2011-2012年肇庆市禽流感职业暴露人群及外环境病毒分布监测分析

目的了解肇庆市禽类职业暴露人群禽流感病毒感染状况以及外环境禽流感病毒的分布情况。方法采集禽类职业暴露人员血清样本,用红细胞血凝抑制试验（HI）检测H5N1流感抗体;采集外环境样本,用荧光定量PCR法检测禽流感病毒FluA、H5、H7和H9核酸。结果2011-2012年共采集职业暴露人员血清样本400份,检测H5N1抗体均为阴性;共采集外环境有效样本202份,检出FluA阳性25份,阳性率为12．38％,其中AH9亚型阳性14份（56．00％）。AH7亚型阳性1份（4．00％）,A未分型10份（40．00％）,未检出AH5亚型。结论肇庆市职业暴露人群尚未发现感染高致病性禽流感H5N1病毒．夕h环境存在禽流感病毒的污染,H9亚型是主要的病原体。     

人冠状病毒229E、OC43与SARS-CoV血清学相关性的研究

目的 检测正常人和SARS患者血清中3种人冠状病毒（229E、OCA3和SARS-CoV）特异性抗体．分析3种冠状病毒血清学相关性。方法 采用免疫印迹、免疫荧光和ELISA方法检测100例健康献血员、34例SARS患者恢复期以及11例SARS患者双份血清中229E、OCA3和SARS-CoV3种冠状病毒核衣壳（N）蛋白抗体。结果 用免疫荧光方法检测100例健康献血员血清中229E、OCA3和SARS-CoV IgG阳性率分别为98％、100％和1％，34例SARS患者恢复期血清中3种冠状病毒IgG的阳性率均为100％；免疫印迹检测100例健康献血员血清中229E、OCA3和SARS-CoVN蛋白IgG阳性率分别9r7％、99％和2％，34例SARS患者恢复期血清中229E、OCA3和SARS-CoVN蛋白IgG阳性率分别97％、100％和100％；11例SARS患者的急性期和恢复期双份血清中，免疫荧光检测有5例出现229E IgG滴度4倍或以上升高，10例出现OC43 IgG滴度4倍或以上升高，ELISA检测2例出现229EN蛋白IgG滴度4倍以上升高，没有一例出现OCA3N蛋白抗体滴度升高。结论 正常人群中普遍存在229E和OCA3两种人冠状病毒抗体，SARS-CoV感染者存在对人冠状病毒229E和OCA3血清学交叉反应，提示核衣壳蛋白不是引起血清学交叉反应的主要抗原，结果对研究SARS溯源有重要意义。     

斑点ELISA与环幼沉淀试验诊断旋毛虫病的研究

目的比较斑点ELISA法（Dot—ELISA）和玻片环幼沉淀试验（CPT）检测感染旋毛虫大鼠血清抗体的敏感性和特异性。方法采用Dot-ELISA和CPT两种免疫学诊断技术检测实验感染旋毛虫大鼠血清特异性抗体。结果Dot．ELISA和CPT法检测阳性率分别为97．5％和95．O％,差异无统计学意义（x2=0．3463,P〉0．05）。同时用此两种血清学方法检测正常大鼠、斯氏狸殖吸虫感染大鼠、日本血吸虫感染兔及蛔虫病人血清,除1例蛔虫病人CPT阳性外,其他均为阴性。从第2周开始旋毛虫感染大鼠Dot—ELISA和CPT均能测出抗体,第5周达高峰。结论Dot．ELISA和CPT对旋毛虫特异性IgG抗体的检测均有较好的敏感性和特异性。     

EBV4型IgA/VCA IgA/EA IgG/EA IgG/ZEBRA抗体在鼻咽癌普查和早期诊断中的应用

目的摸索以疱疹病毒4型（EBV）IgG／ZEBRA为捕捉抗原的间接酶联免疫吸附试验（ELISA）条件,为大量人群普查奠定基础。方法将纯化的ZEBRA抗原用于对鼻咽癌（NPC）患者血清及健康人血清IgG／ZEBRA抗体的ELISA检测。结果检测NPC患者血清288份,其中ELISA实验显示阳性262份,敏感度91％,检测正常人血清96份,其中阳性5份,特异度94．8％。其结果显示NPC组的阳性率与健康对照组的数据之间差异有统计学意义（P〈0．001）。本研究在此基础上对广东惠州5463份和广西桂平2017份血清进行检测,检出早期鼻咽癌患者5例。并将结果与免疫酶法检测IgA／VCA、IgA/EA、IgG／EA比较。结论以EBV早期抗原ZEBRA为捕捉抗原的间接ELISA方法具有较高的特异性和敏感性,可以用于大量人群的NPC早期筛查和早期诊断。     

