本文選題：野鳥 + 禽流感　； 參考：《北京協(xié)和醫(yī)學(xué)院》2014年博士論文

【摘要】：禽流感(Avian Influenza, A1)是由甲型流感病毒所引起的各種家禽及野生禽類感染疾病綜合征。根據(jù)致病性不同可劃分為高致病性禽流感和低致病性禽流感。近兒年來,屢有禽流感病毒特別是低致病性禽流感突破種間屏障作用,直接感染人類或其它哺乳動物,甚至致人死亡事件的情況發(fā)生。野鳥作為儲存宿主在禽流感病毒傳播中的作用一直受到高度關(guān)注：H5N1亞型禽流感隨野鳥遷徙在不到兩年的時間里傳播到14個國家40多個地區(qū)；感染人的H7N9亞型禽流感病毒致少有2個基因節(jié)片源于野鳥攜帶病毒的變異；導(dǎo)致江西感染病例的人H10N8亞型禽流感病毒來自于野鳥。這些事件說明禽流感特別是高致病性禽流感已經(jīng)嚴(yán)重危及到人類的健康與生命安全,并有進一步突破種間屏障引發(fā)人間流感大流行的潛在可能。在野生禽流感監(jiān)測中,方面,發(fā)達(dá)國家己將野鳥禽流感監(jiān)測列入疫病監(jiān)測中的常態(tài)化監(jiān)測工作。中國林業(yè)局自2008年起開展了主動監(jiān)測工作。因野鳥采樣極為困難,國家投入相對較少,我國對野鳥禽流感的監(jiān)測工作還相對落后,如2013年在發(fā)生H7N9人感染事件前,周邊的日韓等國均已在野鴨中發(fā)生該病毒的前體病毒,而我國缺少相關(guān)毒株的監(jiān)測,無法有效溯源。另一方面,H5、H6、H7、H9、H10等多亞型禽源病毒向人類跨種傳播的現(xiàn)象提示我們可能有更多亞型的禽流感病毒會對人類健康構(gòu)成威脅,因此,深入了解作為儲存宿主的野鳥中禽流感病毒的分布情況和野鳥源病毒對哺乳動物的跨種傳播作用,對于加深我們對流感病毒的認(rèn)知,做好禽流感的防控工作有著重要的意義。 本研究采集了14省市48個地區(qū)的15152份野鳥樣品,進行了流感病毒的分離鑒定,分離得到57株病毒,進行一步分析表明野鳥流感病毒攜帶率約為0.38%,秋季樣品分離率高于春季。拭子樣品分離率約為0.42%,糞便(環(huán)境)樣品分離率為0.40%,組織樣品分離率為0.18%。分離的57株病毒包括8種HA和7種NA亞型,其中H3、H4亞型為優(yōu)勢流行毒株,但野鳥攜帶高致病性H5亞型禽流感在病毒的比例較高(17.5%),說明防控形勢依然嚴(yán)峻。為進一步了解這些毒株的起源及與國內(nèi)流行株(包括人、豬流行株)之間的關(guān)系,本研究對30株代表病毒進行了全基因測序。HA基因進化分析表明,除H3亞型具有宿主特異性外,其余亞型均存在禽、豬或禽、人或三者基因重組現(xiàn)象。NA基因進化關(guān)系更加復(fù)雜,但與HA進化關(guān)系無協(xié)同或一致性。相比于外部基因,內(nèi)部基因(PB2、PB、PA、NP、M、NS)進化關(guān)系更是錯綜復(fù)雜,均存在禽、豬、人宿主間基因節(jié)片交換現(xiàn)象。以上結(jié)果表明野鳥是流感病毒天然的貯存庫和基因庫,為不同亞型或不同宿主的流感病毒提供基因片段。此外,首次在國內(nèi)野鳥中分離到H13N6和H5N8病毒,提示進一步加強候鳥監(jiān)測工作將有助于提前了解可能進入家禽中的病毒。 為了解這些病毒對哺乳動物的致病性,本研究以小鼠為模型對28株病毒進行感染與致病能力研究。所用用的28株病毒均能在小鼠的肺臟、鼻甲骨中復(fù)制,但病毒滴度差別較大,以H5N8最高,可達(dá)105.7EID50/ml。 H5N1、H5N8、H4N6、H3N2、H7、H9N2業(yè)型毒株對小鼠均有不同程度的致死性,其中H5N8的致死率達(dá)100%,其余H3和H6亞型的病毒雖然能使小鼠感染,但并無特別的臨床特征。本結(jié)果提示我們上述源于野鳥的病毒已不需要適應(yīng)就可能直接感染哺乳動物。 為進一步評估毒株在哺乳動物間水平傳播的能力,分別進行了受體結(jié)合特性分析和豚鼠間水平傳播能力評估。受體結(jié)合特性測定結(jié)果表明H3業(yè)型病毒均具有SAα-2,6Gal(人樣流感病毒受體)結(jié)合特性,H7、H9亞型也具有一定的SAα-2,6Gal結(jié)合特性。本研究選取H3和H9亞型毒株在豚鼠間進行了接觸傳播研究,結(jié)果表明S89(H3N2)、ZH47(H3N3)和JL-2(H9N2)均具有一定的豚鼠間傳播能力,其中以S89(H3N2)傳播能力最高(2/3)。利用反向遺傳學(xué)技術(shù)分析了決定S89(H3N2)與SAα-2,6Gal結(jié)合的關(guān)鍵位點,發(fā)現(xiàn)L274I影響病毒受體結(jié)合特性,表明除已發(fā)現(xiàn)的系列位點外,受體結(jié)合區(qū)域的其它位點同樣會影響病毒受體識別能力。 為進一步分析野鳥、野生動物乃致一些家養(yǎng)動物攜帶病原體的背景,我們建立了病毒性病原體宏基因組學(xué)分析方法,結(jié)果表明,該方法無樣品類型特殊性差別,對環(huán)境、組織樣品均普遍適用。應(yīng)用該方法首次對野鳥體內(nèi)病毒種類及其豐度進行了研究,檢測到多種病毒(鵝圓環(huán)病毒、雉皰疹病毒、鴨乙型肝炎病毒、鶴乙型肝炎病毒、禽流感病毒等)。在對突發(fā)大規(guī)模死亡的家養(yǎng)豬樣品分析中發(fā)現(xiàn)豬流行性腹瀉病毒和新型豬嵴病毒。吉林省鼠出血熱監(jiān)測表明,宿主動物攜帶的漢坦病毒仍以漢城病毒為主,其遺傳進化呈現(xiàn)多樣性。這些研究表明該方法對新型或未知病毒性病原體分析具有重要應(yīng)用價值。
