的定義依92/40/EEC條款，修訂如下 （一）具H5或H7型，靜脈接種病原性指數（Intravenous pathogenicity index, IVPI）＞1.2。 （二）具H5或H7型，IVPI＜1.2，但HA抗原裂解點含多個鹼性氨基酸。 三、HA氨基酸序列決定流感病毒的病原性 （一）HA的構造 HA有5個抗原決定位，蓋住球狀構造的表面，皆具有中和反應。球狀構造亦存有病毒與細胞的受器接合部位（receptor-binding site）形如袋狀位於每一次單位的遠端。此接合部位不受抗體影響，且組成的氨基酸序列呈高度保留的現象。即在不同亞形毒株皆相同（Tyr98,Trp 153, His 183, Glu 190, Leu194，氨基酸簡寫之後為其在HA的位置），然而不同動物別就不同，即其有動物別的特異性，因此人、鳥、馬之間的病毒"不會"互相感染。但以人的病毒人工感染鴨，可在鴨呼吸上皮繁殖但不會在腸道繁殖。 （二）HA被切開才具感染力 HA0被切開成HA1及HA2。其HA在粗內質網（rER）合成，N端有疏水性的引導序列，HA0經粗內質網醣化，形成三合體，運送至細胞膜時被切為HA1（分子量約47,000）及HA2（分子量約為29,000），被切開的HA才具有病原性，在酸性環境中形成游離HA2 N端的疏水性融合蛋白（fusion peptide），與細胞膜接觸而進行融合。細胞內的酶要將HA0切開與酶辨識HA1 C端的氨基酸序列有關，高病原性毒株，其HA1的C端含4～6個鹼性氨基酸。非高病原性毒株只含單個Arg，前者可被很多種細胞的酶切開，因此，病毒可散佈至全身，而導致死亡。後者只為trypsin胰蛋白酶切開，而trypsin胰蛋白酶只在呼吸道及消化道上皮細胞有之，所以病毒只局限在呼吸道及消化道繁殖，無法擴及全身，而為非高病原性毒株。 （三）HA氨基酸序列決定流行性感冒病毒的病原性 無病原性毒株的HA蛋白質切割位（HA1C端）只含1-2個鹼性氨基酸（basic amino acid），而有病原性毒株含多個鹼性氨基酸. HPAI毒株在HA1C端裂解點含多個鹼性氨基酸，使HA蛋白被很多種細胞內蛋白酶分解，使病毒散佈全身。 Non-HPAI只為trypsin-like protease切，而此類胰蛋白酶只存在呼吸道及消化道上皮，病毒無法擴及全身。一般而言BXBR（B=代表鹼性氨基酸，如argine or lysine, X=任一種, R=arginine）是高病原性病毒株所必備的最基本的構造。若與細菌共同感染可使病毒HA抗原分解，促病毒增殖擴散，因此野外發現病例比人工感染者症狀嚴重。 大部分的弱毒株HA蛋白切割位的序列為RETR*GLF（*HA1與HA2之間的切割點，GLF為HA2的N端融合蛋白的起始），大部分的美國弱毒株為RKTR*GLF，此BBXR為弱毒（R與K為鹼性氨基酸），1983年賓州的弱毒株A/Penn/1/83雖為KKKR*GLF，有4個鹼性氨基酸但被第11個Asp的醣基蓋住，所以為低病原性。BXBR為最基本的條件的實例，如1999年義大利的高病原性毒株H7切割位的序列為GSRVRR*GLF，如前述美國的弱毒株；然而一般強毒株大都有4個鹼性氨基酸以上，如H7的PEIPKKKKR*GLF。 這種多個鹼性氨基酸的理論可能只適用於H5及H7，因為殺死2000多萬人以上的1918西班牙流感H1N1相關的序列為PSIQSR*GLF，只有一個鹼性氨基酸R，但其毒力卻超強。 （四）HA突變而影響病原性的實例 1993年H5N2家禽流行性感冒病毒引起墨西哥雞隻發病，產蛋下降，死亡率升高，至1994年5月才首次分離出病毒但此分離株對SPF雞無病原性，且其HA切位氨基酸序列為弱毒株，此時此病毒已散佈至全國各地。至1995年初病毒HA切割位插入2個鹼性氨基酸，轉變為強毒，並已擴散至各地，造成嚴重的損失。 禽流感病毒各段基因很容易發生交換（reassortment），但發生重組（recombination）則甚少見，但智利的毒株就發生重組而變為高病原性的例子。2002智利低病原性毒珠H7N3的HA基因PEKPKTR*GLF，由NP基因得到10個氨基酸，形成PEKPKTCSPLSRCRETR*GLF，而轉變為高病原性（劃底線者為插入的氨基酸。 除了上述HA1 C端的氨基酸序列影響外，HA1 N端的氨基酸醣化亦會影響毒力，如1983年賓州發生高病原性家禽流行性感冒H5N2病例，非高病原性的毒株在HA第11個氨基酸為Asn，含一醣化支鏈，此支鏈在立體位置靠近切割點，因此干擾細胞蛋白的切割，使病毒呈低病原性的。第11個氨基酸Asn要被醣基化，受其後第2個氨基酸影響，需為Ser或Thr才會促使11個氨基酸Asn醣基化，6個月後，毒株因第13個位發生點突變，由原來的Thr變成為Lys，因此使第11個氨基酸Asn失去醣化而沒有蓋住切割位，切割位易被蛋白酶切割，因此轉變為高病原性毒株。此次暴爆發使美國撲殺1千7百萬隻雞，損失6千萬美元。但若切割位的鹼性氨基酸夠多，此醣化之有無則與病原性無關係，如FP/34（H7）與Turkey／1keland／83（H5），在此部位序列雖含有醣基，但仍為高病原性毒株。另有其他與病原性有關的序列，如HA1第17個位置若為Arg而不是His也可促進切割而呈現高病原性。禽流感病毒在HA蛋白球型的頂部有一個醣基化氨基酸也與高病原性有關。由演化樹分析，高病原性毒株並未自行成立一分支，而與個別的低病原性毒株在同一分支，由此可知高病原性與低病原性毒株可以互相轉變。 四、其他基因 NA蛋白質切除HA的sialic acid 而促進病毒的移動。 NA與病毒釋放有關，NA少的病毒由細胞釋放出的效能降低，HA與NA量的平衡與病原性有關， NA與plasminogen結合能力越高導致HA易被切開病毒在組織的分怖越廣，對白鼠的病原性越高。 NA蛋白質C端有 Lys及第146個氨基酸無醣基化，可以促進HA的切割，因此增強病毒的病原性。 NA的莖部有氨基酸缺失的為高病原性毒株，但台灣的H6N1的NA莖部雖有氨基酸缺失但卻是低病原性的。 其他與病原性有關的基因尚包括M, PB1, PB2等（表1）。 表1. 由人分離的H5N1毒株基因序列與病原性的關係（J Virol 74:10807） 病原性    基因（位置）    NA（223）  M1（15）  PB1（198） PB1（317）PB2（355） 高    I*     I   K     I     K 低    T     V     R     M     Q * NA（223）由I變為T發生醣基化使其頂端酶的活性增加。 I: Ileu, K: Lys, T: Thr, V: Val, R: Arg,M: Met, Q: Gln. 由人分離的H5N1毒株與毒力有關的基因尚有 NS基因的第92氨基酸為Glu 。 五、不同鳥禽對禽流感的不同感受性 雞及火雞的感受性最高，其次為雞，鴨鵝等水禽則有抵抗力。但2002-2004由香港、中國的分離株已突變成可使鴨、鵝、蒼鷺等致死的毒株。有關珍禽方面，流行性感冒病毒亦曾由各國檢疫的珍禽分離到，可能與原產地的發生有關。雀科鳥類（passerine birds）亦可能傳播病毒。不同毒株感染珍禽引起的症狀差異很大，由小型鸚鵡（parakeets）分離到的多為H5,H7亞型，病鳥羽毛逆立，綠色下痢，昏睡及神經症狀，強毒株死亡率可達30％。鴿子有抵抗力，感染機乎不發病。以香港H5N1毒株攻擊，感受性依序為火雞、雞、鵪鶉、雉雞、雀類、鸚鵡等，鴯（emu）、麻雀次之，而鴨、鵝、海鷗、鴿子、鼠、兔子、椋鳥（starling）則有抵抗性（AD 47:956）。 六、流行性感冒病毒的宿主範圍 流行性感冒病毒 由HA決定 宿主範圍，因HA需與宿主細胞的接受器接合才可進入細胞中，同樣是A型感冒病毒依HA及NA的不同而分為很多亞型，感染人類者只有H1、H2及H3。感染禽類類而引起高死亡率的為H5或H7。HA或NA的突變可導致抗原性改變的變異株產生，人體內的抗體無法保護這些變異株的感染而造成流行及死亡。存在鳥類及其他動物的17種HA亞型及9種NA亞型會與人類A型病毒互相交換基因片斷。鳥類來源的病毒不會在人體內繁殖及造成疾病，1997年以前只有H7引起的2個病例報告，在中國南方，農民血清抗體對雞源病毒的陽性率由1％至38％，其中對H5亞型抗體佔7％。反之，1983年美國賓州暴發雞瘟時飼養的雞農沒有對H5亞型的抗體。1997年香港發生的H5N1則是首次引起世界注意的大事件。 七、禽流感病毒提供基因給人類 歷史上人類發生幾次流行性感冒大流行都與鳥類有關，因人類來源的病毒由鳥類來源的病毒發生基因交換而產生新的變種，最主要交換病毒的來源為野生水禽類； 如1918大流行的西班牙型感冒病毒（H1N1）可能包含了8段鳥類來源的基因；或由禽鳥類傳給豬再傳給人，在其基因的前部和后部是人類流感病毒的編碼，而在基因的中段則是豬流感病毒的編碼。1957年大流行的亞洲型流感（H2N2）包含了3段鳥類來源的基因 1968大流行的香港型流感（H3N2）包含了2段鳥類來源的基因。 所以在人類病毒的演變上鳥類是扮演重要的角色，這些基因可能在豬混合而來。 八、禽流感病毒感染人類 事實上禽類的Ａ型病毒是不會輕易感染人類的，因為人類的細胞沒有該病毒的接受器。一般感染人類的Ａ型病毒毒是不會引起雞隻大量發病死亡；因此同為Ａ型流行性感冒病毒，人是人、雞是雞，分屬不同病毒。1997年香港及2004年發生人感染H5N1亞型為原本感染禽類的病毒，世界衛生組織進行該病毒的基因分析，發現該病毒的全部基因皆由禽鳥而來，沒有像1918年、1957年和1968年發生大流行的Ａ型流感病毒混合了人類與鳥類的病毒基因。雖已證實此禽病毒沒有與人類流感病毒基因發生交換的現象，但是否禽病毒經由點突變而產生與人接受器接合部位改變的突變種則有待證實。 H5N1禽流感是透過與活家禽近距離接觸而傳播，而人類之間的傳播能力十分低。H5N1禽流感的徵狀與普通流感差不多，但較易導致高燒、肺炎、呼吸衰竭、多種器官衰竭，以致死亡。最近多個亞洲國家報告雞隻中出現禽流感，主要影響家禽，但人類亦有受感染個案。 九、豬流感 由動科所的調查，台灣地區豬群中存在的類人型（Human-like）H3N2亞型病毒及近來出現的豬流感新興變異病毒株（(HlN2、H3N1)帶有曾經於1980年至1982年間在臺灣地區人類族群中流行過的A/Bangkokjl/79（H3N2）病毒基因, 因而具有由豬隻傳回人類族群的可能性。由於A/Bangkok/1/79（H3N2）病毒已在臺灣地區人類族群中消失很長一段時間，年輕一代的青少年及兒童對此種病毒已幾乎不其兔疫力， 倘若此類具有人類流成病毒基因片段之病毒變異株由豬隻傳回人類族群，進而可在人群之中蔓延流傳，即有可能造成另一波全球性流感大流行。 又鑑於豬隻對豬型、禽型及人型流行性感冒病毒皆具感受性 , 可成為各型病毒基因交換重新排列（Reassortment）的混合場所（Mixing vessel），向來被認為是傳播禽流感病毒基因進入人流感病毒基因的媒介 , 因此倘若目前在亞洲各國肆虐的高病原性 H5Nl 亞型禽流成病毒或臺灣地區出現的低病原性 H5N2亞型禽流成病毒成染到臺灣豬群，重組出帶有禽流感病毒基因的豬流感病毒變異株，將更大大提高侵害人類的威脅，亦有可能造成另一波全球性流感大流行。 所幸，臺灣地區在養豬群中至今尚未發現禽流感病毒或帶有禽流感病毒基因的豬流感病毒變異株，亦未有與禽豬密切接觸的相關工作人員出現感染病例，但仍不可輕忽禽豬等動物流成病毒對人類可能的威脅。 十、預防流感 預防流行性感冒預防流行性感冒的最好方法，是增強自己的抵抗力，要有充足的睡眠和休息，飲食均衡、適量運動、注意空氣流通，和切勿吸煙。如果患有感冒徵狀，宜留在家中休息，避免前往擠迫和空氣不流通的公共場所。 染病的活鳥和家禽的糞便中可能會帶有病毒，故應盡量避免接觸活鳥和家禽及其糞便。如曾接觸活鳥或家禽，要立刻用洗手。如家中飼養雀鳥，應避免和牠有親密接觸，並每次在接觸牠或替牠處理糞便後用水洗手。學校及幼兒院舍亦應採取措施防止兒童接觸活鳥及家禽。出外旅遊時應避免接觸活鳥及家禽。進食家禽肉類和蛋類時應徹底煮熟。如果您的工作與動物或其排泄物有接觸的機會，請於每年流感盛行季節（每年9月起），到衛生所或醫院施打流感疫苗，可以保護自己也保護別人受到流感病毒感染。如果您出現感冒、發燒、肌肉酸痛、頭痛與極度倦怠等類流感症狀，請戴上口罩後，儘速至疾病管制局合約之採檢醫療機構就醫，並告訴醫師您的職業，您將會是使用流感抗病毒藥劑的優先使用對象。。

禽流感H5N1及流感

禽流感H5N1及流感 流行性感冒係經由"飛沬"或直接"接觸"病者的分泌物，透過呼吸道傳染，可發生在所有年齡層，其重要性在於爆發流行快速、散播範圍廣泛以及併發症嚴重，如肺炎重症等。爆發流行時，重症及死亡者多見於"老年人"、"嬰幼兒"及"免疫功能低下者"。 流感病毒可以分為 A、B、C三型，其中A型流感病毒變異快速，歷史上有三次流感大流行，皆由A型病毒引發，並造成慘重傷亡。禽流感亦為A型流感病毒，原來只在禽鳥類間傳播，但香港於1997年開始出現人類禽流感案例。其後又陸續在東南亞及中東等國出現人類疫情，至目前(2012）為止，全球約有近600個人類案例，雖然目前尚未大量傳播，但死亡率高達五成，且禽流感病毒可能正逐漸調適，有潛力發展為可有效地人傳人的病毒，而造成大流行。目前每年施打的流感疫苗，對老年人或免疫力較差的高危險群可減少罹病或出現嚴重併發症的機會，但對禽流感則無預防作用。抗病毒藥物包含M2 protein抑制劑及神經胺酸酵素(neuraminidase)抑制劑兩大類，前者只對A型流感有效，後者可對抗A及B型流感。至於治療禽流感，只有神經胺酸酵素抑制劑可能有療效。值得注意的是，有關流感及禽流感病毒出現抗藥性的報導正不斷地增加中。 前　言 流行性感冒(influenza)，一種不斷變異、無國界、傳染性強、傳播速度快的疾病，與人類併存的關係由來已久，流感近百年來數次大流行造成極大的震撼，其中1918年的流行性感冒造成全球四、五千萬人死亡，死亡人數超過第一、二次世界大戰及二十世紀所有戰爭。雖然這麼高的死亡人數可能與當時的醫藥科學不及今日發達有關，然而歷史上血淋淋的教訓似乎仍無法給人深刻的警覺，如今早已被人們所淡忘。1997年自香港爆發人類禽流感以來，歷史又再一次提醒人們：如果我們稍有懈怠，一場比嚴重急性呼吸道症候群(severe acute respiratory syndrome; SARS)更兇猛的疾病大流行就會再次向我們襲來，所造成的感染及死亡人數難以估計，對人類社會秩序及經濟活動將形成莫大的衝擊。 流行性感冒 流行性感冒(簡稱流感)是指由流行性感冒病毒(influenza virus)所引起的呼吸道傳染性疾病，它有別於一般感冒。所造成的症狀從輕微到很嚴重都有可能，有時可導致死亡。流感可發生在所有的年齡層，但以老年人（65以上）、嬰幼兒（5歲以下）或免疫力缺陷者較易出現嚴重的併發症及死亡。 一、流感的症狀 流感的潛伏期約1-4日，在潛伏期就可能將病毒傳染給他人，典型的流感症狀包括：發燒(通常為高燒)、頭痛、咳嗽、喉嚨痛、鼻塞、流鼻水、全身肌肉酸痛、疲倦及腸胃道症狀等。一般認為流感的症狀可以比普通感冒（鼻病毒為主）來得嚴重，持續時間也較久。大部份人都能在5天至一星期內自行痊癒，但是，年老者（65歲以上）及5歲以下幼兒，慢性疾病患者(如：心臟病、慢性呼吸道疾病患者)，則有較大機會出現併發 症，例如：支氣管炎、肺炎。根據過去的文獻，並無法單從臨床症狀的輕重來區分病患是否得到流感或是一般感冒，尤其在非流感流行期間，上述各項症狀用來預測流感的準確性更差，所以確診仍需參考當時流行趨勢並依賴病毒培養（要2週）或分子生物學等診斷工具。 二、病毒學 流行性感冒病毒屬於正黏液病毒(orthomyxovirus)，在電子顯微鏡下呈球狀，其直徑大約為100 nm，是一種單股RNA病毒，它的RNA總長14000Kb,分成8個節段，而分別製造11種不同的蛋白質。病毒的外表有兩種重要的抗原(糖蛋白）：血球凝集素(hemagglutinin, H抗原)及神經胺酸酶(neuraminidase, N抗原)。 流感病毒依核蛋白（RNP)的不同，可分為A、B、C三型，其中只有A型與B型可以引起大規模的季節性流行，C型流感在臨床輕症故不太重要，成人血清證實7成都得過。A型流感病毒依表面抗原血球凝集素 (H抗原，H1-17），17最近在蝙蝠體內分離出來，神經胺酸酵素(N抗原，N1-9)的不同，還可分為許多亞型，如H1N1、H3N2等；而B型流感病毒則不區分亞型。A型流感病毒除了感染哺乳動物、禽類，如豬、馬、雞、鴨等，而B型及C型則至今只曾出現在人類。自1977年開始，A型H1N1、H3N2及B型流感病毒不斷循環出現在全球人類的季節性流感。 流感病毒的命名格式如下: 型別/來源區域/病毒株號/分離年份(HN)。如：A/Taiwan/1/1986(H1N1)，表示它是1986年在台灣分離出而具有H1N1亞型的A型流感病毒，其病毒株編號為1。 B/Hong Kong/330/2001 ，表示它是2001年在香港分離出的B型流感病毒，其病毒株編號為330。 流感病毒之遺傳物質為RNA，發生突變機率較DNA高，每1萬分之一出錯一次！流感病毒基因為14000個核苷，故每複製一次、約出現1至2個錯誤核苷！在一個病人身上分離出之流感病毒基因的相異性可達4%之多！人和猩猩基因差異性只1%！當病毒產生抗原微變(antigenic drift)，即病毒RNA發生點變異，這樣的情形會發生在A及B型流感病毒，所以每年都會有流感的小流行出現，其中B型只感染人，只會在複製過程不斷累積錯誤核苷，直到較大變異時，才造成季節性流感！較A型發生變異的速度來得慢。抗原巨變(antigenic shift)主要發生在A型流感病毒，因其宿主有人、其他哺乳動物、及禽類！是指RNA節段發生基因重組，因人類普遍對新抗原缺乏免疫力，這樣的變化可能釀成大流行。 對病毒抗原尤其是血球凝集素所產生的免疫反應，可以減少被感染的可能性，即使被感染也可減輕疾病嚴重度[5]。但是對某一病毒株或其亞型產生的抗體，對其他病毒株或其亞型並無法提供或僅能提供部分保護作用[6]。同樣的，如果病毒出現新的變異，原有的抗體也無法完全發揮保護作用。 三、歷史上的大流行 人類近代史上有三次流感大流行，皆由A型流感病毒引發，每一次都造成龐大的死傷。(1)1918年西班牙流感：1918年至1919年，H1N1型流感分別在法國、美國波士頓及非洲塞外理昂開始爆發，在全球傳播，導致罕見的嚴重性及致死率，死亡人數約4,000萬。死亡者有許多是二十至三十歲，身體健壯的年輕人，十八個月後疾病突然消失。(2)1957年亞洲流感：1957年至1958年的H2N2型流感，死亡人數估計為200萬。(3)1968年香港流感：1968年至1969年的H3N2型流感，死亡人數估計為100萬。隨著科技的進步，目前已有能力還原出1918年西班牙流感的病毒基因序列，科學家從三位受害者保留下來的肺部組織中，將史上死傷最慘重的1918年流感病毒在實驗室內重生(resurrection)，並成功的感染實驗動物 及造成100%的致死率，於是拼湊出罪魁禍首的完整面貌[7]。重現這種病毒，讓科學家得以研究它高致死率的原因，並且追查其起源。 四、新型流感 流感病毒之基因容易突變，並可存在於多重宿主，當不同來源的流感病毒發生重組，常使抗原發生重大改變，或因不明原因，造成症狀及感染宿主發生變化之新病毒，即為「新型流行性感冒(novel influenza)」之概念。在防治上，「新型流感」係指民眾就醫後，其臨床症狀、流行病學資料符合採檢條件，並經檢驗結果確認其為A型流感病毒，且亞型為H1、H3以外者。人類感染禽流感亦屬於新 型流感之一。 五、流感之實驗室診斷 (1)病毒培養：傳統上，取鼻咽或喉頭拭子檢體於雞胚胎培養，缺點為費時，約需3至10天。 (2)檢測病毒RNA：利用反轉錄聚合酉每鏈反應(RT-PCR)等分子生物學檢驗方法，可較快且準確地作出診斷。結果可在1天內得知，是目前最受歡迎的檢測方法。 (3)快速抗原診斷法: 取鼻咽或喉頭拭子檢體，檢測流感病毒抗原，可在30分鐘內得知結果，但其敏感性及專一性較差[1,8]。 (4)檢測血中抗體：必須檢測急性期與恢復期血清，比對是否抗體陽轉或4倍以上上升，需兩週以上才可得知結果，因此通常只用於流行病學之調查[9]。 禽流感 禽流感是由禽型流行性感冒病毒感染所引起，所有的禽型流行性感冒病毒都是A型流感病毒，宿主有雞、鴨、鵝、水鳥、鯨魚、海豹及水貂等。病毒在自然宿主野鴨的消化道表皮細胞繁殖，但不致病，僅在糞便內排出病毒，且可達兩個星期之久。其他家禽若接觸到此種糞便，便可能吸入病毒至呼吸道內而發病。一般而言，禽流感病毒可分為高致病性和低致病性兩種，受高致病性禽流感病毒感染的鳥類，家禽致死率可高達80%以上。最近被高度注意的高致病性禽流感病毒主要為H5N1流感病毒，此禽流感病毒亞型最初於1961年在南非從鳥類(燕鷗)中首次分離。H5N1病毒在鳥類中傳染性非常強。 H5N1病毒在人類身上的增生效率不佳, 直接傳給人類相當罕見。然而近年來情況卻悄悄地發生了變化。禽流感的傳播已經跨越了原先的物種範圍，開始侵襲人類，而其高致死率也引起廣泛的注意。人類感染禽流感後，潛伏期一般為2-5天，症狀與其它流感相似，主要為發燒、流鼻涕、鼻塞、咳嗽、咽痛、頭痛、全身不適，有些會有噁心、腹痛、腹瀉、稀水便等消化道症狀，超過6成的患者可見肝指數 異常，病情嚴重者會發展成進行性肺炎、急性呼吸窘迫症、肺出血、腎衰竭、敗血症休克等多種併發症，甚至死亡。 禽流感病毒在禽類造成的大流行在文獻上並不少見，而造成人類的感染則是到1996年才首次被發表出來。隔年(1997年)，在香港即發生18例人類感染禽流感病毒(H5N1)，其中6人死亡。1999年，香港有2個孩子被確認感染禽流感H9N2病毒。證據顯示家禽是感染源，傳染的主要模式是從鳥傳人。1998-99年間，中國大陸報告了更多人感染H9N2的病例。2003年，去中國旅行的一香港家庭的兩個成員發生禽流感(H5N1)感染，一人死亡。2003年，在荷蘭家禽爆發禽流感期間，家禽工作人員和其家庭成員被證實感染禽流感(H7N7)者，共有80多個病例，1個患者死亡。2003年，一個兒童在香港被證實感染H9N2。兒童住院治療，但恢復了健康。最近的也是最嚴重的禽流感病毒H5N1疫情，始自2003年12月，韓國首先在禽類爆發疫情；2004年1月起，越南、日本、泰國、柬埔寨、寮國、印尼、中國以及馬來西亞等亞洲國家接續爆出禽類感染的疫情，其中越南及泰國甚至爆發人類感染的病例。2004年10月泰國動物園爆發老虎感染群突發，2005年4月大量野鳥在青海死亡，並在一些候鵝身上分離出H5N1禽流感病毒，暗示禽流感病毒可經由候鳥遷移路線傳播。同年7月起，果真陸續在俄羅斯的西伯利亞、哈薩克、土耳其、羅馬尼亞及克羅埃西亞等歐陸國家爆出禽類感染的疫情。2005年，除了越南及泰國，柬埔寨、印尼、中國也發生人類感染禽流感病毒H5N1的病例。2006年1-4月，土耳其、伊拉克、亞塞拜然、埃及也陸續出現H5N1感染及死亡的案例；迄2011年止，總計有15國566例人類感染禽流感病毒H5N1的病例，其中233例死亡。Studies that used the WHO criteria included 7,304 study participants. Rates of seropositivity were from 0 – 5.3%, with one study reporting 11.7% positivity. The meta-analysis yielded a seropositivity rate of 1.2% (95% confidence interval 0.6% – 2.1%). When only poultry workers were considered, the seropositivity rate was 1.4%. Other studies were separately analyzed that did not utilize WHO guidelines; these included 6,774 participants and yielded a seropositivity rate of 1.9% (95% confidence interval 0.5 – 3.4%). A total of 12,677 study participants from 20 studies were included in this meta-analysis, of which 1-2% had evidence for prior H5N1 infection. 流行性感冒疫苗 目前流感疫苗可分為兩種，一為不活化疫苗，僅含有抗原成份而沒有病毒殘餘的活性；一為活性減毒疫苗，這是無致病力的活性病毒。流感疫苗之選用，具有全球一致性，係世界衛生組織(World Health Organization;WHO)依據每年於全球83個國家地區，超過110個監測點所偵側之流感病毒，每年2月中召集會議研商選定病毒株，公開宣佈推介，由製造廠商據以生產供應給各國使用，全世界完全相同，台灣亦涵蓋其中。最近幾年，因為一直有A型H1N1與A型H3N2流感病毒的同時流行，所以疫苗的成分都包括了這兩種A型流感病毒與一種B型流感病毒。目前我國所使用的疫苗屬於不活化疫苗。 根據國外研究報告顯示：流感疫苗對健康的年輕人有70%防護效果。對老年人則可減少50%罹患流感之嚴重性及其併發症，並可減少80%之死亡率[13-15]。 接種後最常見的副作用反應是接種部位局部疼痛、紅腫，另有極少數出現全身性反應，例如發燒、肌肉痛、倦怠感等。過敏及神經系統等反應則罕見[16-18]。疫苗之保護力約可持續1年，且由於流感病毒的變異性極大，幾乎每年均會發生變異，原施打疫苗對不同抗原型之病毒並不具免疫力，致保護效果降低。即使病毒未發生變異，疫苗成分相同，其保護效果亦約只能維持1年，因此建議每年均須接種1次。 對於成人及大於36個月的兒童，其接種劑量為施打0.5mL一劑；對於介於6個月至35個月齡的兒童，則為施打0.25mL一劑。但若該兒童(小於8歲)過去並未接種過流感疫苗或未曾感染過流感，則應在間隔4星期後施打第二劑。在下列情況不能接種流感疫苗： 1.已知對蛋之蛋白質(egg-protein)或疫苗其他成份有過敏性休克反應者。 2.年齡六個月以下者。 3.過去注射曾經發生不良反應者。 4.發燒或急性疾病，宜予延後接種。 5.其他經醫師評估不適合接種者，不予接種。 直至目前為止，仍未有可供人類使用而能有效預防禽流感H5N1的疫苗上市。而流感疫苗不能預防禽流感，但有助減低因感染流感而引致併發症及住院的可能性，故建議年老者及患有慢性疾病的人士接種流感疫苗。接種流感疫苗亦能減低人們同時感染人及禽流感病毒而出現基因重組的機會，避免將來出現能引起流感大流行的新病毒種類。 抗病毒藥物 抗流感病毒藥物主要包含兩大類，一類為M2 protein抑制劑，另一類為neuraminidase抑制劑。M2 protein抑制劑用於預防與治療A型流感病毒感染症，而neuraminidase抑制劑可用於預防與治療A、B型流感病毒感染症。 (1) M2 protein抑制劑：包括amantadine與rimantadine，均可作用於病毒外膜上由M2 protein組成的離子通道(ion channel)。當病毒進入細胞內時，氫離子會進入病毒的M2 protein離子通道，並引發病毒複製之後續機序。一旦amantadine與rimantadine進入M2 protein離子通道，則將阻斷氫離子進入病毒的M2 protein離子通道，抑制病毒於細胞內複製。但這些藥物只對A型流感病毒有效，且伴存一些不良反應 (神經及胃腸副作用)。目前已經有許多具有抗藥性的病毒產生，因此這類藥物被用來治療流感之角色已大為降低。 (2) 神經胺酸酵素(neuraminidase)抑制劑：包括zanamivir (Relenza，瑞樂莎)與oseltamivir (Tamiflu，克流感)。A型流感表面具有hemagglutinin及neuraminidase兩種醣蛋白分子。N-acetylneuraminic acid (即sialic acid)為流感病毒接受體成份之一，病毒顆粒表面之hemagglutinin會與之接合，而neuraminidase負責切斷此接合部位，可讓已複製完成之病毒自宿主細胞中釋出，感染其他健康細胞。Sialic acid同時為呼吸道分泌物中的具保護作用之黏蛋白成分之一，被neuraminidase破壞後，呼吸道表皮細胞即失去自然屏障，而讓病毒有機可趁。藉由抑制neuraminidase可預防疾病之發生，並降低病毒感染之嚴重性，減輕症狀與縮短病程。 這類藥物不只對A型流感病毒有效，對B型流感病毒也有治療效果。Zanamivir為乾粉吸入劑型，投與途徑為經口吸入呼吸道，約有78%會沉積於口咽部，約只有15%會到達支氣管與肺，其中5-15%被吸收，並由尿液排除，身體可用率僅2%。Osel-tamivir為口服藥，約有75%的藥物會進入全身循環中，半衰期是6-10小時，由腎臟排出，腎衰竭病人必須調整劑量。 這類藥物必須盡早投與，病人感染流感病毒後36-48小時內接受zanamivir或oseltamivir，可在1-2天內減輕症狀。目前已知"成人流感病毒"約有0.4%對neuraminidase產生"抗藥性"，小孩"感染之流感病毒"抗藥性"更可"高達4%"。禽流感對amantadine類藥物已經具有抗藥性[20-21]，而oseltamivir對禽流感可能具有潛在的治療或預防效果。但在越南發表的研究報告顯示，H5N1病毒已經出現對oseltamivir的抗藥性[22]。檢驗結果顯示，在其中兩名患者體內，病毒對oseltamivir已經產生抗藥性；一名婦女和她十幾歲的女兒在感染禽流感的早期階段，接受了醫生推薦的oseltamivir劑量治療，但仍然死亡。想用neuraminidase抑制劑治療禽流感，可能需要更高劑量和更長療程才能見效。 結　語 由全球的案例看來，禽鳥病毒"極不容易"傳染給其它物種，包括豬和人類。但近來對總數超過12000人的20個研究，經由這些人血清中存在針對H5N1抗體的比例達1至2%。表示禽流感早已感染人類，且重症比例并不如之前顯示的高！"絕大多數"禽流感病毒"不會"傳給人類，但H5N1禽流感病毒是一種具有大流行潛力的病毒株，因為它極有可能變異為一種能在人類中傳染的病毒株，如果H5N1型病毒發展為像普通流感一樣可以有效率地人傳人，並且在人類產生大規模群突發，後果極可能會是「災難性」的。根據歷史上流感大流行的時間表來看，目前人類世界可能處 在另一次新型流感大流行的邊緣。而目前在治療藥物存量不足，以及效用仍有疑慮的情形下，若新型流感爆發大流行，在擔心死亡的恐懼下，民眾將產生非理性的恐慌，屆時將對各項產業產生全面性衝擊。因此，無論是政府、廠商或民眾，都應做最壞的打算以及最好的準備，因應各種情況，進行沙盤推演，以建構對應計畫，避免措手不及。若能及早進行危機處理與應變準備，將可望使疫情的影響降至最低

2012Review of Influenza

