本文選題：甲型流感病毒 + 禽流感病毒　； 參考：《中國人民解放軍軍事醫(yī)學科學院》2014年博士論文

【摘要】：人感染禽流感病毒（avian influenza viruses, AIVs）的事件近年來報道愈加頻繁。自從禽流感病毒H5N1于1997年被報道能夠感染并致人死亡以來，已在15個國家造成658人感染，病死率近60%；禽流感病毒H7N7也于2003年在荷蘭引起1人死亡；其他禽流感病毒如H9N2、H7N3等感染人事件在香港、加拿大等地也有報道。2013年初，禽流感病毒H7N9人感染病例又在我國東南部出現(xiàn)，截止2014年2月28日H7N9已經(jīng)造成375人感染，115人死亡。因此，我國禽間AIVs感染及人群AIVs感染狀況是備受關(guān)注的科學問題。 進行H7N9禽流感病毒的現(xiàn)場調(diào)查，檢測方法是關(guān)鍵一環(huán)。為了選擇能夠應用于現(xiàn)場H7N9禽流感病毒的快速、高效的檢測方法，我們對5種用于檢測人甲型流感感染的商品化試劑盒（Life Technologies VetMAXTM-Gold SIV DetectionKit、Quidel Molecular Influenza A+B assay、Remel Xpect Flu AB assay、QuidelQuickvue influenza assay以及Quidel Sofia Influenza A+B assay）進行了評價。首先從江蘇省無錫市和南京市采集的健康活禽肛拭子樣本提取RNA，用世界衛(wèi)生組織（World Health Organization，WHO）推薦的實時熒光定量PCR（qReal-timePCR，qRT-PCR）influenza A assay和H7N9assay進行甲型流感病毒和禽流感病毒H7N9檢測，篩選出27個H7N9陽性標本、70個H7N9及甲型流感陰性標本。然后對這97個標本使用上述5種試劑盒進行盲法檢測以及WHO qRT-PCR H7N9assay的復核。檢測時，根據(jù)試劑盒的不同試驗原理，核酸分子檢測方法每種重復3次，抗原檢測方法每種重復2次。最后以WHO qRT-PCR H7N9assay作金標準，計算這幾種方法的敏感度和特異度及其可信區(qū)間（confidence interval，CI）。結(jié)果：（1）WHO qRT-PCR H7N9assay第一輪和第二輪結(jié)果有很好的一致性。（2）Quidel Quickvue influenza assay對97份標本檢測結(jié)果全部顯示陰性。其他四種試驗方法均有很好的特異度（96-100%）。（3）Life Technologies VetMAXTM-GoldSIV Detection Kit特異度最高，為100%（95%CI0.87-1.00），假陽性率4%（95%CI1-12%）。Quidel Molecular Influenza A+B assay敏感度較高，達85%（95%CI66-96%），假陽性率3%（95%CI0-10%）。 為掌握AIVs在無錫地區(qū)家禽中的感染狀況及其遺傳進化特征，我們于2013年7月-12月每月在無錫市活禽交易市場和家禽養(yǎng)殖場的家禽中采集活禽及其相關(guān)環(huán)境拭子標本，以拭子標本提取的RNA為模板，運用WHO qRT-PCR influenzaA assay對樣品進行甲型流感病毒篩查，對篩查陽性標本采用一步法反轉(zhuǎn)錄PCR進行流感病毒全基因組PCR擴增以及基因克隆和測序。測序所得的序列進行系統(tǒng)發(fā)育分析，同時對甲型流感病毒陽性標本進行MDCK細胞及雞胚分離培養(yǎng)和鑒定。結(jié)果：（1）共采集標本305份，甲型流感病毒陽性率為28.5%（87/305）。家禽交易市場樣本甲型流感病毒陽性率高于養(yǎng)殖場（31.9%vs.10.4%，χ2=9.16，P=0.002）；（2）共有6個樣本可進行分型，分別為H9N2（3個），H5N8（1個），H11N2（1個）和H5N1（1個）。系統(tǒng)發(fā)育分析表明H5N8和H11N2有不同程度的重組。（3）共分離到3株禽流感病毒：A/Chicken/1/Wuxi/2013(H9N2)、A/Chicken/2/Wuxi/2013(H9N2)和A/Chicken/1/Wuxi/2013(H5N1)。 禽間AIVs感染情況復雜多樣，而禽類飼養(yǎng)、加工等職業(yè)暴露人群是感染AIVs的高危人群。除了感染AIVs發(fā)病甚至死亡之外，他們當中還存在諸如H5、H7、H9、H11等亞型AIVs的隱性感染者。豬能夠感染豬流感病毒（swine influenzavirus，SIV），也能感染多種禽源和人源流感病毒，在“禽-豬-人”的種間傳播鏈中扮演著流感病毒中間宿主及多重宿主的作用。研究已證明豬可感染禽流感病毒H5N1和H9N2。廣東、福建、浙江地區(qū)約1%的豬職業(yè)暴露人群中有H9N2感染。因此，為了解禽和豬職業(yè)暴露人群甲型流感病毒的感染狀況，通過分析感染與一般人口社會學情況和流行病學特征的關(guān)系，確定感染AIVs的危險因素，為人感染AIVs的防治策略提供依據(jù)，我們在無錫地區(qū)禽（豬）養(yǎng)殖場、屠宰場、市場和散養(yǎng)戶集中地的暴露人群中開展了甲型流感病毒血清學現(xiàn)況調(diào)查。