高致病性人禽流感H5N1疫苗滴鼻免疫应答效果的初步研究

目的 通过高致病性人禽流感H5N1全病毒-MF59佐剂疫苗滴鼻免疫Balb/c小鼠,评价该疫苗所诱导的系统免疫与黏膜免疫应答效果.方法 以不同剂量抗原按比例与MF59佐剂配伍制成粘膜疫苗,滴鼻免疫Balb/c小鼠,二免2周采血检测血清IgG、IgM效价及血清中HAI(HA inhibitor)的中和抗体效价,同时收集鼻、肺灌洗液,检测其lgG和slgA抗体效价.结果 H5NI+MF59组血清抗体效价较H5NI组有显著升高(P＜0.01);在各剂量组中,随着剂量的增加抗体效价呈上升趋势.12μg腭后抗体效价呈下降趋势,以HSNI+MF59(12μg)组效价最高;肺鼻灌洗液中,均可检测到特异性分泌型IrA、IsG,其中特异性分泌型IgA效价略高于IgG;抗体亚型的分布以IgG1、IgG2b为主.结论 灭活高致病性禽流感全病毒H5N1在佐剂MF59作用下可诱导机体产生体液免疫应答,同时还可以在黏膜局部产生特异性分泌型IgA、IsG,为高致病人禽流感病毒I-15N1黏膜疫苗的研制奠定了基础.     

用重组SAG1抗原检测弓形虫抗体

目的探讨重组SAG1抗原对弓形虫IgG和IgM抗体的检测效果。方法用rSAG1作抗原建立免疫印迹方法（rSAG1-WB）,与玻片虫体过氧化物酶免疫染色试验（TSHE）平行检测不同来源血清。结果15例病原学检查阳性小鼠血清和5例免疫兔血清的IgG抗体均为阳性,30例正常小鼠血清和10例正常兔血清均未出现阳性反应。rSAG1-WB检测可疑弓形虫病患者血清阳性率为60．3％（38／63）,献血员血清阳性率为6％（3／50）,与TSHE检测结果（65．1％和4％）均无统计学差异（P〉0．05）。1例IgM强阳性血清和13例IgM弱阳性血清在Western—blot检测中分别出现相应的强阳性与弱阳性反应,50例献血员血清均未出现IgM阳性反应,结果与TSHE一致。结论rSAG1-WB检测弓形虫IgG和IgM抗体均具有高度的敏感性和特异性．与TSHE的符合率高。     

人博卡病毒母婴感染的临床前瞻性研究

目的调查本地区人博卡病毒母婴感染情况。方法用ELISA检测母婴血清人博卡病毒IgG抗体及用荧光定量PCR检测母婴血清人博卡病毒DNA。结果316例母婴配对标本中，孕妇血清人博卡病毒I蜘抗体阳性的有127例，阳性率为40．20％（127／316）；新生儿脐带血人博卡病毒IgG抗体阳性有93例，阳性率为29．43％（93／316）。新生儿人博卡病毒IgG抗体阳性占母亲人博卡病毒IgG抗体阳性的百分比为73．23％（93／127）。母婴同时人博卡病毒IgG抗体阳性为93例。316例母婴配对标本人博卡病毒DNA均为阴性。结论人博卡病毒IgG抗体可从孕妇经胎盘传给新生儿，但是否存在人博卡病毒母婴垂直传播尚有待于进一步研究。     

甘肃省SARS抗体血清流行病学调查

目的 了解甘肃省严重急性呼吸综合征(SARS)患者、密切接触者和正常人群血清SARS冠状病毒特异性IgG抗体水平。方法 利用酶联免疫吸附试验(ELISA)法检测SARS病毒IgG抗体水平。检测对象包括甘肃省9例SARS患者的急性期和(或)恢复期系列血清，l109例直接护理SARS患者的医生、护士、实验室检测人员、疾控人员和曾与患者有接触的人员以及978例正常人血清。结果 9例临床诊断病例SARS冠状病毒特异性IgG抗体中6例为阳性，疾病恢复后12个月血清抗体仍为阳性；密切接触者1例特异性IgG抗体阳性；正常人3例特异性IgG抗体阳性。结论 病例抗体阳性符合临床诊断，密切接触者和正常人的抗体阳性数较低，提示可能不存在隐性感染。     