Influenza is a myxovirus belonging to the family of viruses known as Orthomyxoviridae. The virus was originally confined to aquatic birds, but it made the transition to humans 6000–9000 years ago, coinciding with the rise of farming, animal husbandry and urbanisation.流感是一種黏液病毒屬於正黏液病毒屬（family),本只局限在水禽、但在六至九千年前傳播至人類，正好和農耕、畜牧及都市化的時間點吻合。 These changes in human behavior and population density provided the ecological niche that enabled influenza, as well as a number of other infectious agents such as the viruses that cause measles and smallpox, to move from animals and adapt to a human host.人類行為的改變、人口的集中提供病毒、像流感、麻疹、天花、從動物變成可適應、感染人類。 Influenza as a disease has been recognised for centuries, even though the viruses which cause it were not correctly identified until the early 1930s, first in the UK and then in the USA. 流感數世紀以來廣為認知，但真正證實是病毒致病則是在1930年代的英國。Indeed the name itself is derived from an Italian word meaning ‘influence’, and reflected the widespread belief in medieval times that the disease was caused by an evil climatic influence due to an unfortunate alignment of the stars.流感英文為influenza,是源自義大利文，意思是影響、在中古世紀認為是因星球不正常的排列、造成惡劣氣候的影響。 Our current understanding – that infectious diseases are caused by infectious agents – is so ingrained that such mystical causes for an illness now seem absurd.現在看來這種想法當然荒謬。 However, even during the Middle Ages, people had a sound idea of infection and realised that some diseases could be passed from one individual to another and others could not. For example, the use of quarantine for a disease such as plague, but not for many other illnesses, shows that people could distinguish infectious diseases from non-infectious diseases even if the causative agent and the method of transmission were obscure.但即使在中古世紀、人們也認知到有些疾病會一個傳一個 The idea that influenza is caused by the influence of the stars, though not a satisfactory explanation of how the disease spread, does identify an important feature of flu – that serious epidemics of the disease occur at irregular intervals. For example, in the twentieth century there were at least five major epidemics of flu that spread around the world (a pandemic), and there were less serious epidemics in most years. In times when people believed in the spontaneous generation of life, the stars would have seemed a reasonable explanation for unpleasant and unexpected epidemics. 1.1 Defining influenza How would you define ‘influenza’? Reveal answer You may well have defined influenza as an infection caused by an influenza virus. However, you may have defined it according to its symptoms: an infection that starts in the upper respiratory tract, with coughing and sneezing, spreads to give aching joints and muscles, and produces a fever that makes you feel awful; but usually it has gone in 5–10 days and most people make a full recovery. The first answer here is the biological definition and, in the Open University course SK320, diseases are defined according to the infectious agent which produces them. This is because different infections can produce the same symptoms, and the same infectious agent can produce quite different symptoms in different people, depending on their age, genetic make-up or the tissue of the body that becomes infected. Here a distinction is made between the infectious disease caused by a particular agent and the disease symptoms. Unfortunately there is a lot of confusion in common parlance about different diseases. Often, people say that they have ‘a bit of flu’ when they have an infection with some other virus, or a bacterium that produces flu-like symptoms. Such loose terminology is understandable, since most people are firstly concerned with the symptoms of their disease. But to treat and control disease requires accurate identification of the causative agent, so this is the starting point for considering any infectious disease. Attributing cause to a disease The difficulties encountered in assigning a particular pathogen to a disease are well-illustrated by influenza. During the influenza pandemic that occurred in 1890, the microbiologist Pfeiffer isolated a novel bacterium from the lungs of people who had died of flu. The bacterium was named Haemophilus influenzae and since it was the only bacterium that could be regularly cultivated from these individuals at autopsy, it was assumed that H. influenzae was the causative agent of flu. Again, in the 1918 flu pandemic, the bacterium could be regularly cultivated from people who had died of flu with pneumonia. So it was thought that flu was caused by the bacterium, and H. influenzae came to be called the ‘influenza bacillus’. The role of H. influenzae was only brought into question in the early 1930s, when Smith, Andrews and Laidlaw showed that it was possible to transfer a flu-like illness from the nasal washings of an infected person to ferrets, using a bacteria-free filtrate. These studies demonstrated that the pathogen was in fact much smaller than any known bacterium and paved the way to the identification of influenza viruses (Figure 1). Figure 1 Influenza viruses are small and use RNA as their genetic material. They have irregular shapes and the outer envelope (the dark-striped outer layer of each virus in this electron micrograph), which is derived from the plasma membrane of the host cell, contains viral proteins. Why do you suppose that H. influenzae was incorrectly identified as the causative agent of flu? Reveal answer The bacterium fulfils two of Koch’s postulates: it is regularly found in serious flu infections and it can be cultured in pure form on artificial media. Moreover, at that time no-one knew what a virus was, and everyone was thinking in terms of bacterial causes for infectious diseases. Although the precise role of H. influenzae in the 1890 and 1918 flu pandemics is not clear, it is likely that the bacteria were present and acting in concert with the flu virus to produce the pneumonia experienced. Such synergy between virus and bacteria was demonstrated by Shope in 1931. He infected pigs with a bacterial-free filtrate (containing swine influenza virus) with or without the bacteria, and showed that the disease produced by the bacteria and filtrate together was more severe than that produced by either one alone (Van Epps, 2006). In its role of co-pathogen, H. influenzae is only one of a number of bacteria that can exacerbate the viral infection. This highlights a very important point. In the tidy world of a microbiology or immunology laboratory, scientists typically examine the effect of one infectious agent in producing disease. In the real world, people often become infected with more than one pathogen. Indeed, infection with one agent often lays a person open to infection with another, as immune defences become overwhelmed. For this reason, a particular disease as seen by physicians may be due to a combination of pathogens. 1.2 Influenza infection in humans Influenza is an acute viral disease that affects the respiratory tract in humans. The virus is spread readily in aerosol droplets produced by coughing and sneezing, which are symptoms of the illness. Other symptoms include fatigue, muscle and joint pains and fever. Following infection, the influenza virus replicates in the cells lining the host’s upper and lower respiratory tract. Virus production peaks 1–2 days later, and virus particles are shed in secretions over the following 3–4 days. During this period, the patient is infectious and the symptoms are typically at their most severe. After one week, virus is no longer produced, although it is possible to detect viral antigens for up to 2 weeks. Immune responses are initiated immediately after the virus starts to replicate, and antibodies against the virus start to appear in the blood at 3–4 days post infection. These continue to increase over the following days and persist in the blood for many months. In a typical flu infection, the virus is completely eliminated from the host’s system within 2 weeks. This is sterile immunity: the virus cannot be obtained from the patient after recovery from the disease. Figure 2 Time course of a typical flu infection Production of the virus (purple bar) starts early after infection and is maximal within two days. The infection is contained during this period by various immune defences. Specific antibody production starts to appear by day 3 (yellow bar), and this contributes to the elimination of the virus. Symptoms (red bar) coincide with the production of the virus. Long description For infants, older people, and those with other underlying diseases (e.g. of the heart or respiratory system) an infection with flu may prove fatal. However, the severity of a flu epidemic and the case fatality rate depend on the strain of flu involved and the level of immunity in the host population. During a severe epidemic, there are typically thousands more deaths than would normally be expected for that time of year, and these can be attributed to the disease. Although older people are usually most at risk from fatal disease, this is not always so. In the 1918 flu pandemic there was a surprisingly high death rate in people aged 20–40 and this was also the case for the 2009 ‘swine flu’ pandemic. Figure 3 Mortality according to age in the 1918 USA flu epidemic: 1917 (red) and 1918 (blue) rates in males and females. This epidemic was notable because it particularly affected people aged 20–40 year Older people are often most severely affected during infectious disease outbreaks because they may have a less effective immune response than younger people, or a reduced capacity to repair and regenerate tissue damaged by the infection. However, there are circumstances where older people may be more resistant to infection than younger people because they may have already encountered the disease (in their youth) and could retain some immunity and so be less susceptible than younger people who have not encountered the disease before. 1.3 Influenza infection in other species Influenza viruses infect a wide range of species, including pigs, horses, ducks, chickens and seals. In most of these other species the virus produces an acute infection. For example, in most of the mammals the symptoms are very similar to those in humans: an acute infection of the respiratory tract, which is controlled by the immune response although fatal infections occur in some species. However, in wild ducks and other aquatic birds the virus primarily infects the gut and the birds do not appear to have any physical symptoms. Despite this, ducks may remain infected for 2–4 weeks and during this time they shed virus in their faeces. Potentially this is a very important reservoir of infection; although flu viruses do not often cross the species barrier, the pool of viruses present in other species is an important genetic reservoir for the generation of new flu viruses that do infect humans. This reservoir becomes particularly important in certain farming communities or in crowded conditions where animals (especially pigs and ducks) are continuously in close proximity with humans (Branswell, 2010). Although such conditions occur in many agricultural communities throughout the world, they are typically observed in South-East and East Asia thereby contributing to these geographical areas often being the source of radically new ‘hybrid’ strains of influenza that incorporate genes from different species-specific strains. (The genetics of influenza are discussed in Section 2.3.) When strategies for controlling a disease are considered, awareness of the possible presence of an animal reservoir of infection is very important. For example, an immunisation programme against flu would substantially reduce the incidence of the current strain in humans but, because there is always a reservoir of these viruses in other animals, and these viruses are constantly mutating, another strain would inevitably emerge and be unaffected by immunisation. It is useful to distinguish diseases such as rabies, which primarily affect other vertebrates and occasionally infect humans (zoonoses), from diseases such as flu where different strains of the virus can affect several species including humans. Identify a fundamental difference between the way that zoonoses (e.g. rabies) are transmitted, and the way in which flu is transmitted. Reveal answer Flu can be transmitted from one human being to another, whereas most zoonoses, including rabies, are not transmitted between people. 2 Influenza viruses Viruses have very diverse genomes. Whereas the genomes of bacteria, plants and animals are of double-stranded DNA, the genomes of viruses can be constituted from either DNA or RNA and may be double- or single-stranded molecules. Usually, DNA is a double-stranded molecule with paired, complementary strands (dsDNA) and RNA is a single stranded molecule (ssRNA). However, some viruses have single-stranded DNA genomes (ssDNA) and some have double-stranded RNA genomes (dsRNA). The type of nucleic acid found in the genome depends on the group of viruses involved. RNA encodes protein in all living things, and the sequence of bases in the RNA determines the sequence of amino acids in the protein. A strand of RNA which has the potential to encode protein is said to be ‘positive sense’ (+). If a strand of RNA is complementary to this, then it is ‘negative sense’ (–). Negative-sense RNA must first be copied to a complementary positive-sense strand of RNA before it can be translated into protein. The description of the influenza genome as negative-sense ssRNA means that its RNA cannot be translated without copying first. This copying is performed by influenza’s viral RNA polymerase, a small amount of which is packaged with the virus, ready to begin copying the viral genome once it enters a host cell. Viral RNA polymerase consists of three subunits: PB1, PB2, and PA, encoded separately by the first three viral RNA strands. Understanding the way in which different viruses replicate is important, since it allows the identification of particular points in their life-cycle that may be susceptible to treatment with antiviral drugs. Classification Viruses are classified into different families, groups and subgroups in much the same way as are species of animals or plants. As you have already read, the influenza viruses are (–)ssRNA organisms (Baltimore group V) and belong to a family called the Orthomyxoviruses . They fall into three groups: influenza A, B and C. Type A viruses are able to infect a wide variety of endothermic (warm-blooded) animals, including mammals and birds, and analysis of their viral genome indicates that all strains of influenza A originated from aquatic birds. By contrast, types B and C are mostly confined to humans. At any one time, a number of different strains of virus may be circulating in the human population. Families, groups and strains of virus Viruses were originally classified into different groups according to similarities in their structure, mode of replication and disease symptoms. For example, the Orthomyxoviruses include viruses that cause different types of influenza, while Paramyxoviruses include the viruses that cause measles and mumps. Such large groupings are often called a family of viruses. The families can be subdivided into smaller groups, such as influenza A, B and C. Even within a single such group of viruses there can be an enormous level of genetic diversity, and this is the basis of the different strains. As an example, two HIV particles from the same individual may be 4% different in their genome; compare this with the 1% difference between the genomes of humans and chimpanzees, which are different species. 