Development of a specific latex agglutination test based on a recombinant hemagglutinin protein to detect antibodies to H5 avian influenza viruses

A latex agglutination test (LAT) was developed for rapid detection of antibodies to H5 avian influenza viruses (AIVs). The hemagglutinin protein of H5 AIV was covalently linked to carboxylated latex by ethyl-dimethyl-amino-propyl carbodiimide to prepare the sensitized latex beads. The LAT was evaluated with the hemagglutination inhibition (HI) assay as the reference test. The H5-LAT showed a sensitivity of 87.0% and specificity of 88.9% in detecting 126 serum samples from experimentally infected chickens and a sensitivity of 82.5% and specificity of 86% in detecting 587 field chicken serum samples from mostly vaccinated chickens. The agreement ratio between H5-LAT and HI was found to be 87.3% and 83.1% for the two groups of samples, respectively. Difficulty with background agglutination in stored chicken sera was overcome by serum pretreatment with either dried chicken liver powder or dilution buffer containing detergent Tween-20. The H5-LAT has advantages over a previously reported whole-virus LAT in terms of biosafety in preparation, chemical stability, and higher specificity. It is a rapid and simple test suitable for field monitoring of antibodies to H5-type AIV.     

Commercial immunoassay kits for the detection of influenza virus type A: evaluation of their use with poultry

Five antigen capture immunoassay test kits, Directigen Flu A (Becton Dickinson), QuickVue Influenza test kit (Quidel), FLU OIA (ThermoBiostar), Zstat Flu (ZymeTx, Inc.) and NOW FLU A Test (Binax) were used to detect avian influenza virus (AIV) in clinical specimens as per manufacturers' protocols. Each kit was shown to be specific for AIV propagated in embryonating chicken eggs (ECE); other respiratory viruses of poultry tested gave negative results. The Directigen Flu A kit proved to be 10-fold more sensitive than the other kits, capable of detecting 10(4.7) mean embryo lethal dose (ELD50)/ml in allantoic fluid; this is more sensitive than the hemagglutination test using chicken erythrocytes. None of the kits proved to be sufficiently sensitive to reliably detect AIV in oropharyngeal and cloacal swabs collected from chickens experimentally infected with AIV subtype H6N2. In two different experiments, individual swabs and pools of five or six swabs were tested. By virus isolation, 39 individual oropharyngeal swabs tested positive for AIV, but Directigen and Flu OIA only detected 2/39 and NOW FLU A 1/39. Zstat and QuickVue did not detect any. Five individual cloacal swabs positive by virus isolation were negative with all five kits. In a second experiment using pools of five swabs, 26 swab pools were positive by virus isolation and 5/26 were positive by Directigen, the only kit to provide any positive results. Five cloacal swab pools were also positive by virus isolation and 1/5 was positive by Directigen; all other test kits were negative. All of these experiments were performed using the H6N2 subtype of AIV. The results are disappointing, as the kits have proven to be insensitive for detecting AIV when compared with the gold standard, virus isolation. This limits their use in diagnostic field investigations. Individual or groups of chickens could be assumed to be positive for AIV if positive by any of the kits, but a negative result with any of the kits would not prove that birds were AIV free.     

高效表达H9亚型禽流感病毒HA基因的重组鸡痘病毒的构建及免疫效力试验

将禽流感病毒血凝素 H9A基因克隆入插入载体 p FG11S中 ,通过酶切鉴定获得了正向转移载体 p FG11SHA;将其与禽痘病毒疫苗株 (w FPV)共转染鸡成纤维细胞 (CEF) ,通过蓝白斑筛选纯化得到重组病毒 r FPV- Ps- HA;以间接免疫荧光法证实 HA基因得到了表达。将该病毒经颈部皮下免疫 1日龄 SPF鸡 ,免疫后 15 d以 H9亚型禽流感病毒 F株翅静脉攻毒 ,攻毒后第 5天采集泄殖腔棉拭子样品进行病毒分离。将此重组病毒与以痘苗病毒 P7.5启动子表达相同基因的重组病毒 r FPV- P7.5 - HA作比较 ,结果表明 ,r FPV- Ps- HA相对于 r FPV- P7.5 - HA明显抑制了病毒的排出 ;攻毒后第 2、5、7、9、11天分别对 r FPV- Ps- HA、油乳剂灭活苗免疫鸡进行泄殖腔、气管排毒规律的检测 ,发现疫苗组均能很好地抑制排毒 ,攻毒对照组泄殖腔的排毒率明显高于气管排毒率     

