焦测序法检测禽流感病毒

以焦测序技术为检测平台,在研究禽流感病毒基因特性的基础上,建立一种检测禽流感病毒及确定其是否为高致病性禽流感病毒的序列测定法。首先,选择一段保守的M基因序列及一段包含裂解位点的HA基因序列为研究对象,采用聚合酶链反应(polymerase chain reaction,PCR)扩增技术初步判断其是否为禽流感病毒及病毒亚型;然后采用焦测序法检测目的片段序列;最后,对焦测序法检测序列进行分析,从基因序列上判断其是否为禽流感病毒,并进一步判断病毒的亚型以及是否为高致病性禽流感病毒。研究结果表明,当焦测序反应中三磷酸酰苷双磷酸酶(Apyrase)的浓度为1.6U/mL时,能有效抑制错误信号的产生;当Klenow的浓度为90U/mL时,可读序列长度为33个碱基。采用优化的焦测序反应体系测定了4个样本,其中1个样本被判断为H5N1亚型禽流感病毒,具有潜在的高致病性;另外3个样本为H9N2型禽流感病毒,具有低致病性。本方法具有准确、快速和实时检测等优点。     

Recovery of known T-cell epitopes by computational scanning of a viral genome

A new computational method (EpiDock) is proposed for predicting peptide binding to class I MHC proteins, from the amino acid sequence of any protein of immunological interest. Starting from the primary structure of the target protein, individual three-dimensional structures of all possible MHC-peptide (8-, 9- and 10-mers) complexes are obtained by homology modelling. A free energy scoring function (Fresno) is then used to predict the absolute binding free energy of all possible peptides to the class I MHC restriction protein. Assuming that immunodominant epitopes are usually found among the top MHC binders, the method can thus be applied to predict the location of immunogenic peptides on the sequence of the protein target. When applied to the prediction of HLA-A^*0201-restricted T-cell epitopes from the Hepatitis B virus, EpiDock was able to recover 92% of known high affinity binders and 80% of known epitopes within a filtered subset of all possible nonapeptides corresponding to about one tenth of the full theoretical list.The proposed method is fully automated and fast enough to scan a viral genome in less than an hour on a parallel computing architecture. As it requires very few starting experimental data, EpiDock can be used: (i) to predict potential T-cell epitopes from viral genomes (ii) to roughly predict still unknown peptide binding motifs for novel class I MHC alleles.     

Pharmacophore modelling,virtual screening and molecular docking studies on PLD1 inhibitors

Lipid metabolism plays a significant role in influenza virus replication and subsequent infection. The regulatory mechanism governing lipid metabolism and viral replication is not properly understood to date, but both Phospholipase D (PLD1 and PLD2) activities are stimulated in viral infection. In vitro studies indicate that chemical inhibition of PLD1 delays viral entry and reduction of viral loads. The current study reports a three-dimensional pharmacophore model based on 35 known PLD1 inhibitors. A sub-set of 25 compounds was selected as the training set and the remaining 10 compounds were kept in the test set. One hundred and twelve pharmacophore models were generated; a six-featured pharmacophore model (AADDHR.57) with survival score (2.69) produced a statistically significant three-dimensional quantitative structure–activity relationship model with r² = 0.97 (internal training set), r² = 0.71 (internal test set) and Q² = 0.64. The predictive power of the pharmacophore model was validated with an external test set (r² = 0.73) and a systematic virtual screening work-flow was employed showing an enrichment factor of 23.68 at the top 2% of the dataset (active and decoys). Finally, the model was used for screening of the filtered PubChem database to fetch molecules which can be proposed as potential PLD1 inhibitors for blocking influenza infection.     