2.1 Structure of influenza The structure of influenza A is shown schematically in Figure 4. The viral genomic RNA, which consists of 8 separate strands , is enclosed by its associated nucleoproteins to make a ribonucleoprotein complex (RNP), and this is contained in the central core of the virus (the capsid). The nucleoproteins are required for viral replication and packing of the genome into the new capsid, which is formed by M1-protein (or matrix protein). The M1-protein is the most abundant component of the virus, constituting about 40% of the viral mass; it is essential for the structural integrity of the virus and to control assembly of the virus. Figure 4 Structure of an influenza A virus. The capsid, formed by M1-protein, also contains the viral genome and a number of enzymes required for viral replication. The viral envelope is a lipid bilayer formed from the plasma membrane of the host cell, which contains two virus-encoded proteins, haemagglutinin and neuraminidase. Orthomyxoviruses have a capsid surrounded by a phospholipid bilayer derived from the plasma membrane of the cell that produced the virus. This layer is the virus’s envelope. Two proteins, haemagglutinin and neuraminidase, are found on the viral envelope. These proteins are encoded by the viral HA and NA genes respectively and are inserted into the plasma membrane of the infected cell before the newly-produced viruses bud off from the cell surface. The haemagglutinin can bind to glycophorin, a type of polysaccharide that contains sialic acid residues, and which is present on the surface of a variety of host cells. The virus uses the haemagglutinin to attach to the host cells that it will infect. Antibodies and drugs against haemagglutinin are therefore particularly important in limiting the spread of the virus, since they prevent it from attaching to new host cells. Neuraminidase is an enzyme that cleaves sialic acid residues from polysaccharides. It has a role in clearing a path to the surface of the target cell before infection, namely, digesting the components of mucus surrounding epithelial cells in the respiratory system. Similarly, neuraminidase also promotes release of the budding virus from the cell surface after infection. The structures of influenza B and influenza C are broadly similar to that of Type A, although in influenza C the functions of the haemagglutinin and the neuraminidase are combined in a single molecule, haemagglutinin esterase. This molecule binds and cleaves a less common type of sialic acid. Influenza C does not normally cause clinical disease or epidemics, so the following discussion is confined to influenza A and B. 2.2 Designation of strains of influenza A considerable number of genetically different strains of influenza A have been identified, and these are classified according to where they were first isolated and according to the type of haemagglutinin and neuraminidase they express. For example ‘A/Shandong/9/93(H3N2)’ is an influenza A virus isolated in the Shandong province of China in 1993 – the ninth isolate in that year – and it has haemagglutinin type 3 and neuraminidase type 2. At the start of the twenty-first century, the major circulating influenza A strains are H1N1 (‘swine flu’) and H3N2. At least 16 major variants of haemagglutinin and 9 variants of neuraminidase have been recognised, but to date most of these have only been found in birds. The designation for influenza B is similar, but omits the information on the surface molecules, for example: ‘B/Panama/45/90’. As you will see later, accurate identification of different strains of flu is crucial if we are to control epidemics by vaccination programmes. 2.3 Genomic diversity of influenza The genome of flu viruses consists of around 14 000 nucleotides of negative-sense single-stranded RNA. Compare this number to the approximately 3 billion nucleotides found in the human genome or the 150 billion nucleotides of the genome of the marbled lungfish (the largest genome known in vertebrates). The genome of influenza viruses is segmented, into 8 distinct fragments of RNA containing 11 genes and encoding approximately 14 proteins (see Table 1 below). This structure has significance for the spread of the virus and the severity of disease symptoms. Cases of influenza generally arise in two main ways: by provoking seasonal annual outbreaks or epidemics and, less commonly, through global pandemics. As you will see shortly, both of these phenomena occur as consequences of the fact that the virus uses RNA as its genetic template and that this RNA genome is segmented into discrete strands. Table 1 The genome of influenza virus. Note that a single RNA segment may encode for more than one protein due to alternative reading frames. Gene name RNA strand (segment number) Function(s) of protein encoded by this gene PB2 (polymerase basic 2) 1 A subunit of viral RNA polymerase involved in cleaving the cap structure of host cell mRNA and generating primers that are subverted for use in the synthesis of viral RNA. PB1 (polymerase basic 1) 2 Core subunit of viral RNA polymerase. Required for polymerase assembly. PB1-F2 2 Binds to components of the host mitochondria, sensitising the cell to apoptosis and contributing to pathogenicity. PA (polymerase acidic) 3 A subunit of viral RNA polymerase which also has protease activity of unknown function. HA (haemagglutinin) 4 Antigenic glycoprotein used for binding to (infecting) the host cell. NP (nucleoprotein) 5 RNase resistant protein. Binds viral genomic RNA to form stable ribonucleoproteins and targets these for export from the host nucleus into the cytosol. Also involved in viral genome packaging and viral assembly. NA (neuraminidase) 6 Cleaves sialic acid. Important for releasing viral particles from host cell. M1 (matrix 1) 7 Binds viral genomic RNA and forms a coat inside the viral envelope in virions. Inside the host cell, it starts forming a layer under patches of the membrane rich in viral HA, NA, and M2 and so facilitates viral assembly and budding from the host cell. M2 (matrix 2) 7 Transmembrane ion channel protein. Allows protons into the virus capsid, acidifying the interior, destabilising binding of M1 to the viral genomic RNA which leads to uncoating of the viral particle inside the host cell. NS1 (non-structural 1) 8 Inhibits nuclear export of the host’s own mRNA, thereby giving preference to viral genomic RNA. Blocks the expression of some host inflammatory mediators (interferons) and interferes with T cell activation*. NS2/NEP (non-structural 2/ nuclear export protein) 8 Mediates the export of viral genomic RNA from the host nucleus to the cytoplasm. * Interferons and T cells are involved in the immune response to pathogens. The influenza virus is a successful pathogen because it is constantly changing. How might having a segmented genome promote the evolution of new strains of influenza virus? answer: If a cell is infected with more than one strain of virus at the same time, then a new strain can be generated simply by mixing RNA strands from different viruses. 2.3.1 Creation of new viral strains Part of the success of influenza as a pathogen is because segmented genome improves the virus’s potential to evolve into new strains through the combination of different RNA stands. This mixing of the genetic material from different viral strains to produce a new strain is termed genetic reassortment. For instance, the virus that caused the 2009 H1N1 ‘swine flu’ pandemic comprises a quadruple reassortment of RNA strands from two swine virus, one avian virus, and one human influenza virus: the surface HA and NA proteins derive from two different swine influenzas (H1 from a North American swine influenza and N1 from a European swine influenza) the three components of the RNA polymerase derive from avian and human influenzas (PA and PB2 from the avian source, PB1 from the human 1993 H3N2 strain) the remaining internal proteins derive from the two swine influenzas (MacKenzie, 2009). This does not necessarily mean that all four viruses infected the same animal at once. The new strain was likely the result of a reassortment of two swine influenza viruses, one from North America and one from Europe. The North American virus may itself have been the product of previous reassortments, containing a human PB1 gene since 1993 and an avian PA and PB2 genes since 2001. The presence of avian influenza RNA polymerase genes in this virus was especially worrying, since the avian polymerase is thought to be more efficient than human or swine versions, allowing the virus to replicate faster and thus making it more virulent. Similar avian RNA polymerase genes are what make H5N1 bird flu extremely virulent in mammals and what made the 1918 human pandemic virus so lethal in people. This mixing of genes from two or more viruses (whether from the same host species or from different species) can cause major changes in the antigenic surface proteins of a virus, such that it is no longer recognised by the host’s immune system. This antigenic shift is described in more detail in Section 3 (specifically, Box 2). In contrast to the major genetic changes caused by reassortment, influenza viruses also undergo constant, gradual, genetic changes due to errors made by their RNA polymerases. 2.4 Infection and replication Influenza RNA polymerase lacks the ability to recognise and repair any errors that occur during genome duplication, resulting in mistakes in copying its viral RNA about once in every 10 000 nucleotides. Because the influenza genome only contains approximately 14 000 nucleotides, this means that, on average, each new virus produced differs by 1 or 2 nucleotides from its ‘parent’. The slow accumulation of random genetic changes, especially in the antigenic surface proteins, explains why antibodies that were effective against the virus one year may be less effective against it in subsequent years. This gradual change in the nature of viral antigens is known as antigenic drift. The replication cycle of influenza is illustrated in Figure 5 Figure 5 Replication cycle of a flu virus. Long description Influenza is spread in aerosol droplets that contain virus particles (or by desiccated viral nuclei droplets), and infection may occur if these come into contact with the respiratory tract. Viral neuraminidase cleaves polysaccharides in the protective mucus coating the tract, which allows the virus to reach the surface of the respiratory epithelium. The haemagglutinin now attaches to glycophorins (sialic-acid-containing glycoproteins) on the surface of the host cell, and the virus is taken up by endocytosis into a phagosome. Acidic lysosomes fuse with the phagosome to form a phagolysosome and the pH inside the phagolysosome falls. This promotes fusion of the viral envelope with the membrane of the phagolysosome, triggering uncoating of the viral capsid and release of viral RNA and nucleoproteins into the cytosol. The viral genomic RNA then migrates to the nucleus where replication of the viral genome and transcription of viral mRNA occur. These processes require both host and viral enzymes. The viral negative-stranded RNA is replicated by the viral RNA-dependent RNA polymerase, into a positive-sense complementary RNA (cRNA), and these positive and negative RNA strands associate to form double-stranded RNA (dsRNA). The cRNA strand is subsequently replicated again to produce new viral genomic negative-stranded RNA. Some of the cRNA is also processed into mRNA for translation of viral proteins. The infection cycle is rapid and viral molecules can be detected inside the host cell within an hour of the initial infection. The envelope glycoproteins (haemagglutinin and neuraminidase) are translated in the endoplasmic reticulum, processed and transported to the cell’s plasma membrane. The viral capsid is assembled within the nucleus of the infected cell. The capsid moves to the plasma membrane, where it buds off, taking a segment of membrane containing the haemagglutinin and neuraminidase, and this forms the new viral envelope. Influenza virus budding from the surface of an infected cell is shown in Figure 6. Figure 6 Flu virus particles budding from the surface of an infected cell. Long description From the description above, identify a process or element in the replication cycle which is characteristic of the virus, and which would not normally occur in a mammalian cell. Answer: The replication of RNA on an RNA template with the production of double-stranded RNA would never normally occur in a mammalian cell. Double stranded RNA is therefore a signature of a viral infection. Significantly, cells have a way of detecting the presence of dsRNA, and this activates interferons: molecules involved in limiting viral replication. 