選擇了禽職業(yè)暴露人群498名，豬職業(yè)暴露人群608名和無職業(yè)暴露的對照人群165名，共1271名研究對象。通過血凝抑制（hemagglutination inhibition, HI）試驗和微量中和（microneutralization，MN）試驗對研究對象血清標本進行了人甲型季節(jié)性流感H1N1（seasonal influenza A/H1N1，sH1N1）、sH3N2，及禽流感病毒H5N1、H9N2和H7N1血清抗體的檢測。結(jié)果：（1）禽類職業(yè)暴露、豬職業(yè)暴露和對照人群的sH1N1HI抗體陽性率分別為17.7%（88/498），10.9%（66/608）和27.3%（45/165），三組sH1N1HI抗體陽性率差異有統(tǒng)計學意義（χ2=29.00，df=2，P=0.000）。sH3N2HI抗體陽性率分別為17.3%（86/498），6.4%（39/608）和8.5%（14/165），三組sH3N2HI抗體陽性率差異有統(tǒng)計學差異（χ2=34.29，df=2，P=0.000）。（2）H5N1在禽類職業(yè)暴露人群當中MN滴度≥1:80和≥1:160的分別占14.6%和8.4%，在豬職業(yè)暴露人群分別占0.2%和0.2%，對照人群中只有1名（0.6%）對象H5N1MN滴度為1:80，沒有更高滴度的對象。多因素logistic回歸分析表明，工作場所為市場的禽類職業(yè)暴露人員（OR=57.3，95%CI7.0-466.5）、慢性呼吸道疾�。∣R=12.8，95%CI1.6-102.5）以及年齡40-60歲（OR=3.1，95%CI1.5-6.6）為H5N1MN滴度≥1:80的危險因素。（3）對H5N1MN滴度≥1:80的76份血清再做HI試驗，最終按WHO MN滴度≥1:80且HI滴度≥1:160的確診病例標準判定有3名H5N1感染者，為2名市場賣禽人員和1名規(guī)�；B(yǎng)雞場工人，占禽暴露人群的0.6%（3/498），這3人中有2人否認近一年內(nèi)有流感樣癥狀。（4）H9N2試驗結(jié)果有5名研究對象MN滴度≥1:10，其中2名是市場賣禽人員，2名是市場賣豬肉人員，1名是豬屠宰場工人。H9N2在禽職業(yè)暴露人群和豬暴露人群的MN抗體陽性率分別為0.4%（2/498）和0.5%（3/608）。對照人群中未發(fā)現(xiàn)H9N2MN抗體陽性者。（5）H7N1HI試驗結(jié)果未發(fā)現(xiàn)抗體滴度≥1:10者。 H7N9禽流感病毒2013年初在中國東南部出現(xiàn)人感染病例，并在短期內(nèi)呈迅速增長態(tài)勢�；钋菁跋嚓P(guān)環(huán)境（包括活禽市場）暴露被認為是感染H7N9的首要危險因素。在H7N9疫情延續(xù)期間，禽暴露人群是否有隱性感染？活禽市場的活禽從業(yè)人員和非活禽從業(yè)人員，以及活禽市場以外的禽類從業(yè)人員相比，感染的風險是否相同？感染與社會人口學、基礎疾病、職業(yè)行為等因素又有什么樣的關(guān)系？基于這些科學問題，我們在無錫地區(qū)禽職業(yè)暴露人群中開展了H7N9的血清學現(xiàn)況調(diào)查，共納入對象1588名，，包括1178名活禽市場從業(yè)人員（300名活禽從業(yè)人員和878名非活禽從業(yè)人員），245名活禽市場以外的活禽從業(yè)人員和165名當?shù)亟】稻用�。實驗方法選擇火雞血HI檢測A/Anhui/1/2013(H7N9)血清抗體，HI滴度≥1:20的再用MN試驗復核，MN滴度≥1:20的判為陽性。為考察可能的交叉反應，同時檢測sH1N1、sH3N2以及LPAI H7N1抗體，結(jié)果按二分類變量形式納入H7N9感染危險因素的單因素logistic回歸檢驗。結(jié)果：（1）HI實驗結(jié)果有27份標本（1.7%）HI抗體滴度≥1:20，再由MN復核，結(jié)果有8份標本（0.5%）MN滴度≥1:20。這8份抗-H7N9中和抗體陽性標本所對應的研究對象分別為無錫7個區(qū)（縣）8個不同活禽市場的從業(yè)人員，其中7名活禽從業(yè)人員，1名為非活禽從業(yè)人員，H7N9抗體陽性率在活禽市場活禽從業(yè)人員中為2.3%（7/300），在非活禽從業(yè)人員中為0.1%（1/878）。這8名研究對象均否認一年內(nèi)有流感樣癥狀。（2）所有研究對象禽流感病毒H7N1的HI結(jié)果均為陰性。（3）二分類單因素logistic回歸分析顯示，活禽市場活禽從業(yè)人員感染H7N9的風險高于活禽市場非活禽從業(yè)人員（OR=20.95，95%CI2.57-171.01）。性別之間、不同年齡組之間、不同文化程度和不同收入水平之間抗體陽性率差異無統(tǒng)計學意義。sH1N1、sH3N2感染與H7N9感染也無統(tǒng)計學關(guān)聯(lián)。 結(jié)論：（1）Life Technologies VetMAXTM-Gold SIV Detection Kit和QuidelMolecular Influenza A+B assays對活禽肛拭子進行禽流感病毒H7N9檢測的效果與WHO推薦的H7N9qRT-PCR assay基本一致。