Immuno-PCR for one step detection of H5N1 avian influenza virus and Newcastle disease virus using magnetic gold particles as carriers

Detecting avian influenza virus (AIV) and Newcastle disease virus (NDV) at low concentrations from tracheal and cloacal swabs of avian influenza- and Newcastle disease-infected poultry was carried out using a highly sensitive immunological-polymerase chain reaction (immuno-PCR) method. Magnetic gold particles were pre-coated with a capture antibody, either a monoclonal anti-AIV/H5 or monoclonal anti-NDV/F and viruses serially diluted ten-fold from 10(2) to 10(-5)EID(50)/ml. A biotinylated detection antibody bound to the viral antigen was then linked via a streptavidin bridge to biotinylated reporter DNA. After extensive washing, reporter DNA was released by denaturation, transferred to PCR tubes, amplified, electrophoresed and visualized. An optimized immuno-PCR method was able to detect as little as 10(-4)EID(50)/ml AIV and NDV. To further evaluate the specificity and the clinical application of this IPCR assay for AIV H5N1 and NDV, the tracheal swab specimens, taken from chickens which were infected with H5N1/AIV, H9N2/AIV, H7N2/AIV, NDV, IBDV, IBV/H(120), were detected by IPCR. Our data demonstrated that this monoclonal antibody-based immuno-PCR method provides a platform capable of rapid screening of clinical samples for trace levels of AIV H5 and NDV in one step.     

Investigation of H7N2 avian influenza outbreaks in two broiler breeder flocks in Pennsylvania, 2001-02

An avian influenza (AI) outbreak occurred in meat-type chickens in central Pennsylvania from December 2001 to January 2002. Two broiler breeder flocks were initially infected almost simultaneously in early December. Avian influenza virus (AIV), H7N2 subtype, was isolated from the two premises in our laboratory. The H7N2 isolates were characterized as a low pathogenic strain at the National Veterinary Services Laboratories based on molecular sequencing of the virus hemagglutinin cleavage site and virus challenge studies in specific-pathogen-free leghorn chickens. However, clinical observations and pathologic findings indicated that this H7N2 virus appeared to be significantly pathogenic in meat-type chickens under field conditions. Follow-up investigation indicated that this H7N2 virus spread rapidly within each flock. Within 7 days of the recognized start of the outbreak, over 90% seroconversion was observed in the birds by the hemagglutination inhibition test. A diagnosis of AI was made within 24 hr of bird submission during this outbreak using a combination of virus detection by a same-day dot-enzyme-linked immunosorbent assay and virus isolation in embryonating chicken eggs. Follow-up investigation revealed that heavy virus shedding (90%-100% of birds shedding AIV) occurred between 4 and 7 days after disease onset, and a few birds (15%) continued to shed virus at 13 days post-disease onset, as detected by virus isolation on tracheal and cloacal swabs. AIV was not detected in or on eggs laid by the breeders during the testing phase of the outbreak. The two flocks were depopulated at 14 days after disease onset, and AIV was not detected on the two premises 23 days after depopulation.     

PCR制备地高辛标记的探针检测禽流感病毒核酸

用聚合酶链反应（PCR）技术,制备了广东禽流感无致病力分离株A/goose/China/24/96(H7N3）核蛋白基因片段（NPc）的地高辛标记的cDNA探针。建立并优化了检测禽流感病毒核酸的探针杂交法,探针杂交法能鉴别出非免疫鸡胚和SPF鸡胚尿囊液中的病毒,攻毒后第3天的SPF和非免疫鸡泄殖腔拭子中AIV的最大检出率为1/10,对临床样品中的AIV的最大检出率为1/7,而直接HA和HI法及AGP试验检不出临床样品的AIV。该探针具有较好的特异性和敏感性,为从分子水平探讨AIV的发病机理、临床早期快速诊断提供了新的研究手段。     