2.5 Cellular pathology of influenza infection Flu viruses can infect a number of different cell types from different species. This phenomenon is partly because the cellular glycoproteins which are recognised by viral haemagglutinin are widely distributed in the infectious agent. What is the term for the property of viruses that allows them to only replicate in particular cell types? Reveal answer A second reason why the virus can infect a variety of cell types is that the replication strategy of flu is relatively simple: ‘infect the cell, replicate as quickly as possible and then get out again’. This is the cytopathic effect of the virus. Cell death caused directly by the virus can be distinguished from cell death caused by the actions of the immune system as it eliminates infected cells. The effects of cell death Cell death impairs the function of an infected organ and often induces inflammation, a process that brings white cells (leukocytes) and molecules of the immune system to the site of infection. In the first instance, the leukocytes are involved in limiting the spread of infection; later they become involved in combating the infection, and in the final phase they clear cellular debris so that the tissue can repair or regenerate. The symptoms of flu experienced by an infected person are partly due to the cytopathic effect of the virus, partly due to inflammation and partly a result of the innate immune response against the virus. The severity of the disease largely depends on the rate at which these processes occur. In most instances, the immune response develops sufficiently quickly to control the infection and patients recover. If viral replication and damage outstrip the development of the immune response then a fatal infection can occur. In severe flu infections, the lungs may fill with fluid as the epithelium lining the alveoli (air sacs) is damaged by the virus. The fluid is ideal for the growth of bacteria, and this can lead to a bacterial pneumonia, in which the lungs become infected with one or more types of bacteria such as Haemophilus influenzae. Damage to cells lining blood vessels can cause local bleeding into the tissues, and this form of ‘fulminating disease’ was regularly seen in post-mortem lung tissues of people who died in the 1918 pandemic. 3 Patterns of disease In humans, pigs and horses, flu viruses circulate through populations at regular intervals. The disease is endemic in tropical regions for all of these host groups (i.e. it is continually present in the community). In temperate latitudes, infections are usually seasonal or epidemic, with the greatest numbers occurring in the winter months (Figure 7). Epidemics also occur sporadically in sea mammals and poultry, and in these species high mortality is typical. Figure 7 Epidemic patterns of flu in temperate latitudes. The graph shows notifications of flu in the USA each week from 1994–1997. Long description In most years, flu in humans affects a minority of the population, the disease course is not very severe and the level of mortality is not great. In such years the influenza virus is slightly different from the previous year due to antigenic drift, which results in the accumulation of genetic mutations that cause the molecules present on the surface of the virus change progressively. In this scenario, the virus is not significantly different from the previous year so that the host’s immune system can more easily mount an effective response than it could to a completely new strain. However, at irregular intervals the virus undergoes an antigenic shift. This process only occurs in influenza A viruses, typically every 10–30 years, and it is associated with severe pandemics, serious disease and high mortality (see Box 2). In Section 2.3 you read that strains of influenza are differentiated and designated using a simple system of numbers and letters that depend on their surface antigens. More commonly, however, strains responsible for pandemics are often given a common name according to the area in the world from which they were thought to originate, or the species they mainly affected before becoming transferred to humans (see Table 2). Evidence suggests, however, that in the twentieth century the major flu pandemics all originated in China, with the exception of the 1918 pandemic, which first occurred in the USA. Major flu pandemic strains of the twentieth century. Year Designation Common name 1900 H3N8 (none) 1918 H1N1 Spanish flu西班牙 1957 H2N2 Asian flu亞洲 1968 H3N2 Hong Kong flu香港 1977 H1N1 Russian flu蘇聯 1997 H5N1 Avian flu禽 The rise of H5N1 – an example of antigenic shift In 1997, a new strain of influenza A, H5N1, was identified in Hong Kong. The strain was rife in chickens and a few hundred people had become infected. Mortality in these individuals was very high, (6 of 18 died), and so there was serious concern that it marked the beginning of a new pandemic. The authorities in Hong Kong responded by a mass cull of poultry in the region and about 1.5 million chickens were slaughtered. H5N1 did not spread easily from person to person and no further cases were reported in people following the slaughter. Whether the H5N1 outbreak was an isolated incident of a strain spreading from chicken to humans, or whether it was the start of a major pandemic which was nipped in the bud, cannot be known. Subsequent analysis showed that the high virulence of the new strain could partly be related to the new variant of haemagglutinin (H5), and partly to a more efficient viral polymerase. This outbreak clearly demonstrates the way in which bird influenza can act as a source of new viral strains, and shows that such new strains may be very dangerous to humans. Since the discovery of the influenza virus in the 1930s it has been possible to isolate and accurately identify each of the epidemic strains, but, as earlier strains of virus have now died out, it has been necessary to infer their identity by examining the antibodies in the serum of affected people. Antibodies and the ability of the immune system to respond to a strain of flu are much more persistent than the virus itself. It is thus possible to analyse antibodies to determine which types of haemagglutinin and neuraminidase they recognise long after the virus itself has gone. One can then deduce which type of influenza virus that person contracted earlier in their life (as explained in Section 5.2). 3.1 Tracking the emergence of new strains Influenza is one of several diseases monitored by the WHO Global Alert and Response   (GAR) network (WHO, 2011a), comprising 110 ‘sentinel’ laboratories in 82 countries. The organisation’s surveillance and monitoring of the disease then forms part of their Global Influenza Programme (GIP), and they use data gathered from participating countries to: provide countries, areas and territories with information about influenza transmission in other parts of the world to allow national policy makers to better prepare for upcoming seasons provide data for decision making regarding recommendations for vaccination and treatment describe critical features of influenza epidemiology including risk groups, transmission characteristics, and impact monitor global trends in influenza transmission inform the selection of influenza strains for vaccine production (WHO, 2011b). The influenza data from the sentinel laboratories is fed into a global surveillance programme, started by the WHO in 1996, called FluNet (WHO, 2011c), which is one of the tools that facilitates the actions described above. Typically a flu vaccine contains material from the main influenza A strains and an influenza B strain, so that an immune response is induced against the most likely infections. Usually the scientists predict correctly and immunised people are effectively protected against the current strains (>90% protection). However, the prediction is occasionally incorrect, or a new strain develops during the time that the vaccine is being manufactured. In this case the vaccine generally provides poor protection. What can you deduce about immunity against flu infection from the observations on vaccination above? answer The immune response is strain-specific. If you are immunised against the wrong strain of flu, then the response is much less effective and you are more likely to contract the disease. 3.2 Immune responses to influenza The immune system uses different types of immune defence against different types of pathogen. The responses against flu are typical of those which are mounted against an acute viral infection, but different from the responses against infection by bacteria, worms, fungi or protist parasites. When confronted with an acute viral infection, the immune system has two major challenges: The virus replicates very rapidly, killing the cells it infects. Since a specific immune response takes several days to develop, the body must limit the spread of the virus until the immune defences can come into play. Viruses replicate inside cells of the body, but they spread throughout the host in the blood and tissue fluids. Therefore, the immune defences must recognise infected cells (intracellular virus) and destroy them. But the immune system must also recognise and eradicate free virus in the tissue fluids (extracellular virus) in order to prevent the virus from infecting new cells.. 3.2.1 Summary of the response How does the body act quickly to limit viral spread? When a virus infects a cell of the body, the molecular machinery for protein synthesis within the cell is usurped as the virus starts to produce its own nucleic acids and proteins. The cell detects the flu dsRNA and other viral molecules and releases interferons, which bind to receptors on neighbouring cells and cause them to synthesise antiviral proteins. If a virus infects such cells they resist viral replication, so fewer viruses are produced and viral spread is delayed. Also, in the earliest stages of a virus infection the molecules on the cell surface change. Cells lose molecules that identify them as normal ‘self’ cells. At the same time they acquire new molecules encoded by the virus. A group of large, granular lymphocytes recognise these changes and are able to kill the infected cell. This function is called ‘natural killer’ cell action and the lymphocytes that carry it out are termed NK cells. Non-adaptive and adaptive responses The actions of both interferons and NK cells in combating infection by influenza occur early in an immune response, and are not specific for the flu virus. These defences occur in response to many different kinds of viral infection, and they are part of our natural, or non-adaptive, immune responses. Note that immunologists use the term non-adaptive to indicate a type of response that does not improve or adapt with each subsequent infection. This is quite different to its use in evolutionary biology, where it means ‘not advantageous’. Such non-adaptive immune responses slow the spread of an infection so that specific, or adaptive, immune defences can come into play. The key features of an adaptive immune response are specificity and memory. The immune response is specific to a particular pathogen, and the immune system appears to ‘remember’ the infection, so that if it occurs again the immune response is much more powerful and rapid. Because an immune response is highly specific to a particular pathogen it often means that a response against one strain of virus is ineffective against another – if a virus mutates then the lymphocytes that mediate adaptive immunity are unable to recognise the new strain. There are two principal arms of the adaptive immune system, mediated by different populations of lymphocytes. One group, called T-lymphocytes, or T cells (which develop in the thymus gland, overlying the heart), recognises antigen fragments associated with cells of the body, including cells which have become infected. A set of cytotoxic T cells (Tc) specifically recognises cells which have become infected and will go on to kill them. In this sense they act in a similar way to NK cells. However they differ from NK cells in that Tc cells are specific for one antigen or infectious agent, whereas NK cells are non-specific. The second group of lymphocytes are B cells (that differentiate in the bone marrow), which synthesise antibodies that recognise intact antigens, either in body fluids or on the surface of other cells. Activated B cells progress to produce a secreted form of their own surface antibody. Antibodies that recognise the free virus act to target it for uptake and destruction by phagocytic cells. Therefore the T cells and NK cells deal with the intracellular phase of the viral infection, while the B cells and antibodies recognise and deal with the extracellular virus. The two reactions described above are illustrated in Figure 8. Figure 8 Immune defences against flu viruses. Antibodies can block the spread of the virus by preventing them from attaching to host cells. Infected cells release interferon, which signals to neighbouring cells to induce resistance. Natural killer cells (NK) and cytotoxic T cells (Tc) recognise and kill virally-infected cells. Long description You might ask why it takes the adaptive immune response so long to get going. The answer is that the number of T cells and B cells that recognise any specific pathogen is relatively small, so first the lymphocytes which specifically recognise the virus must divide so that there are sufficient to mount an effective immune response. This mechanism is fundamental to all adaptive immune responses. How do pandemic strains of influenza A come about? Answer Question 2 Pandemic strains of influenza A normally arise by simultaneous infection of a non-human host (typically poultry or pigs) with two or more strains of influenza A. Reassortment of the eight viral segments from each virus allows the generation of a new hybrid virus type, with a completely novel surface structure that has never been seen before by a host immune system (antigenic shift). 