（2）江蘇省無錫地區(qū)禽間AIVs感染情況復雜多樣，有H5N1、H5N8、H9N2和H11N2等，H5N8和H11N2很可能是重組毒株。（3）職業(yè)暴露人群AIVs感染普遍，禽暴露人群中存在較高水平的禽流感病毒H5N1感染以及低水平的H7N9和H9N2感染，豬暴露人群中也存在低水平的H5N1和H9N2感染。這些感染者很可能是隱性或輕癥感染。（4）工作場所為市場的禽類職業(yè)暴露人員、慢性呼吸道疾病以及年齡40-60歲為H5N1的感染危險因素。（5）活禽市場的活禽從業(yè)人員感染H7N9的危險高于活禽市場非活禽從業(yè)人員。因此，禽類職業(yè)暴露人群，特別是市場禽類從業(yè)人群，以及有呼吸道慢性疾病禽類暴露人群是AIVs的防控重點人群。加強活禽市場的管理、環(huán)境消毒和人員防護是控制AIVs傳播的必要手段。對禽類和職業(yè)暴露人群AIVs感染狀況進行系統(tǒng)、連續(xù)地監(jiān)測有著重要的公共衛(wèi)生意義。 本研究的創(chuàng)新點包括：（1）用HI和MN兩種方法串聯(lián)，證實了活禽市場禽暴露人群中存在禽流感病毒H7N9隱性感染，而活禽市場以外禽暴露人群中沒有發(fā)現(xiàn)H7N9隱性感染；（2）在同一禽暴露人群中發(fā)現(xiàn)多種AIVs隱性感染，包括H5N1、H7N9和H9N2感染；（3）發(fā)現(xiàn)禽暴露人群中禽流感病毒H5N1感染率較高；（4）發(fā)現(xiàn)豬暴露人群中存在低水平的禽流感病毒H5N1和H9N2感染；（5）發(fā)現(xiàn)活禽市場禽間AIVs感染復雜多樣，并存在重組毒株。 本研究的主要局限是血清學橫斷面調(diào)查無法確定研究對象AIVs感染發(fā)生的時間點，只能證明調(diào)查人群中可能存在既往感染。
[Abstract]:Human infection of avian influenza viruses (AIVs) has been reported more frequently in recent years. Since the avian influenza virus H5N1 was reported to be infected and killed in 1997, 658 people have been infected in 15 countries and the fatality rate is nearly 60%; the avian influenza virus H7N7 also caused 1 deaths in Holland in 2003; and other avian influenza viruses. The virus, such as H9N2, H7N3 and other infectious events in Hongkong, Canada and other places were reported in early.2013, and the case of avian influenza virus H7N9 infection appeared in the southeast of China. As of February 28, 2014, H7N9 had caused 375 people and 115 people died. Therefore, the AIVs infection among birds in China and the status of AIVs infection in the population are the scientific problems of concern.
In order to choose the fast and efficient detection method that can be applied to the field H7N9 avian influenza virus, we have 5 commercialized kits (Life Technologies VetMAXTM-Gold SIV DetectionKit, Quidel Molecular Influenza A+) to select the fast and efficient detection methods that can be used in the field of avian influenza virus in the field. B assay, Remel Xpect Flu AB assay, QuidelQuickvue influenza assay, and Quidel Sofia were evaluated first from the healthy live bird swab samples collected from the city of Wuxi and Nanjing, Jiangsu. QRT-PCR) influenza A assay and H7N9assay were tested for influenza A virus