Inhibition of Influenza Virus Ribonucleic Acid Polymerase by Ribavirin Triphosphate

Ribavirin 5′-triphosphate (RTP), derived from the broad-spectrum antiviral compound ribavirin (Virazole), can selectively inhibit influenza virus ribonucleic acid polymerase in a cell-free assay. Ribavirin and its 5′-monophosphate have no effect on the polymerase. The inhibition is competitive with respect to adenosine 5′-triphosphate and guanosine 5′-triphosphate. RTP also inhibits ApG- and GpC-stimulated influenza virus ribonucleic acid polymerase. Since ribavirin is phosphorylated in the cell, the inhibition of influenza multiplication in the cell may also be caused by RTP.     

Comparative Pathogenesis of an Avian H5N2 and a Swine H1N1 Influenza Virus in Pigs

Pigs are considered intermediate hosts for the transmission of avian influenza viruses (AIVs) to humans but the basic organ pathogenesis of AIVs in pigs has been barely studied. We have used 42 four-week-old influenza naive pigs and two different inoculation routes (intranasal and intratracheal) to compare the pathogenesis of a low pathogenic (LP) H5N2 AIV with that of an H1N1 swine influenza virus. The respiratory tract and selected extra-respiratory tissues were examined for virus replication by titration, immunofluorescence and RT-PCR throughout the course of infection. Both viruses caused a productive infection of the entire respiratory tract and epithelial cells in the lungs were the major target. Compared to the swine virus, the AIV produced lower virus titers and fewer antigen positive cells at all levels of the respiratory tract. The respiratory part of the nasal mucosa in particular showed only rare AIV positive cells and this was associated with reduced nasal shedding of the avian compared to the swine virus. The titers and distribution of the AIV varied extremely between individual pigs and were strongly affected by the route of inoculation. Gross lung lesions and clinical signs were milder with the avian than with the swine virus, corresponding with lower viral loads in the lungs. The brainstem was the single extra-respiratory tissue found positive for virus and viral RNA with both viruses. Our data do not reject the theory of the pig as an intermediate host for AIVs, but they suggest that AIVs need to undergo genetic changes to establish full replication potential in pigs. From a biomedical perspective, experimental LP H5 AIV infection of pigs may be useful to examine heterologous protection provided by H5 vaccines or other immunization strategies, as well as for further studies on the molecular pathogenesis and neurotropism of AIVs in mammals.     

The Special Neuraminidase Stalk-Motif Responsible for Increased Virulence and Pathogenesis of H5N1 Influenza A Virus

The variation of highly pathogenic avian influenza H5N1 virus results in gradually increased virulence in poultry, and human cases continue to accumulate. The neuraminidase (NA) stalk region of influenza virus varies considerably and may associate with its virulence. The NA stalk region of all N1 subtype influenza A viruses can be divided into six different stalk-motifs, H5N1/2004-like (NA-wt), WSN-like, H5N1/97-like, PR/8-like, H7N1/99-like and H5N1/96-like. The NA-wt is a special NA stalk-motif which was first observed in H5N1 influenza virus in 2000, with a 20-amino acid deletion in the 49^th to 68^th positions of the stalk region. Here we show that there is a gradual increase of the special NA stalk-motif in H5N1 isolates from 2000 to 2007, and notably, the special stalk-motif is observed in all 173 H5N1 human isolates from 2004 to 2007. The recombinant H5N1 virus with the special stalk-motif possesses the highest virulence and pathogenicity in chicken and mice, while the recombinant viruses with the other stalk-motifs display attenuated phenotype. This indicates that the special stalk-motif has contributed to the high virulence and pathogenicity of H5N1 isolates since 2000. The gradually increasing emergence of the special NA stalk-motif in H5N1 isolates, especially in human isolates, deserves attention by all.     

