甲型流感病毒流行毒株检测和分型基因芯片的研制

【目的】研制一种可同时对甲型流感病毒H1N1、H1N2、H3N2、H5N1和H9N2等5种流行亚型进行检测和分型的基因芯片。【方法】根据National Center for Biotechnology Information中Influenza Virus Resource数据库,针对H1N1、H1N2、H3N2、H5N1和H9N2等5种亚型甲型流感病毒的HA和NA基因设计46条特异性寡核苷酸探针和1条质控探针,点制成基因芯片。利用通用引物扩增流感病毒HA和NA基因,使用Klenow酶对扩增产物进行荧光标记和片段化,将标记后产物和芯片杂交,清洗、扫描后根据荧光信号判定检测结果。用18株不同种属来源的甲型流感病毒分离毒株和186份咽拭子对芯片特异性、敏感性和临床应用进行初步评价。【结果】所有18株分离毒株均能被芯片准确检测并分型,芯片检测灵敏度能达约1×104个病毒基因拷贝。同时8份咽拭子检测结果为H1N1阳性,4份咽拭子为H3N2阳性。【结论】研究表明该芯片具有较高的特异性和灵敏度,可为甲型流感病毒的监测提供一种有效的方法。     

Microarray for diagnostics of human pathogenic influenza-a virus subtypes

An oligonucleotide microarray was developed for diagnostics of human pathogenic influenza-A virus subtypes. It contained discriminating probes for H1, H2, H3, H5, H7, and H9 subtypes of hemagglutinin and for N1, N2, and N7 subtypes of neuraminidase. An additional set of probes was used for revealing the M-gene of the influenza-A virus. The proposed microarray was tested on samples of pathogenic H5N1 avian influenza virus, pandemic H1N1 swine influenza virus, and seasonal H1N1 and H3N2 influenza viruses. The microarray can be used for the analysis both of cultivated strains and clinical specimens.     

利用基因芯片技术区分禽流感病毒主要亚型

[目的]研制可同时区分AIV的H5、H7、H9血凝素亚型及N1、N2神经氨酸酶亚型的基因诊断芯片.[方法]分别克隆了禽流感病毒的M基因,H5、H7、H9亚型HA基因,N1、N2亚型NA基因以及看家基因GAPDH的重组质粒.以重组质粒为模板,用PCR方法扩增制备探针,纯化后点于氨基修饰的片基上,制备基因芯片.在PCR过程中对待检样品进行标记,然后与芯片杂交,洗涤,扫描并进行结果分析.[结果]结果显示检测探针可特异性的与相应的标记样品进行杂交,呈现较强的杂交信号,且无交叉杂交.同时用RT-PCR、鸡胚接种和基因芯片方法对H1-H15亚型AIV参考毒株、30份人工感染样品、21份现地疑似样品进行检测,结果发现,对人工感染样品芯片检测方法与鸡胚接种和RT-PCR的符合率分别为100%和96%,现地样品符合率为100%.[结论]研究表明该方法可用于同步鉴别部分主要流行的禽流感亚型,是一种有效的新方法.     

Subtyping of influenza virus A hemagglutinine with hybridization microarray

An oligonucleotide microarray for influenza A hemagglutinine subtyping was presented. The number of probes for determination of each subtype hemagglutinine (H1-H13, H15, H16, pandemic flu H1N1)varied from 13 to 28. When testing of the microarray using 40 type A influenza virus isolates the hemagglutinin subtypes were unambiguously determined for 36 specimens.     

Subtyping of influenza virus A hemagglutinin with a hybridization microarray

An oligonucleotide microarray for influenza A hemagglutinin subtyping was presented. The number of probes for the determination of each subtype of hemagglutinin (H1-H13, H15, H16, pandemic flu H1N1) varied from 13 to 28. When testing the microarray using 40 type-A influenza virus isolates, the hemagglutinin subtypes were unambiguously determined for 36 specimens.     

Universal oligonucleotide microarray for sub-typing of Influenza A virus

A universal microchip was developed for genotyping Influenza A viruses. It contains two sets of oligonucleotide probes allowing viruses to be classified by the subtypes of hemagglutinin (H1-H13, H15, H16) and neuraminidase (N1-N9). Additional sets of probes are used to detect H1N1 swine influenza viruses. Selection of probes was done in two steps. Initially, amino acid sequences specific to each subtype were identified, and then the most specific and representative oligonucleotide probes were selected. Overall, between 19 and 24 probes were used to identify each subtype of hemagglutinin (HA) and neuraminidase (NA). Genotyping included preparation of fluorescently labeled PCR amplicons of influenza virus cDNA and their hybridization to microarrays of specific oligonucleotide probes. Out of 40 samples tested, 36 unambiguously identified HA and NA subtypes of Influenza A virus.     