4 Antiviral treatments Two classes of antiviral drugs are used to combat influenza: neuraminidase inhibitors and M2 protein inhibitors. Why are antibiotics not used to combat influenza? Reveal answer Neuraminidase inhibitors neuraminidase is an enzyme that is present on the virus envelope and cleaves sialic acid groups found in the polysaccharide coating of many cells (especially the mucus coating of the respiratory tract). Neuraminidase is used to clear a path for the virus to a host cell and facilitates the shedding of virions from an infected cell. Inhibition of neuraminidase therefore helps prevent the spread of virus within a host and its shedding to infect other hosts. The two main neuraminidase inhibitors currently in clinical use are zanamivir (trade name Relenza) and oseltamivir (trade name Tamiflu). These are effective against influenza A and B, but not influenza C which exhibits a different type of neuraminidase activity that only cleaves 9-O-acetylated sialic acid. M2 inhibitors Recall from Table 1 that the influenza M2 protein forms a pore that allows protons into the capsid, acidifying the interior and facilitating uncoating. Drugs such as amantadine (trade name Symmetrel) and rimantadine (trade name Flumadine) block this pore, preventing uncoating and infection. However, their indiscriminate use in ‘over-the-counter’ cold remedies and farmed poultry has allowed many strains of influenza to develop resistance. Influenza B has a different type of M2 protein which is largely unaffected by these drugs. 5 Diagnosis of influenza Many diseases produce symptoms similar to those of influenza; in fact, ‘flu-like’ is a term that is frequently used to describe several different illnesses. Since influenza spreads rapidly by airborne transmission and is a life-threatening condition in certain vulnerable groups, it is important that cases of the disease are identified as quickly as possible, so that preventative measures may be taken. Most viral infections are not treated, although antiviral drugs such as zanamivir are used for potentially life-threatening cases or where the risk of transmission is high (as occurs during a pandemic). Which sites in the body should be sampled for diagnosis? answer The influenza virus infects the respiratory tract and is spread by coughing and sneezing, so specimens should be taken from the nose, throat or trachea. In practice, the best specimens are nasal aspirates or washes, but swabs of the nose or throat may be used if they are taken vigorously enough to obtain cells. Ideally, samples should be taken within three days of the onset of illness, and all specimens need to be preserved in a transport medium and kept chilled until they reach the clinical microbiology laboratory. 5.1 Initial identification of influenza infection Oral swabs or nasal aspirates are initially screened for the presence of a variety of respiratory viruses. This is done by extracting RNA from the sample and subjecting it to a reverse-transcription polymerase chain reaction (RT-PCR) as described below: Initially the RNA sample is reverse transcribed into complementary DNA (cDNA), using a commercially-available reverse transcriptase enzyme. The cDNA is then used in a standard PCR reaction to detect and amplify a short sequence of nucleotides specific to the virus. Multiple DNA sequences, each specific for a different type of virus, can be amplified in the same reaction, provided that these sequences are of different lengths. Each of the different amplified sequences is separated from the others when the entire sample is subjected to gel electrophoresis (an analytical technique in which molecules of different sizes move at various rates through a gel support in an applied electric field, thus making it possible to identify specific molecules.) PCR technique. Typically, nucleotide sequences specific to 5 types of virus are searched for in each sample: influenza A, influenza B, respiratory syncytial virus (Baltimore group V, (–)ssRNA virus, and a major cause of respiratory illness in young children), adenoviruses and enteroviruses. Those samples that test positive for influenza in the RT-PCR reaction are inoculated into cells in culture. Sufficient virus for a limited number of tests can be produced from such cultures within 24 hours, but they are often maintained for up to a week. 5.2 Determining the subtype of influenza Immunofluorescence Confirmation of a case of influenza is usually achieved by performing tests on some of the inoculated cultured cells using reference fluorescent-labelled antisera provided by the WHO. A reference antiserum is a sample serum known to contain antibodies specific for the molecule to be assayed (in this case, haemagglutinin or neuraminidase). These antisera are prepared using purified haemagglutinin and neuraminidase and are monospecific, each antibody reacting only with one epitope e.g. H1 or H3. For the test, an antibody is added to a sample of inoculated cells. Following a wash step, if the antibody remains bound to the cells then they fluoresce under appropriate illumination, indicating the presence of viral antigen on the cell surface. This diagnostic technique can identify influenza virus on infected cells in as little as 15 minutes. A positive result not only confirms the RT-PCR data, but gives additional information on the subtype of the virus. Further PCR analyses Standardised RT-PCR protocols exist to look for the presence of different haemagglutinin and neuraminidase subtypes, chiefly H1, H3, H5, N1 and N2. (Poddar, 2002). If the PCR analysis indicates a dangerous strain of influenza A e.g. H5N1, then it is instantly sent to a WHO reference laboratory for further tests. Haemagglutination assays Influenza has haemagglutinins protruding from its viral envelope, which it uses to attach to host cells prior to entry. These substances form the basis of a haemagglutination assay, in which viral haemagglutinins bind and cross-link (agglutinate) red blood cells added to a test well, causing them to sink to the bottom of the solution as a mat of cells. If agglutination does not occur, then the red blood cells are instead free to roll down the curved sides of the tube to form a tight pellet. A related test called a haemagglutination-inhibition assay (HAI), incorporates antibodies against different subtypes of viral haemagglutinin. The antibodies bind and mask the viral haemagglutinin, preventing it from attaching to and cross-linking red blood cells. A HAI assay can be set up in one of two ways: either a known reference antibody is added to an unknown virus sample, or known reference viral haemagglutinin is added to a sample of patient serum containing antibodies against influenza. This second version of the HAI assay can therefore be used long after the infection has passed, when virions are no longer present. Haemagglutination and HAI assays have the advantage that they are simple to perform and require relatively cheap equipment and reagents. However, they can be prone to false positive or false negative results, if the sample contains non-specific inhibitors of haemagglutination (preventing agglutination) or naturally occurring agglutinins of red blood cells (causing agglutination). If a confirmed influenza A isolate reacts weakly or not at all in HAI then this indicates an unknown variant of influenza A and the sample is immediately sent to a WHO reference laboratory for further tests. Neuraminidase inhibition assay Typing influenza isolates in terms of their neuraminidase makes use of the enzyme activity of this glycoprotein. The neuraminidase inhibition assay is performed in two parts. The first part determines the amount of neuraminidase activity in a patient influenza sample, as outlined in Figure 9a. A substrate (called fetuin) that is rich in sialic acid residues is added to a sample of the influenza virus, and the viral neuraminidase enzyme cleaves the substrate to produce free sialic acid. Addition of a substance that inactivates the neuraminidase stops the reaction, and a chromogen (a colourless compound that reacts to produce a coloured end-product) that turns pink in the presence of free sialic acid is added. The intensity of the pink colour is proportional to the amount of free sialic acid and can be measured using a spectrophotometer. This assay of neuraminidase activity allows the appropriate amount of virus sample to be determined, and this quantity is then used in the second part of the assay. If too much or too little virus is used, the resulting changes, and therefore the neuraminidase, may be undetectable. In the second part of the assay (Figure 9b), viral samples from the patient are incubated with anti-neuraminidase reference antisera. Each of the reference antisera used for this test has antibodies that bind one particular neuraminidase variant, e.g. N1 or N2. How can these antisera be used to type the neuraminidase variant? Figure 9 The neuraminidase inhibition assay. (a) Assay of neuraminidase activity. (b) The inhibition of the assay itself. 6 Summary of the unit . A single pathogen can produce different types of disease in different people. Genetic variation in a pathogen can also affect the type of disease it produces. To understand this we need to know something of the genetic and social differences in the host population, and of the diversity of the pathogen. The symptoms of a particular disease may be produced by different pathogens or by a combination of pathogens. To understand this requires some knowledge of pathology and cell biology. Some diseases, such as flu, affect humans and several other animal species, whereas others are more selective in their host range. The basic biology of different pathogens underlies these differences. Flu is a disease that can be contracted several times during a lifetime, but many other infectious diseases are only ever contracted once. To understand this we need to look at how the immune system reacts to different pathogens, and how responses vary depending on the pathogen. Outbreaks of flu occur regularly, but some epidemics are much more serious than others. This requires an understanding of aspects of virology, immunology, evolutionary biology and epidemiology. In 1892, German bacteriologist Richard Pfeiffer isolated what he thought was the causative agent of influenza. The culprit, according to Pfeiffer, was a small rod-shaped bacterium that he isolated from the noses of flu-infected patients (1). He dubbed it Bacillus influenzae (or Pfeiffer's bacillus). Few doubted the validity of this discovery, in large part because bacteria had been shown to cause other human diseases, including anthrax, cholera, and plague. Figure 1 Richard Shope, 1936. The filtration question When history's deadliest influenza pandemic began in 1918, most scientists believed that Pfeiffer's bacillus caused influenza. With the lethality of this outbreak (which killed an estimated 20 to 100 million worldwide) came urgency—researchers around the world began to search for Pfeiffer's bacillus in patients, hoping to develop antisera and vaccines that would protect against infection. In many patients, but not all, the bacteria were found. Failures to isolate B. influenzae (now known as Haemophilus influenzae) were largely chalked up to inadequate technique, as the bacteria were notoriously difficult to culture (2). The first potential blow to Pfeiffer's theory came from Peter Olitsky and Frederick Gates at The Rockefeller Institute. Olitsky and Gates took nasal secretions from patients infected with the 1918 flu and passed them through Berkefeld filters, which exclude bacteria. The infectious agent—which caused lung disease in rabits—passed through the filter, suggesting that it was not a bacterium (3, 4). Although the duo had perhaps isolated the influenza virus (which they nevertheless referred to as an atypical bacterium called Bacterium pneumosintes), other researchers could not reproduce their results. One of the doubters was Oswald Avery (Rockefeller Institute), who developed a culture media—chocolate agar—that optimized the growing conditions for B. influenzae and thus minimized false negative results from patient samples. Thus, the idea that flu was transmitted by a filterable agent (or virus) was dismissed. Insights from pigs Olitsky and Gates would not be vindicated until a decade later, when Shope—a young physician from Iowa then working on hog cholera at the Rockefeller Institute—turned his attention to swine influenza. Pig farmers in Iowa had reported two outbreaks—one in 1918 and another in 1929—of a highly contagious, influenza-like disease among their animals. The disease bore such a remarkable resemblance to human flu that it was named swine influenza. Shope and his mentor Paul Lewis took mucus and lung samples from the infected pigs and attempted to isolate the disease-causing agent. They quickly isolated a bacterium that looked exactly like Pfeiffer's human bacterium (and was thus called B. influenzae suis), but when they injected the bacteria into pigs, it caused no disease (5). Shope then filtered the samples and, like Olitsky and Gates, found that the filtrate contained the infectious agent. Shope's filtrate caused a highly contagious, influenza-like disease in pigs—albeit a more mild one than seen in naturally-infected pigs. Mixing the filtrate with the bacterium reproduced the severe disease. He concluded—correctly—that the filterable agent caused the infection, which then facilitated secondary infection with the bacterium (6). Shope published his results in a series of papers in The Journal of Experimental Medicine (5, 6). Using Shope's technique, Wilson Smith, Christopher Andrewes, and Patrick Laidlaw (National Institute for Medical Research, UK) soon isolated the virus from humans (7), laying to rest any lingering doubts about the nature of the flu-inducing agent. Both Shope and the British trio later demonstrated that sera from humans that were infected with the 1918 flu virus could neutralize the pig virus, leading them to conclude that the swine virus was a surviving form of the 1918 human pandemic virus (8, 9). In fact, a related strain of flu still circulates among pigs today.