Lucidone Suppresses Hepatitis C Virus Replication by Nrf2-Mediated Heme Oxygenase-1 Induction

Upon screening of plant-derived natural products against hepatitis C virus (HCV) in the replicon system, we demonstrate that lucidone, a phytocompound, isolated from the fruits of Lindera erythrocarpa Makino, significantly suppressed HCV RNA levels with 50% effective concentrations of 15 ± 0.5 μM and 20 ± 1.1 μM in HCV replicon and JFH-1 infectious assays, respectively. There was no significant cytotoxicity observed at high concentrations, with a 50% cytotoxic concentration of 620 ± 5 μM. In addition, lucidone significantly induced heme oxygenase-1 (HO-1) production and led to the increase of its product biliverdin for inducing antiviral interferon response and inhibiting HCV NS3/4A protease activity. Conversely, the anti-HCV activity of lucidone was abrogated by blocking HO-1 activity or silencing gene expression of HO-1 or NF-E2-related factor 2 (Nrf2) in the presence of lucidone, indicating that the anti-HCV action of lucidone was due to the stimulation of Nrf-2-mediated HO-1 expression. Moreover, the combination of lucidone and alpha interferon, the protease inhibitor telaprevir, the NS5A inhibitor BMS-790052, or the NS5B polymerase inhibitor PSI-7977, synergistically suppressed HCV RNA replication. These findings suggest that lucidone could be a potential lead or supplement for the development of new anti-HCV agent in the future.     

Treating Critical Illness Caused by the 2009 H1N1 Influenza A Virus

Background

The 2009 H1N1 influenza A pandemic has set the world spinning, unexpectedly producing significant morbidity and mortality in young, otherwise healthy patients.

Discussion

As the virus spreads across the Northern Hemisphere, emergency physicians are confronted with the challenging task of caring for the many that become critically ill from this pathogen. With the exception of a few observational studies and case reports, there is little information to guide the emergency physician in resuscitating and delivering critical care to a rapidly deteriorating patient. Many moribund patients with 2009 H1N1 influenza A infection require non-conventional critical care therapies.

Conclusion

In this article, we describe the case of a critically ill patient with confirmed 2009 H1N1 influenza A infection. After a brief review of the unique characteristics of this virus, we discuss the management of critically ill patients burdened by infection with 2009 H1N1 influenza A.     

A Drug-Disease Model Describing the Effect of Oseltamivir Neuraminidase Inhibition on Influenza Virus Progression

A population drug-disease model was developed to describe the time course of influenza virus with and without oseltamivir treatment and to investigate opportunities for antiviral combination therapy. Data included viral titers from 208 subjects, across 4 studies, receiving placebo and oseltamivir at 20 to 200 mg twice daily for 5 days. A 3-compartment mathematical model, comprising target cells infected at rate β, free virus produced at rate p and cleared at rate c, and infected cells cleared at rate δ, was implemented in NONMEM with an inhibitory Hill function on virus production (p), accounting for the oseltamivir effect. In congruence with clinical data, the model predicts that the standard 75-mg regimen initiated 2 days after infection decreased viral shedding duration by 1.5 days versus placebo; the 150-mg regimen decreased shedding by an additional average 0.25 day. The model also predicts that initiation of oseltamivir sooner postinfection, specifically at day 0.5 or 1, results in proportionally greater decreases in viral shedding duration of 5 and 3.5 days, respectively. Furthermore, the model suggests that combining oseltamivir (acting to subdue virus production rate) with an antiviral whose activity decreases viral infectivity (β) results in a moderate additive effect dependent on therapy initiation time. In contrast, the combination of oseltamivir with an antiviral whose activity increases viral clearance (c) shows significant additive effects independent of therapy initiation time. The utility of the model for investigating the pharmacodynamic effects of novel antivirals alone or in combination on emergent influenza virus strains warrants further investigation.     

