本文選題：豬源甲型H1N1流感病毒 + 分離鑒定��； 參考：《廣西大學(xué)》2015年碩士論文

【摘要】：2009年3月新甲型H1N1流感病毒在墨西哥暴發(fā),同年4月第1株2009甲型H1N1流感病毒被分離,之后有越來(lái)越多的報(bào)道表明2009甲型H1N1流感病毒可以由人向豬傳播。研究證明,2009甲型H1N1流感病毒是混合了豬源流感病毒、禽源流感病毒和人源流感病毒三者特征的重組病毒,給養(yǎng)豬業(yè)帶來(lái)巨大經(jīng)濟(jì)損失,也對(duì)人類的健康構(gòu)成威脅。因此對(duì)豬群中H1N1流感病毒進(jìn)行流行病學(xué)調(diào)查和監(jiān)測(cè)具有重要的公共衛(wèi)生意義。本實(shí)驗(yàn)從2013年1月～2014年12月對(duì)廣西省部分養(yǎng)豬場(chǎng)及發(fā)病豬群進(jìn)行樣品采集和病毒的分離,目的為研究該區(qū)域豬群中流行的流感病毒亞型、變異規(guī)律和發(fā)病情況,為豬流感的研究和防控提供參考。實(shí)驗(yàn)成果如下：1.從發(fā)病豬群分離鑒定得到12株H1N1亞型豬流感病毒,其中1株為2009甲型H1N1流感病毒。本研究從2013年1月～2014年12月采集至廣西8個(gè)市(地區(qū))發(fā)病豬群的豬組織病料共1609份進(jìn)行病毒分離。經(jīng)9-11日齡SPF雞胚分離得到12株豬流感病毒株。運(yùn)用RT-PCR的方法對(duì)12株病毒的HA和NA基因進(jìn)行擴(kuò)增和序列測(cè)定分析,鑒定12株病毒均為H1N1亞型,其中1株為2009甲型H1N1流感病毒。將這株豬源2009甲型H1N1流感病毒株作為本實(shí)驗(yàn)研究的對(duì)象,并命名為A/swine/Guangxi/18/2013(H1N1)。2.遺傳進(jìn)化分析表明分離株8個(gè)基因片段與其他2009甲型H1N1流感病毒的進(jìn)化關(guān)系一致。對(duì)A/swine/Guangxi/18/2013(H1N1)全基因組的8個(gè)基因片段進(jìn)行RT-PCR擴(kuò)增、克隆及序列測(cè)定,運(yùn)用MegAlign軟件進(jìn)行核苷酸和氨基酸的比較分析,再通過(guò)MEGA5.0軟件構(gòu)建遺傳進(jìn)化樹(shù)。進(jìn)化分析結(jié)果表明分離株與2009甲型H1N1流感毒株處于同一個(gè)進(jìn)化枝,與A/California/04/2009(H1N1)親緣關(guān)系很近,起源于同一祖先。其基因來(lái)源情況為：HA、NS和NP基因來(lái)源于古典型H1N1豬源譜系,NA和M基因來(lái)源于類禽型H1N1豬源譜系,PA和PB2基因來(lái)源于禽源譜系,PB1基因來(lái)源于H3N2人源譜系。分離株8個(gè)基因片段在抗原位點(diǎn)、受體結(jié)合位點(diǎn)、糖基化位點(diǎn)以及耐藥性位點(diǎn)等多處氨基酸發(fā)生了變異,表明其抗原特性、宿主適應(yīng)性、致病力以及抗藥性發(fā)生了變化。3.動(dòng)物實(shí)驗(yàn)發(fā)現(xiàn)分離株對(duì)BALB/c小鼠具有致病性。對(duì)BALB/c小鼠的攻毒實(shí)驗(yàn)結(jié)果顯示病毒可感染小鼠,并能在肺臟與鼻甲骨組織中進(jìn)行有效復(fù)制,感染小鼠后引起明顯的臨床癥狀,體重迅速下降造成小鼠平均體重最高降幅為13.02%。
[Abstract]:A new influenza A (H1N1) virus broke out in Mexico in March 2009, and the first strain of influenza A (H1N1) virus 2009 was isolated in April of the same year. After that, more and more reports showed that the 2009 A (H1N1) influenza virus could be transmitted from human to pig. It has been proved that the 2009 A (H1N1) influenza virus is a recombinant virus mixed with swine influenza virus, avian influenza virus and human influenza virus, which brings huge economic loss to pig industry and poses a threat to human health. Therefore, epidemiological investigation and surveillance of swine H1N1 influenza virus have important public health significance. From January 2013 to December 2014, samples were collected and virus isolated from some pig farms and infected pigs in Guangxi province. To provide reference for swine flu research and prevention and control. The results of the experiment are as follows: 1. Twelve strains of swine influenza A (H1N1) subtype were isolated and identified from the infected pigs, one of which was A (H1N1) virus. From January 2013 to December 2014, a total of 1609 samples were collected from pigs infected in 8 cities (regions) of Guangxi for virus isolation. Twelve strains of swine flu virus were isolated from 9-11 day old SPF chicken embryo. The HA and na genes of 12 viruses were amplified and sequenced by RT-PCR. All the 12 strains were identified as H1N1 subtypes, one of which was A (H1N1) virus. The swine strain of influenza A (H1N1) 2009 was named as A / swine / 18 / 2013H _ 1N _ 1 / 2. Genetic evolution analysis showed that 8 gene fragments of the isolated strain were consistent with the evolution of other influenza A (H1N1) viruses. Eight gene fragments were amplified by RT-PCR, cloned and sequenced. The nucleotide and amino acids were compared with MegAlign software, and then the genetic evolution tree was constructed by MEGA5.0 software. The results of evolutionary analysis showed that the isolated strain was in the same phylogenetic branch as the influenza A (H1N1) strain 2009, and was closely related to A / California / 04 / 2009 H1 / N1, and originated from the same ancestor. The gene sources were as follows: 1. The gene sources of Ham NS and NP genes were from na and M genes of the classical H1N1 swine lineage. The PA and PB2 genes of the avian H1N1 swine lineage were derived from the avian origin lineage and PB1 gene from the H3N2 human lineage. The amino acids of 8 gene fragments of the isolated strain were mutated at antigenic sites, receptor binding sites, glycosylation sites and drug resistance sites, indicating that the antigenic characteristics, host adaptability, pathogenicity and drug resistance changed .3. Animal experiments showed that the isolated strain was pathogenicity to BALB / c mice. The results of virus attack on BALB / c mice showed that the virus could infect mice and replicate effectively in the lungs and turbinate bone tissues. After infection, the mice caused obvious clinical symptoms, and rapid weight loss resulted in the highest average weight reduction of 13.02% in mice.
【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】：2015
【分類號(hào)】：S852.65

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