Selection of DNA aptamers that bind to influenza A viruses with high affinity and broad subtype specificity

Many cases of influenza are reported worldwide every year. The influenza virus often acquires new antigenicity, which is known as antigenic shift; this results in the emergence of new virus strains, for which preexisting immunity is not found in the population resulting in influenza pandemics. In the event a new strain emerges, diagnostic tools must be developed rapidly to detect the novel influenza strain. The generation of high affinity antibodies is costly and takes time; therefore, an alternative detection system, aptamer detection, provides a viable alternative to antibodies as a diagnostic tool. In this study, we developed DNA aptamers that bind to HA1 proteins of multiple influenza A virus subtypes by the SELEX procedure. To evaluate the binding properties of these aptamers using colorimetric methods, we developed a novel aptamer-based sandwich detection method employing our newly identified aptamers. This novel sandwich enzyme-linked aptamer assay successfully detected the H5N1, H1N1, and H3N2 subtypes of influenza A virus with almost equal sensitivities. These findings suggest that our aptamers are attractive candidates for use as simple and sensitive diagnostic tools that need sandwich system for detecting the influenza A virus with broad subtype specificities.     

H5N1禽流感病毒环介导等温扩增快速检测方法的建立

目的：建立H5N1禽流感病毒环介导等温扩增（LAMP）快速检测方法。方法：从GenBank中获得H5N1禽流感病毒血凝素（HA）基因序列,应用DNAStar软件MegAlign程序分析其序列,利用PrimerExplorerV3软件在序列保守区域设计LAMP引物,即外引物、内引物和环引物,同时以所克隆的阳性质粒为模板,对试验中的几个重要参数进行优化。结果：LAMP检测方法对H5N1禽流感病毒的灵敏度达到4～6个拷贝,其引物对于H1、H9亚型禽流感病毒和新城疫病毒无非特异性扩增,表现出良好的H5亚型特异性。结论：建立的H5N1禽流感病毒环介导等温扩增快速检测方法灵敏度高、特异性强、重复性好,为快速检测禽流感病毒提供了新方法和新思路。     

Recognition of cloned influenza virus hemagglutinin gene products by cytotoxic T lymphocytes.

The influenza A virus hemagglutinin (HA) is an integral membrane glycoprotein expressed in large quantities on infected cell surfaces and is known to serve as a target antigen for influenza virus-specific cytotoxic T lymphocytes (CTL). Despite the fact that HAs derived from different influenza A virus subtypes are serologically non-cross-reactive, the HA has been implicated by previous experiments to be a target antigen for the subset of T cells capable of lysing cells infected with any human influenza A subtype (cross-reactive CTL). To directly determine whether the HA is recognized by cross-reactive CTL, we used vaccinia virus recombinants containing DNA copies of the PR8 (A/Puerto Rico/8/34) (H1N1) or JAP (A/JAP/305) (H2N2) HA genes. When these viruses were used to stimulate HA-specific CTL and to sensitize target cells for lysis by HA-specific CTL, we found no evidence for HA recognition by cross-reactive CTL aside from a relatively small degree of cross-reactivity between H1 and H2 HAs. Results of unlabeled target inhibition studies were consistent with the conclusion that the HA is, at most, only a minor target antigen for cross-reactive CTL.     

季节性流感病毒H1N1实时荧光定量PCR检测方法的建立

目的建立一种快速定量检测季节性流感病毒H1N1核酸的实时荧光定量PCR检测方法及试剂盒。方法选择季节性流感病毒H1N1的保守基因NP基因作为检测靶目标,应用Clustal W软件进行序列同源性比对分析,筛选出季节性流感病毒H1N1特异性的保守序列作为引物候选区域,然后应用Primer Express及PrimerPremier 5.0软件包对候选引物进行进一步配对及筛选,得到最优特异性检测引物。同时,由病毒全长cDNA扩增出NP基因,琼脂糖凝胶电泳检测NP基因的扩增情况并对目的条带进行切胶回收及纯化,对回收后的NP全长基因进行核酸浓度测定,并换算成拷贝数,作为定量标准品。结果应用ABI公司的Power SYBR Green PCR MasterMix及StepOne实时荧光定量PCR仪,该检测系统灵敏度可达102 copies/μL,不同梯度标准品间线性关系（R2）达0.999,斜率为-0.3433,扩增效率为95.572%,所有标准品均在83.2℃出现尖且窄的特异性熔解峰。结论利用该检测系统可以快速定量检测季节性流感病毒H1N1,灵敏度高,可用作基础及临床实验室对季节性流感病毒H1N1感染的辅助诊断方法和临床效果的监测手段,对实验操作者要求相对较低,具有实际的应用价值。     