MicroRNA-103-1 Selectively Downregulates Brain NCX1 and Its Inhibition by Anti-miRNA Ameliorates Stroke Damage and Neurological Deficits

Na⁺/Ca2+ exchanger (NCX) is a plasma membrane transporter that, by regulating Ca2+ and Na⁺ homeostasis, contributes to brain stroke damage. The objectives of this study were to investigate whether there might be miRNAs in the brain able to regulate NCX1 expression and, thereafter, to set up a valid therapeutic strategy able to reduce stroke-induced brain damage by regulating NCX1 expression. Thus, we tested whether miR-103-1, a microRNA belonging to the miR-103/107 family that on the basis of sequence analysis might be a potential NCX1 regulator, could control NCX1 expression. The role of miR-103-1 was assessed in a rat model of transient cerebral ischemia by evaluating the effect of the correspondent antimiRNA on both brain infarct volume and neurological deficits. NCX1 expression was dramatically reduced when cortical neurons were exposed to miR-103-1. This alleged tight regulation of NCX1 by miR-103-1 was further corroborated by luciferase assay. Notably, antimiR-103-1 prevented NCX1 protein downregulation induced by the increase in miR-103-1 after brain ischemia, thereby reducing brain damage and neurological deficits. Overall, the identification of a microRNA able to selectively regulate NCX1 in the brain clarifies a new important molecular mechanism of NCX1 regulation in the brain and offers the opportunity to develop a new therapeutic strategy for stroke.     

甲型H1N1流感病毒临床实验室诊断策略

目的 探讨用于甲型H1N1流感患者临床实验室诊断的策略.方法 采用Dot-ELISA法检测流感患者中的甲型流感病毒的抗原,初步明确为甲型流感或排除非甲型流感;采用real-time RT-PCR法检测甲型流感病毒抗原阳性患者中的甲型H1N1流感病毒特异性核酸,进一步确定甲型H1N1流感病毒.结果 对44 448例在西安地区就诊的发热伴有流感样症状者的鼻咽腔取分泌物进行甲型流感病毒抗原筛查,其阳性筛检率为28.25%;对甲型流感病毒抗原筛查阳性的17 714例患者进行甲型H1N1流感病毒核酸检测,其阳性检出率为41.92%.结论 首先用甲型流感病毒抗原筛查,排除非甲型流感病毒;进而在甲型流感病毒的范围内进行甲型H1N1流感病毒的检测,即加快了甲型流感病毒的排查效率,又减轻了甲型H1N1流感病毒核酸检测的压力;进而又减轻了大部分非甲型H1N1流感患者核酸检测的经济负担.该文认为该检测策略为甲型H1N1流感病毒的临床实验室诊断提供了参考,可为甲型H1N1流感病毒感染的控制、诊断,以及合理用药和针对性治疗提供依据.     

甲型h1n1肺炎的ct影像特征

目的:探讨甲型H1N1肺炎CT表现,提高对该病的认识.材料与方法:回顾性分析25例经临床、实验室证实的甲型H1N1肺炎胸部CT表现.25例中男10例,女15例.年龄11～ 33岁,平均25.5岁.白血球计数2.1 × 10^9/L ～ 11.9 × 10^9/L,中性粒细胞百分数27.1％ ～93.1％,淋巴细胞百分数3.6％～57.4％.结果:25例中累及5个肺叶11例(占44.0％),累及4个肺叶6例(占24.0％),累及3个肺叶5例(占20.0％),累及2个肺叶3例(占12.0％).磨玻璃样影8例(占32.0％),表现为小片状及云雾样淡薄影,边缘模糊;实变影10例(占40.0％),表现为团片状、斑片状及棉球样影,密度较高;磨玻璃影合并实变影7例(占28.0％),表现为云雾样淡薄影与棉球样实变影同时存在.25例中实变影中可见支气管充气征8例(占32.0％);病灶内合并网格样改变7例(占28.0％);合并小结节影5例(占20.0％);合并两侧胸腔少量积液2例(占8.0％).结论:甲H1N1肺炎病变以多发,分布范围广,病灶变化快,CT表现具有一定特征.     