陕西、青海、新疆三省(区)部分人群庚型肝炎病毒血清学调查

目的：调查陕西、青海、新疆三省（区）部分人群庚型肝炎病毒（HGV／GBV）血清学特征。方法：采用ELISA方法，共调查三省（区）1469份血清中GBV－IgG抗体。结果：少数民族血清GBV－IgG抗体的阳性率（藏族4．11％，蒙古族5．36％，维吾尔族4．55％，回族4．00％）略高于汉族（1．36％－1．73％），但差异无显著性（P＞0．05）；吸毒人群GBV－IgG阳性率（11．30％，34／301）明显高于正常人群（2．44％，18／736），（P＜0．01）；献血员GBV－IgG阳性率为1．02％－7．68％。结论：三省区民族间GBV－IgG抗体阳性差异无显著性，血源性传播是其重要途径，应加强对献血员及吸毒人员的监管。     

EBVTK激酶基因在原核细胞中的表达及其在NPC诊断中的意义

目的 探索以Epstein-Barr病毒（EBV）TK激酶为抗原，建立简便、快速、敏感和特异的鼻咽癌（NPC）诊断方法。方法 构建原核表达载体pRSET-TK，在原核细胞BL21（DF3）中获得高效表达的TK激酶，以Western blot检测证明其抗原的特异性和应用于NPC检测的可行性。以纯化的TK激酶为抗原初步建立ELISA检测方法，检测NPC患者血清中TK／IgG抗体。结果 在NPC患者血清中含有特异的针对TK激酶的IgG抗体，Western blot TK/IgG检测的敏感度和特异度均为100％。结论 初步建立了ELISA TK／IgG诊断NPC方法，具有较高的敏感性和特异性。     

Detection of antibody to avian influenza A (H5N1) virus in human serum by using a combination of serologic assays

From May to December 1997, 18 cases of mild to severe respiratory illness caused by avian influenza A (H5N1) viruses were identified in Hong Kong. The emergence of an avian virus in the human population prompted an epidemiological investigation to determine the extent of human-to-human transmission of the virus and risk factors associated with infection. The hemagglutination inhibition (HI) assay, the standard method for serologic detection of influenza virus infection in humans, has been shown to be less sensitive for the detection of antibodies induced by avian influenza viruses. Therefore, we developed a more sensitive microneutralization assay to detect antibodies to avian influenza in humans. Direct comparison of an HI assay and the microneutralization assay demonstrated that the latter was substantially more sensitive in detecting human antibodies to H5N1 virus in infected individuals. An H5-specific indirect enzyme-linked immunosorbent assay (ELISA) was also established to test children's sera. The sensitivity and specificity of the microneutralization assay were compared with those of an H5-specific indirect ELISA. When combined with a confirmatory H5-specific Western blot test, the specificities of both assays were improved. Maximum sensitivity (80%) and specificity (96%) for the detection of anti-H5 antibody in adults aged 18 to 59 years were achieved by using the microneutralization assay combined with Western blotting. Maximum sensitivity (100%) and specificity (100%) in detecting anti-H5 antibody in sera obtained from children less than 15 years of age were achieved by using ELISA combined with Western blotting. This new test algorithm is being used for the seroepidemiologic investigations of the avian H5N1 influenza outbreak.     

Virulent influenza A viruses induce apoptosis in chickens

Virulent avian influenza A viruses produce lethal disease in chickens. Since cell death can be caused by either necrosis or apoptosis, we investigated the types of cell death that occur in natural hosts, chickens, infected with virulent avian viruses. Using biochemical methods, we demonstrate that virulent avian influenza viruses induce apoptosis of vascular endothelial cells in liver, kidney, and brain. Viral antigens were also detected in these organs, suggesting that viral replication induces apoptosis in infected chickens. These results indicate that apoptosis does occur in virulent avian influenza virus infection in a natural host, and may contribute to the lethality of the virus.     

