Evaluation of monoclonal antibodies for subtyping of currently circulating human type A influenza viruses.

The hemagglutinin subtype specificities of six monoclonal antibodies (MAbs) to influenza type A viruses were evaluated in a rapid culture assay by immunoperoxidase staining. Confluent monolayers of MDCK cells in multiwell plates were inoculated with (i) 23 reference viruses, (ii) 200 isolates collected during the influenza season 1995 to 1996, and (iii) 28 clinical specimens previously found to be influenza virus positive. After overnight incubation, the cells were fixed and stained with MAbs IVA1/B10, IIF4/D3, 12L/5, 13L/6, 18L/1, or 18L/4. Type-specific MAbs were included as controls. All antibodies gave intensive cytoplasmic staining with infected cells in the absence of any reaction with uninfected cells. MAbs 12L/5, 13L/6, 18L/1, and 18L/4 exclusively reacted with viruses of the subtype H1, and the antibodies IVA1/B10 and IIF4/D3 exclusively reacted with viruses of the subtype H3. None of these MAbs reacted with viruses of the H2 subtype or with influenza type B viruses. Of the 200 recent isolates, 63 were identified as influenza virus type A, subtype H1, 95 were identified as type A, subtype H3, and 41 were identified as type B. One isolate contained a mixture of a type A (H3) and a type B influenza virus. Of the 28 previously positive clinical specimens, 15 contained an influenza virus A, subtype H3, 1 contained an influenza virus A, subtype H1, and 9 contained an influenza B virus. The subtype of a very weakly positive specimen could not be determined, and two specimens remained negative. The MAbs described here allow for a rapid typing and subtyping of influenza virus isolates and for the type- and subtype-specific detection of influenza viruses in clinical specimens.     

流感及H5N1亚型禽流感病毒液相检测芯片的制备

目的 建立利用液相芯片技术检测甲、乙型流感和H5N1亚型高致病性禽流感病毒的方法,并对该方法进行评价。方法 对GenBank中甲型流感病毒的NP、乙型流感病毒的HA以及高致病性禽流感病毒（H5N1）的H5、N1基因片段序列进行同源性比对,根据保守序列,设计针对各基因的简并引物和寡核苷酸探针,制备探针偶联微球,将样本核酸多重PCR扩增产物与微球进行杂交,以Bio-Plex液相芯片检测系统进行芯片检测。结果 该方法可以对甲型流感病毒的NP基因、乙型流感病毒的HA基因以及高致病性禽流感病毒（H5N1）的H5、N1基因同时进行检测,病毒核酸的最低检出量为1pg,检测特异性高。结论 成功构建了甲、乙型流感病毒和H5N1亚型高致病性禽流感病毒液相芯片检测系统,为流感、禽流感的快速检测、诊断奠定了基础。     

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.     

A multiplex real-time RT-PCR for detection and identification of influenza virus types A and B and subtypes H5 and N1

A multiplex real-time RT-PCR method for the simultaneous detection of influenza virus types A and B and identification of subtypes H5 and N1 in a single tube is described. The method was developed with four sets of primers and probes which were specific to influenza virus (sub)types A, B, H5, and N1, and evaluated by using a total of 40 influenza virus reference strains, including 17 avian influenza A (12 H5N1, 1 H1N1, 1 H3N2, 1 H4N6, 1 H7N3, and 1 H9N2), 18 human influenza A (11 H3N2, 6 H1N1 and 1 H5N1) and 5 influenza B viruses. The method exhibited a high specificity and sensitivity of approximately 10(1)-10(2)copies/microl for each (sub)type and a high reproducibility with intra- and inter-assay CV from 0.13 to 4.24%. In an analysis of 189 clinical samples from patients during the year 2004 and 2005, the method identified 81 positive samples (42.9%) and identified simultaneously 14 type B samples and 11 subtype N1 samples, in comparison only 46 positive samples (24.3%) identified by the conventional culturing method. The method would be a useful molecular diagnostic tool for large-scale screening of clinical samples for influenza virus.     

Typing (A/B) and subtyping (H1/H3/H5) of influenza A viruses by multiplex real-time RT-PCR assays

In this study, a specific and sensitive one-step multiplex real-time RT-PCR was developed in two assays by using primers and a number of specific locked nucleic acid (LNA)-mediated TaqMan probes which increase the thermal stability of oligonucleotides. The first assay consisted of primers and probes specific to the matrix (M1) gene of influenza A virus, matrix (M1) gene of influenza B virus and GAPDH gene of host cells for typing of influenza virus and verification by an internal control, respectively. The other assay employed primers and probes specific to the hemagglutinin gene of H1, H3 and H5 subtypes in order to identify the three most prominent subtypes of influenza A capable of infecting humans. The specificity results did not produce any cross reactivity with other respiratory viruses or other subtypes of influenza A viruses (H2, H4 and H6-H15), indicating the high specificity of the primers and probes used. The sensitivity of the assays which depend on the type or subtype being detected was approximately 10 to 10(3)copies/microl that depended on the types or subtypes being detected. Furthermore, the assays demonstrated 100% concordance with 35 specimens infected with influenza A viruses and 34 specimens infected with other respiratory viruses, which were identified by direct nucleotide sequencing. In conclusion, the multiplex real-time RT-PCR assays have proven advantageous in terms of rapidity, specificity and sensitivity for human specimens and thus present a feasible and attractive method for large-scale detection aimed at controlling influenza outbreaks.     

