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.     

Oligonucleotide microarray for subtyping of influenza virus A neuraminidase

Microarray for influenza A neuraminidase subtyping was presented. Selection of oligoprobes proceeded in two steps. First step included selection of peptides specific for each subtype of neuraminidase. At the second step oligoprobes were calculated using found peptides structures with the subsequent additional selection of the most specific and representative probes. From 19 to 24 probes were used for determination of each subtype of neuraminidase. Microchip testing for 19 samples with the most widespread types (N1 and N2) specifies in unequivocal definition 18 of them and only one isolate has not been identified.     

A compact microarray for subtyping of influenza a virus

A version of the universal oligonucleotide hybridization microchip with the size of 6 × 5 spots (4 × 4 mm) has been proposed, which operates on the principle of “one spot-one subtype.” This microchip may be the prototype of a biosensor for fixation of influenza A virus and typing of 15 subtypes of hemagglutinin and 9 subtypes of neuraminidase.     

A second generation universal microarray for subtyping influenza a virus

The microchip for influenza A subtyping working on the ??one spot-one subtype?? principle was developed. Each spot contains a set of oligonucleotide probes specific for particular subtypes of hemagglutinin, neuraminidase and matrix protein (influenza A marker). Reliability of the proposed chip is the same as for the full-size microchip for separate hemagglutinin and neuraminidase typing which was created in our group earlier. The image was visualized by labeling the analyzed nucleic acid by either fluorescent dye or biotin with the following fixation in streptavidin-gold nanoparticles and development by silver precipitation. In the second case, the image was analyzed using an ordinary scanner that essentially simplifies influenza A subtyping.     

禽流感病毒分型基因芯片的研制

[目的]禽流感病毒是一种全球重要的人和动物呼吸道病病原,快速确定其不同亚型对于全球流感监测具有重要的意义.本研究意在研制一种可同时鉴定禽流感病毒所有亚型的方法.[方法]根据GenBank上已发表的禽流感病毒不同亚型(16个HA亚型和9个NA亚型)的基因序列,设计合成了25对特异性引物和1对通用引物,然后以各亚型病毒的参考株RNA作为模板,建立扩增不同亚型的多重RT-PCR方法.参考各亚型病毒靶cDNAs区域的保守序列设计了52条亚型特异的探针,进而利用扩增的各亚型病毒的靶cDNAs对其特异性进行评价.在此基础上,将设计好的探针点制到处理好的玻片上,制备了禽流感病毒分型鉴定基因芯片,结合所建立的扩增不同亚型的多重RT-PCR方法,开发了禽流感病毒亚型鉴定基因芯片试剂.利用收集自49个地区的2653份标本对其特异性和敏感性进行了初步评价.[结果]用于评价的各亚型参考毒株均出现良好的特异性杂交信号,检测的敏感度可达2.47 PFU/mL或2.5 ng靶DNA片段,而且与禽类常见的IBV、NDV等6种病毒均无交叉反应.[结论]证明该病毒分型基因芯片具有良好的特异性、敏感性.     

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     

Signature patterns revealed by microarray analyses of mice infected with influenza virus A and Streptococcus pneumoniae

We used cDNA microarrays to identify differentially expressed genes in mice in response to infections with influenza virus A/PR/8/34 (H1N1) and Streptococcus pneumoniae. Expression microarray analysis showed up-regulation and down-regulation of many genes involved in the defense, inflammatory response and intracellular signaling pathways including chemokine, apoptosis, MAPK, Notch, Jak-STAT, T-cell receptor and complement and coagulation cascades. We have revealed signature patterns of gene expression in mice infected with two different classes of pathogens: influenza virus A and S. pneumoniae. Quantitative real-time RT-PCR results confirmed microarray results for most of the genes tested. These studies document clear differences in gene expression profiles between mice infected with influenza virus A and S. pneumoniae. Identification of genes that are differentially expressed after respiratory infections can provide insights into the mechanisms by which the host interacts with different pathogens, useful information about stage of diseases and selection of suitable targets for early diagnosis and treatments. The advantage of this novel approach is that the detection of pathogens is based on the differences in host gene expression profiles in response to different pathogens instead of detecting pathogens directly.     

Oligonucleotide microarray for the subtyping of influenza virus a neuraminidase

A microarray for the subtyping of influenza A neuraminidase is presented. The selection of oligoprobes proceeded in two steps. The first step included the selection of peptides specific for each subtype of neuraminidase. At the second step, the oligoprobes were calculated using the found peptide structures with the subsequent additional selection of the most specific and representative probes. From 19 to 24 probes were used for the determination of each neuraminidase subtype. The microarray testing for 19 samples with the most widespread types (N1 and N2) specifies in an unequivocal definition 18 of them and only 1 isolate has not been identified.     

