Relationship between pathogenicity and sequences of H6N1 avian influenza viruses and vertical transmission test

家禽流行性感冒是由A型流行性感冒病毒所引起，可感染多種禽鳥，顯現不同臨床病症。目前台灣家禽流行性感冒多以H6血清亞型為主，西元2000年，一台灣H6N1亞型AIV被分離出，為探究病毒病原性與基因序列的相關性，利用極限稀釋及3次雞胚胎繼代分離得一較低病原性毒株2838DN，以接觸感染方式，分離出一株更高毒力的毒株2838K。IVPI測得此兩病毒株分別為0.16及0.72。我們設計針對HA，NA，M，NS，NP，PA，PB1，PB2等8段基因的引子，作RT-PCR增幅及以TA-cloning方式，定出此兩株病毒之八段基因序列，發現在HA序列有7個胺基酸變異，在NA基因序列桿狀部位有12個氨基酸的缺失，此缺失對病原性影響，並無明確之關聯性；NS基因有1個氨基酸變異；PB1基因序列有2個氨基酸變異，新發現的PB1-F2有90個氨基酸，其序列並無變異；PB2基因有5個氨基酸變異；PA基因有2個氨基酸變異。另一方面，儘管家禽流行性感冒病毒的傳播方式以水平傳播為主，關於它是否會垂直傳播的證據仍缺乏，相關paper研究也很少，臨床上AIV會造成雞隻的產蛋率下降，曾於發病雞場的蛋之卵黃、卵白及卵殼分離到病毒，但這和垂直傳播上的關聯如何無法直接證得。為了解禽流感病毒是否會垂直傳播，我們利用人工點鼻定量接種較高毒力之此台灣H6N1 禽流感病毒株於產蛋母雞，產蛋後犧牲母雞取臟器作分離，從雞隻產下的蛋孵出的小雞，於孵出一日齡時，解剖取其氣管、腎臟、卵黃囊，作成乳劑分離病毒，以RT-PCR方式偵測病毒。另以1.5-2.5 EID50/0.1ml劑量，攻此毒株於9-11日齡SPF 雞胚胎尿囊腔，追蹤孵出小雞帶毒情形部分，，經過攻毒152顆雞胚胎尿囊腔中所孵出之91隻小雞，皆未分離偵測出病毒存在。點鼻接種的9隻母雞體內，從母雞氣管、腎臟、卵巢、輸卵管，可成奶擢鬙X所給予的AIV病毒，並從孵出的12隻小雞中，有6隻成弘輕Abstract The virulence of avian influenza virus is polygenic, and the hemagglutinin (HA) glycoprotein plays as a determinant of pathogenicity. In Taiwan H6 subtype AIV is the most occurrence subtype. The purpose of this study was to recognize the relationship between pathogenenicity and sequences of H6N1 avian influenza viruses in Taiwan. By 3 rounds of limiting dilution and plaque purification, a H6N1 low pathogenetic AIV strain, 2838DN, was cloned from a field isolate, 2838/00. By contact infection, a more virulent strain named 2838K was obtained from the kidneys of the dead chicks. The IVPI of 2838DN and 2838K was 0.16 and 0.72, respectively. Cytopathic effect also revealed the increased virulence of 2838K compared to 2838DN strain. We have cloned the whole genome of eight genes of AIV 2838DN and 2838K successfully. By comparing the amino acid of HA, replacement of Alanine by Threonine at the –5th and glutamic acid by lysine at the 304th may increase the virulence. 12 amino acid deletion in the stalk of NA gene was found in both clones. There are 6 amino acid mutations in HA gene, 1 in NA and NS gene, 2 in PA and PB1 gene, 5 in PB2 gene, 7 in NP gene of 2838K compared to 2838DN strain. Although horizontal transmission of AI viruses commonly occurs, proof of vertical transmission is lacking and information concerning the potential for egg-borne transmission of the virus is limited. To clearify whether H6N1 AIV vertical transmission vertically transmit or not. The representative virus strain 2838K as 0.5-2.5 EID50/0.1ml was inoculated into the allantoic cavity of twenty 9-11 day-old SPF embryonating chicken eggs per test and 8 repeats had been conducted. Among 152 inoculated SPF embryonating eggs, we were unable to isolate AIV from 91 chicks. In the other way, by intranasal inoculation of 2838K to 9 laying hens, we detected AIV isolated among 6 hatchings from inoculated hens successfully.目錄 目錄---------------------------------------------------------------------------------------------------Ⅰ 表次---------------------------------------------------------------------------------------------------Ⅳ 圖次---------------------------------------------------------------------------------------------------Ⅴ 英文摘要---------------------------------------------------------------------------------------------Ⅶ 中文摘要---------------------------------------------------------------------------------------------Ⅷ 第一章 文獻探討-----------------------------------------------------------------------------------1 第一節 家禽流行性感冒的歷史背景與流行病學--------------------------------------1 第二節 家禽流行性感冒病毒的性狀-----------------------------------------------------4 2.1 家禽流行性感冒病毒的型態構造與基因結構-------------------------------5 2.2 家禽流行性感冒病毒的蛋白質------------------------------------------------- 6 2.3 家禽流行性感冒病毒的複製與釋放------------------------------------------ 11 2.4 病毒對理化因子的特性--------------------------------------------------------- 11 2.5 傳播途徑---------------------------------------------------------------------------12 第三節 家禽流行性感冒病毒的宿主、致病機制-------------------------------------13 