麻黄水提液抑制呼吸道合胞病毒作用实验研究

目的 研究麻黄水提液在细胞水平抑制呼吸道合胞病毒(RSV)的作用及其作用机制,为我国中药的临床应用提供实验依据. 方法 采用细胞培养技术,以利巴韦林注射液为阳性对照,观察麻黄水提液抗RSV作用. 结果 麻黄水提液有抗RSV作用且存在剂量-反应关系(P＜0.01),麻黄水提液除2.00 mg/ml剂量组外其它剂量组在8h内给药对RSV都有抑制作用(P＜0.05),麻黄水提液对RSV穿入和吸附过程都有明显抑制作用(P＜0.01). 结论 麻黄水提液在体外实验中对呼吸道合胞病毒有一定抑制作用.     

Immunomodulatory Activity of Red Ginseng against Influenza A Virus Infection

Ginseng herbal medicine has been known to have beneficial effects on improving human health. We investigated whether red ginseng extract (RGE) has preventive effects on influenza A virus infection in vivo and in vitro. RGE was found to improve survival of human lung epithelial cells upon influenza virus infection. Also, RGE treatment reduced the expression of pro-inflammatory genes (IL-6, IL-8) probably in part through interference with the formation of reactive oxygen species by influenza A virus infection. Long-term oral administration of mice with RGE showed multiple immunomodulatory effects such as stimulating antiviral cytokine IFN-γ production after influenza A virus infection. In addition, RGE administration in mice inhibited the infiltration of inflammatory cells into the bronchial lumens. Therefore, RGE might have the potential beneficial effects on preventing influenza A virus infections via its multiple immunomodulatory functions.     

匍扇藻多糖抗病毒活性的初步研究(英文)

目的:研究褐藻匍扇藻(Lobophora variegata)多糖的抗病毒活性。方法:匍扇藻多糖的粗提物经沸水提取和乙醇沉淀后获得,粗多糖经离子交换柱层析法分离以获取较纯的多糖片段。它们的抗病毒活性采用细胞病变效应减少法和空斑减少检测法检测。粗多糖的细胞毒性采用 MTT 法测定。结果:匍扇藻粗多糖具有显著的抗单纯疱疹病毒Ⅰ型和Ⅱ型的活性,它能抑制不同的单纯疱疹病毒毒株,包括标准株、阿昔洛韦抗性株和临床病毒株。通过空斑减少法测得它对阿昔洛韦抗性株和 HSV-Ⅱ型临床株的半数有效浓度(EC50)分别为 18.2 和 6.25 μg/ml。因为它的细胞毒性很低,因此具有较大的选择性系数。这种粗多糖有一定的抗呼吸道合胞病毒(RSV)活性,但对流感病毒没有抑制作用。该粗多糖经DEAE离子交换柱层析分离得到两个不同的多糖,它们也都显示出抗单纯疱疹病毒的活性。结论:匍扇藻含有具抗病毒活性的多糖并且对单纯疱疹病毒显示出较强的抑制作用。     

溶葡萄球菌复合酶体外抗流感病毒效果研究

目的：研究溶葡萄球菌复合酶体外抗流感病毒效果，探寻预防、治疗流感病毒感染的新药和给药途径。方法：应用细胞病变抑制试验和直接免疫荧光法．经不同给药途径、作用时间和剂量，研究溶葡萄球菌复合酶体外抗流感病毒效果。并与已被普遍公认有效的抗流感药物磷酸奥司他韦比较。结果：两种试验方法都显示，两种药物在不同给药方式下，大部分实验组对病毒的抑制作用与对照组相比均有统计学意义（P〈0．05）。溶葡萄球菌复合酶1：5稀释后与病毒作用30min和磷酸奥司他韦治疗组的抗病毒效果最显著，且两者间差别无统计学意义（P〉0．05）。结论：溶葡萄球菌复合酶在体外抗流感病毒中具有较明显的作用，部分实验组的效果和公认的高效抗流感药物磷酸奥司他韦没有差别。在预防流感流行中具有一定的应用前景。     