禽流感病毒HA蛋白与人受体的分子对接

血凝素(hemagglutinin,HA)是位于禽流感病毒表面的糖蛋白。在病毒感染过程中,HA与禽类宿主细胞表面受体结合,介导病毒膜与宿主核内体膜的融合,在传染过程中发挥关键作用。自然界中的禽流感病毒处于不断演化之中,其HA的禽受体结合位点常常发生氨基酸变异。因此,当HA变异体与人受体结合能力较强时,禽流感病毒往往会发生跨种传播而感染人。为预防禽流感的跨种传播,人们迫切需要发展大规模快速检测或预测HA变异体与人受体结合亲和力的方法,以评估各种新发禽流感病毒的跨种传播能力,提前筛选出有潜在危险的病毒株。针对此问题,本研究以H7N9亚型的HA蛋白H7为研究对象,发展了一种运用分子对接的计算方法,预测HA变异体与人受体的结合亲和力。该方法的计算结果表明,H7与人受体的结合亲和力普遍弱于有较强传染人能力的H1,说明H7N9亚型病毒的跨种传播能力普遍较弱;但是,计算分析也揭示,部分新发的H7N9毒株的HA有强的人受体结合亲和力,提示在自然演化过程中,H7N9病毒有可能演化出具有较强的感染人能力的新毒株,这与2013年禽流感疫情的实际发生情况相一致。因此,本文所发展的计算方法可用于快速预测新发禽流感病毒HA与人受体的结合亲和力,为新发禽流感病毒的跨种传播风险评估提供理论依据。     

Pharmacophore Mode lingand Virtual Screening to Design the Potential Influenza Virus Endonuclease Inhibitors

Influenza virus endonuclease is an attractive target for antiviral therapy in the treatment of influenza infection. The purpos e of this study is to design a novel antiviral agent with improved biological activities against the influenza virus endonuclease. In this study, chemical feature‐based 3D pharmacophore models were developed from 41 known influenza virus endonuclease inhibitors. The best quantitative pharmacohore model (Hypo 1), which consists of two hydrogen‐bond acceptors and two hydrophobic features, yields the highest correlation coefficient (R = 0.886). Hypo 1 was further validated by the cross validation method and the test set compounds. The application of this model for predicting the activities of 11 known influenza virus endonuclease inhibitors in the test set shows great success. The correlation coefficient of 0.942 and a cross validation of 95;% confidence level prove that this model is reliable in identifying structurally diverse compounds for influenza virus endonuclease inhibition. The most active compound (compound 1) from the training set was docked into the active site of the influenza virus endonuclease as an additional verification that the pharmacophore model is accurate. The docked conformation showed important hydrogen bond interactions between the compound and two amino acids, Lys 134 and Lys 137. After validation, this model was used to screen the NCI chemical database to identify new influenza virus endonuclease inhibitors. Our study shows that the to pranking compound out of the 10 newly identified compounds using fit value ranking has an estimated activity of 0.049 μM. These newly identified lead compounds can be further experimentally validated using in vitro techniques.     

Inhibition of Influenza Virus Replication in MDCK Cells by Circular Dumbbell RNA/DNA Chimeras with Closed Alkyl Loop Structures

We have demonstrated that a new type of circular dumbbell RNA/DNA chimeric oligonucleotide (CDRDON) with two closed nucleotide or alkyl loop structures (hexa‐ethylene glycol) inhibits influenza virus A replication in MDCK cells. The enzymatic synthesis of circular dumbbell RNA/DNA chimeric oligonucleotides was achieved by enzymatically ligating a self‐complementary phosphorylated oligonucleotide with T4‐RNA ligase. The CDRDON‐Al, with two closed alkyl loop structures, showed higher nuclease resistance, hybridization, and cellular uptake than the anti‐S‐ODN and the CDRDON, with two closed nucleotide hairpin‐loop structures. The circular dumbbell RNA/DNA chimeric oligonucleotide (CDRDON‐Al‐PB2‐as), containing an AUG initiation‐codon sequence as the target of PB2, showed highly inhibitory effects on influenza A virus RNA expression. The limited toxicity of unmodified phosphodiester oligonucleotides and the sequence‐specific binding to target mRNA indicate that circular dumbbell RNA/DNA chimeric phosphodiester oligonucleotides can be used with intact cells, and may prevent viral replication in culture.     

