本文選題：H9N2亞型流感病毒 + 蛋白數(shù)據(jù)分析庫�。� 參考：《浙江大學》2013年博士論文

【摘要】：1998年,香港地區(qū)報告首起人感染H9N2亞型禽流感病毒(簡稱H9N2病毒)病例以后,中國大陸也相繼出現(xiàn)了人感染H9N2病毒病例。有研究表明,H9N2病毒在我國禽類從業(yè)人群中的感染率高達15%左右,并引起急性呼吸道感染癥狀。當前,H9N2病毒在我國家禽中持續(xù)傳播,并出現(xiàn)了大規(guī)模基因重組,H9N2病毒已經(jīng)成為人類呼吸道感染的重要病原之一,并作為一個潛在的流感大流行候選病毒而引起了全球的重視。 本研究對2011年100份來自浙江省家禽糞便樣本進行了病毒分離,對獲得的3株H9N2病毒進行全基因序列測定和進化分析；應用生物信息學軟件,對網(wǎng)絡數(shù)據(jù)庫中H9N2病毒蛋白信息,進行了收集處理,構建一個實用、快速和高效的,能用于H9N2病毒生物學特征分析的蛋白數(shù)據(jù)分析庫；分別在小鼠和人細胞水平上建立了感染模型,通過對Toll樣受體(TLR)、NF-κB和IL-6表達水平之間的相互關系進行分析,初步探討H9N2病毒對人類的致病機制。 首先,在2011年2月至6月期間,對來源于浙江省活禽市場中健康家禽的糞便樣本進行收集,獲得100份樣本,并集中進行病毒分離,將獲得的3株H9N2和9株H1亞型病毒株進行了基因擴增、序列測定和同源性分析。結果表明,浙江省家禽中存在H9N2、H1N2和H1N3病毒,均為歐亞系；H9N2病毒出現(xiàn)了對金剛烷胺類藥物耐藥的S31N突變位點。病毒進化分析表明,出現(xiàn)了H9N2和H1N2病毒重組現(xiàn)象,提示需加強家禽中流感病毒監(jiān)測。本研究在中國范圍內首次報道了H1N3亞型流感病毒,為H1亞型流感流行病學研究提供了重要資料。 應用生物信息學軟件,對網(wǎng)絡數(shù)據(jù)庫中所有2013株H9N2病毒的蛋白信息,進行了全面收集和重新處理,建立了一個總共包含PB2、PB1、PB1-F2、PA、HA、 NP、NA、M1、M2、NS1和NS2等11個流感病毒蛋白的數(shù)據(jù)分析庫,共包含了8948條序列信息。分析發(fā)現(xiàn),禽類是H9N2病毒的主要自然宿主(占97.47%),主要分離于1997至2012年期間(占96.32%),主要來自亞洲地區(qū)(占96.37%)。有78.10%的H9N2病毒HA蛋白234位氨基酸為亮氨酸,具有與人類細胞優(yōu)先結合的特性；有9.3%的H9N2病毒M2蛋白出現(xiàn)了對金剛烷胺類藥物耐藥的S31N突變點。該蛋白數(shù)據(jù)分析庫,能快速進行有針對性的病毒生物特征分析,為有效預測H9N2病毒的感染性、致病性和耐藥性提供參考。 為了評價H9N2病毒對哺乳動物的致病性,本研究首先在小鼠水平上建立了H9N2病毒誘導急性肺損傷的動物模型,對正常小鼠和受病毒感染小鼠的外周血和肺臟中的IL-6、SOD、TLR4、TLR7和NF-κB表達水平進行檢測。結果表明,H9N2病毒浙江株A/chicken/Zhejiang/329/2011(Zj329)能感染小鼠,出現(xiàn)典型的急性臨床癥狀。病毒感染小鼠后,小鼠肺組織細胞中TLR4、TLR7和NF-κB mRNA水平發(fā)生了明顯上調,并引起外周血中炎性因子IL-6大量表達,在感染后第6d,這些參數(shù)值都達到最高。外周血中SOD表達水平在病毒感染后的2-6d內發(fā)生下降,在第4d達到最低值。 進一步在人肺A549細胞水平上建立了H9N2病毒(Zj329)的細胞感染模型,對Zj329病毒感染后細胞中和細胞上清中TLR4、TLR7、NF-κB和IL-6表達水平進行了檢測。結果表明,Zj329病毒能有效感染A549細胞,出現(xiàn)典型的細胞病變現(xiàn)象。病毒感染A549細胞后,細胞中TLR4和TLR7mRNA水平都發(fā)生了明顯上調,在病毒感染后12h達到最高值,同時NF-κB和IL-6表達活性也發(fā)生了明顯上調,在24h內隨時間增加而增強。 綜上所述,本研究得出以下結論： 1、浙江省家禽中存在H9N2病毒,均為歐亞系。 2、首次發(fā)現(xiàn)H9N2和H1N2亞型流感病毒之間出現(xiàn)了重組現(xiàn)象。 3、H9N2病毒浙江株能有效感染小鼠和人A549細胞,發(fā)生肺臟損傷和細胞病變,TLR4、TLR7、NF-κB和IL-6介導了H9N2病毒感染哺乳動物而引起的免疫損傷過程。 總之,在近年來禽流感病毒感染人類時有發(fā)生的情況下,本研究構建的具有實用和快速的H9N2病毒蛋白數(shù)據(jù)分析庫,為有效分析H9N2病毒的感染性、致病性和耐藥性提供參考。通過本研究的開展,獲得了H9N2病毒浙江株的相關信息,并在中國范圍內首次報道了H1N3亞型流感病毒,發(fā)表了相關SCI論文3篇,不僅豐富了我國流感病毒的分子流行病學資料,也將為人感染禽流感防治工作的開展提供科學依據(jù)。同時,本研究對于闡明H9N2病毒在感染人類呼吸道過程中的致病機制,具有重要意義。
[Abstract]:In 1998, the first human infection of the H9N2 subtype of avian influenza virus (H9N2 virus) was reported in Hongkong area, and the human infection of H9N2 virus has also appeared in the mainland of China. Some studies have shown that the infection rate of H9N2 virus in our poultry workers is about 15%, and the symptoms of acute respiratory infection are cited. At present, the H9N2 virus is in me. H9N2 has become one of the important pathogens of human respiratory infection, and has attracted worldwide attention as a potential influenza pandemic candidate virus.
In this study, 100 avian feces samples from Zhejiang province were isolated in 2011. The total gene sequencing and evolution analysis of 3 H9N2 viruses were carried out. The information of H9N2 virus protein in the network database was collected and processed by using bioinformatics software to construct a practical, fast and efficient way to be used for H9N2 disease. The protein data analysis library of the analysis of toxic biological characteristics; the infection model was established on the level of mice and human cells, and the relationship between the expression level of Toll like receptor (TLR), NF- kappa B and IL-6 was analyzed, and the pathogenic mechanism of H9N2 virus to human was preliminarily discussed.
First, from February 2011 to June, the fecal samples from healthy poultry from the live poultry market in Zhejiang province were collected, 100 samples were collected, and virus isolation was concentrated. 3 strains of H9N2 and 9 H1 subtypes were amplified, sequenced and homologous analysis. The results showed that there were H9N2, H1 in poultry in Zhejiang province. The N2 and H1N3 viruses were all Eurasian, and the H9N2 virus appeared in the S31N mutation site of drug resistance to amantadine. The analysis of virus evolution showed that the recombination of H9N2 and H1N2 viruses appeared, which suggested that the monitoring of influenza virus in poultry should be strengthened. This study reported the first influenza virus of H1N3 subtype for the first time in China, which was the epidemic of H1 subtype influenza. The study of disease studies provides important information.