Virus-specific T cells as correlate of (cross-)protective immunity against influenza

Since inactivated influenza vaccines mainly confer protective immunity by inducing strain-specific antibodies to the viral hemagglutinin, these vaccines only afford protection against infection with antigenically matching influenza virus strains. Due to the continuous emergence of antigenic drift variants of seasonal influenza viruses and the inevitable future emergence of pandemic influenza viruses, there is considerable interest in the development of influenza vaccines that induce broader protective immunity. It has long been recognized that influenza virus-specific CD8⁺ T cells directed to epitopes located in the relatively conserved internal proteins can cross-react with various subtypes of influenza A virus. This implies that these CD8⁺ T cells, induced by prior influenza virus infections or vaccinations, could afford heterosubtypic immunity. Furthermore, influenza virus-specific CD4⁺ T cells have been shown to be important in protection from infection, either via direct cytotoxic effects or indirectly by providing help to B cells and CD8⁺ T cells. In the present paper, we review the induction of virus-specific T cell responses by influenza virus infection and the role of virus-specific CD4⁺ and CD8⁺ T cells in viral clearance and conferring protection from subsequent infections with homologous or heterologous influenza virus strains. Furthermore, we discuss vector-based vaccination strategies that aim at the induction of a cross-reactive virus-specific T cell response.     

Isolation and characterization of influenza C virus inhibitors in rat serum

Two inhibitors against haemagglutination by influenza C virus were isolated from pooled sera of normal rats by sequential chromatography on Blue Sepharose CL 6B, Ultrogel AcA 22, and DEAE-cellulose. The two inhibitors were identified as α₁-macroglobulin and murinoglobulin by comparison with the authentic samples. These inhibitors abolished the haemagglutination by influenza C virus strains but did not affect the haemagglutination by influenza A and B virus strains. Haemagglutination inhibition activity of both inhibitors was completely destroyed by incubation with neuraminidase from Arthrobacter ureafaciens. By contrast, no activity was lost after treatment with neuraminidase from Vibrio cholerae. These results suggest that the sialic acid residue(s) which is excised by the former neuraminidase but not by the latter is essential for the haemagglutination inhibition. The two inhibitors were inactivated by treating with sodium hydroxide and methylamine but not with sodium metaperiodate.     

CC及CXC趋化因子在流感病毒感染过程中调控不同免疫细胞的作用研究进展

流行性感冒(简称流感)是由流感病毒感染引起的急性呼吸道传染病。流感病毒经呼吸道侵袭人体后,呼吸道上皮细胞首先作出反应,产生多种细胞因子诱导机体发挥免疫应答,其中CC趋化因子及CXC趋化因子在流感病毒感染过程中调控多种免疫细胞,在流感病毒感染早期控制炎症反应,维持机体稳态过程中发挥重要作用。分析宿主体内趋化因子调控免疫细胞在流感病毒感染过程中的作用机制,从而为治疗流感提供新的策略。本文主要针对流感病毒感染过程中发挥主要作用的CC趋化因子、CXC趋化因子及其相关受体对各类型免疫细胞的调节作用进行综述。     

Echinacea purpurea aerial extract alters course of influenza infection in mice

Influenza infection is a major clinical problem and Echinacea purpurea, a widely consumed botanical product, is purported to alter the course of respiratory infections including influenza. Mice infected with WSN influenza A and treated with E. purpurea polysaccharide extract had less weight loss than untreated mice but similar pulmonary viral titers. Echinacea-treated mice had lower systemic and pulmonary KC and IL-10 levels and lower systemic IFN-γ levels following influenza infection. These suggest that E. purpurea alters the clinical course of influenza infection in mice through modulation of cytokines and not direct antiviral activity.     