and avian influenza virus H7N9, and 27 positive specimens of H7N9, 70 H7N9 and influenza a negative specimens were screened. Then the 97 specimens were blinded by the above 5 kits and the WHO qRT-PCR H7N9assay was rechecked. Test principle, nucleic acid molecular detection method is repeated 3 times each, the method of antigen detection is repeated 2 times. Finally, WHO qRT-PCR H7N9assay is used as gold standard to calculate the sensitivity and specificity of these methods and their confidence interval (confidence interval, CI). Results: (1) the results of WHO qRT-PCR H7N9assay first and second rounds are in good agreement. (2) the results of Quidel Quickvue influenza assay were all negative for the test results of 97 specimens. The other four test methods had good specificity (96-100%). (3) Life Technologies VetMAXTM-GoldSIV Detection Kit specificity was the highest, 100% (95%CI0.87-1.00), false positive rate 4% (95%CI1-12%) sensitivity The rate was 85% (95%CI66-96%), and the false positive rate was 3% (95%CI0-10%).
In order to grasp the infection status and genetic evolution characteristics of AIVs in poultry in Wuxi area, we collected live fowl and its related environmental swabs in poultry and poultry farm of Wuxi city in July 2013 in -12 month, using RNA as a template extracted from swab specimens and using WHO qRT-PCR influenzaA assay to carry out the samples. The screening of influenza A virus was carried out by one step reverse transcriptional PCR for PCR amplification and gene cloning and sequencing of the influenza virus. Sequence analysis was carried out, and MDCK cells and chicken embryos were isolated and identified for the positive specimens of influenza A virus. Results: (1) collection of specimens. 305, the positive rate of influenza A virus was 28.5% (87/305). The positive rate of influenza A virus in poultry trading market samples was higher than that of farms (31.9%vs.10.4%, Chi 2=9.16, P=0.002); (2) there were 6 samples to be typed, respectively H9N2 (3), H5N8 (1), H11N2 (1) and H5N1 (1). Phylogenetic analysis showed that H5N8 and H11N2 had different degrees. Recombinant (3) a total of 3 strains of avian influenza virus were isolated: A/Chicken/1/Wuxi/2013 (H9N2), A/Chicken/2/Wuxi/2013 (H9N2) and A/Chicken/1/Wuxi/2013 (H5N1).