Oligodeoxynucleotide hybridization inhibition of HIV and influenza virus.

Synthetic oligodeoxyribonucleotides have been found to inhibit the replication and expression of HIV and of Influenza virus in growing tissue cultures. Nuclease resistant oligomers have been more effective than unmodified oligomers. In mouse and rat studies, labeled oligomers, on intravenous or intraperitoneal injection, leave the blood stream rapidly and enter numerous organs of the body. The half life in these organs is 24-48 hours in the case of the phosphorothioate oligomers.     

Chaotic mixer improves microarray hybridization

Hybridization is an important aspect of microarray experimental design which influences array signal levels and the repeatability of data within an array and across different arrays. Current methods typically require 24h and use target inefficiently. In these studies, we compare hybridization signals obtained in conventional static hybridization, which depends on diffusional target delivery, with signals obtained in a dynamic hybridization chamber, which employs a fluid mixer based on chaotic advection theory to deliver targets across a conventional glass slide array. Microarrays were printed with a pattern of 102 identical probe spots containing a 65-mer oligonucleotide capture probe. Hybridization of a 725-bp fluorescently labeled target was used to measure average target hybridization levels, local signal-to-noise ratios, and array hybridization uniformity. Dynamic hybridization for 1h with 1 or 10ng of target DNA increased hybridization signal intensities approximately threefold over a 24-h static hybridization. Similarly, a 10- or 60-min dynamic hybridization of 10ng of target DNA increased hybridization signal intensities fourfold over a 24h static hybridization. In time course studies, static hybridization reached a maximum within 8 to 12h using either 1 or 10ng of target. In time course studies using the dynamic hybridization chamber, hybridization using 1ng of target increased to a maximum at 4h and that using 10ng of target did not vary over the time points tested. In comparison to static hybridization, dynamic hybridization reduced the signal-to-noise ratios threefold and reduced spot-to-spot variation twofold. Therefore, we conclude that dynamic hybridization based on a chaotic mixer design improves both the speed of hybridization and the maximum level of hybridization while increasing signal-to-noise ratios and reducing spot-to-spot variation.     

Peptide-mediated interference with influenza A virus polymerase

The assembly of the polymerase complex of influenza A virus from the three viral polymerase subunits PB1, PB2, and PA is required for viral RNA synthesis. We show that peptides which specifically bind to the protein-protein interaction domains in the subunits responsible for complex formation interfere with polymerase complex assembly and inhibit viral replication. Specifically, we provide evidence that a 25-amino-acid peptide corresponding to the PA-binding domain of PB1 blocks the polymerase activity of influenza A virus and inhibits viral spread. Targeting polymerase subunit interactions therefore provides a novel strategy to develop antiviral compounds against influenza A virus or other viruses.     

A seven-segmented influenza A virus expressing the influenza C virus glycoprotein HEF

Influenza viruses are classified into three types: A, B, and C. The genomes of A- and B-type influenza viruses consist of eight RNA segments, whereas influenza C viruses only have seven RNAs. Both A and B influenza viruses contain two major surface glycoproteins: the hemagglutinin (HA) and the neuraminidase (NA). Influenza C viruses have only one major surface glycoprotein, HEF (hemagglutinin-esterase fusion). By using reverse genetics, we generated two seven-segmented chimeric influenza viruses. Each possesses six RNA segments from influenza virus A/Puerto Rico/8/34 (PB2, PB1, PA, NP, M, and NS); the seventh RNA segment encodes either the influenza virus C/Johannesburg/1/66 HEF full-length protein or a chimeric protein HEF-Ecto, which consists of the HEF ectodomain and the HA transmembrane and cytoplasmic regions. To facilitate packaging of the heterologous segment, both the HEF and HEF-Ecto coding regions are flanked by HA packaging sequences. When introduced as an eighth segment with the NA packaging sequences, both viruses are able to stably express a green fluorescent protein (GFP) gene, indicating a potential use for these viruses as vaccine vectors to carry foreign antigens. Finally, we show that incorporation of a GFP RNA segment enhances the growth of seven-segmented viruses, indicating that efficient influenza A viral RNA packaging requires the presence of eight RNA segments. These results support a selective mechanism of viral RNA recruitment to the budding site.     