With the application of bioinformatics software, the protein information of all 2013 H9N2 viruses in the network database was collected and retreated. A data analysis library including 11 influenza viruses, including PB2, PB1, PB1-F2, PA, HA, NP, NA, M1, M2, NS1 and NS2, was set up. The total of 8948 sequences of information were included. The main natural host of the virus (97.47%), mainly from 1997 to 2012 (96.32%), mainly from Asia (96.37%). 78.10% of the H9N2 virus HA protein 234 amino acids are leucine, with the characteristics of the combination of human cells, and 9.3% of the H9N2 virus M2 protein appears to be S31N resistant to the amantadine drug S31N The protein data analysis library can quickly carry out targeted viral biometric analysis to provide a reference for the effective prediction of H9N2 virus infection, pathogenicity and drug resistance.
In order to evaluate the pathogenicity of H9N2 virus to mammals, this study first established an animal model of H9N2 virus induced acute lung injury at the mouse level. The expression of IL-6, SOD, TLR4, TLR7 and NF- kappa B in the peripheral blood and lung of normal mice and infected mice was detected. The results showed that the H9N2 virus Zhejiang strain A/chicken/ was found to be A/chicken/. Zhejiang/329/2011 (Zj329) can infect mice and have typical acute clinical symptoms. After the virus infected mice, the levels of TLR4, TLR7 and NF- kappa B mRNA in the lung tissue of mice were obviously up-regulated, and the inflammatory factor IL-6 was expressed in large numbers in the peripheral blood. The values of these parameters reached the highest level after infection. The SOD expression level in peripheral blood was the highest. It decreased within 2-6d after viral infection and reached the lowest level at 4D.
A cell infection model of H9N2 virus (Zj329) was established on the level of human lung A549 cells, and the expression of TLR4, TLR7, NF- kappa B and IL-6 in the cells and cell supernatant after Zj329 virus infection was detected. The results showed that the Zj329 virus could effectively infect A549 cells and present typical cytopathic phenomena. The virus infected A549 cells after the virus infection. The levels of TLR4 and TLR7mRNA in the cell were obviously up-regulated, and 12h reached the highest value after the virus infection. Meanwhile, the expression of NF- kappa B and IL-6 also increased obviously, and increased with time in 24h.
To sum up, this study draws the following conclusions:
1, there are H9N2 viruses in poultry in Zhejiang Province, all of which are Eurasian.
2, it was the first time that there was a recombination between H9N2 and H1N2 influenza viruses.
3, H9N2 virus Zhejiang strain can effectively infect mice and human A549 cells and have lung injury and cytopathic lesions. TLR4, TLR7, NF- kappa B and IL-6 mediate the immune damage caused by H9N2 virus infection in mammals.
In a word, in the case of the outbreak of avian influenza virus infection in recent years, the practical and rapid data analysis library of H9N2 virus was constructed to provide a reference for the effective analysis of the infection, pathogenicity and drug resistance of the H9N2 virus. Through this study, the related information of the Zhejiang virus of the H9N2 virus was obtained, and in China The H1N3 subtype influenza virus was reported for the first time, and 3 articles related to SCI were published, which not only enriched the molecular epidemiological data of the influenza virus in China, but also provided a scientific basis for the development of the prevention and control of avian influenza. At the same time, this study clarifies the pathogenesis of H9N2 virus in the infection of human respiratory tract. Significance.