[Abstract]:Avian influenza (Avian Influenza, A1) is a variety of poultry and wild avian infectious diseases caused by influenza A virus. According to the pathogenicity, it can be divided into highly pathogenic avian influenza and low pathogenic avian influenza. In recent years, many avian influenza viruses, especially low pathogenic avian influenza, have broken through the interspecific barrier and directly infected people. The role of wild birds as storage hosts in the transmission of avian influenza viruses has been highly concerned: H5N1 subtype avian influenza spread to more than 40 regions in less than two years with the migration of wild birds in less than two years; the H5N1 avian influenza virus (H7N9) of the infected human is 2 less. The H10N8 subtype of avian influenza virus, which causes Jiangxi infection cases, comes from wild birds. These events indicate that avian influenza, especially highly pathogenic avian influenza, has seriously endangered human health and life safety, and further breaks through the interspecific barrier to cause the potential of the human influenza pandemic. In the field of wild bird flu monitoring, in the field of wild bird flu surveillance, the monitoring work of wild bird bird flu is included in the monitoring of epidemic disease. China Forestry Bureau has carried out active monitoring since 2008. Because of the very difficult sampling of wild birds and relatively less state input, the monitoring work of wild bird and bird flu in China is still relatively backward, for example, 20 13 years before the outbreak of H7N9 infection, Japan and South Korea and other countries in Japan and South Korea have had the virus precursor in wild ducks, but our country lacks the monitoring of related strains and can not be traced effectively. On the other hand, the spread of H5, H6, H7, H9, H10 and other subtypes of avian viruses suggests that we may have more subtype of avian influenza virus. It is a threat to human health. Therefore, it is of great significance to understand the distribution of avian influenza virus in wild birds as storage hosts and the cross species transmission of wild bird virus to mammals. It is of great significance to deepen our understanding of influenza virus and do a good job in the prevention and control of avian influenza.