Assessment of Pandemic and Seasonal Influenza A (H1N1) Virus Susceptibility to Neuraminidase Inhibitors in Three Enzyme Activity Inhibition Assays

The neuraminidase inhibitors (NAIs) zanamivir and oseltamivir are currently the only antiviral drugs effective for the treatment and prophylaxis of 2009 pandemic influenza A (H1N1) virus infections. The proven potential of these viruses to acquire NAI resistance during treatment emphasizes the need to assess their NAI susceptibility. The 50% inhibitory concentrations (IC₅₀s) are known to vary depending on the neuraminidase inhibition (NI) test used; however, few side-by-side comparisons of different NI assays have been done. In the present study, a panel of 11 isolates representing 2009 seasonal and pandemic influenza H1N1 viruses, including oseltamivir-resistant H275Y variants, were tested in three functional NI assays: chemiluminescent (CL), fluorescent (FL), and colorimetric (CM). The sensitivities of the viruses to zanamivir, oseltamivir, and three investigational NAIs (peramivir, R-125489, and A-315675) were assessed. All isolates with the exception of H275Y variants were sensitive to all five NAIs by all three NI assays. The H275Y variants showed substantially elevated IC₅₀s against oseltamivir and peramivir. The three NI assays generally yielded consistent results; thus, the choice of NI assay does not appear to affect conclusions based on drug susceptibility surveillance. Each assay, however, offers certain advantages compared to the others: the CL assay required less virus volume and the FL assay provided the greatest difference in the IC₅₀s between the wild type and the variants, whereas the IC₅₀s obtained from the CM assay may be the most predictive of the drug concentrations needed to inhibit enzyme activity in humans. It would be desirable to develop an NI assay which combines the advantages of all three currently available assays but which lacks their shortcomings.For the treatment and chemoprophylaxis of infections caused by influenza A viruses, the U.S. Food and Drug Administration (FDA) has approved four drugs: amantadine and rimantadine as well as zanamivir and oseltamivir. These drugs belong to two classes, adamantanes (i.e., M2 ion-channel blockers) and neuraminidase (NA) inhibitors (NAIs), respectively. In recent years, the effectiveness of M2 blockers has been greatly compromised, which limits their usefulness in clinical practice. This is largely due to the rapid emergence and widespread circulation of adamantane-resistant influenza viruses (1, 5, 6, 7, 14, 17). More recently, the emergence and worldwide spread of seasonal H1N1 viruses resistant to oseltamivir, currently the most widely used drug against influenza infections, became a considerable public health concern (15, 21, 25, 32). Monitoring the NAI resistance of influenza viruses is an ongoing public health issue since the emergence in 2009 of pandemic viruses that are resistant to M2 blockers.Cell culture-based assays are typically not used for assessment of virus sensitivity to NAIs because of the unpredictable effect of hemagglutinin (HA) receptor binding (2, 34). Instead, drug susceptibility can be monitored by functional (biochemical) NA inhibition (NI) assays, and subsequent genotypic methods are generally required to identify the molecular marker(s) of resistance in the NA. The principle underlying the functional methods relies on the enzymatic nature of the NA, a viral surface glycoprotein and antigen. NA acts by cleaving the terminal neuraminic acid (also called sialic acid) from receptors recognized by influenza viral HA, thus facilitating the release of progeny virions from infected cells and preventing self-aggregation (29). Structurally, NAIs mimic the natural substrate, neuraminic acid, and produce tight interactions, with conserved residues of the NA active site competing with neuraminic acid for binding (11, 23). Preincubation of virus with NAIs leads to the inhibition of enzyme activity, which is detected after the addition of enzyme substrate. Most NI assays commonly used for virus surveillance utilize as substrates small synthetic conjugates that produce either a luminescent or a fluorescent signal upon cleavage by the NA enzyme. The chemiluminescent (CL) assay uses the 1,2-dioxetane derivative of neuraminic acid substrate in the influenza neuraminidase inhibitor resistance detection (NA-Star) kit (8), while the fluorescent (FL) assay employs 2′-O-(4-methylumbelliferyl)-N-acetylneuraminic acid substrate (MUNANA) (30). The results of the NI assays are expressed as the 50% inhibitory concentration (IC₅₀), which represents the NAI concentration that inhibits 50% of the enzyme activity of the virus. As the NA activity of clinical specimens is usually insufficient for determining the IC₅₀ due to a low viral content, NI assays, using either the substrate provided with the NA-Star kit or the MUNANA substrate, require virus propagation in cell cultures or embryonated chicken eggs. It is noteworthy that IC₅₀s are specific to the virus type/subtype