A new method for analyzing H5N1 avian influenza virus

In this paper a novel method for phylogenetic analysis of H5N1 avian influenza virus has been proposed. At first we provide a mapping of virus protein sequence. Based on this mapping, we propose a new distance measure and make use of the corresponding similarity matrix to construct phylogenic tree without requiring multiple alignment. As an application, we construct phylogenic tree for 123 species of H5N1 avian influenza virus. The phylogeny obtained is generally consistent with evolutionary trees constructed in previous studies.     

Inhibitory effect and possible mechanism of action of patchouli alcohol against influenza A (H2N2) virus

In the present study, the anti-influenza A (H2N2) virus activity of patchouli alcohol was studied in vitro, in vivo and in silico. The CC?? of patchouli alcohol was above 20 μM. Patchouli alcohol could inhibit influenza virus with an IC?? of 4.03 ± 0.23 μM. MTT assay showed that the inhibition by patchouli alcohol appears strongly after penetration of the virus into the cell. In the influenza mouse model, patchouli alcohol showed obvious protection against the viral infection at a dose of 5 mg/kg/day. Flexible docking and molecular dynamic simulations indicated that patchouli alcohol was bound to the neuraminidase protein of influenza virus, with an interaction energy of -40.38 kcal mol?1. The invariant key active-site residues Asp151, Arg152, Glu119, Glu276 and Tyr406 played important roles during the binding process. Based on spatial and energetic criteria, patchouli alcohol interfered with the NA functions. Results presented here suggest that patchouli alcohol possesses anti-influenza A (H2N2) virus properties, and therefore is a potential source of anti-influenza agents for the pharmaceutical industry.     

Multivalent neuraminidase hydrolysis resistant triazole-sialoside protein conjugates as influenza-adsorbents

Triazole-sialoside tailored proteins with high hemagglutinin (HA) and neuraminidase (NA) binding affinity are prepared. Dynamic light scattering shows that these pseudo-sialylated proteins are ideal virus capture macromolecules.     

Proximity Ligation‐Based Fluorogenic Imaging Agents for Neuraminidases

Reagents to visualize and localize neuraminidase activity would be valuable probes to study the role of neuraminidases in normal cellular processes as well as during viral infections or cancer development. Herein, a new class of neuraminidase‐imaging probes that function as proximity ligation reagents by releasing a highly reactive fluorophore that tags nearby cellular material is described. It is further demonstrated that it is possible to create an influenza virus‐specific reagent, which can specifically detect influenza virus infections in mammalian cells. These reagents have potential use as specific histological probes independent of viral antigenicity and, therefore, offer some advantages over commonly used anti‐neuraminidase antibodies.     

Urolithin M5 from the Leaves of Canarium album (Lour.) DC. Inhibits Influenza Virus by Targeting Neuraminidase

Ganlanye (GLY), the leaf of Canarium album (Lour.) DC., is a traditional Chinese medicinal herb for warm disease treatment. We found that its aqueous extract could inhibit the influenza A virus. To find and characterize anti-influenza virus phytochemicals from GLY, we performed (1) bioassay-guided isolation, (2) a cell and animal assay, and (3) a mechanism study. Bioassay-guided isolation was used to identify the effective components. Influenza virus-infected MDCK cell and BALB/c mouse models were employed to evaluate the anti-influenza virus activities. A MUNANA assay was performed to find the NA inhibitory effect. As a result, urolithin M5 was obtained from the crude extract of GLY. It inhibited influenza virus activities in vitro and in vivo by suppressing the viral NA activity. In the MDCK cell model, urolithin M5 could inhibit an oseltamivir-resistant strain. In a PR8-infected mouse model, 200 mg/kg/d urolithin M5 protected 50% of mice from death and improved lung edema conditions. GLY was recorded as a major traditional herb for warm disease treatment. Our study identified GLY as a potent anti-influenza herb and showed urolithin M5 as the active component. We first report the in vivo activity of urolithin M5 and support the anti-influenza application of GLY.     

