One-step multiplex RT-PCR for detection and subtyping of swine influenza H1, H3, N1, N2 viruses in clinical samples using a dual priming oligonucleotide (DPO) system

The swine influenza virus (SIV) H1N1, H1N2, and H3N2 subtypes circulate in Korean farm. A novel multiplex RT-PCR (m-RT-PCR) was developed to detect and subtype swine influenza viruses. This m-RT-PCR assay could identify H1, H3, N1 and N2 from clinical samples in single tube reaction using DPO system. Korean SIVs are closely related to the United States influenza viruses, and primers were developed for SIV from North American viruses and recently Korean isolates. The sensitivity of the m-RT-PCR was 10TCID(50)/ml for H1N1, H1N2 or H3N2. The lowest viral concentrations detected by single PCR were 1TCID(50)/ml for each subtype. Non-specific reactions were not observed when other viruses and bacteria were used to assess the m-RT-PCR. The results of m-RT-PCR were more effective than virus isolation or hemagglutination (HA) test. This assay using a DPO system provides a rapid, sensitive, and cost-effective laboratory diagnosis for detecting and subtyping of SIV in pigs.     

抗禽流感病毒H7亚型血凝素特异性单克隆抗体的研制

目的:研制禽流感病毒H7亚型血凝素特异性单克隆抗体(mAb)。方法:以H7亚型禽流感诊断抗原为免疫原免疫6～8周雌性BALB/c小鼠,末次加强免疫后取其脾细胞与骨髓瘤细胞Sp2/0-Ag-14进行融合。通过HA和HI试验筛选阳性克隆。应用HI试验和Western blot试验测定mAb的反应性和特异性。结果:共获得4株分泌抗AIVH7亚型HAmAbs的杂交瘤细胞株,分别命名为2E2、2A4、5F5、7G5。这些mAb的腹水HI效价在5×27～5×211之间,其中2E2属于IgM亚类,2A4属于IgG1亚类,5F5、7G5属于IgG2a亚类。Western blot分析结果显示,4株AIVH7亚型HAmAb能与AIVH7蛋白在Mr75000处反应,但不与新城疫病毒(NDV)蛋白发生反应,表明这些mAb能特异性识别AIVH7亚型HA。mAbHI反应性测定结果表明:4株mAb中,2E2、5F5、7G5只与H7亚型AIV发生特异性HI反应,而不与其他亚型AIV以及NDV、传染性支气管炎病毒(IBV)反应,显示出良好的特异性;而2A4除了与H7亚型AIV反应外,还与H15N8标准株发生低水平交叉反应。结论:这些mAb不仅为H7亚型AIV的HA结构分析提供了工具,而且为建立快速廉价的H7亚型禽流感诊断方法提供了核心试剂。     

Rapid pathotyping of recent H5N1 highly pathogenic avian influenza viruses and of H5 viruses with low pathogenicity by RT-PCR and restriction enzyme cleavage pattern (RECP)

Rapid and accurate diagnosis of avian influenza virus infections of poultry and humans comprises detection, subtyping, and, in case of subtypes H5 and H7, pathotyping of such viruses. Reliable methods for pathotyping of H5 avian influenza viruses (AIV) are based on determination of the intravenous pathogenicity index (IVPI) in specific pathogen free (SPF) chickens and on characterization of the hemagglutinin (HA) gene cleavage site by sequencing. The number of basic amino acids (arginine and lysine) at the cleavage site is an important indicator of pathogenicity. In this paper, a new rapid method for pathotyping of H5 subtype avian influenza viruses is described which is based on RT-PCR and restriction enzyme cleavage pattern (RECP) assay using the MboII restriction enzyme. Validation of the method using 28 H5 subtype reference isolates from different animal species revealed good performance characteristics regarding sensitivity and specificity, especially when targeting recent highly pathogenic AIV (HPAIV) of subtype H5N1 and Asian origin. In addition, RECP results were validated by testing 47 field samples from different sources and by sequencing of their RT-PCR products. This approach for H5 AIV pathotyping proved to be fast, reliable, and comparatively sensitive and is suitable especially for laboratories lacking sequencing or in vivo pathotyping facilities.     