In this study, 15152 samples of wild birds were collected from 48 regions of 14 provinces and cities. The isolation and identification of influenza viruses were carried out and 57 viruses were isolated. One step analysis showed that the carrying rate of wild bird flu virus was about 0.38%, the separation rate of samples in autumn was higher than that in spring. The separation rate of swab samples was about 0.42%, and the separation rate of fecal (environment) samples was 0.40%, tissue sample was organized. 57 strains of virus isolated by 0.18%. include 8 HA and 7 NA subtypes, of which H3 and H4 subtypes are the dominant strains, but the proportion of high pathogenic H5 subtype of avian influenza in wild birds is higher (17.5%), indicating that the prevention and control situation is still severe. In this study, the genetic analysis of the whole gene sequencing of 30 representative viruses showed that, except for the host specificity of the H3 subtype, the other subtypes had avian, pig, or avian, human or three gene recombination, the.NA gene evolution was more complex, but there was no synergism or consistency with HA, compared with the external gene. The evolutionary relationships of the genes (PB2, PB, PA, NP, M, NS) are more complex, and there are gene segments exchange between birds, pigs and human hosts. The above results show that the wild bird is a natural storage and gene pool of influenza viruses for influenza viruses in different subtypes or different hosts. In addition, it is the first time to separate H13N6 and H5N8 in domestic wild birds. The virus suggests that further monitoring of migratory birds will help advance understanding of viruses that may enter poultry.
In order to understand the pathogenicity of these viruses to mammals, this study used mice as a model to study the infection and pathogenicity of 28 strains of virus. The 28 viruses used in this study could be replicated in the lungs and nasal turbinate of mice, but the titer of the virus was different, with the highest H5N8, up to 105.7EID50/ml. H5N1, H5N8, H4N6, H3N2, H7, H9N2 industry strains to small The lethality of mice to varying degrees, of which the lethal rate of H5N8 is 100%, and the other H3 and H6 subtypes can make mice infected, but there is no special clinical feature. The results suggest that the virus from wild birds that have not needed adaptation may directly infect mammals.
In order to further evaluate the ability of the strains to spread among mammals, the receptor binding characteristics and the evaluation of the horizontal transmission ability between guinea pigs were carried out respectively. The results of receptor binding characterization showed that all H3 virus had SA alpha -2,6Gal (human like influenza virus receptor) binding characteristics, H7, H9 subtype also had a certain SA alpha -2,6Gal binding specificity. In this study, H3 and H9 subtypes were selected for contact transmission between guinea pigs. The results showed that S89 (H3N2), ZH47 (H3N3) and JL-2 (H9N2) had a certain ability of transmission between guinea pigs, and the ability of S89 (H3N2) was the highest (2/3). 274I affects the receptor binding characteristics of the virus, suggesting that apart from the serial loci found, other loci in the receptor binding region also affect the ability of viral receptor recognition.
In order to further analyze the background of wild birds and wild animals causing some domestic animals to carry pathogens, we have established a method of macrogenomics analysis of viral pathogens. The results show that the method has no specific differences in sample types, and is generally applicable to environment and tissue samples. The species and abundance of virus in wild birds should be first used for the first time. A variety of viruses (goose ring virus, pheasant herpes virus, duck hepatitis B virus, duck hepatitis B virus, avian influenza virus, avian influenza virus, etc.) were detected. The swine epidemic diarrhea virus and new swine crista virus were found in the analysis of domestic pig samples of sudden mass death. The surveillance of rat hemorrhagic fever in Jilin province showed that the host animal carried the Han Dynasty. Tana virus is still dominated by Seoul virus, and its genetic evolution presents diversity. These studies show that the method is of great value for the analysis of new or unknown viral pathogens.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R511.7

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