Seroprevalence of avian influenza H9N2 among poultry workers in Shandong Province, China

H9N2 avian influenza virus has been circulating widely in birds, with occasional infection among humans. Poultry workers are considered to be at high risk of infection with avian influenza due to their frequent exposure to chickens, but the frequency of H9N2 avian influenza virus infections among them is still indistinct. This study was carried out in order to identify the seroprevalence of H9N2 avian influenza virus among poultry workers in Shandong, China. During the period from December 2011 to February 2012, a total of 482 subjects took part in this study, including 382 poultry workers and 100 healthy residents without occupational poultry exposure. Serum samples were collected and tested for the presence of antibodies against H9N2 avian influenza virus by hemagglutination inhibition (HI) and microneutralization (MN) assays. Nine subjects (9/382?=?2.3 %) were positive for antibodies against H9N2 avian influenza virus among poultry workers by either HI or MN assays using ≥40 cut-off, while none of the 100 healthy residents were seropositive. In conclusion, our study identified H9N2 avian influenza infections among poultry workers in Shandong, China, and continuous surveillance of H9N2 avian influenza virus infection in humans should be carried out to evaluate the threat to public health.     

Generation and evaluation of an H9N1 influenza vaccine derived by reverse genetics that allows utilization of a DIVA strategy for control of H9N2 avian influenza

H9N2 avian influenza viruses have circulated widely in domestic poultry around the world, and their outbreaks have resulted in heavy morbidity and mortality. In addition, H9N2 avian influenza viruses were transmitted directly from birds to humans in Hong Kong and mainland China during 1998 and 2003, which prompted the public health authorities to seek protective strategies to control H9N2 influenza viruses. In this study, we attempted to develop a DIVA (differentiating infected and vaccinated animals) strategy for H9N2 avian influenza viruses. This strategy does not interfere with serological monitoring and allows effective control of H9N2 avian influenza. We generated a reassortant H9N1 influenza vaccine strain by reverse genetics and employed an enzyme-linked immunosorbent assay (ELISA) with a truncated N1 antigen expressed in E. coli to differentiate between vaccinated and naturally infected animals. Immunization of BALB/c mice with the inactivated reassortant H9N1 vaccine conferred protection against lethal challenge with H9N2 viruses. Meanwhile, the ELISA can be used to distinguish between vaccination and natural infection quickly and easily. Therefore, this study has opened up a new avenue for the control of H9N2 avian influenza.     

Serologic evidence of avian influenza virus infections among Nigerian agricultural workers

Nigeria has had multiple incursions of highly pathogenic avian influenza A (HPAI) H5N1 virus into its poultry population since 2006. This study aimed to determine if Nigerians exposed to poultry had evidence of avian influenza virus transmission to man. Between 2008 and 2010, 316 adult farmers and open market workers and 54 age‐group matched, non‐animal exposed controls were enrolled in a prospective, population‐based study of zoonotic influenza transmission in four towns in southeastern Nigeria. Questionnaire data and sera obtained at the time of enrollment were examined for evidence of previous infection with 10 avian influenza virus strains. Serologic studies on sera collected at the time of enrollment showed modest evidence of previous infection with three avian‐origin influenza viruses (H5N1, H5N2, and H11N1) and one avian‐like H9N2 influenza virus, with eight (2.4%) of animal‐exposed subjects and two (3.7%) unexposed subjects having elevated microneutralization assay antibody titer levels (ranging from 1:10 to 1:80). Statistical analyses did not identify specific risk factors associated with the elevated antibody titers observed for these zoonotic influenza viruses. These data suggested only occasional virus transmission to humans in areas thought to have been enzootic for avian influenza virus. Prospective data from this cohort will help the authors to better understand the occurrence of zoonotic infections due to avian influenza viruses in Nigeria. J. Med. Virol. 85:670–676, 2013. © 2013 Wiley Periodicals, Inc.     

Human Health Implications of Avian Influenza Viruses and Paramyxoviruses

Among avian influenza viruses and avian paramyxoviruses are the aetiological agents of two of the most devastating diseases of the animal kingdom: (i) the highly pathogenic form of avian influenza, caused by some viruses of the H5 and H7 subtypes, and (ii) Newcastle disease, caused by virulent strains of APMV type 1. Mortality rates due to these agents can exceed 50% in naïve bird populations, and, for some strains of AI, nearly 100%. These viruses may also be responsible for clinical conditions in humans. The virus responsible for Newcastle disease has been known to cause conjunctivitis in humans since the 1940s. The conjunctivitis is self-limiting and does not have any permanent consequences. Until 1997, reports of human infection with avian influenza viruses were sporadic and frequently associated with conjunctivitis. Recently, however, avian influenza virus infections have been associated with fatalities in human beings. These casualties have highlighted the potential risk that this type of infection poses to public health. In particular, the pathogenetic mechanisms of highly pathogenic avian influenza viruses in birds and the possibility of reassortment between avian and human viruses in the human host represent serious threats to human health. For this reason, any suspected case should be investigated thoroughly.This article is subject to British Crown Copyright with British Crown user rights reserved. Permission requests should be directed to enquiries@vla.defra.gsi.gov.uk     