and to the individual NAI tested (8, 19, 20, 24, 32, 37). The IC₅₀s obtained can be used for assessment of virus susceptibility to NAIs, including detection of resistant viruses, as well as for comparing the potencies of antiviral drugs belonging to the NAI class. Although both the CL and FL assays allow reliable detection of NAI resistance, the more recently developed CL assay was reported to be about 70 times more sensitive in detecting NA activity and has a greater linear range than the FL assay (8). The CL assay was also selected for use in the global drug susceptibility surveillance program by the Neuraminidase Inhibitor Susceptibility Network (NISN) (37, 39) and by other surveillance laboratories (28, 32). It should also be noted that IC₅₀s may vary even for the same virus when the NI assay is done using the NA-Star substrate (CL assay) and the MUNANA substrate (FL assay), according to reports on seasonal viruses (37). Whether one of the two assays, the CL or FL assay, more reliably predicts the level of resistance and the drug concentration required for the NA activity inhibition in vivo are key points of interest and remain to be elucidated.A third assay, the colorimetric (CM) assay, which utilizes fetuin as the substrate of the NA, is typically used to determine the titer of anti-NA antibodies because small substrates do not effectively compete with antibodies (3, 31). This assay is not widely used for antiviral susceptibility testing. Unlike the NA-Star and MUNANA synthetic substrates, fetuin is a large, natural, and soluble bovine glycoprotein that contains abundant neuraminic acids at the ends of its oligosaccharide moiety (which include the presence of two residues of α2,3-linked sialic acid and one residue of α2,6-linked sialic acid) (4, 33) and has been used as a substrate in NA-catalyzed reactions (3). Given that NAIs compete with the enzyme substrate for binding to the active site, the structure of the substrate can potentially influence the outcome of the competition and, as a result, the IC₅₀. In this respect, fetuin may represent a better natural substrate for the enzyme-neuraminic acid attached via an α2,3 or α2,6 linkage to oligosaccharide chains on the cell surface. Furthermore, since the cleavage of each neuraminic acid is chemically converted, the CM assay can be a quantifiable method from which the resulting IC₅₀s would correlate more closely to the NA activity of the virus tested. Despite these apparent advantages to the use of fetuin, the CM method relies on chemical reactions that are time-consuming, cumbersome, and impractical for high-throughput use. In addition, the assay requires concentrated virus stocks for testing. Thus, fetuin is still considered an undefined substrate that does not confer sufficient sensitivity or specificity for use in routine NAI susceptibility assays (34). The potential usefulness of a large substrate such as fetuin for assessment of the NAI susceptibilities of novel H1N1 viruses or novel inhibitors remains largely unexplored.Resistance to NAIs is not defined as clearly as that to adamantanes. In NI assays, a drug-resistant virus should have IC₅₀s consistently greater than the threshold value that is determined for each viral type/subtype and drug tested (27, 32, 37). Since the 2007-2008 influenza season, about a decade after the introduction of NAIs into clinical use, an NA framework mutation, H275Y (H274Y in N2 numbering), was consistently and most commonly detected in oseltamivir-resistant H1N1 viruses isolated worldwide (15, 21, 25, 32). Although the H275Y substitution represents the most-defined oseltamivir resistance marker of influenza viruses carrying the NA of the N1 subtype (35), novel NAI resistance-associated mutations—determined by elevated IC₅₀s in NI assays—continue to be revealed (21, 22, 32). Importantly, oseltamivir-resistant viruses from the ongoing H1N1 pandemic have been detected and reported around the world (9, 10, 26, 38). Seasonal and 2009 pandemic H1N1 viruses have the same phylogenetically distant NA gene ancestors (16), which necessitates the comprehensive assessment of the drug susceptibilities of the new pandemic viruses. Therefore, it is necessary to evaluate existing NI assays in order to better understand which assay may be the most sensitive for the detection of NAI resistance and/or the most predictive of virus susceptibility to NAIs in vivo.In the present study, we assessed the susceptibilities of a panel of seasonal and pandemic H1N1 influenza viruses, including virus variants bearing the established oseltamivir resistance mutation, H275Y in the NA, against five NAIs: two FDA-approved NAIs, zanamivir and oseltamivir, and three investigational NAIs, peramivir, R-125489 (the bioactive metabolite of the prodrug CS-8958 [laninamivir]), and A-315675 (a bioactive form of the prodrug A-322278). In order to better characterize and assess the consistency of IC₅₀s and levels of susceptibility, these viruses were tested in the widely used CL and FL assays, as well as with the CM method.     