野生鸟类禽流感病毒感染情况的调查

为了解野生鸟类禽流感病毒(AIV)的携带感染情况,2006年～2010年,本研究在湖南省主要候鸟迁徙地收集115只野鸟组织或拭子样品、75份野鸟的新鲜粪便样品和72份血清样品。组织或拭子样品采用RT-PCR方法检测和鸡胚接种病毒分离鉴定,血清样品分别进行H5(含Re-5和Re-4)、H6、H7、H9、H10和H11抗体检测。结果表明,从斑鸠和绿头鸭组织中分别分离到H5N1亚型和H3N2亚型AIV;72份血清中有17份抗体为阳性,其中H5(Re-5)亚型5份、H5(Re-4)亚型1份、H6亚型1份、H7亚型2份和H9亚型8份,阳性率分别为6.94%、1.39%、1.39%、2.78%和11.11%。H10和H11亚型未检测到抗体阳性。     

H5N1亚型禽流感病毒感染海兰白鸡模型的建立

为建立H5N1亚型禽流感病毒感染海兰白鸡模型,本研究选取1株鹅源H5N1高致病性禽流感病毒A/goose/guangdong/1/96(H5N1)(简称GD1/96),测定其对4周龄海兰白鸡的半数致死量.感染模型试验中,将30只4周龄海兰白鸡随机分成3组,每组10只,5只直接感染,5只同居,试验组设置一个重复,将病毒液稀释至10^4.5EID₅₀,滴鼻、点眼各0.1 mL,对照组接种PBS,感染后24 h放入同居鸡;感染后连续观察14 d,记录死亡时间,每天采集咽喉拭子和泄殖腔拭子;感染组和同居组第3、5 天各剖解3只鸡,采集气管、肺脏、脑、脾脏、肾脏和十二指肠,进行病毒分离;qRT-PCR法分析感染组和同居组第3、5 天鸡肺组织中IFN-α和TNF-α的相对表达量.结果显示,GD1/96株的鸡胚半数感染量(EID₅₀)为10^-8.167/0.1 mL,对4周龄海兰白鸡的半数致死量为10^4.5 EID₅₀.感染模型试验结果显示,以10^4.5EID₅₀的攻毒剂量感染海兰白鸡,感染组鸡在感染后8 d全部死亡;在感染和同居3 d后,各组鸡的咽喉拭子和泄殖腔拭子均可检测到病毒;感染和同居后第3、5 天,各组鸡的6种组织中均可分离到高滴度的病毒;IFN-α和TNF-α在感染组和同居组的鸡肺脏组织中的表达量均显著增加(P <0.05).本试验建立了海兰白鸡的H5N1亚型禽流感病毒感染模型,为H5N1亚型禽流感病毒的致病机理及表达抗流感基因转基因鸡的研究奠定了基础.     

The occurrence and transmission characteristics of airborne H9N2 avian influenza virus

To better understand the transmission route of H9N2 avian influenza virus (AIV), two duplicate trials were conducted to observe the process of aerosol infection and direct contact in specific pathogen free chickens. Fifteen chickens (G1) were inoculated with H9N2 AIV and housed together with another 15 chickens (G2) in the same positive-negative-pressure isolator (A). Fifteen chickens (G3) were bred in another isolator (B) which was connected with A so that air could flow unidirectionally from A to B. Air, oropharyngeal and cloacal swabs, and blood samples were collected for the detection of aerosolized virus, virus shedding, and seroconversion. AIV aerosols were initially detected at day 2-3 post inoculation (dpi), reaching peak concentrations at 7 dpi. Virus shedding was detected in all chickens of G2, but only in a part in G3 (T1: 87%, T2: 80%). Antibodies were initially detected at 4-5 dpi, peaking at 14-21 dpi. The results showed that H9N2 AIV could be transmitted by both aerosol exposure and direct contact.     

Nephrotropic properties demonstrated by A/chicken/Alabama/75 (H4N8) following intravenous challenge of chickens.