Automated screening using microfluidic chip-based PCR and product detection to assess risk of BK virus-associated nephropathy in renal transplant recipients

The cost-effective detection of viral particles in bodily fluids could enable more effective responses to viral outbreaks, whether isolated clinical cases, or influenza epidemics. In renal transplant recipients, complications arising from high levels of BK virus can lead to graft dysfunction, graft loss, and/or reduced patient survival. We describe a microfluidic system for the sensitive analysis of BK virus (viral load) in unprocessed urine samples that are applied directly onto the chip, thus avoiding labor-intensive processing and sources of inter-assay variability. Integration of small volume genetic amplification (PCR) and electrophoretic analysis detects as few as 1-2 viral copies, distinguishes between high, medium and low levels of virus and reliably identifies viral loads requiring clinical intervention. As a first step to wider application in the clinic and in the field, the present work presents an entirely microchip-based system, validated against conventional clinical methods using clinical samples.     

Rapid Identification of the Receptor‐Binding Specificity of Influenza A Viruses by Fluorogenic Glycofoldamers

The re‐emergence of influenza raises a global concern that viral pandemics can unpredictably occur. However, effective approaches that can probe the infection risk of influenza viruses for humans are rare. In this work, we develop a glycofoldamer that can rapidly identify the glycan‐receptor specificity of influenza viruses in a high‐throughput manner. The coupling of glycan receptors that can be recognized by hemagglutinin (a surface protein on the virion capsid of influenza) to a fluorogenic‐dye foldamer produces the glycofoldamers with minimal fluorescence in aqueous solution. After interaction with human‐infecting virus strains for only five minutes, the fluorescence intensity of the glycofoldamer is remarkably enhanced with a blue‐shifted emission peak. The probes have also proven effective for the rapid identification of 1) the human‐ or bird‐infecting properties of influenza viruses in a high‐throughput manner and 2) the receptor‐specificity switch of a virus strain by mutations.     

Proviral load determination of different feline immunodeficiency virus isolates using real-time polymerase chain reaction: influence of mismatches on quantification

Lentiviruses are associated not only with immunodeficiency but also with malignancies. The mechanisms involved in tumorigenesis are still not fully understood. Cats infected with feline immunodeficiency virus (FIV) in the wild represent one model in which the role of viral load in the pathogenesis can be studied, since tumors, especially lymphomas, are quite often observed in cats infected with FIV. To be able to compare the viral load data among cats infected with different FIV isolates, the method used to obtain the viral load has to be unaffected by isolate-specific differences. This is especially true for the real-time polymerase chain reaction (PCR), a new method for viral load determination, since nucleotide sequence mismatches have been used for allelic discrimination with this method. To investigate the influence of these mismatches on PCR efficiency, we have used an FIV-specific real-time PCR and determined the influence of nucleotide sequence variation in several characterized FIV isolates as well as unknown isolates from naturally infected cats. We could demonstrate that minor mismatches, such as point mutations in the primer or the probe region, decrease overall PCR efficiency but do not abolish the quantification, in contrast to major mismatches of three or four nucleotides, which lead to complete inhibition of the real-time PCR detection. Based on these results, it will be possible to design real-time PCR systems allowing the quantification of a broad range of isolates, which is a prerequisite for the investigation of the impact of viral load in tumorigenesis.     

Synthesis of adenylyl-(3'----5')-guanosine and some analogues as probes to explore the molecular mechanism of stimulation of influenza virus RNA polymerase

Influenza virus mRNA synthesis is primed by a capped oligonucleotide which is cleaved off from a cellular mRNA by a viral protein. The dinucleotide A3'p5'G can be used as a primer for the viral RNA polymerase mediated RNA synthesis in a cell-free system. Analogues of A3'p5'G have therefore been synthesized using the phosphotriester approach, and their priming ability for the influenza virus mRNA synthesis has been determined. An absence of the 2'-hydroxyl function in the guanosine residue in the dinucleotide, as in A3'p5'dG, drastically decreased its priming ability. Similarly, an alteration of the 3'----5' phosphate linkage to a 2'----5' phosphodiester linkage affected the priming ability quite severely. However a dinucleotide, with the 2'-hydroxyl function omitted in the adenosine moiety, as in dA3'p5'G, could still stimulate the mRNA synthesis. None of the modified dinucleotides inhibited A3'p5'G or globin mRNA primed influenza mRNA synthesis.     

