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.     

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.     

Evaluation of twenty rapid antigen tests for the detection of human influenza A H5N1, H3N2, H1N1, and B viruses

Twenty rapid antigen assays were compared for their ability to detect influenza using dilutions of virus culture supernatants from human isolates of influenza A H5N1 (clade 1 and 2 strains), H3N2 and H1N1 viruses, and influenza B. There was variation amongst the rapid antigen assays in their ability to detect different influenza viruses. Six of the 12 assays labeled as distinguishing between influenza A and B had comparable analytical sensitivities for detecting both influenza A H5N1 strains, although their ability to detect influenza A H3N2 and H1N1 strains varied. The two assays claiming H5 specificity did not detect either influenza A H5N1 strains, and the two avian influenza‐specific assays detected influenza A H5N1, but missed some influenza A H3N2 virus supernatants. Clinical trials of rapid antigen tests for influenza A H5N1 are limited. For use in a pandemic where novel influenza strains are circulating (such as the current novel influenza A H1N1 09 virus), rapid antigen tests should ideally have comparable sensitivity and specificity for the new strains as for co‐circulating seasonal influenza strains. J. Med. Virol. 81:1918–1922, 2009. © 2009 Wiley‐Liss, Inc.     

儿童甲型H1N1流感和季节性流感患者的病毒载量及相关临床症状研究

目的 分析并比较儿童甲型H1N1流感和季节性流感患者咽拭子标本的病毒载量及相关临床症状.方法 应用荧光PCR方法对采集的咽拭子标本进行检测,并通过建立核酸标准品,绘制标准曲线,测定标本中的病毒载量,同时结合所收集的患者临床症状数据资料应用随机区组方差和卡方检验方法进行统计分析.结果 2009年9月至2010年9月期间收集的1,040份咽拭子标本中,共检出甲型H1N1流感病毒阳性标本120份,甲型H3N2流感病毒阳性标本61份,乙型流感病毒阳性标本99份;对收集的流感阳性标本病毒载量测定结果显示:不同型别,不同发病时间流感患者咽拭子标本的病毒载量差异无统计学意义(P＞0.05);甲型H1N1流感、季节性流感感染者的性别比例差异无统计学意义,甲型H1N1流感感染者出现咳嗽,流涕临床症状者明显高于与乙型流感感染者.结论 甲型H1N1流感患者咳嗽,流涕症状比季节性乙型流感患者多见,而甲型H1N1流感和季节性流感患者咽拭子标本的病毒载量无显著性差异.     

Multiplex RT-PCR assay for differentiating European swine influenza virus subtypes H1N1, H1N2 and H3N2

In Europe, three major swine influenza viral (SIV) subtypes (H1N1, H1N2 and H3N2) have been isolated in pigs. Developing a test that is able to detect and identify the subtype of the circulating strain rapidly during an outbreak of respiratory disease in the pig population is of essential importance. This study describes two multiplex RT-PCRs which distinguish the haemagglutinin (HA) gene and the neuraminidase (NA) gene of the three major subtypes of SIV circulating in Europe. The HA PCR was able to identify the lineage (avian or human) of the HA of H1 subtypes. The analytical sensitivity of the test, considered to be unique, was assessed using three reference viruses. The detection limit corresponded to 1×10(-1) TCID(50)/200μl for avian-like H1N1, 1×10(0) TCID(50)/200μl for human-like H1N2 and 1×10(1) TCID(50)/200μl for H3N2 SIV. The multiplex RT-PCR was first carried out on a collection of 70 isolated viruses showing 100% specificity and then on clinical samples, from which viruses had previously been isolated, resulting in an 89% positive specificity of the viral subtype. Finally, the test was able to identify the viral subtype correctly in 56% of influenza A positive samples, from which SIV had not been isolated previously. It was also possible to identify mixed viral infections and the circulation of a reassortant strain before performing genomic studies.     

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%.     

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.     

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.     

Single-step multiplex reverse transcription-polymerase chain reaction (RT-PCR) for influenza A virus subtype H5N1 detection

Influenza A virus subtype H5N1 causes a rapidly fatal systemic disease in domestic poultry and spreads directly from poultry to humans. The aim of this study was to develop a rapid, cost-saving and effective method for influenza A virus subtype H5N1 detection. The selected primer set was used in single-step RT-PCR for simultaneous detection in multiplex format of the 276-, 189-, and 131-bp fragments, corresponding to sequences specific for M, H5 and N1. The amplified DNA fragments were clearly separated by agarose gel electrophoresis. The sensitivity of this assay was about 10(3) copies/microL. Moreover, this method can be applied to detect not only avian but also human influenza A virus subtype H5N1. In conclusion, the highlights of this particular method are its rapidity and cost-effectiveness, thus rendering it feasible and attractive for large-scale screening at times of influenza A virus subtype H5N1 outbreak.     

2009甲型H1N1流感大流行期间北京儿童的流感监测

目的 了解2009年甲型H1N1流感大流行期间北京地区儿童中流感流行的情况.方法 采用WHO推荐的实时荧光定量RT-PCR和国家流感中心推荐的分型方法,对2009年甲型H1N1流感大流行期间因流感样症状来首都儿科研究所附属儿童医院就诊患儿的咽拭子标本进行流感病毒核酸检测.结果 2009年6月1日至2010年2月28日期间共检测了4363份咽拭子标本,其中623例为甲型H1N1阳性,阳性率为14.3%,657例为其他甲型流感病毒阳性(15.1%),所有甲型流感病毒的总阳性率为29.3%.623例中有23例为危重症病例(占阳性患者的3.7%),其中5例死亡.618例信息完整的甲型H1N1病例中,患儿年龄为14天～16岁,性别比例为男比女为1.3:1.1～3岁儿童占25.2%,3～6岁学龄前儿童和6～12岁学龄儿童所占比例相近,各约占30%.在监测期间,仅呈现了一个甲型H1N1的流行波.2009年11月达到最高峰,随后减弱,2010年2月快速下降至2.7%.对监测期间每周20～30份临床标本同时进行季节性流感的监测显示,季节性H3N2、甲型H1N1和乙型流感交替流行.呼吸道合胞病毒(RSV)在甲型H1N1流行趋势减缓后逐渐流行成为流行优势株.结论 2009年6月至2010年2月北京地区儿童中出现甲型H1N1的流行,主要累及学龄前和学龄儿童.季节性流感和RSV与甲型H1N1交替流行.