Serum antibody responses in naturally occurring influenza A virus infection determined by enzyme-linked immunosorbent assay, hemagglutination inhibition, and complement fixation

Serum antibody responses to influenza A virus infection were examined in 388 normal subjects during a trial of chemoprophylaxis in an outbreak of influenza A in 1980-1981 in which both A/H1N1 and A/H3N2 viruses circulated. Paired serum specimens obtained over a 6-week period were tested for antibodies to both A/H1N1 and A/H3N2 viruses by conventional hemagglutination inhibition, complement fixation, and an enzyme-linked immunosorbent assay (ELISA). Antibody responses detected by ELISA were determined by calculation of the area generated between titration curves of paired sera (area method), as well as by a conventional endpoint dilution method (endpoint method). Forty-two significant antibody rises were detected; 42 by ELISA (area method), 33 by ELISA (endpoint method), 32 by hemagglutination inhibition, and 13 by complement fixation. ELISA (area method) detected rises more frequently than either ELISA (endpoint method) (P less than 0.01), hemagglutination inhibition (P less than 0.005), or complement fixation (P less than 0.001). Another sensitive assay, the microneutralization test, detected significantly fewer rises (33, P less than 0.025) than the ELISA (area method). In the 42 subjects with ELISA (area method) rises, corroborating evidence of influenza A infection by other techniques (virus isolation, microneutralization, hemagglutination inhibition, or complement fixation tests) were available for 39 (93%). ELISA (area method) rises were subtype specific in all serum pairs in which other documentation of subtype-specific infection was available (38 of 38). Thus, ELISA (area method) was the single most sensitive assay for detection of serum antibody rises in this setting and possessed a high degree of subtype specificity.     

Expression and purification of recombinant H5HA1 protein of H5N1 avian influenza virus in E. coli and its application in indirect ELISA

The PCR amplified HA1 fragment of H5N1 (H5HA1) avian influenza virus (AIV) hemagglutinin gene was cloned into pET28a (+) expression vector and expressed in Rosetta Blue (DE3) pLysS cells. The recombinant H5HA1 (rH5HA1) protein purified by passive gel elution after SDS-PAGE of the inclusion bodies reacted specifically with H5N1 serum in Western blot analysis. A subtype specific indirect enzyme linked immunosorbent assay (iELISA) using the rH5HA1 protein as the coating antigen was developed for detecting antibodies to H5 subtype of AIV. The assay had 89.04% sensitivity and 95.95% specificity when compared with haemagglutination inhibition test. The Kappa value of 0.842 indicated a perfect agreement between the tests. The iELISA developed can be used for serosurveillance of avian influenza in chickens.     

2016—2018年长沙市人群及活禽市场环境H5N6亚型禽流感病毒监测分析

目的:开展2016—2018年长沙市人群感染和活禽市场(live poultry markets, LPMs)环境污染H5N6亚型禽流感病毒(avian influenza virus, AIV)监测,为防控人感染H5N6亚型AIV提供实验室数据。方法:采集2016—2018年长沙市流感样病例和不明原因肺炎病例咽拭子6...     

Development of an immunochromatographic strip for rapid detection of H9 subtype avian influenza viruses

An immunochromatographic strip was developed for the detection of the H9 subtype of avian influenza viruses (H9AIVs) in poultry, using two monoclonal antibodies (MAb), 4C4 for H9AIV hemagglutinin (HA) and 4D4 for nucleoprotein. The 4C4 MAb was labeled with colloidal gold as the detection reagent, and the 4D4 MAb was blotted on the test line while a goat anti-mouse antibody was used on the control line of the nitrocellulose membrane. In comparison with the HA and HA inhibition (HI) tests, the strip was specific for the detection of H9AIV, with a sensitivity at 0.25 HA units within 10 min. Storage of the strips at room temperature for 6 months or at 4°C for 12 months did not change their sensitivity and specificity. Evaluation of the strip with experimental tracheal and cloacal swab samples collected from H9N2-infected chickens revealed that the strip detected the H9N2 viruses on day 3 postinoculation, earlier than the appearance of clinical symptoms. Application of the strip for the analysis of 157 tracheal or cloacal samples from potentially infected chickens on five poultry farms showed that four farms had chickens that were infected with H9AIV. Further characterization of 10 positive and 30 negative randomly selected samples showed that no single sample was false positive or negative, as determined by the standard virus isolation and HI assays. Therefore, the immunochromatographic strip for the detection of H9AIVs has high specificity, sensitivity, and stability. This finding, together with the advantages of rapid detection and easy operation and without the requirement for special skills and equipment, makes the strip suitable for onsite detection and the differentiation of H9AIVs from other viruses in poultry.     

Development of rHA1-ELISA for specific and sensitive detection of H9 subtype influenza virus

A recombinant antigen-based single serum dilution ELISA was developed for simultaneous detection and subtyping of influenza viruses. Recombinant baculovirus encoding the hemagglutinin (HA₁ subunit) of H9N2 virus was generated. To evaluate the rHA1-ELISA, microplates were coated with purified HA1 protein and tested with reference control sera. Subsequently, 92 field sera collected from chickens suspected to be infected with H9N2 AIV were employed to test the efficacy of the rHA1-ELISA. The sera were tested simultaneously by HI and a commercial AIV ELISA kit. The rHA1-ELISA appeared to be highly specific and sensitive for direct detection of H9N2 antibodies in serum samples.     

