本文選題：H4亞型禽流感病毒 + 分離鑒定�。� 參考：《廣西大學(xué)》2017年碩士論文

【摘要】：H4亞型禽流感病毒(avian influenza virus,AIV)是一種比較常見(jiàn)的低致病性AIV(low pathogenic,LPAIV),尤其在我國(guó)南方地區(qū),H4亞型AIV在流感病毒監(jiān)測(cè)中的分離率較高,且常與H1、H3、H5、H6和H9等多種亞型的AIV混合感染,較易發(fā)生病毒的重排和變異。因此,了解H4亞型AIV的遺傳進(jìn)化關(guān)系及其致病性對(duì)我國(guó)禽流感的防控具有重要意義。本研究對(duì)2015-2016年廣西活禽市場(chǎng)H4亞型AIV進(jìn)行分離與鑒定及其生物學(xué)研究,并對(duì)分離株進(jìn)行病毒全基因組序列分析。結(jié)果共分離出6株H4亞型AIV,均為L(zhǎng)PAIV,同時(shí)具備SAα2,3和SAα2,6受體的親和特性,但其結(jié)合SAα2,3受體的能力更高。遺傳進(jìn)化分析表明6株廣西H4亞型AIV分離株的8個(gè)基因均屬于歐亞譜系,不同基因組之間的來(lái)源比較復(fù)雜,其中HA基因的核苷酸序列主要與蒙古A/duck/Mongolia/OIE-7438/2011(H4N6)分離株具有較高的同源性。H4亞型AIV分離株主要與N2和N8亞型組合,其中H4N2亞型的NA基因與蒙古的A/duck/Mongolia/154/20 1 5(H 1N2)和浙江的 A/duck/Zhejiang/727042/2014(H6N2)分離株的NA基因核苷酸序列具有較高的同源性;H4N8亞型的NA基因與越南A/duck/Vietnam/OIE-2577/2011(H3N8)分離株NA基因核苷酸序列具有較高同源性,表明廣西H4亞型AIV分離株極有可能與上述不同國(guó)家的分離株發(fā)生重組,推測(cè)其可能是由于候鳥(niǎo)遷徙等原因?qū)е碌牟《局亟M。為了建立快速H4亞型AIV檢測(cè)方法,根據(jù)GenBank中H4亞型AIV的HA基因保守序列,設(shè)計(jì)1對(duì)特異性引物,通過(guò)優(yōu)化反應(yīng)條件,建立了 H4亞型AIVSYBRGreenI實(shí)時(shí)熒光定量檢測(cè)方法。并以β-actin作為內(nèi)參基因?qū)Ω鹘M織樣品中細(xì)胞的起始數(shù)量進(jìn)行校正,為準(zhǔn)確確定不同組織樣品中H4亞型AIV的拷貝數(shù)提供依據(jù)。為了掌握H4亞型AIV分離株對(duì)雞群的致病情況,篩選了 6株H4亞型AIV分離株。對(duì)4周齡SPF雞以106 EID50/只進(jìn)行滴鼻感染,結(jié)果6株H4亞型AIV分離株均不需要提前適應(yīng)即可在SPF雞體內(nèi)進(jìn)行有效地復(fù)制,且6株H4亞型AIV分離株均可向外界排毒。其中水禽源毒株 A/duck/Guangxi/101DI8/2011(H4N6)、A/duck/Guangxi/030E93/2015(H4N2)和A/goose/Guangxi/220G30/2016(H4N2)均具有水平傳播的能力。整個(gè)試驗(yàn)過(guò)程中病毒未引起雞群的全身性感染或者死亡,感染3 d后剖檢部分感染雞可見(jiàn)肺臟和腎臟有輕微的充血,脾臟有輕微的腫大,其余器官無(wú)明顯的解剖學(xué)病變;病理切片觀察可見(jiàn)氣管、肺臟、肝臟、心臟和腎臟出現(xiàn)淋巴細(xì)胞浸潤(rùn),肺臟和腎臟出現(xiàn)充血等組織學(xué)病變。同時(shí)用雞胚對(duì)氣管、胸腺、腦、肝臟、脾臟、心臟、肺臟、胰腺、腎臟、法氏囊和盲腸扁桃體11個(gè)不同組織進(jìn)行病毒滴定,結(jié)果顯示在氣管和肺臟中病毒滴度較高,呈現(xiàn)出較強(qiáng)的組織嗜性。H4亞型AIV屬于LPAIV,目前也尚未引起家禽發(fā)病及流行的情況,但當(dāng)家禽群中存在H4亞型AIV和其它亞型AIV混合感染時(shí),不同亞型AIV的基因極易發(fā)生交換及重排產(chǎn)生新亞型AIV。因此加_7H4亞型AIV的監(jiān)測(cè),對(duì)AIV的防控有重要意義。
[Abstract]:H4 subtype avian influenza virus (Ave) is a common low pathogenic AIV(low pathogenetic AIV, especially in southern China, where the isolation rate of H4 subtype AIV in influenza virus surveillance is high, and it is often co-infected with H1H3, H5, H6, H9 and other subtypes of AIV. It is easy to rearrange and mutate the virus. Therefore, it is important to understand the genetic and evolutionary relationship of H4 subtype AIV and its pathogenicity for the prevention and control of avian influenza in China. In this study, the AIV of H4 subtype in Guangxi live poultry market from 2015 to 2016 was isolated and identified, and the whole genome sequence of the virus was analyzed. Results A total of 6 H4 subtype AIVs were isolated, all of them were LPAIV.The affinity of SA 偽 2C 3 and SA 偽 2O 6 receptors was also found, but the ability of binding SA 偽 2O 3 receptor was higher. Genetic evolution analysis showed that the 8 genes of 6 AIV isolates of H4 subtype in Guangxi belonged to Eurasian lineage, and the origin of different genomes was complex. The nucleotide sequence of HA gene was highly homologous