Influenza is a myxovirus belonging to the family of viruses known as Orthomyxoviridae. The virus was originally confined to aquatic birds, but it made the transition to humans 6000–9000 years ago, coinciding with the rise of farming, animal husbandry and urbanisation.流感是一種黏液病毒屬於正黏液病毒屬（family),本只局限在水禽、但在六至九千年前傳播至人類，正好和農耕、畜牧及都市化的時間點吻合。 These changes in human behavior and population density provided the ecological niche that enabled influenza, as well as a number of other infectious agents such as the viruses that cause measles and smallpox, to move from animals and adapt to a human host.人類行為的改變、人口的集中提供病毒、像流感、麻疹、天花、從動物變成可適應、感染人類。 Influenza as a disease has been recognised for centuries, even though the viruses which cause it were not correctly identified until the early 1930s, first in the UK and then in the USA. 流感數世紀以來廣為認知，但真正證實是病毒致病則是在1930年代的英國。Indeed the name itself is derived from an Italian word meaning ‘influence’, and reflected the widespread belief in medieval times that the disease was caused by an evil climatic influence due to an unfortunate alignment of the stars.流感英文為influenza,是源自義大利文，意思是影響、在中古世紀認為是因星球不正常的排列、造成惡劣氣候的影響。 Our current understanding – that infectious diseases are caused by infectious agents – is so ingrained that such mystical causes for an illness now seem absurd.現在看來這種想法當然荒謬。 However, even during the Middle Ages, people had a sound idea of infection and realised that some diseases could be passed from one individual to another and others could not. For example, the use of quarantine for a disease such as plague, but not for many other illnesses, shows that people could distinguish infectious diseases from non-infectious diseases even if the causative agent and the method of transmission were obscure.但即使在中古世紀、人們也認知到有些疾病會一個傳一個 The idea that influenza is caused by the influence of the stars, though not a satisfactory explanation of how the disease spread, does identify an important feature of flu – that serious epidemics of the disease occur at irregular intervals. For example, in the twentieth century there were at least five major epidemics of flu that spread around the world (a pandemic), and there were less serious epidemics in most years. In times when people believed in the spontaneous generation of life, the stars would have seemed a reasonable explanation for unpleasant and unexpected epidemics. 1.1 Defining influenza How would you define ‘influenza’? Reveal answer You may well have defined influenza as an infection caused by an influenza virus. However, you may have defined it according to its symptoms: an infection that starts in the upper respiratory tract, with coughing and sneezing, spreads to give aching joints and muscles, and produces a fever that makes you feel awful; but usually it has gone in 5–10 days and most people make a full recovery. The first answer here is the biological definition and, in the Open University course SK320, diseases are defined according to the infectious agent which produces them. This is because different infections can produce the same symptoms, and the same infectious agent can produce quite different symptoms in different people, depending on their age, genetic make-up or the tissue of the body that becomes infected. Here a distinction is made between the infectious disease caused by a particular agent and the disease symptoms. Unfortunately there is a lot of confusion in common parlance about different diseases. Often, people say that they have ‘a bit of flu’ when they have an infection with some other virus, or a bacterium that produces flu-like symptoms. Such loose terminology is understandable, since most people are firstly concerned with the symptoms of their disease. But to treat and control disease requires accurate identification of the causative agent, so this is the starting point for considering any infectious disease. Attributing cause to a disease The difficulties encountered in assigning a particular pathogen to a disease are well-illustrated by influenza. During the influenza pandemic that occurred in 1890, the microbiologist Pfeiffer isolated a novel bacterium from the lungs of people who had died of flu. The bacterium was named Haemophilus influenzae and since it was the only bacterium that could be regularly cultivated from these individuals at autopsy, it was assumed that H. influenzae was the causative agent of flu. Again, in the 1918 flu pandemic, the bacterium could be regularly cultivated from people who had died of flu with pneumonia. So it was thought that flu was caused by the bacterium, and H. influenzae came to be called the ‘influenza bacillus’. The role of H. influenzae was only brought into question in the early 1930s, when Smith, Andrews and Laidlaw showed that it was possible to transfer a flu-like illness from the nasal washings of an infected person to ferrets, using a bacteria-free filtrate. These studies demonstrated that the pathogen was in fact much smaller than any known bacterium and paved the way to the identification of influenza viruses (Figure 1). Figure 1 Influenza viruses are small and use RNA as their genetic material. They have irregular shapes and the outer envelope (the dark-striped outer layer of each virus in this electron micrograph), which is derived from the plasma membrane of the host cell, contains viral proteins. Why do you suppose that H. influenzae was incorrectly identified as the causative agent of flu? Reveal answer The bacterium fulfils two of Koch’s postulates: it is regularly found in serious flu infections and it can be cultured in pure form on artificial media. Moreover, at that time no-one knew what a virus was, and everyone was thinking in terms of bacterial causes for infectious diseases. Although the precise role of H. influenzae in the 1890 and 1918 flu pandemics is not clear, it is likely that the bacteria were present and acting in concert with the flu virus to produce the pneumonia experienced. Such synergy between virus and bacteria was demonstrated by Shope in 1931. He infected pigs with a bacterial-free filtrate (containing swine influenza virus) with or without the bacteria, and showed that the disease produced by the bacteria and filtrate together was more severe than that produced by either one alone (Van Epps, 2006). In its role of co-pathogen, H. influenzae is only one of a number of bacteria that can exacerbate the viral infection. This highlights a very important point. In the tidy world of a microbiology or immunology laboratory, scientists typically examine the effect of one infectious agent in producing disease. In the real world, people often become infected with more than one pathogen. Indeed, infection with one agent often lays a person open to infection with another, as immune defences become overwhelmed. For this reason, a particular disease as seen by physicians may be due to a combination of pathogens. 1.2 Influenza infection in humans Influenza is an acute viral disease that affects the respiratory tract in humans. The virus is spread readily in aerosol droplets produced by coughing and sneezing, which are symptoms of the illness. Other symptoms include fatigue, muscle and joint pains and fever. Following infection, the influenza virus replicates in the cells lining the host’s upper and lower respiratory tract. Virus production peaks 1–2 days later, and virus particles are shed in secretions over the following 3–4 days. During this period, the patient is infectious and the symptoms are typically at their most severe. After one week, virus is no longer produced, although it is possible to detect viral antigens for up to 2 weeks. Immune responses are initiated immediately after the virus starts to replicate, and antibodies against the virus start to appear in the blood at 3–4 days post infection. These continue to increase over the following days and persist in the blood for many months. In a typical flu infection, the virus is completely eliminated from the host’s system within 2 weeks. This is sterile immunity: the virus cannot be obtained from the patient after recovery from the disease. Figure 2 Time course of a typical flu infection Production of the virus (purple bar) starts early after infection and is maximal within two days. The infection is contained during this period by various immune defences. Specific antibody production starts to appear by day 3 (yellow bar), and this contributes to the elimination of the virus. Symptoms (red bar) coincide with the production of the virus. Long description For infants, older people, and those with other underlying diseases (e.g. of the heart or respiratory system) an infection with flu may prove fatal. However, the severity of a flu epidemic and the case fatality rate depend on the strain of flu involved and the level of immunity in the host population. During a severe epidemic, there are typically thousands more deaths than would normally be expected for that time of year, and these can be attributed to the disease. Although older people are usually most at risk from fatal disease, this is not always so. In the 1918 flu pandemic there was a surprisingly high death rate in people aged 20–40 and this was also the case for the 2009 ‘swine flu’ pandemic. Figure 3 Mortality according to age in the 1918 USA flu epidemic: 1917 (red) and 1918 (blue) rates in males and females. This epidemic was notable because it particularly affected people aged 20–40 year Older people are often most severely affected during infectious disease outbreaks because they may have a less effective immune response than younger people, or a reduced capacity to repair and regenerate tissue damaged by the infection. However, there are circumstances where older people may be more resistant to infection than younger people because they may have already encountered the disease (in their youth) and could retain some immunity and so be less susceptible than younger people who have not encountered the disease before. 1.3 Influenza infection in other species Influenza viruses infect a wide range of species, including pigs, horses, ducks, chickens and seals. In most of these other species the virus produces an acute infection. For example, in most of the mammals the symptoms are very similar to those in humans: an acute infection of the respiratory tract, which is controlled by the immune response although fatal infections occur in some species. However, in wild ducks and other aquatic birds the virus primarily infects the gut and the birds do not appear to have any physical symptoms. Despite this, ducks may remain infected for 2–4 weeks and during this time they shed virus in their faeces. Potentially this is a very important reservoir of infection; although flu viruses do not often cross the species barrier, the pool of viruses present in other species is an important genetic reservoir for the generation of new flu viruses that do infect humans. This reservoir becomes particularly important in certain farming communities or in crowded conditions where animals (especially pigs and ducks) are continuously in close proximity with humans (Branswell, 2010). Although such conditions occur in many agricultural communities throughout the world, they are typically observed in South-East and East Asia thereby contributing to these geographical areas often being the source of radically new ‘hybrid’ strains of influenza that incorporate genes from different species-specific strains. (The genetics of influenza are discussed in Section 2.3.) When strategies for controlling a disease are considered, awareness of the possible presence of an animal reservoir of infection is very important. For example, an immunisation programme against flu would substantially reduce the incidence of the current strain in humans but, because there is always a reservoir of these viruses in other animals, and these viruses are constantly mutating, another strain would inevitably emerge and be unaffected by immunisation. It is useful to distinguish diseases such as rabies, which primarily affect other vertebrates and occasionally infect humans (zoonoses), from diseases such as flu where different strains of the virus can affect several species including humans. Identify a fundamental difference between the way that zoonoses (e.g. rabies) are transmitted, and the way in which flu is transmitted. Reveal answer Flu can be transmitted from one human being to another, whereas most zoonoses, including rabies, are not transmitted between people. 2 Influenza viruses Viruses have very diverse genomes. Whereas the genomes of bacteria, plants and animals are of double-stranded DNA, the genomes of viruses can be constituted from either DNA or RNA and may be double- or single-stranded molecules. Usually, DNA is a double-stranded molecule with paired, complementary strands (dsDNA) and RNA is a single stranded molecule (ssRNA). However, some viruses have single-stranded DNA genomes (ssDNA) and some have double-stranded RNA genomes (dsRNA). The type of nucleic acid found in the genome depends on the group of viruses involved. RNA encodes protein in all living things, and the sequence of bases in the RNA determines the sequence of amino acids in the protein. A strand of RNA which has the potential to encode protein is said to be ‘positive sense’ (+). If a strand of RNA is complementary to this, then it is ‘negative sense’ (–). Negative-sense RNA must first be copied to a complementary positive-sense strand of RNA before it can be translated into protein. The description of the influenza genome as negative-sense ssRNA means that its RNA cannot be translated without copying first. This copying is performed by influenza’s viral RNA polymerase, a small amount of which is packaged with the virus, ready to begin copying the viral genome once it enters a host cell. Viral RNA polymerase consists of three subunits: PB1, PB2, and PA, encoded separately by the first three viral RNA strands. Understanding the way in which different viruses replicate is important, since it allows the identification of particular points in their life-cycle that may be susceptible to treatment with antiviral drugs. Classification Viruses are classified into different families, groups and subgroups in much the same way as are species of animals or plants. As you have already read, the influenza viruses are (–)ssRNA organisms (Baltimore group V) and belong to a family called the Orthomyxoviruses . They fall into three groups: influenza A, B and C. Type A viruses are able to infect a wide variety of endothermic (warm-blooded) animals, including mammals and birds, and analysis of their viral genome indicates that all strains of influenza A originated from aquatic birds. By contrast, types B and C are mostly confined to humans. At any one time, a number of different strains of virus may be circulating in the human population. Families, groups and strains of virus Viruses were originally classified into different groups according to similarities in their structure, mode of replication and disease symptoms. For example, the Orthomyxoviruses include viruses that cause different types of influenza, while Paramyxoviruses include the viruses that cause measles and mumps. Such large groupings are often called a family of viruses. The families can be subdivided into smaller groups, such as influenza A, B and C. Even within a single such group of viruses there can be an enormous level of genetic diversity, and this is the basis of the different strains. As an example, two HIV particles from the same individual may be 4% different in their genome; compare this with the 1% difference between the genomes of humans and chimpanzees, which are different species. 2.1 Structure of influenza The structure of influenza A is shown schematically in Figure 4. The viral genomic RNA, which consists of 8 separate strands , is enclosed by its associated nucleoproteins to make a ribonucleoprotein complex (RNP), and this is contained in the central core of the virus (the capsid). The nucleoproteins are required for viral replication and packing of the genome into the new capsid, which is formed by M1-protein (or matrix protein). The M1-protein is the most abundant component of the virus, constituting about 40% of the viral mass; it is essential for the structural integrity of the virus and to control assembly of the virus. Figure 4 Structure of an influenza A virus. The capsid, formed by M1-protein, also contains the viral genome and a number of enzymes required for viral replication. The viral envelope is a lipid bilayer formed from the plasma membrane of the host cell, which contains two virus-encoded proteins, haemagglutinin and neuraminidase. Orthomyxoviruses have a capsid surrounded by a phospholipid bilayer derived from the plasma membrane of the cell that produced the virus. This layer is the virus’s envelope. Two proteins, haemagglutinin and neuraminidase, are found on the viral envelope. These proteins are encoded by the viral HA and NA genes respectively and are inserted into the plasma membrane of the infected cell before the newly-produced viruses bud off from the cell surface. The haemagglutinin can bind to glycophorin, a type of polysaccharide that contains sialic acid residues, and which is present on the surface of a variety of host cells. The virus uses the haemagglutinin to attach to the host cells that it will infect. Antibodies and drugs against haemagglutinin are therefore particularly important in limiting the spread of the virus, since they prevent it from attaching to new host cells. Neuraminidase is an enzyme that cleaves sialic acid residues from polysaccharides. It has a role in clearing a path to the surface of the target cell before infection, namely, digesting the components of mucus surrounding epithelial cells in the respiratory system. Similarly, neuraminidase also promotes release of the budding virus from the cell surface after infection. The structures of influenza B and influenza C are broadly similar to that of Type A, although in influenza C the functions of the haemagglutinin and the neuraminidase are combined in a single molecule, haemagglutinin esterase. This molecule binds and cleaves a less common type of sialic acid. Influenza C does not normally cause clinical disease or epidemics, so the following discussion is confined to influenza A and B. 2.2 Designation of strains of influenza A considerable number of genetically different strains of influenza A have been identified, and these are classified according to where they were first isolated and according to the type of haemagglutinin and neuraminidase they express. For example ‘A/Shandong/9/93(H3N2)’ is an influenza A virus isolated in the Shandong province of China in 1993 – the ninth isolate in that year – and it has haemagglutinin type 3 and neuraminidase type 2. At the start of the twenty-first century, the major circulating influenza A strains are H1N1 (‘swine flu’) and H3N2. At least 16 major variants of haemagglutinin and 9 variants of neuraminidase have been recognised, but to date most of these have only been found in birds. The designation for influenza B is similar, but omits the information on the surface molecules, for example: ‘B/Panama/45/90’. As you will see later, accurate identification of different strains of flu is crucial if we are to control epidemics by vaccination programmes. 