The use of FTA® filter papers for diagnosis of avian influenza virus

Avian influenza viruses (AIVs) infect a wide range of host species including domestic poultry and wild birds; also, AIVs may infect humans in whom some highly pathogenic viruses (HPAIV) may cause acute fatal disease. Accurate laboratory diagnosis of AIV infections requires time-consuming and logistically complex precautionary measures for shipment of specimens or viruses to avoid biohazard exposure. The feasibility was investigated of the Flinders Technology Associates filter paper (FTA? card) for infectivity of AIVs and to preserve viral RNA for detection by RT-qPCR, sequencing and by DNA microarray assay. The infectivity of AIV subtype H6N2 and HPAIV subtype H5N1 was inactivated completely within one hour after adsorption to the FTA card at room temperature. FTA-adsorbed viral RNA remained stable for five months. Swab samples obtained from chickens infected experimentally with H5N1 virus and spotted directly onto the FTA? cards allowed a sensitive and straightforward diagnosis by RT-qPCR. FTA? cards were also suitable for examination of field samples, although AIV RNA was detected with reduced sensitivity in comparison to direct examination of swab fluids. The use of FTA? cards will facilitate safe transport of samples for molecular diagnosis of AIV avoiding the need for an uninterrupted cold storage.     

Preparation of anti-idiotypic antibody against avian influenza virus subtype H9

To generate monoclonal anti-idiotypic antibodies(mAb2)against avian influenza virus subtype H9(H9 AⅣ),BALB/c mice were immunized with purified chicken anti-H9-AⅣ IgG and the splenocytes of immunized mice werefused with myeloma cells NS-1.Hybridoma cells were screened by indirect enzyme-linked immunosorbent assayswith both chicken and rabbit anti-H9-AⅣ IgG as coating antigens.One hybridoma cell clone secreting monoclonalantibody against idiotypes shared by both chicken and rabbit anti-H9-AⅣ IgG was established.Experimentsdemonstrated the mAb2 was able to inhibit the binding of hemagglutinin to anti-H9-AⅣ IgG and to inducechickens to generate hemagglutination inhibition antibodies,indicating this anti-species-sharing-idiotypic antibodybore the internal image of hemagglutinin on avian influenza virus.Cellular & Molecular Immunology.2005;2(2):155-157.     

Chimaeric VP2 proteins from infectious bursal disease virus containing the N-terminal M2e of H9 subtype avian influenza virus induce neutralizing antibody responses to both viruses

Subunit vaccines capable of inducing antibody against both infectious bursal disease virus (IBDV) and H9 subtype avian influenza virus (AIV) were developed. The VP2 protein of IBDV was used as a cargo protein to display a 12-amino-acid immunodominant epitope derived from the N-terminal M2 extracellular domain (nM2e) of the H9 subtype AIV. Two chimaeric proteins were constructed by insertion of one copy of the nM2e into the P_BC region (VP2_BCnM2e(H9)) or by fusing four copies of nM2e to the carboxyl terminal (VP2-4nM2e(H9)) of VP2. Genes that encoded the VP2 chimaeras were subsequently cloned into a baculovirus vector and expressed in Spodoptera frugiperda cells. The recombinant proteins were used to vaccinate chickens at day 0 and again after 4 weeks. Blood was collected at 2-week intervals after primary and secondary vaccination to detect the antibody titre against VP2 or the nM2e via indirect enzyme-linked immunosorbent assay. Virus neutralization tests were also performed to measure anti-IBDV or anti-H9 AIV neutralizing antibodies in chick embryo fibroblasts. Oropharyngeal and cloacal swabs were collected 3, 5 and 7 days post H9 subtype AIV infection for virus isolation. Vaccination with VP2-4nM2e(H9) induced higher levels of antibody responses against IBDV or H9 subtype AIV, and provided better protection against an IBDV virulent challenge compared with vaccination with VP2BCnM2e(H9) vaccine, the wild-type VP2 subunit vaccine or the IBDV subunit commercial vaccines. Both chimaeric VP2 vaccines showed poor efficacy in inhibiting H9 virus replication post challenge. In summary, chimaeric proteins that contain the nM2e epitope were able to induce both IBDV and H9 subtype AIV-neutralizing antibody responses.     

An outbreak of avian influenza subtype H9N8 among chickens in South Korea.