【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2013
【分類號】：R511.7

【參考文獻】

相關期刊論文 前9條

1 牛旭艷;張春晶;顧立剛;;毒熱平注射液對甲型流感病毒H3N2體外感染細胞中TLR7信號通路的影響[J];中華中醫(yī)藥雜志;2010年02期

2 呂衛(wèi)華;黃衛(wèi)良;;急性支氣管肺炎患兒血清SOD及相關細胞因子測定的意義[J];放射免疫學雜志;2010年03期

3 黃麗;郭顯蓉;屠宴會;宋德祿;楊浩;尹洪超;;A549細胞和Hep-2細胞表面糖鏈類型鑒定及其與H5N1型禽流感病毒結合的特性[J];基礎醫(yī)學與臨床;2010年12期

4 仇保豐;劉武杰;彭大新;胡順林;劉秀梵;;近年來華東地區(qū)家鴨中禽流感病毒的亞型分布[J];微生物學報;2008年10期

5 黃升海;魏偉;云云;;呼吸道合胞病毒感染上調Toll樣受體7和3基因的早期表達(英文)[J];微生物學報;2009年02期

6 徐明舉;利凱;崔紅玉;魏東;張瑞華;王存連;李寸欣;徐彤;;H3N2豬流感病毒誘導的小鼠急性肺損傷與SOD、NO、MDA和OH·變化的相關性[J];中國病理生理雜志;2011年04期

7 郭元吉,溫樂英,王敏,李梓,張燁,郭俊峰;我國豬群中H9N2亞型毒株HA和NA基因特性的研究[J];中華實驗和臨床病毒學雜志;2004年01期

8 郭元吉,李建國,程小雯,王敏,鄒毅,李釧華,蔡訪潺,廖華樂,張燁,郭俊峰,黃瑞敏,貝東;禽H9N2亞型流感病毒能感染人的發(fā)現(xiàn)[J];中華實驗和臨床病毒學雜志;1999年02期

9 楊式芹;張彩云;謝劍鋒;翁育偉;;福建省2004-2008年職業(yè)暴露人群H9亞型禽流感病毒抗體血清流行病學調查[J];中國人獸共患病學報;2010年12期

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