Subtype identification of the novel A H1N1 and other human influenza A viruses using an oligonucleotide microarray

A novel strain of influenza A (H1N1) virus was isolated in Mexico and the US in March and April 2009. This novel virus spread to many countries and regions in a few months, and WHO raised the level of pandemic alert from phase 5 to phase 6 on June 11, 2009. The accurate identification of H1N1 virus and other human seasonal influenza A viruses is very important for further treatment and control of their infections. In this study, we developed an oligonucleotide microarray to subtype human H1N1, H3N2 and H5N1 influenza viruses, which could distinguish the novel H1N1 from human seasonal H1N1 influenza viruses and swine H1N1 influenza viruses. The microarray utilizes a panel of primers for multiplex PCR amplification of the hemagglutinin (HA), neuraminidase (NA) and matrix (MP) genes of human influenza A viruses. The 59-mer oligonucleotides were designed to distinguish different subtypes of human influenza A viruses. With this microarray, we accurately identified and correctly subtyped the reference virus strains. Moreover, we confirmed 4 out of 39 clinical throat swab specimens from suspected cases of novel H1N1.     

A new and rapid genotypic assay for the detection of neuraminidase inhibitor resistant influenza A viruses of subtype H1N1, H3N2, and H5N1

The neuraminidase of influenza viruses is the target of the inhibitors oseltamivir and zanamivir. Recent reports on influenza viruses with reduced susceptibility to neuraminidase inhibitors (NAI) are a cause for concern. Several amino acid substitutions, each as a consequence of one single nucleotide mutation, are known to confer resistance to NAI. An increase of NAI-resistant viruses appears to be likely as a result of a wider application of NAI for treatment and prophylaxis of seasonal influenza infections. Monitoring the occurrence and spread of resistant viruses is an important task. Therefore, RT-PCR assays were developed with subsequent pyrosequencing analysis (PSQ-PCR). These assays allow a rapid, high-throughput and cost-effective screening of subtype A/H1N1, A/H3N2, and A/H5N1 viruses. Various specimens such as respiratory swabs, allantoic fluid, or cell-propagated viruses can be used and results are available within hours. Several A/H1N1, A/H3N2, and A/H5N1 viruses isolated from human and avian specimens were tested to evaluate the method. Positive controls encoding resistance-associated mutations were created using site-directed mutagenesis. The results obtained with these controls showed that the assay can discriminate clearly the wild-type virus from a mutant virus. The detection limit of minor virus variants within the viral quasispecies amounts to 10%.     

H5N1 Oseltamivir-resistance detection by real-time PCR using two high sensitivity labeled TaqMan probes

A single amino acid substitution, from histidine to tyrosine at position 274 of the neuraminidase gene has converted Oseltamivir sensitive H5N1 influenza A virus into a resistant strain. Currently, Oseltamivir is being stockpiled in many countries potentially affected by the influenza A virus subtype H5N1 epidemic. To identify this change in Oseltamivir-treated patients, a method based on real-time PCR using two labeled TaqMan probes was developed for its rapid detection. In order to validate the method, Oseltamivir specimen from treated (Oseltamivir-resistant strain from a Vietnamese patient, two Oseltamivir-treated tigers) and untreated subjects have been used for this study. The results thus obtained as well as those derived from clone selection and sequencing showed that TaqMan probes could clearly discriminate wild type H274 from the mutant 274Y variant. The sensitivity of this assay was as low as 10 copies/microl and allowed the detection of the mutation in a mixture of wild type and mutant. Overall, the assay based on real-time PCR with two labeled TaqMan probes described here should be useful for detecting Oseltamivir-resistant H274Y H5N1 influenza A virus in many species and various sources of specimens with high sensitivity and specificity. Such studies can address potential differences in the diagnostic outcomes between patients who develop detectable Oseltamivir resistance and those who retain only the wild type strain of H5N1.     

多重RT-PCR方法检测甲型流感病毒

目的建立一种快速、敏感、特异的多重RT-PCR,同时检测甲型流感病毒中的3个分型:甲型H1N1流感病毒,季节性H1N1流感病毒,季节性H3N2流感病毒,并将此方法应用到实验室流感病毒核酸检测技术中。方法利用甲型流感病毒3个分型病毒的引物,在同一个RT-PCR反应体系中,对疑似流感咽拭子标本进行检测。结果多重RT-PCR对甲型流感病毒中分型病毒有较高的灵敏度和特异性,可直接从疑似流感标本中同时进行甲型流感病毒分型检测。结论此实验中采用的多重RT-PCR具有与常规RT-PCR一样的特异性和敏感度,而且比普通RT-PCR和病毒分离法更快速,也更简便。     

A simple restriction fragment length polymorphism-based strategy that can distinguish the internal genes of human H1N1, H3N2, and H5N1 influenza A viruses