Tissue tropism properties of A/chicken/Alabama/75 (H4N8) were examined after intravenous inoculation of 5-week-old specific-pathogen-free chickens. From 14 clinically normal chickens euthanatized on days 1-20 postinoculation, the frequencies of virus recovery were highest for cloacal swabs (86%), bursal swabs (64%), and kidney tissues (64%) and lowest for tracheal swabs (14%), thymus tissues (14%), bone-marrow swabs (7%), and brain tissues (0%). Evidence that the high frequency of virus recovery from kidney tissues was associated with virus replication in the kidney tissues was provided by high virus titers, ranging up to 10(9.5) mean embryo infectious dose per gram of kidney tissue, and by identification of intranuclear and intracytoplasmic type A influenza nucleoprotein in kidney cells using immunohistochemistry. Virus-recovery and virus titer results from three chickens that died on days 4 and 5 postinoculation paralleled the results from the clinically normal chickens. These findings indicate that A/chicken/Alabama/75 has nephrotropic properties similar to nephrotropic properties previously reported for waterfowl-origin type A influenza viruses and provide evidence that kidney lesions could be manifestations of systemic influenza infections in commercial laying chickens.     

禽流感油乳剂灭活疫苗的研究

将6种不同亚型的禽流感病毒（AIVH2N9、H3N8、H5N1、H5N2、H7N1、H9N2）分别接种鸡胚,收获尿囊液,经甲醛灭活,以矿物油为佐剂制成油乳剂灭活疫苗。疫苗接种4和8周龄SPF鸡,注苗后均无不良反应。每种亚型疫苗免疫后14天和21天攻毒保护率均达90％-100％。分别用H5N1和H9N2亚型灭活疫苗免疫8周龄SPF鸡、25和28周龄健康商品蛋鸡,免疫后7天产生免疫力,14天保护率达100％,21天后抗体达高峰,仔鸡接苗后最高血凝抑制（HI）几何平均滴度（GMT）为7.3-8.0log2,蛋鸡为8.0-10.5log2。免疫后180天,抗体效价不低于6.5log2。免疫后180天分别以AIV攻击,H5亚型疫苗组,用强毒攻击无一发病和死亡,对照鸡全部发病死亡;H9亚型疫苗组,对照鸡在攻毒后72小时停产,免疫鸡无一发病且产蛋正常,对同源攻毒的保护率达100％。     

Evaluation of the infectivity, length of infection, and immune response of a low-pathogenicity H7N2 avian influenza virus in specific-pathogen-free chickens

The H7N2 subtype of avian influenza virus (AIV) field isolate (H7N2/chicken/PA/3779-2/97), which caused the 1997-98 AIV outbreak in Pennsylvania, was evaluated for its infectivity, length of infection, and immune response in specific-pathogen-free (SPF) chickens. The composite findings of three clinical trials with various concentrations of virus indicated that this H7N2 subtype contained minimal pathogenicity for chickens. The concentration of the virus in the inoculum proved critical in the establishment of a productive infection in a chicken. Seven-day-old SPF chickens were not infected when inoculated with 10(0.7-2.0) mean embryo lethal dose (ELD50) of the H7N2 virus per bird. At this dose level, the immune response to this virus was not detected by the hemagglutination-inhibition (HI) test. Nonetheless, chickens at ages of 5 and 23 wk old tested were successfully infected when exposed to 10(4.7-5.7) ELD50 of H7N2 infectious doses per bird by various routes of administration and also by direct contact. Infected birds started shedding virus as early as 2 days postinoculation, and the period of virus shedding occurred mostly within 1 or 2 wk postinoculation (WPI). This H7N2 subtype of AIV induced a measurable immune response in all birds within 2 wk after virus exposure. Antibody titers were associated with AIV infectious doses and age of exposure of birds. Challenge of these infected birds with the same H7N2 virus at 5 and 10 WPI indicated the infective virus was recoverable from cloacal swabs at 3 days postchallenge and disappeared thereafter. In these challenged birds, the antibody levels as measured by the HI test spiked within 1-2 wk.     