荧光定量RT-PCR快速检测A型流感病毒

10例感染A型流感病毒者随机分为对照组和实验组。对照组5例进行血清学、病毒分离培养等方法进行检测,试验组5例进行荧光定量RT-PCR检测。结果试验组的确诊率和治疗效果均高于对照组,检测时间对照组为4～7d,而试验组可在4h内完成,治疗时间试验组也较短。差异均有统计学意义(P<0.05)。     

Inhibition of Cellular and Viral Protein Synthesis by 3-Methyleneoxindole

The effect of 3-methyleneoxindole (MO) on mengovirus and L-cell protein synthesis was investigated. MO was found to inhibit mengovirus multiplication and the incorporation of radioactive amino acids into both viral and cellular proteins. These results suggest that the antiviral effect of this compound is not specific but rather stems from its inhibition of the cellular translational machinery upon which mengovirus depends. We have also found that MO inhibits natural messenger ribonucleic acid (mengovirus and globin messenger ribonucleic acid) translation in cell-free extracts from Ehrlich ascites tumor cells but has no significant effect on polyuridylic acid translation. Additional data which suggest that MO inhibits protein synthesis at the level of initiation are shown.     

Inhibition of Herpes Simplex Virus Reactivation by Dipyridamole

Herpes simplex virus (HSV) reactivation from latency was investigated. Reactivation of thymidine kinase-negative HSV, which is defective for reactivation, was greatly enhanced by thymidine (TdR). The reactivation-enhancing effect of TdR was blocked by dipyridamole (DPM), a known nucleoside transport inhibitor. DPM also inhibited wild-type HSV reactivation, suggesting potential antiviral use.     

Irreversible Inhibition of Herpes Simplex Virus Replication in BSC-1 Cells by Zinc Ions

A substance inhibiting Escherichia coli motility was isolated by "motilometry" assay from the culture broth of Streptomyces sp. strain NR9GG8 and was found to be identical with desferrioxamine B. Its inhibitory effect was reversed by cyclic adenosine 3',5'-monophosphate (cAMP), while the motility stimulation by cAMP was diminished by this inhibitor. Its effects on various enzymes involved in cAMP metabolism of function of cAMP were examined.     

Specific Interactions between the Viral Coreceptor CXCR4 and the Biguanide-Based Compound NB325 Mediate Inhibition of Human Immunodeficiency Virus Type 1 Infection

The present studies were conducted to better define the mechanism of action of polyethylene hexamethylene biguanide (PEHMB) (designated herein as NB325), which was shown in previous studies to inhibit infection by the human immunodeficiency virus type 1 (HIV-1). Fluorescence-activated flow cytometric analyses of activated human CD4⁺ T lymphocytes exposed to NB325 demonstrated concentration-dependent reductions in CXCR4 epitope recognition in the absence of altered recognition of selected CD4 or CD3 epitopes. NB325 also inhibited chemotaxis of CD4⁺ T lymphocytes induced by the CXCR4 ligand CXCL12. However, NB325 did not cause CXCR4 internalization (unlike CXCL12) and did not interfere with CXCL12 binding. Additional flow cytometric analyses using antibodies with distinct specificities for extracellular domains of CXCR4 demonstrated that NB325 specifically interfered with antibody binding to extracellular loop 2 (ECL2). This interaction was confirmed using competitive binding analyses, in which a peptide derived from CXCR4 ECL2 competitively inhibited NB325-mediated reductions in CXCR4 epitope recognition. Collectively, these results demonstrate that the biguanide-based compound NB325 inhibits HIV-1 infection by specifically interacting with the HIV-1 coreceptor CXCR4.