AIVs infection among birds is complex and diverse, while poultry raising, processing and other occupational exposures are high-risk groups of AIVs infection. In addition to infection of AIVs and even death, there are also latent infections such as H5, H7, H9, H11 and other subtype AIVs. Pigs can infect swine influenza virus (swine influenzavirus, SIV) and can infect a variety of birds. Source and human influenza viruses play the role of influenza virus intermediate host and multihost in the interspecific transmission chain of "avian - pig human". Studies have shown that pigs can infect avian influenza virus H5N1 and H9N2. Guangdong, Fujian, Zhejiang region, about 1% of porcine occupational exposure population have H9N2 infection. The infection status of influenza virus, through the analysis of the relationship between infection and the social and epidemiological characteristics of the general population, determines the risk factors of AIVs infection and provides the basis for the prevention and control strategy of human infection of AIVs. We have carried out influenza A influenza in the exposed population of poultry (pig) farms, slaughterhouses, markets and scattered households in Wuxi. A total of 498 avian occupational exposure, 608 porcine occupational exposures and 165 non occupational exposure controls were selected, and 1271 subjects were selected. The serum samples were tested by hemagglutination inhibition (HI) test and microneutralization (microneutralization, MN) test. H1N1 (seasonal influenza A/H1N1, sH1N1), sH3N2, and avian influenza virus H5N1, H9N2 and H7N1 serum antibodies were detected. Results: (1) occupational exposure in poultry was 17.7% (88/498), 10.9% (66/608) and 27.3%, respectively, and the positive rates of the three groups were different. The positive rates of.SH3N2HI antibody (chi 2=29.00, df=2, P=0.000) were 17.3% (86/498), 6.4% (39/608) and 8.5% (14/165), and there was a significant difference in the positive rate of sH3N2HI antibody in the three groups (x 2=34.29, df=2, P=0.000). (2) 14.6% and 8.4% of H5N1 in the occupational exposure population of poultry, respectively, in the occupational exposure of pigs. The group accounted for 0.2% and 0.2% respectively, and only 1 (0.6%) in the control group had H5N1MN titers of 1:80 and no higher titer. Multiple factor Logistic regression analysis showed that workplace exposed workers (OR=57.3,95%CI7.0-466.5), chronic respiratory disease (OR=12.8,95%CI1.6-102.5) and 40-60 years old (OR=3.1,95%CI1.5) -6.6) for the risk factors of H5N1MN's titer more than 1:80. (3) 76 sera of H5N1MN titer more than 1:80 were re done by HI test, and 3 H5N1 were determined according to the confirmed case standard of WHO MN titer > 1:80 and HI titer > 1:160. It was 2 market poultry personnel and 1 large-scale poultry farm workers, accounting for 0.6% of the poultry exposed population, and 2 of these 3 people. Influenza like symptoms were denied in the last year. (4) the H9N2 test results showed that 5 subjects had the MN titer more than 1:10, of which 2 were market selling poultry, 2 were market selling pork personnel and 1 were pigs slaughterhouse workers.H9N2 in poultry occupational exposure and pigs exposed to MN antibody positive rates of 0.4% (2/498) and 0.5% (3/608) respectively. The control population was not in the control population. H9N2MN antibody positive was found. (5) H7N1HI test results showed no antibody titer was greater than 1:10.