Immunostimulant effect of a mixed herbal extract on infectious bursal disease virus (IBDV) vaccinated chickens in the context of a co-infection model of avian influenza virus H9N2 and IBDV

This study was conducted to assess the comparative effects of a mixed herbal extract (MHE) containing Ocimum sanctum, Withania somnifera, Emblica officinalis, Tinospora cordifolia, Mangifera indica, and Asphaltum (shilajit) on infectious bursal disease virus (IBDV)-vaccinated (VAC) chickens infected with IBDV and avian influenza virus (AIV) H9N2. The experiment included three groups (G1-G3): G1, the negative control group; G2, the VAC + challenged (Ch) group; and G3, the VAC + Ch + MHE group. MHE was orally administered continuously for 5 weeks post-vaccination (PV) with IBDV at 12 days of age, and the chicks were simultaneously challenged with virulent IBDV (intraocularly) and AIV H9N2 (intranasally) at 21 days PV. Blood and tissue samples as well as tracheal and cloacal swabs were gathered at different times PV and post-challenge. Immunological and haematological parameters, histopathological lesions, relative organ weights and final live weights revealed significant differences (P ≤ 0.05) between G2 and G3 groups. Furthermore, in the G3 group, the protection rates, ELISA and HI titers and CD4+/CD8+ ratio were significantly increased, whereas viral shedding titers and the heterophil/lymphocyte ratio were decreased. In conclusion, the oral administration of the mixed herbal extract for 5 weeks can stimulate the immune response to IBDV vaccination and relieves the pathogenicity of an AIV H9N2 and IBDV co-infection in chickens.     

A longitudinal study of a novel dot-enzyme-linked immunosorbent assay for detection of avian influenza virus

A monoclonal antibody (MAb)-based dot-enzyme-linked immunosorbent assay (ELISA) has been developed that detected the epitopes specifically associated with avian influenza virus (AIV). The dot-ELISA detected the antigens of AIV directly from clinical and field specimens. Data obtained from experimentally AIV-infected specific-pathogen-free chickens and also the 2001/02 AIV outbreak of serotype H7N2 positive flocks in Pennsylvania indicated that the mean sensitivity (Se) of the dot-ELISA ranged between 45% and 68% and the mean specificity (Sp), between 85% and 90%. The values were derived from various clinical and field specimens when compared with virus isolation with embryonating chicken eggs. On routine AIV surveillance samples, the dot-ELISA achieved a 92%-100% Sp on the basis of resting over 1500 AIV surveillance samples that were confirmed negative by virus isolation. The dot-ELISA detected AIV antigens with a 5-microl allantoic fluid sample that contained a concentration of 0.4 hemagglutinating units. Furthermore, the dot-ELISA retained its specificity for AIV because no cross-reactions were obtained with various other avian viruses. The findings in this study indicated that the dot-ELISA was highly sensitive and specific and comparable with the commercial Directigen test in the detection of AIV obtained from clinical and field specimens.     

H9N2亚型禽流感病毒中和单克隆抗体的制备与应用

为建立H9N2亚型禽流感病毒（Avian influenza virus,AIV）免疫层析快速检测技术,本研究以差速离心法纯化H9N2亚型AIV免疫BALB/c小鼠,将免疫小鼠脾细胞与骨髓瘤细胞SP2/0进行细胞融合和HAT选择性培养;以H9N2亚型AIV感染MDCK细胞建立异源免疫过氧化物酶单层细胞试验（IPMA）的单克隆抗体检测方法,通过对杂交瘤细胞的IPMA筛选和连续克隆化筛选鉴定抗H9N2亚型AIV中和性单克隆抗体;以胶体金标记HA单克隆抗体,配对HA单克隆抗体和羊抗小鼠IgG为检测线和质控线,制备H9N2亚型AIV快速检测试纸条,测定其特异性和敏感性。结果显示,获得了11株稳定分泌抗H9N2亚型AIV单克隆抗体的杂交瘤细胞,其单克隆抗体腹水IPMA效价在1.28×10^-4至2.56×10^-5之间。单克隆抗体3A2、5H6、6B8、7E10和9G12血凝抑制试验（HI）显示血凝抑制活性,其（HI）效价在6log2~9log2之间。单克隆抗体3A2、6B8和9G12在病毒中和试验中对H9N2亚型AIV有显著病毒中和活性,中和效价分别1∶6 400、1∶25 600和1∶25 600。Western blotting结果提示,该中和单克隆抗体识别HA蛋白线性抗原表位。利用配对单克隆抗体3A2和9G12研制的H9N2亚型AIV检测试纸条检测H9N2亚型AIV尿囊液的效价为9log2,灵敏度与经典血凝试验（HA）相当,与其他亚型AIV （H1、H3、H5、H7）,以及新城疫病毒和鸡传染性法氏囊病病毒等相关病毒均无交叉反应。本研究制备了具有病毒中和活性的抗H9N2亚型AIV单克隆抗体,并初步研制了H9N2亚型AIV检测试纸条,为H9N2亚型AIV新型疫苗研制和快速检测奠定良好的研究基础。     