A simple molecular technique for rapid genotyping was developed to monitor the internal gene composition of currently circulating influenza A viruses. Sequence information from recent H1N1, H3N2, and H5N1 human virus isolates was used to identify conserved regions within each internal gene, and gene-specific PCR primers capable of amplifying all three virus subtypes were designed. Subtyping was based on subtype-specific restriction fragment length polymorphism (RFLP) patterns within the amplified regions. The strategy was tested in a blinded fashion using 10 control viruses of each subtype (total, 30) and was found to be very effective. Once standardized, the genotyping method was used to identify the origin of the internal genes of 51 influenza A viruses isolated from humans in Hong Kong during and immediately following the 1997-1998 H5N1 outbreak. No avian-human or H1-H3 reassortants were detected. Less than 2% (6 of 486) of the RFLP analyses were inconclusive; all were due to point mutations within a restriction site. The technique was also used to characterize the internal genes of two avian H9N2 viruses isolated from children in Hong Kong during 1999.     

H9N2禽流感病毒一步法双重荧光RT-PCR检测方法的建立

目的 建立一种可同时检测禽流感病毒H9N2的HA和NA基因一步法双重荧光RT-PCR方法.方法 针对H9N2禽流感病毒的HA和NA基因保守区,设计相应的特异性引物以及探针,优化检测体系及反应条件,建立一步法双重荧光定量RT-PCR方法.对该方法的灵敏度、特异性、稳定性进行验证与评估,并对家禽粪便标本进行应用检测,以单重实时荧光RT-PCR方法作为参照,检测结果不一致的样本采用测序进行验证.结果 该方法特异性强,与H1、H3、H5、H7亚型禽流感病毒、鸡新城疫及鸡传染性支炎病毒均无交叉反应,对HA和NA基因的最低检出限分别可达50拷贝/μL和25拷贝/μL,组间与组内的变异系数在0.20 ～0.79％之间.对82份粪便标本进行检测,H9N2禽流感病毒的阳性率为8.14％ (7/82),与单重实时荧光RT-PCR法检测结果一致.结论 该方法特异性强、灵敏度高、稳定性好,可应用于临床禽流感样本的检测.     

Detection and identification of human influenza viruses by the polymerase chain reaction

A series of oligonucleotide primers are described which hybridize to conserved regions of influenza virus cDNA and prime DNA synthesis in Taq polymerase catalyzed amplification reactions (PCR). Primers were designed to hybridize as nested pairs and, following a two-step amplification, produce uniquely sized DNA fragments diagnostic for viral type and subtype. Influenza A and B matrix-protein genes and the influenza C haemagglutinin gene were targets for the type-specific primers. Subtype-specific primers targeted conserved sequences within the three haemagglutinin or two neuraminidase subtypes of different human influenza isolates. The utility of this method was demonstrated using computer search methods and by accurately amplifying DNA from a variety of influenza A, B, and C strains. Type-specific primer sets showed a broad type specificity and amplified DNA from viral strains of unknown sequence. Restriction mapping and DNA sequencing showed that fragments amplified in this manner derived from the input template, confirming the accuracy of the method and demonstrating how PCR can be used to quickly derive sufficient sequence information for analysis of viral relatedness. Subtyping primers were able to distinguish accurately between the three haemagglutinin (H1, H2, H3) and two neuraminidase (N1, N2) alleles of human influenza A isolates. Again DNA was amplified from viruses of unknown sequence confirming that most of these primer sets may prove useful as broad range subtyping reagents. In order to simplify the work associated with analysis of many samples, we have also devised a rapid method for the isolation of viral RNA and synthesis of cDNA. Using this 'mini-prep' technique, it is possible to detect, amplify, and identify picogram quantities of influenza virus in a single day, confirming that PCR provides a useful alternative to existing methods of influenza detection.     

Comparative analytical sensitivities of six rapid influenza A antigen detection test kits for detection of influenza A subtypes H1N1, H3N2 and H5N1.

BACKGROUND: Rapid and simple methods for diagnosing human influenza A (H5N1) disease urgently needed. The limited data so far suggest that the currently available rapid antigen detection kits have poor clinical sensitivity for diagnosis of human H5N1 disease. OBJECTIVES: To compare the analytical sensitivity of six commercially available rapid antigen detection kits for the detection of "human" (subtypes H1N1, H3N2) and "avian" (subtype H5N1) influenza A viruses. STUDY DESIGN: Six commercially available test kits for the detection of influenza A were investigated. Analytic sensitivity for the detection of two contemporary H1N1, two H3N2 and three H5N1 viruses was determined using virus culture as a reference method. RESULTS AND CONCLUSIONS: Each test kit detected the H5N1 virus subtypes as efficiently as they detected conventional human viruses of subtypes H1N1 or H3N2. However, limits of detection of influenza viruses of all subtypes by antigen detection kits were >1000-fold lower than virus isolation. Thus, the reportedly poor clinical sensitivity of these antigen detection kits for diagnosis of patients with H5N1 disease is not due to a difference of sensitivity for detecting avian influenza H5N1 compared to human influenza viruses.