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

【摘要】：1998年,香港地區(qū)報(bào)告首起人感染H9N2亞型禽流感病毒(簡(jiǎn)稱H9N2病毒)病例以后,中國(guó)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(guó)禽類從業(yè)人群中的感染率高達(dá)15%左右,并引起急性呼吸道感染癥狀。當(dāng)前,H9N2病毒在我國(guó)家禽中持續(xù)傳播,并出現(xiàn)了大規(guī)�；蛑亟M,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(gè)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(duì)2011年100份來(lái)自浙江省家禽糞便樣本進(jìn)行了病毒分離,對(duì)獲得的3株H9N2病毒進(jìn)行全基因序列測(cè)定和進(jìn)化分析；應(yīng)用生物信息學(xué)軟件,對(duì)網(wǎng)絡(luò)數(shù)據(jù)庫(kù)中H9N2病毒蛋白信息,進(jìn)行了收集處理,構(gòu)建一個(gè)實(shí)用、快速和高效的,能用于H9N2病毒生物學(xué)特征分析的蛋白數(shù)據(jù)分析庫(kù)；分別在小鼠和人細(xì)胞水平上建立了感染模型,通過(guò)對(duì)Toll樣受體(TLR)、NF-κB和IL-6表達(dá)水平之間的相互關(guān)系進(jìn)行分析,初步探討H9N2病毒對(duì)人類的致病機(jī)制。 首先,在2011年2月至6月期間,對(duì)來(lái)源于浙江省活禽市場(chǎng)中健康家禽的糞便樣本進(jìn)行收集,獲得100份樣本,并集中進(jìn)行病毒分離,將獲得的3株H9N2和9株H1亞型病毒株進(jìn)行了基因擴(kuò)增、序列測(cè)定和同源性分析。結(jié)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(duì)金剛烷胺類藥物耐藥的S31N突變位點(diǎn)。病毒進(jìn)化分析表明,出現(xiàn)了H9N2和H1N2病毒重組現(xiàn)象,提示需加強(qiáng)家禽中流感病毒監(jiān)測(cè)。本研究在中國(guó)范圍內(nèi)首次報(bào)道了H1N3亞型流感病毒,為H1亞型流感流行病學(xué)研究提供了重要資料。 應(yīng)用生物信息學(xué)軟件,對(duì)網(wǎng)絡(luò)數(shù)據(jù)庫(kù)中所有2013株H9N2病毒的蛋白信息,進(jìn)行了全面收集和重新處理,建立了一個(gè)總共包含PB2、PB1、PB1-F2、PA、HA、 NP、NA、M1、M2、NS1和NS2等11個(gè)流感病毒蛋白的數(shù)據(jù)分析庫(kù),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(lái)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(xì)胞優(yōu)先結(jié)合的特性；有9.3%的H9N2病毒M2蛋白出現(xiàn)了對(duì)金剛烷胺類藥物耐藥的S31N突變點(diǎn)。該蛋白數(shù)據(jù)分析庫(kù),能快速進(jìn)行有針對(duì)性的病毒生物特征分析,為有效預(yù)測(cè)H9N2病毒的感染性、致病性和耐藥性提供參考。 為了評(píng)價(jià)H9N2病毒對(duì)哺乳動(dòng)物的致病性,本研究首先在小鼠水平上建立了H9N2病毒誘導(dǎo)急性肺損傷的動(dòng)物模型,對(duì)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(dá)水平進(jìn)行檢測(cè)。結(jié)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(xì)胞中TLR4、TLR7和NF-κB mRNA水平發(fā)生了明顯上調(diào),并引起外周血中炎性因子IL-6大量表達(dá),在感染后第6d,這些參數(shù)值都達(dá)到最高。外周血中SOD表達(dá)水平在病毒感染后的2-6d內(nèi)發(fā)生下降,在第4d達(dá)到最低值。 進(jìn)一步在人肺A549細(xì)胞水平上建立了H9N2病毒(Zj329)的細(xì)胞感染模型,對(duì)Zj329病毒感染后細(xì)胞中和細(xì)胞上清中TLR4、TLR7、NF-κB和IL-6表達(dá)水平進(jìn)行了檢測(cè)。結(jié)果表明,Zj329病毒能有效感染A549細(xì)胞,出現(xiàn)典型的細(xì)胞病變現(xiàn)象。病毒感染A549細(xì)胞后,細(xì)胞中TLR4和TLR7mRNA水平都發(fā)生了明顯上調(diào),在病毒感染后12h達(dá)到最高值,同時(shí)NF-κB和IL-6表達(dá)活性也發(fā)生了明顯上調(diào),在24h內(nèi)隨時(shí)間增加而增強(qiáng)。 綜上所述,本研究得出以下結(jié)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(xì)胞,發(fā)生肺臟損傷和細(xì)胞病變,TLR4、TLR7、NF-κB和IL-6介導(dǎo)了H9N2病毒感染哺乳動(dòng)物而引起的免疫損傷過(guò)程。 總之,在近年來(lái)禽流感病毒感染人類時(shí)有發(fā)生的情況下,本研究構(gòu)建的具有實(shí)用和快速的H9N2病毒蛋白數(shù)據(jù)分析庫(kù),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(guò)本研究的開(kāi)展,獲得了H9N2病毒浙江株的相關(guān)信息,并在中國(guó)范圍內(nèi)首次報(bào)道了H1N3亞型流感病毒,發(fā)表了相關(guān)SCI論文3篇,不僅豐富了我國(guó)流感病毒的分子流行病學(xué)資料,也將為人感染禽流感防治工作的開(kāi)展提供科學(xué)依據(jù)。同時(shí),本研究對(duì)于闡明H9N2病毒在感染人類呼吸道過(guò)程中的致病機(jī)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(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R511.7

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