Development of subtype-specific and heterosubtypic antibodies to the influenza A virus hemagglutinin after primary infection in children

Children undergoing primary infection with an H1N1 or H3N2 influenza A virus developed subtype-specific hemagglutination inhibition antibodies and enzyme-linked immunosorbent assay antibodies to purified hemagglutinin (HA) of the infecting virus subtype. They also developed lower titered ELISA antibodies to the noninfecting H1 or H3 HA and to H8 (an avian strain) HA. Thus, after primary infection with an influenza A virus, children develop enzyme-linked immunosorbent assay, but not hemagglutination inhibition, antibodies reactive with heterosubtypic HAs. These heterosubtypic antibodies could influence the response to infection with other wild-type or attenuated vaccine strains of influenza A virus.     

Influenza A viral nucleoprotein detection in isolates from human and various animal species

Summary A double antibody sandwich, enzyme-linked immunosorbent assay (DAS-ELISA) was developed to detect influenza A viral antigen, employing a monoclonal antibody directed against type-specific influenza A nucleoprotein (McAb anti-NP). McAb anti-NP was used to coat ELISA plates as well as to prepare the peroxidase conjugate. Influenza A viruses of avian, equine, swine, and human origin were detected in allantoic fluids of inoculated eggs with higher sensitivity by the DAS-ELISA than by hemagglutination (HA) assays. Minimal concentrations of 8 ng/ml influenza virus protein were detected in Nonidet P40-treated virus preparations. Viral antigen detection in tissues of experimentally infected chickens and pigs was successful, but in pigs yielded a lower positive score than the conventional method of virus isolation in eggs. The test is sensitive, rapid, and easy to perform, but does not permit influenza A subtyping. In avian species, the McAb anti-NP DAS-ELISA differentiates between influenza and Newcastle disease viruses. In pigs, the test distinguishes between influenza and Aujeszky's disease.     

Antibody-binding epitope differences in the nucleoprotein of avian and mammalian influenza A viruses

Abstract Influenza virus nucleoprotein (NP) binds to the viral genome RNA and forms the internal ribonucleoprotein complex of the virus particle. Avian and human influenza virus NP have characteristic differences at several amino acid positions. It is not known whether any of these differences can be recognized by antibodies. In the present study five monoclonal antibodies (MAbs) were produced against NP of A/Duck/Novosibirsk/56/05 (H5N1) influenza virus. Two MAbs discerned human and avian influenza strains on ELISA testing. The NP expressed in a prokaryotic system was used for the analysis of site-specific mutants carrying amino acid substitutions in the relevant positions. Amino acid residues in positions 100 and 101 were shown to be recognized by the MAbs. The residue in position 100 is host-specific, and its recognition by the MAb 2E6 may be useful for the differentiation of human and avian viruses. The data are discussed in view of the effects of amino acid substitutions in influenza virus NP affecting both host range and antibody-binding specificity.     

Evaluation of oral immunization with recombinant avian influenza virus HA1 displayed on the Lactococcus lactis surface and combined with the mucosal adjuvant cholera toxin subunit B

The development of safe and efficient avian influenza vaccines for human and animal uses is essential for preventing virulent outbreaks and pandemics worldwide. In this study, we constructed a recombinant (pgsA-HA1 gene fusion) Lactococcus lactis strain that expresses and displays the avian influenza virus HA1 antigens on its surface. The vectors were administered by oral delivery with or without the addition of cholera toxin subunit B (CTB). The resulting immune responses were analyzed, and the mice were eventually challenged with lethal doses of H5N1 viruses. Significant titers of hemagglutinin (HA)-specific serum IgG and fecal IgA were detected in the group that also received CTB. Cellular immunities were also shown in both cell proliferation and gamma interferon (IFN-γ) enzyme-linked immunospot (ELISpot) assays. Most importantly, the mice that received the L. lactis pgsA-HA1 strain combined with CTB were completely protected from lethal challenge of the H5N1 virus. These findings support the further development of L. lactis-based avian influenza virus vaccines for human and animal uses.