Subtyping animal influenza virus with general multiplex RT-PCR and Liquichip high throughput (GMPLex)

This study developed a multiplex RT-PCR integrated with luminex technology to rapidly subtype simultaneously multiple influenza viruses. Primers and probes were designed to amplify NS and M genes of influenza A viruses HA gene of H1, H3, H5, H7, H9 subtypes, and NA gene of the N1 and N2 subtypes. Universal super primers were introduced to establish a multiplex RT-PCR (GM RT-PCR). It included three stages of RT-PCR amplification, and then the RT-PCR products were further tested by LiquiChip probe, combined to give an influenza virus (IV) rapid high throughput subtyping test, designated as GMPLex. The IV GMPLex rapid high throughput subtyping test presents the following features: high throughput, able to determine the subtypes of 9 target genes in H1, H3, H5, H7, H9, N1, and N2 subtypes of the influenza A virus at one time; rapid, completing the influenza subtyping within 6 hours; high specificity, ensured the specificity of the different subtypes by using two nested degenerate primers and one probe, no cross reaction occurring between the subtypes, no non-specific reactions with other pathogens and high sensitivity. When used separately to detect the product of single GM RT-PCR for single H5 or N1 gene, the GMPLex test showed a sensitivity of 10⁻⁵(= 280ELD₅₀) forboth tests and the Luminex qualitative ratio results were 3.08 and 3.12, respectively. When used to detect the product of GM RT-PCR for H5N1 strain at the same time, both showed a sensitivity of 10⁻⁴(=2800 ELD₅₀). The GMPLex rapid high throughput subtyping test can satisfy the needs of influenza rapid testing.Key words: Influenza Virus, General multiplex RT-PCR, Iuminex assay, Subtyping, HA and NA genes     

Microarray Multiplex Assay for the Simultaneous Detection and Discrimination of Influenza A and Influenza B Viruses

In this study, we present a microarray approach for the typing of influenza A and B viruses, and the subtyping of H1 and H3 subtypes. We designed four pairs of specific multiplex RT-PCR primers and eight specific oligonucleotide probes and prepared microarrays to identify the specific subtype of influenza virus. Through amplification and fluorescent marking of the multiplex RT-PCR products on the M gene of influenza A and B viruses and the HA gene of subtypes H1 and H3, the PCR products were hybridized with the microarray, and the results were analyzed using a microarray scanner. The results demonstrate that the chip developed by our research institute can detect influenza A and B viruses specifically and identify the subtypes H1 and H3 at a minimum concentration of 1 × 10² copies/μL of viral RNA. We tested 35 clinical samples and our results were identical to other fluorescent methods. The microarray approach developed in this study provides a reliable method for the monitoring and testing of seasonal influenza.     

Rapid detection and subtyping of human influenza A viruses and reassortants by pyrosequencing

Background

Given the continuing co-circulation of the 2009 H1N1 pandemic influenza A viruses with seasonal H3N2 viruses, rapid and reliable detection of newly emerging influenza reassortant viruses is important to enhance our influenza surveillance.

Methodology/Principal Findings

A novel pyrosequencing assay was developed for the rapid identification and subtyping of potential human influenza A virus reassortants based on all eight gene segments of the virus. Except for HA and NA genes, one universal set of primers was used to amplify and subtype each of the six internal genes. With this method, all eight gene segments of 57 laboratory isolates and 17 original specimens of seasonal H1N1, H3N2 and 2009 H1N1 pandemic viruses were correctly matched with their corresponding subtypes. In addition, this method was shown to be capable of detecting reassortant viruses by correctly identifying the source of all 8 gene segments from three vaccine production reassortant viruses and three H1N2 viruses.

Conclusions/Significance

In summary, this pyrosequencing assay is a sensitive and specific procedure for screening large numbers of viruses for reassortment events amongst the commonly circulating human influenza A viruses, which is more rapid and cheaper than using conventional sequencing approaches.     

Human histone genes are interspersed with members of the Alu family and with other transcribed sequences

We have isolated a series of recombinant λCh4A phages containing human histone genes. Histone H2A, H2B, H3 and H4 genes have been found to be clustered, but are not present in any simple repeat pattern. Hybridization of a blot containing phage DNA with S phase polysomal cDNA indicates the presence of additional sequences complementary to HeLa polysomal RNA sequences. Northern blot analysis using these clones as probes has also shown the presence of sequences complementary to non-histone-coding RNAs, some of which accumulate differentially in different stages of the cell cycle. We have also found, by hybridization with appropriate probes, that histone genes are interspersed with several copies of the Alu DNA family; however, not all of the histone genes are associated with an Alu DNA sequence.     