Low pathogenic avian influenza subtype H9N8 was diagnosed on a Korean native chicken farm in Gyeonggi province, South Korea, in late April 2004. Clinical signs included moderate respiratory distress, depression, mild diarrhoea, loss of appetite and a slightly elevated mortality (1.4% in 5 days). Pathologically, mucopurulent tracheitis and air sacculitis were prominently found with urate renal deposition. The isolated A/chicken/Kr/164/04 (H9N8) had an Ala-Ser-Gly-Arg (A/S/G/R) motif at the cleavage site of haemagglutinin, which has been commonly found in H9N2 isolated from Korean poultry. Phylogenetic analysis of the haemagglutinin and neuraminidase genes of the H9N8 avian influenza virus (AIV) isolate showed that reassortment had occurred. Its haemagglutinin gene was similar to that of Korean H9N2 AIVs, but its neuraminidase gene was closely related to that of A/WBF/Kr/KCA16/03 (H3N8) isolated from the faeces of wild birds in Korea. The pathogenicity of the isolate was tested on 6-week-old specific pathogen free chickens. The inoculated virus (H9N8) was recovered from most tested organs, including the trachea, lung, kidney, spleen, and caecal tonsil. This is the first report of an outbreak of low pathogenic avian influenza in chickens caused by AIV subtype H9N8.     

Development of an antigen-capture enzyme-linked immunosorbent assay using monoclonal antibodies for detecting H6 avian influenza viruses

The H6 subtype of avian influenza virus (AIV) infection occurs frequently in wild and domestic birds. AIV antigen detection is preferred for controlling AIV as birds are infected before they produce antibodies. The purpose of this study was to develop an early diagnostic method for AIV detection. Six monoclonal antibodies (mAbs) developed from a field H6N1 AIV strain were tested for their ability to bind to viruses. The two that showed the greatest binding ability to AIVs were used for antigen detection. An antigen-capture enzyme-linked immunosorbent assay (ELISA) to detect H6 AIVs was developed using these mAbs. One mAb was coated onto an ELISA plate as the capture antibody. The other mAb was used as the detector antibody after labeling with horseradish peroxidase. The antigen-capture ELISA detected H6N1 AIVs but not H5 AIVs, human H1N1, H3N2 influenza or other viruses. This antigen-capture ELISA could be used to specifically detect H6N1 AIV.     

Detection of Nonhemagglutinating Influenza A(H3) Viruses by Enzyme-Linked Immunosorbent Assay in Quantitative Influenza Virus Culture

To assess the efficacy of novel antiviral drugs against influenza virus in clinical trials, it is necessary to quantify infectious virus titers in respiratory tract samples from patients. Typically, this is achieved by inoculating virus-susceptible cells with serial dilutions of clinical specimens and detecting the production of progeny virus by hemagglutination, since influenza viruses generally have the capacity to bind and agglutinate erythrocytes of various species through their hemagglutinin (HA). This readout method is no longer adequate, since an increasing number of currently circulating influenza A virus H3 subtype (A[H3]) viruses display a reduced capacity to agglutinate erythrocytes. Here, we report the magnitude of this problem by analyzing the frequency of HA-deficient A(H3) viruses detected in The Netherlands from 1999 to 2012. Furthermore, we report the development and validation of an alternative method for monitoring the production of progeny influenza virus in quantitative virus cultures, which is independent of the capacity to agglutinate erythrocytes. This method is based on the detection of viral nucleoprotein (NP) in virus culture plates by enzyme-linked immunosorbent assay (ELISA), and it produced results similar to those of the hemagglutination assay using strains with good HA activity, including A/Brisbane/059/07 (H1N1), A/Victoria/210/09 (H3N2), other seasonal A(H1N1), A(H1N1)pdm09, and the majority of A(H3) virus strains isolated in 2009. In contrast, many A(H3) viruses that have circulated since 2010 failed to display HA activity, and infectious virus titers were determined only by detecting NP. The virus culture ELISA described here will enable efficacy testing of new antiviral compounds in clinical trials during seasons in which nonhemagglutinating influenza A viruses circulate.     

禽流感病毒H5N1 单克隆抗体的生物学特性鉴定

目的:探讨禽流感病毒单克隆抗体的生物学特性。方法:制备H5N1禽流感病毒的单克隆抗体,鉴定其亚型、效价、血凝抑制活性以及与其他亚型流感病毒的交叉反应性。免疫印迹方法验证单抗与H5N1抗原的结合,人体正常组织芯片的免疫组织化学染色法观察单抗的组织学反应。结果:发现两株禽流感病毒H5N1的单抗(H5-32和H5-35)分别与人体的肾、胰腺组织存在阳性结合。结论:禽流感病毒H5N1与人体组织间可能存在某种关联,这或许可为禽流感病毒感染和致病研究提供参考。