2.3 Genomic diversity of influenza The genome of flu viruses consists of around 14 000 nucleotides of negative-sense single-stranded RNA. Compare this number to the approximately 3 billion nucleotides found in the human genome or the 150 billion nucleotides of the genome of the marbled lungfish (the largest genome known in vertebrates). The genome of influenza viruses is segmented, into 8 distinct fragments of RNA containing 11 genes and encoding approximately 14 proteins (see Table 1 below). This structure has significance for the spread of the virus and the severity of disease symptoms. Cases of influenza generally arise in two main ways: by provoking seasonal annual outbreaks or epidemics and, less commonly, through global pandemics. As you will see shortly, both of these phenomena occur as consequences of the fact that the virus uses RNA as its genetic template and that this RNA genome is segmented into discrete strands. Table 1 The genome of influenza virus. Note that a single RNA segment may encode for more than one protein due to alternative reading frames. Gene name RNA strand (segment number) Function(s) of protein encoded by this gene PB2 (polymerase basic 2) 1 A subunit of viral RNA polymerase involved in cleaving the cap structure of host cell mRNA and generating primers that are subverted for use in the synthesis of viral RNA. PB1 (polymerase basic 1) 2 Core subunit of viral RNA polymerase. Required for polymerase assembly. PB1-F2 2 Binds to components of the host mitochondria, sensitising the cell to apoptosis and contributing to pathogenicity. PA (polymerase acidic) 3 A subunit of viral RNA polymerase which also has protease activity of unknown function. HA (haemagglutinin) 4 Antigenic glycoprotein used for binding to (infecting) the host cell. NP (nucleoprotein) 5 RNase resistant protein. Binds viral genomic RNA to form stable ribonucleoproteins and targets these for export from the host nucleus into the cytosol. Also involved in viral genome packaging and viral assembly. NA (neuraminidase) 6 Cleaves sialic acid. Important for releasing viral particles from host cell. M1 (matrix 1) 7 Binds viral genomic RNA and forms a coat inside the viral envelope in virions. Inside the host cell, it starts forming a layer under patches of the membrane rich in viral HA, NA, and M2 and so facilitates viral assembly and budding from the host cell. M2 (matrix 2) 7 Transmembrane ion channel protein. Allows protons into the virus capsid, acidifying the interior, destabilising binding of M1 to the viral genomic RNA which leads to uncoating of the viral particle inside the host cell. NS1 (non-structural 1) 8 Inhibits nuclear export of the host’s own mRNA, thereby giving preference to viral genomic RNA. Blocks the expression of some host inflammatory mediators (interferons) and interferes with T cell activation*. NS2/NEP (non-structural 2/ nuclear export protein) 8 Mediates the export of viral genomic RNA from the host nucleus to the cytoplasm. * Interferons and T cells are involved in the immune response to pathogens. The influenza virus is a successful pathogen because it is constantly changing. How might having a segmented genome promote the evolution of new strains of influenza virus? answer: If a cell is infected with more than one strain of virus at the same time, then a new strain can be generated simply by mixing RNA strands from different viruses. 2.3.1 Creation of new viral strains Part of the success of influenza as a pathogen is because segmented genome improves the virus’s potential to evolve into new strains through the combination of different RNA stands. This mixing of the genetic material from different viral strains to produce a new strain is termed genetic reassortment. For instance, the virus that caused the 2009 H1N1 ‘swine flu’ pandemic comprises a quadruple reassortment of RNA strands from two swine virus, one avian virus, and one human influenza virus: the surface HA and NA proteins derive from two different swine influenzas (H1 from a North American swine influenza and N1 from a European swine influenza) the three components of the RNA polymerase derive from avian and human influenzas (PA and PB2 from the avian source, PB1 from the human 1993 H3N2 strain) the remaining internal proteins derive from the two swine influenzas (MacKenzie, 2009). This does not necessarily mean that all four viruses infected the same animal at once. The new strain was likely the result of a reassortment of two swine influenza viruses, one from North America and one from Europe. The North American virus may itself have been the product of previous reassortments, containing a human PB1 gene since 1993 and an avian PA and PB2 genes since 2001. The presence of avian influenza RNA polymerase genes in this virus was especially worrying, since the avian polymerase is thought to be more efficient than human or swine versions, allowing the virus to replicate faster and thus making it more virulent. Similar avian RNA polymerase genes are what make H5N1 bird flu extremely virulent in mammals and what made the 1918 human pandemic virus so lethal in people. This mixing of genes from two or more viruses (whether from the same host species or from different species) can cause major changes in the antigenic surface proteins of a virus, such that it is no longer recognised by the host’s immune system. This antigenic shift is described in more detail in Section 3 (specifically, Box 2). In contrast to the major genetic changes caused by reassortment, influenza viruses also undergo constant, gradual, genetic changes due to errors made by their RNA polymerases. 2.4 Infection and replication Influenza RNA polymerase lacks the ability to recognise and repair any errors that occur during genome duplication, resulting in mistakes in copying its viral RNA about once in every 10 000 nucleotides. Because the influenza genome only contains approximately 14 000 nucleotides, this means that, on average, each new virus produced differs by 1 or 2 nucleotides from its ‘parent’. The slow accumulation of random genetic changes, especially in the antigenic surface proteins, explains why antibodies that were effective against the virus one year may be less effective against it in subsequent years. This gradual change in the nature of viral antigens is known as antigenic drift. The replication cycle of influenza is illustrated in Figure 5 Figure 5 Replication cycle of a flu virus. Long description Influenza is spread in aerosol droplets that contain virus particles (or by desiccated viral nuclei droplets), and infection may occur if these come into contact with the respiratory tract. Viral neuraminidase cleaves polysaccharides in the protective mucus coating the tract, which allows the virus to reach the surface of the respiratory epithelium. The haemagglutinin now attaches to glycophorins (sialic-acid-containing glycoproteins) on the surface of the host cell, and the virus is taken up by endocytosis into a phagosome. Acidic lysosomes fuse with the phagosome to form a phagolysosome and the pH inside the phagolysosome falls. This promotes fusion of the viral envelope with the membrane of the phagolysosome, triggering uncoating of the viral capsid and release of viral RNA and nucleoproteins into the cytosol. The viral genomic RNA then migrates to the nucleus where replication of the viral genome and transcription of viral mRNA occur. These processes require both host and viral enzymes. The viral negative-stranded RNA is replicated by the viral RNA-dependent RNA polymerase, into a positive-sense complementary RNA (cRNA), and these positive and negative RNA strands associate to form double-stranded RNA (dsRNA). The cRNA strand is subsequently replicated again to produce new viral genomic negative-stranded RNA. Some of the cRNA is also processed into mRNA for translation of viral proteins. The infection cycle is rapid and viral molecules can be detected inside the host cell within an hour of the initial infection. The envelope glycoproteins (haemagglutinin and neuraminidase) are translated in the endoplasmic reticulum, processed and transported to the cell’s plasma membrane. The viral capsid is assembled within the nucleus of the infected cell. The capsid moves to the plasma membrane, where it buds off, taking a segment of membrane containing the haemagglutinin and neuraminidase, and this forms the new viral envelope. Influenza virus budding from the surface of an infected cell is shown in Figure 6. Figure 6 Flu virus particles budding from the surface of an infected cell. Long description From the description above, identify a process or element in the replication cycle which is characteristic of the virus, and which would not normally occur in a mammalian cell. Answer: The replication of RNA on an RNA template with the production of double-stranded RNA would never normally occur in a mammalian cell. Double stranded RNA is therefore a signature of a viral infection. Significantly, cells have a way of detecting the presence of dsRNA, and this activates interferons: molecules involved in limiting viral replication. 2.5 Cellular pathology of influenza infection Flu viruses can infect a number of different cell types from different species. This phenomenon is partly because the cellular glycoproteins which are recognised by viral haemagglutinin are widely distributed in the infectious agent. What is the term for the property of viruses that allows them to only replicate in particular cell types? Reveal answer A second reason why the virus can infect a variety of cell types is that the replication strategy of flu is relatively simple: ‘infect the cell, replicate as quickly as possible and then get out again’. This is the cytopathic effect of the virus. Cell death caused directly by the virus can be distinguished from cell death caused by the actions of the immune system as it eliminates infected cells. The effects of cell death Cell death impairs the function of an infected organ and often induces inflammation, a process that brings white cells (leukocytes) and molecules of the immune system to the site of infection. In the first instance, the leukocytes are involved in limiting the spread of infection; later they become involved in combating the infection, and in the final phase they clear cellular debris so that the tissue can repair or regenerate. The symptoms of flu experienced by an infected person are partly due to the cytopathic effect of the virus, partly due to inflammation and partly a result of the innate immune response against the virus. The severity of the disease largely depends on the rate at which these processes occur. In most instances, the immune response develops sufficiently quickly to control the infection and patients recover. If viral replication and damage outstrip the development of the immune response then a fatal infection can occur. In severe flu infections, the lungs may fill with fluid as the epithelium lining the alveoli (air sacs) is damaged by the virus. The fluid is ideal for the growth of bacteria, and this can lead to a bacterial pneumonia, in which the lungs become infected with one or more types of bacteria such as Haemophilus influenzae. Damage to cells lining blood vessels can cause local bleeding into the tissues, and this form of ‘fulminating disease’ was regularly seen in post-mortem lung tissues of people who died in the 1918 pandemic. 3 Patterns of disease In humans, pigs and horses, flu viruses circulate through populations at regular intervals. The disease is endemic in tropical regions for all of these host groups (i.e. it is continually present in the community). In temperate latitudes, infections are usually seasonal or epidemic, with the greatest numbers occurring in the winter months (Figure 7). Epidemics also occur sporadically in sea mammals and poultry, and in these species high mortality is typical. Figure 7 Epidemic patterns of flu in temperate latitudes. The graph shows notifications of flu in the USA each week from 1994–1997. Long description In most years, flu in humans affects a minority of the population, the disease course is not very severe and the level of mortality is not great. In such years the influenza virus is slightly different from the previous year due to antigenic drift, which results in the accumulation of genetic mutations that cause the molecules present on the surface of the virus change progressively. In this scenario, the virus is not significantly different from the previous year so that the host’s immune system can more easily mount an effective response than it could to a completely new strain. However, at irregular intervals the virus undergoes an antigenic shift. This process only occurs in influenza A viruses, typically every 10–30 years, and it is associated with severe pandemics, serious disease and high mortality (see Box 2). In Section 2.3 you read that strains of influenza are differentiated and designated using a simple system of numbers and letters that depend on their surface antigens. More commonly, however, strains responsible for pandemics are often given a common name according to the area in the world from which they were thought to originate, or the species they mainly affected before becoming transferred to humans (see Table 2). Evidence suggests, however, that in the twentieth century the major flu pandemics all originated in China, with the exception of the 1918 pandemic, which first occurred in the USA. Major flu pandemic strains of the twentieth century. Year Designation Common name 1900 H3N8 (none) 1918 H1N1 Spanish flu西班牙 1957 H2N2 Asian flu亞洲 1968 H3N2 Hong Kong flu香港 1977 H1N1 Russian flu蘇聯 1997 H5N1 Avian flu禽 The rise of H5N1 – an example of antigenic shift In 1997, a new strain of influenza A, H5N1, was identified in Hong Kong. The strain was rife in chickens and a few hundred people had become infected. Mortality in these individuals was very high, (6 of 18 died), and so there was serious concern that it marked the beginning of a new pandemic. The authorities in Hong Kong responded by a mass cull of poultry in the region and about 1.5 million chickens were slaughtered. H5N1 did not spread easily from person to person and no further cases were reported in people following the slaughter. Whether the H5N1 outbreak was an isolated incident of a strain spreading from chicken to humans, or whether it was the start of a major pandemic which was nipped in the bud, cannot be known. Subsequent analysis showed that the high virulence of the new strain could partly be related to the new variant of haemagglutinin (H5), and partly to a more efficient viral polymerase. This outbreak clearly demonstrates the way in which bird influenza can act as a source of new viral strains, and shows that such new strains may be very dangerous to humans. Since the discovery of the influenza virus in the 1930s it has been possible to isolate and accurately identify each of the epidemic strains, but, as earlier strains of virus have now died out, it has been necessary to infer their identity by examining the antibodies in the serum of affected people. Antibodies and the ability of the immune system to respond to a strain of flu are much more persistent than the virus itself. It is thus possible to analyse antibodies to determine which types of haemagglutinin and neuraminidase they recognise long after the virus itself has gone. One can then deduce which type of influenza virus that person contracted earlier in their life (as explained in Section 5.2). 3.1 Tracking the emergence of new strains Influenza is one of several diseases monitored by the WHO Global Alert and Response   (GAR) network (WHO, 2011a), comprising 110 ‘sentinel’ laboratories in 82 countries. The organisation’s surveillance and monitoring of the disease then forms part of their Global Influenza Programme (GIP), and they use data gathered from participating countries to: provide countries, areas and territories with information about influenza transmission in other parts of the world to allow national policy makers to better prepare for upcoming seasons provide data for decision making regarding recommendations for vaccination and treatment describe critical features of influenza epidemiology including risk groups, transmission characteristics, and impact monitor global trends in influenza transmission inform the selection of influenza strains for vaccine production (WHO, 2011b). The influenza data from the sentinel laboratories is fed into a global surveillance programme, started by the WHO in 1996, called FluNet (WHO, 2011c), which is one of the tools that facilitates the actions described above. Typically a flu vaccine contains material from the main influenza A strains and an influenza B strain, so that an immune response is induced against the most likely infections. Usually the scientists predict correctly and immunised people are effectively protected against the current strains (>90% protection). However, the prediction is occasionally incorrect, or a new strain develops during the time that the vaccine is being manufactured. In this case the vaccine generally provides poor protection. What can you deduce about immunity against flu infection from the observations on vaccination above? answer The immune response is strain-specific. If you are immunised against the wrong strain of flu, then the response is much less effective and you are more likely to contract the disease. 3.2 Immune responses to influenza The immune system uses different types of immune defence against different types of pathogen. The responses against flu are typical of those which are mounted against an acute viral infection, but different from the responses against infection by bacteria, worms, fungi or protist parasites. When confronted with an acute viral infection, the immune system has two major challenges: The virus replicates very rapidly, killing the cells it infects. Since a specific immune response takes several days to develop, the body must limit the spread of the virus until the immune defences can come into play. Viruses replicate inside cells of the body, but they spread throughout the host in the blood and tissue fluids. Therefore, the immune defences must recognise infected cells (intracellular virus) and destroy them. But the immune system must also recognise and eradicate free virus in the tissue fluids (extracellular virus) in order to prevent the virus from infecting new cells.. 