检测禽流感病毒抗体的重组核蛋白间接ELISA方法的建立

以大肠杆菌系统表达的H9N2亚型禽流感病毒（AIV）核蛋白（NP）为抗原，建立了禽流感间接酶联免疫吸附试验抗体检测技术（NP—ELISA）。对263份待检血清（包括临床收集的243份血清和20份H9N2亚型AIV免疫鸡阳性血清）进行检测，NP—ELISA与琼脂免疫扩散试验（AGP）的总符合率为83．3％，与血凝抑制试验（HI）的总符合率为92％。特异性试验表明，NP—ELISA方法可以检测H5、H7和H9亚型AIV特异性抗体，检测为阳性的血清样品能够被阳性鸡胚尿囊液阻断。敏感性试验证实，NP—ELISA最早可以检测鸡感染后7d的血清样品，并于感染后10d确定100％血清阳性，而AGP检测直到首免后21～28d才出现部分血清阳性，HI检测直到10～14d才出现部分血清阳性，并且NP-ELISA要比HI敏感4～40倍。试验证明，NP—ELISA是检测AIV血清型特异性抗体的一种特异、敏感、快速、经济的血清学检测技术。     

H5亚型禽流感病毒间接免疫荧光快速诊断方法的建立

本研究以当前严重威胁我国养禽业的高致病性禽流感H5亚型病毒为研究对象，病毒在犬肾细胞（MDCK）上培养增殖，经蔗糖梯度离心对病毒进行纯化，免疫清洁级的新西兰公兔，高免血清经辛酸-硫酸铵法和葡聚糖G50柱纯化，制得第一抗体。以FITC标记的山羊抗兔IgG为第二抗体，通过反应条件的优化，建立了间接免疫荧光快速诊断方法。本法的最佳检测组织为心肌和胰腺，检测时间只需3小时，本法可检出人工感染后36小时尚未表现出临床症状鸡只中的病毒，对禽流感H7亚型、H9亚型病、新城疫、传染性支气管炎和传染性喉气管炎禽出败等病料进行特异性检验结果均为阴性。运用本方法对69个禽场的临床病料进行了检测，检测结果与鸡胚分离法进行比对，9个阳性场（广东省2004年9个原疫点的病料）的9份病料中，检出8份阳性；而鸡胚分离法阴性样品，本法检测结果与之完全相符。本法用于禽流感H5亚型病毒的快速诊断具有快速、简便、敏感、特异、费用低廉和不存在交叉污染等优点，在当前流行的H5亚型高致病性禽流感快速诊断中具有良好的应用前景。     

支气管炎型H9亚型禽流感病毒的分离与鉴定

广东省某鸡场发生一起以严重呼吸困难、急性死亡为特征的病例,5000只鸡在1周内连续死亡400多只。通过病理剖检,几乎所有病死鸡支气管都充满干酪样物质,导致支气管严重阻塞,气管及支气管黏膜严重出血。采集病死鸡的肝脏、脾、肺和气管干酪样物质,病料经处理后通过SPF鸡胚绒毛尿囊膜接种获得一株病毒。通过琼脂凝胶免疫扩散(AGP)试验,该病毒能与禽流感标准阳性血清形成明显沉淀线,初步确定该病毒为禽流感病毒;通过血凝HA试验,该病毒能够凝集鸡红细胞,但此血凝现象不能被抗NDV血清、抗EDSV-76血清、抗H 5亚型流感单因子血清、抗H 7亚型流感单因子血清所抑制,而能被抗H 9亚型禽流感单因子血清所抑制,以及通过H 9亚型流感病毒的RT-PCR分子生物学诊断方法,最终确诊该病毒为H 9亚型禽流感病毒。动物回归试验,攻毒鸡表现与临床发病鸡一致的症状及典型病理变化,患鸡支气管被干酷样物严重阻塞,气管、支气管黏膜较严重出血,通过病料的病毒分离与鉴定,能再次分离到该病毒。