野鸟禽流感监测及哺乳动物跨种传播能力评估

发布时间：2018-06-10 11:32

本文选题：野鸟 + 禽流感　；参考：《北京协和医学院》2014年博士论文

【摘要】：禽流感(Avian Influenza, A1)是由甲型流感病毒所引起的各种家禽及野生禽类感染疾病综合征。根据致病性不同可划分为高致病性禽流感和低致病性禽流感。近儿年来,屡有禽流感病毒特别是低致病性禽流感突破种间屏障作用,直接感染人类或其它哺乳动物,甚至致人死亡事件的情况发生。野鸟作为储存宿主在禽流感病毒传播中的作用一直受到高度关注：H5N1亚型禽流感随野鸟迁徙在不到两年的时间里传播到14个国家40多个地区；感染人的H7N9亚型禽流感病毒致少有2个基因节片源于野鸟携带病毒的变异；导致江西感染病例的人H10N8亚型禽流感病毒来自于野鸟。这些事件说明禽流感特别是高致病性禽流感已经严重危及到人类的健康与生命安全,并有进一步突破种间屏障引发人间流感大流行的潜在可能。在野生禽流感监测中,方面,发达国家己将野鸟禽流感监测列入疫病监测中的常态化监测工作。中国林业局自2008年起开展了主动监测工作。因野鸟采样极为困难,国家投入相对较少,我国对野鸟禽流感的监测工作还相对落后,如2013年在发生H7N9人感染事件前,周边的日韩等国均已在野鸭中发生该病毒的前体病毒,而我国缺少相关毒株的监测,无法有效溯源。另一方面,H5、H6、H7、H9、H10等多亚型禽源病毒向人类跨种传播的现象提示我们可能有更多亚型的禽流感病毒会对人类健康构成威胁,因此,深入了解作为储存宿主的野鸟中禽流感病毒的分布情况和野鸟源病毒对哺乳动物的跨种传播作用,对于加深我们对流感病毒的认知,做好禽流感的防控工作有着重要的意义。本研究采集了14省市48个地区的15152份野鸟样品,进行了流感病毒的分离鉴定,分离得到57株病毒,进行一步分析表明野鸟流感病毒携带率约为0.38%,秋季样品分离率高于春季。拭子样品分离率约为0.42%,粪便(环境)样品分离率为0.40%,组织样品分离率为0.18%。分离的57株病毒包括8种HA和7种NA亚型,其中H3、H4亚型为优势流行毒株,但野鸟携带高致病性H5亚型禽流感在病毒的比例较高(17.5%),说明防控形势依然严峻。为进一步了解这些毒株的起源及与国内流行株(包括人、猪流行株)之间的关系,本研究对30株代表病毒进行了全基因测序。HA基因进化分析表明,除H3亚型具有宿主特异性外,其余亚型均存在禽、猪或禽、人或三者基因重组现象。NA基因进化关系更加复杂,但与HA进化关系无协同或一致性。相比于外部基因,内部基因(PB2、PB、PA、NP、M、NS)进化关系更是错综复杂,均存在禽、猪、人宿主间基因节片交换现象。以上结果表明野鸟是流感病毒天然的贮存库和基因库,为不同亚型或不同宿主的流感病毒提供基因片段。此外,首次在国内野鸟中分离到H13N6和H5N8病毒,提示进一步加强候鸟监测工作将有助于提前了解可能进入家禽中的病毒。为了解这些病毒对哺乳动物的致病性,本研究以小鼠为模型对28株病毒进行感染与致病能力研究。所用用的28株病毒均能在小鼠的肺脏、鼻甲骨中复制,但病毒滴度差别较大,以H5N8最高,可达105.7EID50/ml。 H5N1、H5N8、H4N6、H3N2、H7、H9N2业型毒株对小鼠均有不同程度的致死性,其中H5N8的致死率达100%,其余H3和H6亚型的病毒虽然能使小鼠感染,但并无特别的临床特征。本结果提示我们上述源于野鸟的病毒已不需要适应就可能直接感染哺乳动物。为进一步评估毒株在哺乳动物间水平传播的能力,分别进行了受体结合特性分析和豚鼠间水平传播能力评估。受体结合特性测定结果表明H3业型病毒均具有SAα-2,6Gal(人样流感病毒受体)结合特性,H7、H9亚型也具有一定的SAα-2,6Gal结合特性。本研究选取H3和H9亚型毒株在豚鼠间进行了接触传播研究,结果表明S89(H3N2)、ZH47(H3N3)和JL-2(H9N2)均具有一定的豚鼠间传播能力,其中以S89(H3N2)传播能力最高(2/3)。利用反向遗传学技术分析了决定S89(H3N2)与SAα-2,6Gal结合的关键位点,发现L274I影响病毒受体结合特性,表明除已发现的系列位点外,受体结合区域的其它位点同样会影响病毒受体识别能力。为进一步分析野鸟、野生动物乃致一些家养动物携带病原体的背景,我们建立了病毒性病原体宏基因组学分析方法,结果表明,该方法无样品类型特殊性差别,对环境、组织样品均普遍适用。应用该方法首次对野鸟体内病毒种类及其丰度进行了研究,检测到多种病毒(鹅圆环病毒、雉疱疹病毒、鸭乙型肝炎病毒、鹤乙型肝炎病毒、禽流感病毒等)。在对突发大规模死亡的家养猪样品分析中发现猪流行性腹泻病毒和新型猪嵴病毒。吉林省鼠出血热监测表明,宿主动物携带的汉坦病毒仍以汉城病毒为主,其遗传进化呈现多样性。这些研究表明该方法对新型或未知病毒性病原体分析具有重要应用价值。
[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.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R511.7

【共引文献】