Subtype cross-reactive, infection-enhancing antibody responses to influenza A viruses.

Antibody-dependent enhancement of the uptake of influenza A virus by Fc receptor-bearing cells was analyzed by using virus strains of the three human influenza A virus subtypes, A/PR/8/34 (H1N1), A/Japan/305/57 (H2N2), and A/Port Chalmers/1/73 (H3N2). Immune sera obtained from mice following primary infection with an H1N1, H2N2, or H3N2 subtype virus neutralized only virus of the same subtype; however, immune sera augmented the uptake of virus across subtypes. Immune sera from H1N1-infected mice augmented uptake of the homologous (H1N1) and H2N2 viruses. Antisera to the H2N2 virus augmented the uptake of virus of all subtypes (H1N1, H2N2, or H3N2). Antisera to the H3N2 virus augmented the uptake of the homologous (H3N2) and H2N2 viruses. These results show that subtype cross-reactive, nonneutralizing antibodies augment the uptake of influenza A virus strains of different subtypes. Antibodies to neuraminidase may contribute to the enhanced uptake of viruses of a different subtype, because N2-specific monoclonal antibodies augmented the uptake of both A/Japan/305/57 (H2N2) and A/Port Chalmers/1/73 (H3N2) viruses.     

Identification of reassortant pandemic H1N1 influenza virus in Korean pigs

Since the 2009 pandemic human H1N1 influenza A virus emerged in April 2009, novel reassortant strains have been identified throughout the world. This paper describes the detection and isolation of reassortant strains associated with human pandemic influenza H1N1 and swine influenza H1N2 (SIV) viruses in swine populations in South Korea. Two influenza H1N2 reassortants were detected, and subtyped by PCR. The strains were isolated using Madin- Darby canine kidney (MDCK) cells, and genetically characterized by phylogenetic analysis for genetic diversity. They consisted of human, avian, and swine virus genes that were originated from the 2009 pandemic H1N1 virus and a neuraminidase (NA) gene from H1N2 SIV previously isolated in North America. This identification of reassortment events in swine farms raises concern that reassortant strains may continuously circulate within swine populations, calling for the further study and surveillance of pandemic H1N1 among swine.     

Differential suppressive effect of promyelocytic leukemia protein on the replication of different subtypes/strains of influenza A virus

Promyelocytic leukemia protein (PML) plays an important role in the defense against a number of viruses, including influenza A virus. However, the sensitivity of influenza A virus subtypes/strains to PML is unknown. We investigated the role of PML in the replication of different influenza A virus subtypes/strains using pan-PML knock-down A549 cells and PML-VI-overexpressed MDCK cells. We found that (i) depletion of pan-PML by siRNA rendered A549 cells more susceptible to influenza A virus strains PR8(H1N1) and ST364(H3N2), but not to strains ST1233(H1N1), Qa199(H9N2) and Ph2246(H9N2); (ii) overexpression of PML-VI in MDCK cells conferred potent resistance to PR8(H1N1) infection, while lacked inhibitory activity to ST1233(H1N1), ST364(H3N2), Qa199(H9N2) and Ph2246(H9N2). Our results suggest that the antiviral effect of PML on influenza A viruses is viral subtype/strain specific.     

Origin of the 1918 Spanish influenza virus: a comparative genomic analysis

To test the avian-origin hypothesis of the 1918 Spanish influenza virus we surveyed influenza sequences from a broad taxonomic distribution and collected 65 full-length genomes representing avian, human and "classic" swine H1N1 lineages in addition to numerous other swine (H1N2, H3N1, and H3N2), human (H2N2, H3N2, and H5N1), and avian (H1N1, H4N6, H5N1, H6N1, H6N6, H6N8, H7N3, H8N4, H9N2, and H13N2) subtypes. Amino acids from all eight segments were concatenated, aligned, and used for phylogenetic analyses. In addition, the genes of the polymerase complex (PB1, PB2, and PA) were analyzed individually. All of our results showed the Brevig-Mission/1918 strain in a position basal to the rest of the clade containing human H1N1s and were consistent with a reassortment hypothesis for the origin of the 1918 virus. Our genome phylogeny further indicates a sister relationship with the "classic" swine H1N1 lineage. The individual PB1, PB2, and PA phylogenies were consistent with reassortment/recombination hypotheses for these genes. These results demonstrate the importance of using a complete-genome approach for addressing the avian-origin hypothesis and predicting the emergence of new pandemic influenza strains.