Ribavirin: A clinical overview

Ribavirin, a broad spectrum, non-interferon-inducing virustatic chemotherapeutic agent, demonstrates activity against a wide range of RNA and DNA viruses, including the retrovirus known to cause the acquired immune deficiency syndrome. The drug's proposed mechanism of action, as well as pharmacokinetics are discussed, and preclinical toxicity, safety and clinical efficacy studies are presented.To date, the best success has occurred in the use of ribavirin to treat respiratory syncytial virus infection in infants and young children and to treat influenza A and B virus infections in young adults. Viral infections, particularly viral pneumonia, are often life-threatening in infants with severe combined immunodeficiency disease (SCID), and ribavirin aerosol has been used successfully to treat respiratory syncytial virus and parainfluenza virus infection of immunodeficient children.Special note is taken of ribavirin's clinical benefit in treating severe and life-threatening infections caused by the Lassa fever virus and the significant improvement over either the use of immune plasma or supportive therapy alone. Indeed, ribavirin thus emerges as the first antiviral drug that is able to reduce mortality in a highly lethal systemic disease by more than 90%.Additional studies demonstrate the drug's efficacy in acute viral hepatitis, herpesvirus infections, and measles. Controlled clinical trials are underway to test the drug in patients infected with the AIDS virus.Corresponding author.     

Efficient influenza B virus propagation due to deficient interferon-induced antiviral activity in MDCK cells

Influenza B virus infections are mainly restricted to humans, which is partially caused by the inability of influenza B virus NS1 protein to counteract the innate immune response of other species. However, for cell culture-based influenza vaccine production non-human cells, such as Madin-Darby canine kidney (MDCK) cells, are commonly used. Therefore, the impact of cellular pathogen defence mechanisms on influenza B virus propagation in MDCK cells was analysed in this study. Activation of the cellular antiviral defence by interferon stimulation slowed down influenza B virus replication at early time points but after 48 h the same virus titres were reached in stimulated and control cells. Furthermore, suppression of the antiviral host defence by transient expression of a viral antagonist, the rabies virus phosphoprotein, could not increase influenza B virus replication. Finally, canine Myxovirus resistance (Mx) proteins showed no antiviral activity in an influenza B virus-specific minireplicon assay in contrast to the murine Mx1 protein. Taken together, these results indicate that an insufficient antiviral defence in MDCK cells promotes efficient influenza B virus replication favouring the use of MDCK cells in influenza vaccine production.     

Avian influenza A (H7N9) virus infection in humans: Epidemiology,evolution, and pathogenesis

New human influenza A virus strains regularly emerge causing seasonal epidemics and occasional pandemics. Lately, several zoonotic avian influenza A strains have been reported to directly infect humans. In early 2013, a novel avian influenza A virus (H7N9) strain was discovered in China to cause severe respiratory disease in humans. Since then, over 450 human cases of H7N9 infection have been discovered and 165 of them have died. Multiple epidemiological, phylogenetic, in vivo, and in vitro studies have been done to determine the origin and pathogenesis of novel H7N9 strain. This article reviews the literature related to the epidemiology, evolution, and pathogenesis of the H7N9 strain since its discovery in February 2013 till August 2014. The data available so far indicate that H7N9 was originated by a two-step reassortment process in birds and transmitted to humans through direct contact with live-bird markets. H7N9 is a low-pathogenic avian virus and contains several molecular signatures for adaptation in mammals. The severity of the respiratory disease caused by novel H7N9 virus in humans can be partly attributed to the age, sex, and underlying medical conditions of the patients. A universal influenza vaccine is not available, though several strain-specific H7N9 candidate vaccine viruses have been developed. Further, novel H7N9 virus is resistant to antiviral drug amantadine and some H7N9 isolates have acquired the resistance to neuraminidase-inhibitors. Therefore, constant surveillance and prompt control measures combined with novel research approaches to develop alternative and effective anti-influenza strategies are needed to overcome influenza A virus.     