利用网络药理学方法研究热毒宁注射液抗流感病毒的分子作用机制

采用分子对接、网络分析预测热毒宁注射液抗流感病毒的分子作用机制, 并通过已建立的体外流感病毒神经氨酸酶筛选模型对网络预测结果验证. 结果表明, 热毒宁注射液所含化合物在化学空间上具有类药性质; 网络分析揭示出热毒宁注射液是通过与流感病毒吸附、脱壳、复制以及释放等环节的多个蛋白相互作用发挥抗流感病毒作用的; 对于预测的15 个活性分子而言, 实验结果初步证实, 对A型流感病毒, 木犀草素呈现较强的抑制作用, 槲皮素则呈现较弱的抑制作用, 这也初步证实了预测结果.     

Ultrastructural Damages to H1N1 Influenza Virus Caused by Vapor Essential Oils

Influenza viruses are transmitted from human to human via airborne droplets and can be transferred through contaminated environmental surfaces. Some works have demonstrated the efficacy of essential oils (EOs) as antimicrobial and antiviral agents, but most of them examined the liquid phases, which are generally toxic for oral applications. In our study, we describe the antiviral activity of Citrus bergamia, Melaleuca alternifolia, Illicium verum and Eucalyptus globulus vapor EOs against influenza virus type A. In the vapor phase, C. bergamia and M. alternifolia strongly reduced viral cytopathic effect without exerting any cytotoxicity. The E. globulus vapor EO reduced viral infection by 78% with no cytotoxicity, while I. verum was not effective. Furthermore, we characterized the EOs and their vapor phase by the head-space gas chromatography–mass spectrometry technique, observing that the major component found in each liquid EO is the same one of the corresponding vapor phases, with the exception of M. alternifolia. To deepen the mechanism of action, the morphological integrity of virus particles was checked by negative staining transmission electron microscopy, showing that they interfere with the lipid bilayer of the viral envelope, leading to the decomposition of membranes. We speculated that the most abundant components of the vapor EOs might directly interfere with influenza virus envelope structures or mask viral structures important for early steps of viral infection.     

Exposure to ultraviolet radiation enhances mortality and pathology associated with influenza virus infection in mice

Ultraviolet radiation (UVR) causes systemic immune suppression, decreasing the delayed type and contact hypersensitivity responses in animals and humans and enhancing certain mycobacterial, parasitic and viral infections in mice. This study tests the hypothesis that prior exposure to UVR enhances influenza infections in mice. BALB/c female mice were exposed to 0-8.2 kJ/m2 of UVR. Exposed and unexposed mice were infected intranasally three days later with 150-300 plaque-forming units/mouse (lethal dose (LD)20-LD40) of mouse-adapted Hong Kong Influenza A/68 (H3N2) virus or sham infected with 50 microL Hanks' balanced salt solution/mouse. Mortality from viral infection ranged from 25-50%. UVR exposure increased virus-associated mortality in a dose-dependent manner (up to a two-fold increase at 8.2 kJ/m2). The increased mortality was not associated with bacterial pneumonia. The highest dose of UVR also accelerated the body weight loss and increased the severity and incidence of thymic atrophy associated with influenza infection. However, UVR treatment had little effect on the increase in lung wet weight seen with viral infection, and, to our surprise, did not cause an increase in virus titers in the lung or dissemination of virus. The mice died 5-6 days after infection, too early for adaptive immune responses to have much impact. Also, UVR did not interfere with the development of protective immunity to influenza, as measured by reinfection with a lethal challenge of virus. Also, cells adoptively transferred from UVR or untreated mice were equally protective of recipient mice challenged with a lethal dose of virus. The mice resemble mice succumbing to endotoxin, and influenza infection increased the levels of tumor necrosis factor alpha (TNF-alpha) in bronchoalveolar lavage fluid and serum cortisol levels; however, UVR preexposure did not increase either of these responses to the virus. The results show that UVR increased the morbidity, mortality and pathogenesis of influenza virus in mice without affecting protective immunity to the virus, as measured by resistance to reinfection. The mechanism of enhanced mortality is uncertain, but the data raises concerns that UVR may exacerbate early responses that contribute to the pathogenesis of a primary viral infection.