Binding kinetics of sulfatide with influenza A virus hemagglutinin

Association of a sulfated galactosyl ceramide, sulfatide, with the viral envelope glycoprotein hemagglutinin (HA) delivered to the cell surface is required for influenza A virus (IAV) replication through efficient translocation of the newly synthesized viral nucleoprotein from the nucleus to the cytoplasm. To determine whether the ectodomain of HA can bind to sulfatide, a secreted-type HA (sHA), in which the transmembrane region and cytoplasmic tail were deleted, was generated by using a baculovirus expression system. The receptor binding ability and antigenic structure of sHA were evaluated by a hemagglutination assay, solid-phase binding assay and hemagglutination inhibition assay. sHA showed subtype-specific antigenicity and binding ability to both sulfatide and gangliosides. Kinetics of sHA binding to sulfatide and GD1a was demonstrated by quartz crystal microbalance (QCM) analysis. QCM analysis showed that the sHA bound with the association rate constant (k _on) of 1.41?×?10⁴ M^?1 sec^?1, dissociation rate constant (k _off) of 2.03?×?10^?4 sec^?1 and K _d of 1.44?×?10^?8 M to sulfatide immobilized on a sensor chip. The k _off values of sHA were similar for sulfatide and GD1a, whereas the k _on value of sHA binding to sulfatide was 2.56-times lower than that of sHA binding to GD1a. The results indicate that sulfatide directly binds to the ectodomain of HA with high affinity.     

Label-free electrical detection of DNA hybridization for the example of influenza virus gene sequences

Microarrays based on DNA-DNA hybridization are potentially useful for detecting and subtyping viruses but require fluorescence labeling and imaging equipment. We investigated a label-free electrical detection system using electrochemical impedance spectroscopy that is able to detect hybridization of DNA target sequences derived from avian H5N1 influenza virus to gold surface-attached single-stranded DNA oligonucleotide probes. A 23-nt probe is able to detect a 120-nt base fragment of the influenza A hemagglutinin gene sequence. We describe a novel method of data analysis that is compatible with automatic measurement without operator input, contrary to curve fitting used in conventional electrochemical impedance spectroscopy (EIS) data analysis. A systematic investigation of the detection signal for various spacer molecules between the oligonucleotide probe and the gold surface revealed that the signal/background ratio improves as the length of the spacer increases, with a 12- to 18-atom spacer element being optimal. The optimal spacer molecule allows a detection limit between 30 and 100 fmol DNA with a macroscopic gold disc electrode of 1 mm radius. The dependence of the detection signal on the concentration of a 23-nt target follows a binding curve with an approximate 1:1 stoichiometry and a dissociation constant of KD=13+/-4 nM at 295 K.     

A型流感病毒的基因组包装

A型流行性感冒病毒的负链RNA基因组由编码病毒中12个蛋白质的八个节段组成。在病毒组装的最后阶段,病毒体从细胞顶端胞浆膜突出时将这些基因组的病毒体（v）RNAs吸收进其中。基因组分段赋予了流感病毒进化的优势,但也提出了问题,在病毒体组装时需要八个节段每一个的至少一个复制本以产生完全有传染性的病毒颗粒。历史上一直存在争论：一方赞同确保足额的基因组合并的特异性包装机制;另一方赞同基因组节段被随机选择而不是以充足数量被包装以确保能自行产生合理比例病毒体的替代模式。近年来人们对该问题已达成一致意见：大多数病毒体仅包含八个节段,特异性机制为选择每个vRNA的某一复制本的确发挥了作用。本综述总结了得出这一结论所做的工作,叙述了在识别特异性包装信号方面最新的进展,讨论了这些RNA元素运转的可能机制。     

Chimeric influenza A viruses with a functional influenza B virus neuraminidase or hemagglutinin

Reassortment of influenza A and B viruses has never been observed in vivo or in vitro. Using reverse genetics techniques, we generated recombinant influenza A/WSN/33 (WSN) viruses carrying the neuraminidase (NA) of influenza B virus. Chimeric viruses expressing the full-length influenza B/Yamagata/16/88 virus NA grew to titers similar to that of wild-type influenza WSN virus. Recombinant viruses in which the cytoplasmic tail or the cytoplasmic tail and the transmembrane domain of the type B NA were replaced with those of the type A NA were impaired in tissue culture. This finding correlates with reduced NA content in virions. We also generated a recombinant influenza A virus expressing a chimeric hemagglutinin (HA) protein in which the ectodomain is derived from type B/Yamagata/16/88 virus HA, whereas both the cytoplasmic and the transmembrane domains are derived from type A/WSN virus HA. This A/B chimeric HA virus did not grow efficiently in MDCK cells. However, after serial passage we obtained a virus population that grew to titers as high as wild-type influenza A virus in MDCK cells. One amino acid change in position 545 (H545Y) was found to be responsible for the enhanced growth characteristics of the passaged virus. Taken together, we show here that the absence of reassortment between influenza viruses belonging to different A and B types is not due to spike glycoprotein incompatibility at the level of the full-length NA or of the HA ectodomain.