to the Mongolian A / Duck / Mongolia- / OIE-7438 / 2011H4N6) isolate. H4 subtype AIV was mainly combined with N2 and N8 subtypes. The na gene of the H4N2 subtype was highly homologous to the na gene of the Mongolian A/duck/Mongolia/154/20 15 (H1N2) and Zhejiang A / Duck / Zhejiangr 727042 / 2014 H6N2) isolates, and the na gene of the H4N8 subtype was highly homologous to the na gene sequence of the Vietnam A / Duckr / Vietnam OIE-2577 / 2011H3N8 (A / Duckr / Vietnam OIE-2577 / 2011H3N8) isolate. It was suggested that the AIV isolates of H4 subtype in Guangxi were likely to recombine with the isolates from different countries mentioned above, which might be caused by the migration of migratory birds. In order to establish a rapid detection method for H4 subtype AIV, a pair of specific primers were designed according to the conserved HA gene sequence of H4 subtype AIV in GenBank. A real-time fluorescence quantitative detection method for H4 subtype AIVSYBRGreenI was established by optimizing the reaction conditions. 尾 -actin was used as an internal reference gene to correct the initial number of cells in each tissue sample, which provided the basis for determining accurately the copy number of H4 subtype AIV in different tissue samples. In order to understand the pathogenicity of H4 subtype AIV isolates to chickens, 6 H4 subtype AIV isolates were screened. SPF chickens aged 4 weeks were infected by nasal drip only with 106 EID50/. The results showed that 6 AIV isolates of H4 subtype could be replicated effectively in SPF chickens without prior adaptation, and 6 AIV isolates of H4 subtype could detoxify the environment. Among them, the waterfowl strain A / Duck / Guangxi / 101 / DI8 / 2011 / H4N6 / A / P / 030E93 / 2005 / H4N2) and A / R / G / Guangdong / 220G / 30 / 2016 / H4N _ 2 have the ability to transmit horizontally. During the whole experiment, the virus did not cause systemic infection or death in chickens. After 3 days of infection, some infected chickens showed slight congestion of lung and kidney, slight enlargement of spleen, and no obvious anatomical changes in other organs. Histological changes such as infiltration of lymphocytes in trachea, lung, liver, heart and kidney, congestion in lung and kidney were observed in pathological sections. The titer of virus in trachea, thymus, brain, liver, spleen, heart, lung, pancreas, kidney, bursa of Fabricius and cecal tonsil was also titrated with chicken embryo. H4 subtype AIV belongs to LPAIV.H4 subtype, which has not caused the disease and epidemic of poultry, but when there is mixed infection of H4 subtype AIV and other subtype AIV in poultry population, The genes of different subtypes of AIV are easy to be exchanged and rearranged to produce new subtypes of AIV. Therefore, it is important for the prevention and control of AIV to add the monitoring of subtype 7 H 4 AIV.
【學(xué)位授予單位】：廣西大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S852.65

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