In early 2013, the H7N9 avian influenza virus had been infected with human cases in southeastern China and was growing rapidly in the short term. Exposure to live birds and related environments, including live poultry market, was considered as the primary risk factor for H7N9 infection. In the duration of the H7N9 epidemic, is there a recessive infection in the exposed population of birds and the living fowl practitioners and non people in the live poultry market Is there the same risk of infection among live poultry workers and poultry workers outside the live poultry market? What are the relationships between infection and social demography, basic disease, occupational behavior and other factors? Based on these scientific problems, we conducted a serological survey of H7N9 in the Wuxi avian occupational exposure population. 1588 participants, including 1178 live poultry market practitioners (300 live bird practitioners and 878 non live poultry employees), 245 live poultry workers outside the live poultry market and 165 local health residents, selected Turkey blood HI to detect A/Anhui/1/2013 (H7N9) blood serum antibody, HI titer more than 1:20, MN test recheck, MN titer The sH1N1, sH3N2, and LPAI H7N1 antibodies were examined for the possible cross reaction, and the results of the single factor Logistic regression test of the risk factors of H7N9 infection were examined in the form of two classified variables. Results: (1) the results of the HI test had 27 specimens (1.7%) of HI anti body titer more than 1:20, then rechecked by MN, and 8 specimens (0.5%) M. The research objects of the 8 anti -H7N9 neutralization antibody positive specimens of N titer more than 1:20. were employed in 8 different live poultry markets in 7 districts and counties in Wuxi, of which 7 live poultry workers and 1 non live poultry workers, and the positive rate of H7N9 antibody was 2.3% (7/300) among live poultry workers in live poultry market and in non living poultry workers. 0.1% (1/878). All 8 subjects denied influenza like symptoms in one year. (2) all the HI results of avian influenza virus H7N1 were negative. (3) two classified single factor Logistic regression analysis showed that the risk of living poultry workers infected with H7N9 was higher than that of non live poultry workers in live poultry market (OR=20.95,95%CI2.57-171.01). There was no significant difference in the positive rate of antibody positive rate between different age groups, different age groups, different educational levels and different income levels, but there was no statistically significant correlation between sH3N2 infection and H7N9 infection.
Conclusion: (1) the effect of Life Technologies VetMAXTM-Gold SIV Detection Kit and QuidelMolecular Influenza A+B assays on the H7N9 detection of avian influenza virus in live bird and anal swabs is basically the same as that recommended by WHO. 11N2 is likely to be a recombinant strain. (3) AIVs infection in occupational exposure population is common, high levels of avian influenza virus H5N1 infection and low level of H7N9 and H9N2 infection are present in the exposed population of birds, and low levels of H5N1 and H9N2 infection are also present in the exposed population of pigs. These infected people can be recessive or mild infection. (4) the workplace is the market. Occupational exposures for poultry, chronic respiratory diseases and 40-60 years of age of 40-60 years as risk factors for infection. (5) the risk of living poultry workers in the live poultry market is higher than that of non living poultry workers in the live poultry market. Therefore, occupational exposure to poultry workers, especially in the market birds, and of chronic respiratory diseases in the respiratory tract. The exposed population is the key group of prevention and control of AIVs. Strengthening the management of the live poultry market, environmental disinfection and personnel protection are the necessary means to control the transmission of AIVs. The system of AIVs infection in poultry and occupational exposed population has important public health significance.
The innovative points of this study include: (1) in series with two methods of HI and MN, it confirmed the existence of H7N9 recessive infection of avian influenza virus in the poultry exposed population of live poultry market, and no H7N9 recessive infection was found in the poultry exposed population outside the live poultry market; (2) multiple AIVs infection in the same bird exposed population, including H5N1, H7N9 and H9N2 infection, was found. (3) the H5N1 infection rate of avian influenza virus was found in the exposed population of birds. (4) there was a low level of avian influenza virus H5N1 and H9N2 infection in the exposed population of pigs, and (5) it was found that the AIVs infection among birds in the live poultry market was complex and varied, and there was a recombinant strain.
The main limitation of this study is that the serological cross-sectional survey can not determine the time point of the AIVs infection, and it can only prove that there may be previous infection in the survey population.
【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R511.7

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本文編號：1989891