Infection dynamics and virus-induced apoptosis in cell culture-based influenza vaccine production—Flow cytometry and mathematical modeling

Cell culture-based influenza vaccine manufacturing is of growing importance. Depending on virus strains, differences in infection dynamics, virus-induced apoptosis, cell lysis and virus yields are observed. Comparatively little is known concerning details of virus–host cell interaction on a cellular level and virus spreading in a population of cells in bioreactors. In this study, the infection of MDCK cells with different influenza A virus strains in lab-scale microcarrier culture was investigated by flow cytometry. Together with the infection status of cells, virus-induced apoptosis was monitored. A mathematical model has been formulated to describe changes in the concentration of uninfected and infected adherent cells, dynamics of virus particle release (infectious virions, hemagglutinin content), and the time course of the percentage composition of the cell population.     

Biopolymer encapsulated live influenza virus as a universal CD8+ T cell vaccine against influenza virus

Current influenza virus vaccines primarily elicit antibodies and can be rendered ineffective by antigenic drift and shift. Vaccines that elicit CD8+ T cell responses targeting less variable proteins may function as universal vaccines that have broad reactivity against different influenza virus strains. To generate such a universal vaccine, we encapsulated live influenza virus in a biopolymer and delivered it to mice subcutaneously. This vaccine was safe, induced potent CD8+ T cell immunity and protected mice against heterosubtypic lethal challenge. Safety of subcutaneous (SQ) vaccination was tested in Rag−/−γc−/− double knockout mice which we show cannot control intranasal infection. Biopolymer encapsulation of live influenza virus could be used to develop universal CD8+ T cell vaccines against heterosubtypic and pandemic strains.     

Ginseng and Salviae herbs play a role as immune activators and modulate immune responses during influenza virus infection

We have investigated the adjuvant roles of common herbal medicines (ginseng, Salviae) and their effects on early immune responses during influenza virus infection in a mouse model. Intranasal co-administration with inactivated influenza virus A (PR8) and ginseng or Salviae extract increased the levels of influenza virus specific antibodies and neutralizing activities compared to immunization with PR8 alone, and provided protective immunity. Salviae co-administration significantly enhanced IFN-gamma and IL-2 cytokine producing splenocytes while ginseng induced high levels of IL-4 and IL-5 cytokine producing cells after challenge infection. Cells expressing an early activation marker CD69 and levels of a pro-inflammatory cytokine IL-6 were highly elevated in lungs from na?ve mice during challenge virus infection, which might be a mechanism in lung inflammation leading to death. In contrast, immunized mice that were co-administered ginseng or Salviae modulated CD69 expressing immune cells, did not produce IL-6, and showed significant enhancement of influenza virus specific IgA antibody in lungs after challenge virus infection. Therefore, these results indicate that both ginseng and Salviae play a role as mucosal adjuvants against influenza virus as well as immuno-modulators during influenza virus infection.     

Complete protection against a H5N2 avian influenza virus by a DNA vaccine expressing a fusion protein of H1N1 HA and M2e

Most influenza vaccines target hemagglutinin (HA) in order to protect the host against infection. However, theses vaccines are strain-specific due to major antigenic variations of HA. Since it is difficult to predict epidemic and pandemic strains of influenza virus, the development of effective vaccines against divergent influenza viruses is urgently needed. Although M2e-based vaccines are associated with weaker protection than HA-based vaccines that induce neutralizing antibodies against challenge virus matched-strain, the extracellular domain of Matrix 2 protein (M2e) is one of a potential broad-spectrum immunogen because it contains highly conserved sequences among influenza A viruses. In this study, M2e sequence was fused to H1N1 HA DNA (M2e-HA) and the immunogenicity and antiviral efficacy of this DNA vaccine was evaluated in response to challenge with a heterosubtypic H5N2 avian influenza virus. Compared to vaccination with HA or M2e DNA alone, vaccination with M2e-HA DNA or combination of M2e DNA and HA DNA (M2e DNA + HA DNA) induced a broad immunity without evidence of immune interference. In addition, HA-specific CD8⁺ and M2e-specific T cell responses elicited by M2e-HA DNA vaccination were significantly higher than those of HA or M2e DNA vaccine alone, respectively. Following challenge with a heterosubtypic influenza virus infection, vaccination with M2e-HA DNA conferred complete protection against mortality. In combination, these results suggest that DNA vaccines expressing a fusion protein, M2e-HA, may provide an attractive approach for the development of broad-spectrum influenza vaccines.     