3.2.1 Summary of the response How does the body act quickly to limit viral spread? When a virus infects a cell of the body, the molecular machinery for protein synthesis within the cell is usurped as the virus starts to produce its own nucleic acids and proteins. The cell detects the flu dsRNA and other viral molecules and releases interferons, which bind to receptors on neighbouring cells and cause them to synthesise antiviral proteins. If a virus infects such cells they resist viral replication, so fewer viruses are produced and viral spread is delayed. Also, in the earliest stages of a virus infection the molecules on the cell surface change. Cells lose molecules that identify them as normal ‘self’ cells. At the same time they acquire new molecules encoded by the virus. A group of large, granular lymphocytes recognise these changes and are able to kill the infected cell. This function is called ‘natural killer’ cell action and the lymphocytes that carry it out are termed NK cells. Non-adaptive and adaptive responses The actions of both interferons and NK cells in combating infection by influenza occur early in an immune response, and are not specific for the flu virus. These defences occur in response to many different kinds of viral infection, and they are part of our natural, or non-adaptive, immune responses. Note that immunologists use the term non-adaptive to indicate a type of response that does not improve or adapt with each subsequent infection. This is quite different to its use in evolutionary biology, where it means ‘not advantageous’. Such non-adaptive immune responses slow the spread of an infection so that specific, or adaptive, immune defences can come into play. The key features of an adaptive immune response are specificity and memory. The immune response is specific to a particular pathogen, and the immune system appears to ‘remember’ the infection, so that if it occurs again the immune response is much more powerful and rapid. Because an immune response is highly specific to a particular pathogen it often means that a response against one strain of virus is ineffective against another – if a virus mutates then the lymphocytes that mediate adaptive immunity are unable to recognise the new strain. There are two principal arms of the adaptive immune system, mediated by different populations of lymphocytes. One group, called T-lymphocytes, or T cells (which develop in the thymus gland, overlying the heart), recognises antigen fragments associated with cells of the body, including cells which have become infected. A set of cytotoxic T cells (Tc) specifically recognises cells which have become infected and will go on to kill them. In this sense they act in a similar way to NK cells. However they differ from NK cells in that Tc cells are specific for one antigen or infectious agent, whereas NK cells are non-specific. The second group of lymphocytes are B cells (that differentiate in the bone marrow), which synthesise antibodies that recognise intact antigens, either in body fluids or on the surface of other cells. Activated B cells progress to produce a secreted form of their own surface antibody. Antibodies that recognise the free virus act to target it for uptake and destruction by phagocytic cells. Therefore the T cells and NK cells deal with the intracellular phase of the viral infection, while the B cells and antibodies recognise and deal with the extracellular virus. The two reactions described above are illustrated in Figure 8. Figure 8 Immune defences against flu viruses. Antibodies can block the spread of the virus by preventing them from attaching to host cells. Infected cells release interferon, which signals to neighbouring cells to induce resistance. Natural killer cells (NK) and cytotoxic T cells (Tc) recognise and kill virally-infected cells. Long description You might ask why it takes the adaptive immune response so long to get going. The answer is that the number of T cells and B cells that recognise any specific pathogen is relatively small, so first the lymphocytes which specifically recognise the virus must divide so that there are sufficient to mount an effective immune response. This mechanism is fundamental to all adaptive immune responses. How do pandemic strains of influenza A come about? Answer Question 2 Pandemic strains of influenza A normally arise by simultaneous infection of a non-human host (typically poultry or pigs) with two or more strains of influenza A. Reassortment of the eight viral segments from each virus allows the generation of a new hybrid virus type, with a completely novel surface structure that has never been seen before by a host immune system (antigenic shift). 4 Antiviral treatments Two classes of antiviral drugs are used to combat influenza: neuraminidase inhibitors and M2 protein inhibitors. Why are antibiotics not used to combat influenza? Reveal answer Neuraminidase inhibitors neuraminidase is an enzyme that is present on the virus envelope and cleaves sialic acid groups found in the polysaccharide coating of many cells (especially the mucus coating of the respiratory tract). Neuraminidase is used to clear a path for the virus to a host cell and facilitates the shedding of virions from an infected cell. Inhibition of neuraminidase therefore helps prevent the spread of virus within a host and its shedding to infect other hosts. The two main neuraminidase inhibitors currently in clinical use are zanamivir (trade name Relenza) and oseltamivir (trade name Tamiflu). These are effective against influenza A and B, but not influenza C which exhibits a different type of neuraminidase activity that only cleaves 9-O-acetylated sialic acid. M2 inhibitors Recall from Table 1 that the influenza M2 protein forms a pore that allows protons into the capsid, acidifying the interior and facilitating uncoating. Drugs such as amantadine (trade name Symmetrel) and rimantadine (trade name Flumadine) block this pore, preventing uncoating and infection. However, their indiscriminate use in ‘over-the-counter’ cold remedies and farmed poultry has allowed many strains of influenza to develop resistance. Influenza B has a different type of M2 protein which is largely unaffected by these drugs. 5 Diagnosis of influenza Many diseases produce symptoms similar to those of influenza; in fact, ‘flu-like’ is a term that is frequently used to describe several different illnesses. Since influenza spreads rapidly by airborne transmission and is a life-threatening condition in certain vulnerable groups, it is important that cases of the disease are identified as quickly as possible, so that preventative measures may be taken. Most viral infections are not treated, although antiviral drugs such as zanamivir are used for potentially life-threatening cases or where the risk of transmission is high (as occurs during a pandemic). Which sites in the body should be sampled for diagnosis? answer The influenza virus infects the respiratory tract and is spread by coughing and sneezing, so specimens should be taken from the nose, throat or trachea. In practice, the best specimens are nasal aspirates or washes, but swabs of the nose or throat may be used if they are taken vigorously enough to obtain cells. Ideally, samples should be taken within three days of the onset of illness, and all specimens need to be preserved in a transport medium and kept chilled until they reach the clinical microbiology laboratory. 5.1 Initial identification of influenza infection Oral swabs or nasal aspirates are initially screened for the presence of a variety of respiratory viruses. This is done by extracting RNA from the sample and subjecting it to a reverse-transcription polymerase chain reaction (RT-PCR) as described below: Initially the RNA sample is reverse transcribed into complementary DNA (cDNA), using a commercially-available reverse transcriptase enzyme. The cDNA is then used in a standard PCR reaction to detect and amplify a short sequence of nucleotides specific to the virus. Multiple DNA sequences, each specific for a different type of virus, can be amplified in the same reaction, provided that these sequences are of different lengths. Each of the different amplified sequences is separated from the others when the entire sample is subjected to gel electrophoresis (an analytical technique in which molecules of different sizes move at various rates through a gel support in an applied electric field, thus making it possible to identify specific molecules.) PCR technique. Typically, nucleotide sequences specific to 5 types of virus are searched for in each sample: influenza A, influenza B, respiratory syncytial virus (Baltimore group V, (–)ssRNA virus, and a major cause of respiratory illness in young children), adenoviruses and enteroviruses. Those samples that test positive for influenza in the RT-PCR reaction are inoculated into cells in culture. Sufficient virus for a limited number of tests can be produced from such cultures within 24 hours, but they are often maintained for up to a week. 5.2 Determining the subtype of influenza Immunofluorescence Confirmation of a case of influenza is usually achieved by performing tests on some of the inoculated cultured cells using reference fluorescent-labelled antisera provided by the WHO. A reference antiserum is a sample serum known to contain antibodies specific for the molecule to be assayed (in this case, haemagglutinin or neuraminidase). These antisera are prepared using purified haemagglutinin and neuraminidase and are monospecific, each antibody reacting only with one epitope e.g. H1 or H3. For the test, an antibody is added to a sample of inoculated cells. Following a wash step, if the antibody remains bound to the cells then they fluoresce under appropriate illumination, indicating the presence of viral antigen on the cell surface. This diagnostic technique can identify influenza virus on infected cells in as little as 15 minutes. A positive result not only confirms the RT-PCR data, but gives additional information on the subtype of the virus. Further PCR analyses Standardised RT-PCR protocols exist to look for the presence of different haemagglutinin and neuraminidase subtypes, chiefly H1, H3, H5, N1 and N2. (Poddar, 2002). If the PCR analysis indicates a dangerous strain of influenza A e.g. H5N1, then it is instantly sent to a WHO reference laboratory for further tests. Haemagglutination assays Influenza has haemagglutinins protruding from its viral envelope, which it uses to attach to host cells prior to entry. These substances form the basis of a haemagglutination assay, in which viral haemagglutinins bind and cross-link (agglutinate) red blood cells added to a test well, causing them to sink to the bottom of the solution as a mat of cells. If agglutination does not occur, then the red blood cells are instead free to roll down the curved sides of the tube to form a tight pellet. A related test called a haemagglutination-inhibition assay (HAI), incorporates antibodies against different subtypes of viral haemagglutinin. The antibodies bind and mask the viral haemagglutinin, preventing it from attaching to and cross-linking red blood cells. A HAI assay can be set up in one of two ways: either a known reference antibody is added to an unknown virus sample, or known reference viral haemagglutinin is added to a sample of patient serum containing antibodies against influenza. This second version of the HAI assay can therefore be used long after the infection has passed, when virions are no longer present. Haemagglutination and HAI assays have the advantage that they are simple to perform and require relatively cheap equipment and reagents. However, they can be prone to false positive or false negative results, if the sample contains non-specific inhibitors of haemagglutination (preventing agglutination) or naturally occurring agglutinins of red blood cells (causing agglutination). If a confirmed influenza A isolate reacts weakly or not at all in HAI then this indicates an unknown variant of influenza A and the sample is immediately sent to a WHO reference laboratory for further tests. Neuraminidase inhibition assay Typing influenza isolates in terms of their neuraminidase makes use of the enzyme activity of this glycoprotein. The neuraminidase inhibition assay is performed in two parts. The first part determines the amount of neuraminidase activity in a patient influenza sample, as outlined in Figure 9a. A substrate (called fetuin) that is rich in sialic acid residues is added to a sample of the influenza virus, and the viral neuraminidase enzyme cleaves the substrate to produce free sialic acid. Addition of a substance that inactivates the neuraminidase stops the reaction, and a chromogen (a colourless compound that reacts to produce a coloured end-product) that turns pink in the presence of free sialic acid is added. The intensity of the pink colour is proportional to the amount of free sialic acid and can be measured using a spectrophotometer. This assay of neuraminidase activity allows the appropriate amount of virus sample to be determined, and this quantity is then used in the second part of the assay. If too much or too little virus is used, the resulting changes, and therefore the neuraminidase, may be undetectable. In the second part of the assay (Figure 9b), viral samples from the patient are incubated with anti-neuraminidase reference antisera. Each of the reference antisera used for this test has antibodies that bind one particular neuraminidase variant, e.g. N1 or N2. How can these antisera be used to type the neuraminidase variant? Figure 9 The neuraminidase inhibition assay. (a) Assay of neuraminidase activity. (b) The inhibition of the assay itself. 6 Summary of the unit . A single pathogen can produce different types of disease in different people. Genetic variation in a pathogen can also affect the type of disease it produces. To understand this we need to know something of the genetic and social differences in the host population, and of the diversity of the pathogen. The symptoms of a particular disease may be produced by different pathogens or by a combination of pathogens. To understand this requires some knowledge of pathology and cell biology. Some diseases, such as flu, affect humans and several other animal species, whereas others are more selective in their host range. The basic biology of different pathogens underlies these differences. Flu is a disease that can be contracted several times during a lifetime, but many other infectious diseases are only ever contracted once. To understand this we need to look at how the immune system reacts to different pathogens, and how responses vary depending on the pathogen. Outbreaks of flu occur regularly, but some epidemics are much more serious than others. This requires an understanding of aspects of virology, immunology, evolutionary biology and epidemiology. In 1892, German bacteriologist Richard Pfeiffer isolated what he thought was the causative agent of influenza. The culprit, according to Pfeiffer, was a small rod-shaped bacterium that he isolated from the noses of flu-infected patients (1). He dubbed it Bacillus influenzae (or Pfeiffer's bacillus). Few doubted the validity of this discovery, in large part because bacteria had been shown to cause other human diseases, including anthrax, cholera, and plague. Figure 1 Richard Shope, 1936. The filtration question When history's deadliest influenza pandemic began in 1918, most scientists believed that Pfeiffer's bacillus caused influenza. With the lethality of this outbreak (which killed an estimated 20 to 100 million worldwide) came urgency—researchers around the world began to search for Pfeiffer's bacillus in patients, hoping to develop antisera and vaccines that would protect against infection. In many patients, but not all, the bacteria were found. Failures to isolate B. influenzae (now known as Haemophilus influenzae) were largely chalked up to inadequate technique, as the bacteria were notoriously difficult to culture (2). The first potential blow to Pfeiffer's theory came from Peter Olitsky and Frederick Gates at The Rockefeller Institute. Olitsky and Gates took nasal secretions from patients infected with the 1918 flu and passed them through Berkefeld filters, which exclude bacteria. The infectious agent—which caused lung disease in rabits—passed through the filter, suggesting that it was not a bacterium (3, 4). Although the duo had perhaps isolated the influenza virus (which they nevertheless referred to as an atypical bacterium called Bacterium pneumosintes), other researchers could not reproduce their results. One of the doubters was Oswald Avery (Rockefeller Institute), who developed a culture media—chocolate agar—that optimized the growing conditions for B. influenzae and thus minimized false negative results from patient samples. Thus, the idea that flu was transmitted by a filterable agent (or virus) was dismissed. Insights from pigs Olitsky and Gates would not be vindicated until a decade later, when Shope—a young physician from Iowa then working on hog cholera at the Rockefeller Institute—turned his attention to swine influenza. Pig farmers in Iowa had reported two outbreaks—one in 1918 and another in 1929—of a highly contagious, influenza-like disease among their animals. The disease bore such a remarkable resemblance to human flu that it was named swine influenza. Shope and his mentor Paul Lewis took mucus and lung samples from the infected pigs and attempted to isolate the disease-causing agent. They quickly isolated a bacterium that looked exactly like Pfeiffer's human bacterium (and was thus called B. influenzae suis), but when they injected the bacteria into pigs, it caused no disease (5). Shope then filtered the samples and, like Olitsky and Gates, found that the filtrate contained the infectious agent. Shope's filtrate caused a highly contagious, influenza-like disease in pigs—albeit a more mild one than seen in naturally-infected pigs. Mixing the filtrate with the bacterium reproduced the severe disease. He concluded—correctly—that the filterable agent caused the infection, which then facilitated secondary infection with the bacterium (6). Shope published his results in a series of papers in The Journal of Experimental Medicine (5, 6). Using Shope's technique, Wilson Smith, Christopher Andrewes, and Patrick Laidlaw (National Institute for Medical Research, UK) soon isolated the virus from humans (7), laying to rest any lingering doubts about the nature of the flu-inducing agent. Both Shope and the British trio later demonstrated that sera from humans that were infected with the 1918 flu virus could neutralize the pig virus, leading them to conclude that the swine virus was a surviving form of the 1918 human pandemic virus (8, 9). In fact, a related strain of flu still circulates among pigs today.