3.1宿主----------------------------------------------------------------------------------13 3.2家禽流行性感冒病毒引起的症狀----------------------------------------------15 3.3致病機制與病原性之評估-------------------------------------------------------15 第四節 實驗室診斷-- ----------------------------------------------------------------------18 4.1病毒分離及同定-------------------------------------------------------------------18 4.2直接偵測病毒蛋白或核酸-------------------------------------------------------19 4.3以血清學抗體作診斷-------------------------------------------------------------19 第二章 緒言----------------------------------------------------------------------------------------20 第三章 材料與方法-------------------------------------------------------------------------------22 第一節 台灣家禽流行性感冒病毒H6N1亞型病原性的評估---------------------22 1.1 病毒株之挑選---------------------------------------------------------------------------------------22 1.2 病毒分離及增殖--------------------------------------------------------- 22 1.3 血球凝集試驗 (HA) 及血球凝集抑制試驗 (HI)--------------------------- 23 1.4 病毒之致病力試驗---------------------------------------------------------------- 24 1.5 增幅此兩病毒株之HA、NA及NP、M、NS、PA、 PB1、PB2基因引子---------------------------------------------------------------------------------------- 26 1.6 病毒核酸的萃取 --------------------------------------------------------------- 27 1.7單管反轉錄聚合酶鏈鎖反應(RT-PCR)------------------------------------------ 27 1.8瓊脂醣凝膠電泳------------------------------------------------------------------------ 28 1.9 膠體純化DNA--------------------------------------------------------------------------- 28 1.10用TA-cloning kit 進行病毒核酸之cloning-------------------------------- 29 1.11 RT-PCR產物或菌液的核苷酸定序------------------------------------------- 30 1.12 基因序列分析------------------------------------------------------------------------- 31 第二節 動物試驗: 由雞胚胎攻毒探討孵出之小雞帶毒情形----------------- 31 2.1病毒株之挑選-------------------------------------------------------------------------------- 31 2.2 病毒增殖------------------------------------------------------------------------------------------- 31 2.3實驗設計----------------------------------------------------------------------------------- 31 2.4以雞胚胎繼代進行病毒再回收之分離與鑑定--------------------- 33 第三節 由產蛋母雞直接攻毒偵測孵出小雞帶毒的真正垂直傳播試驗------------- 33 3.1病毒株之挑選---------------------------------------------------------------------- 33 3.2病毒株之力價測定---------------------------------------------------------------- 33 3.3實驗動物---------------------------------------------------------------------------- 34 3.4飼料與飲水------------------------------------------------------------------------- 34 3.5飼養環境---------------------------------------------------------------------------- 34 3.6實驗設計---------------------------------------------------------------------------- 34 3.7採樣、病毒分離與鑑定---------------------------------------------------------- 36 第四章 結果--------------------------------------------------------------------------------------- 37 第一節 台灣家禽流行性感冒病毒H6N1亞型病原性之評估----------------- 37 1.1 病毒株之挑選-----------------------------------------------------------------37 1.2 病毒的病原性試驗-----------------------------------------------------------37 1.3 病毒株的力價測定-----------------------------------------------------------37 1.4兩株病毒基因胺基酸序列差異---------------------------------------------38 第二節 由雞胚胎攻毒探討孵出之一日齡雞感染實驗------------------------------ 39 2.1病毒株之挑選------------------------------------------------------------------39 2.2 SPF雞胚胎組攻毒實驗結果------------------------------------------------39 2.3陰性對照組結果------------------------------------------41 第三節 由產蛋母雞直接攻毒偵測孵出小雞帶毒的真正垂直傳播試驗--------- 42 3.1病毒株之挑選------------------------------------------------------------------42 3.2病毒株之力價測定------------------------------------------------------------42 3.3攻毒组母雞結果---------------------------------------------------------------42 3.4攻毒組母雞產蛋及孵出小雞帶毒結果------------------------------------43 3.5陰性對照組及哨兵雞結果---------------------------------------------------43 第五章 討論---------------------------------------------------------------------------------------66 第六章 參考文獻---------------------------------------------------------------------------------7