Antitumor Effect of the Ethanolic Extract from Seeds of Euphorbia lathyris in Colorectal Cancer

The seeds of Euphorbia lathyris have been used in traditional medicine to treat various medical conditions. However, neither all of their active biocompounds nor the molecular mechanisms underlying their therapeutic effects have been described. A new ethanolic extract of defatted flour from mature seeds of Euphorbia lathyris showed a high total polyphenol content and significant antioxidant activity. Chromatographic analysis showed that esculetin, euphorbetin, gaultherin, and kaempferol-3-rutinoside were the most abundant polyphenolic bioactive compounds. Antiproliferative assays showed a high and selective antitumor activity against colon cancer cell lines (T84 and HCT-15). In addition, a significant antiproliferative activity against glioblastoma multiforme cells was also demonstrated. Its mechanism of action to induce cell death was mediated by the overexpression of caspases 9, 3, and 8, and by activation of autophagy. Interestingly, a reduction in the migration capacity of colon cancer cells and a significant antiangiogenic effect on human umbilical vein endothelial cells were also demonstrated. Finally, the extract significantly reduced the subpopulations of cancer stem cells. This extract could be the basis to develop new therapeutic strategies for the treatment of colon cancer, although further experiments will be necessary to determine its in vivo effects.     

Universal influenza DNA vaccine encoding conserved CD4 T cell epitopes protects against lethal viral challenge in HLA-DR transgenic mice

The goal of the present study was to design a vaccine that would provide universal protection against infection of humans with diverse influenza A viruses. Accordingly, protein sequences from influenza A virus strains currently in circulation (H1N1, H3N2), agents of past pandemics (H1N1, H2N2, H3N2) and zoonotic infections of man (H1N1, H5N1, H7N2, H7N3, H7N7, H9N2) were evaluated for the presence of amino acid sequences, motifs, that are predicted to mediate peptide epitope binding with high affinity to the most frequent HLA-DR allelic products. Peptides conserved among diverse influenza strains were then synthesized, evaluated for binding to purified HLA-DR molecules and for their capacity to induce influenza-specific immune recall responses using human donor peripheral blood mononuclear cells (PBMC). Accordingly, 20 epitopes were selected for further investigation based on their conservancy among diverse influenza strains, predicted population coverage in diverse ethnic groups and capacity to recall influenza-specific responses. A DNA plasmid encoding the epitopes was constructed using amino acid spacers between epitopes to promote optimum processing and presentation. Immunogenicity of the DNA vaccine was measured using HLA-DR4 transgenic mice and the TriGrid™ in vivo electroporation device. Vaccination resulted in peptide-specific immune responses, augmented HA-specific antibody responses and protection of HLA-DR4 transgenic mice from lethal PR8 influenza virus challenge. These studies demonstrate the utility of this vaccine format and the contribution of CD4⁺ T cell responses to protection against influenza infection.     

Synthesis of new C5-(1-substituted-1,2,3-triazol-4 or 5-yl)-2'-deoxyuridines and their antiviral evaluation

The synthesis and antiviral evaluation of a series of C5-(1,4- and 1,5-disubstituted-1,2,3-triazolo)-nucleoside derivatives is described. The key steps of this synthesis are regioselective Huisgen’s 1,3-dipolar cycloaddition, using either copper-catalyzed azide-alkyne cycloaddition (CuAAC) or ruthenium-catalyzed azide-alkyne cycloaddition (RuAAC) under microwave activation. Some compounds among the 5a-l series possess activity against herpes simplex viruses 1 and 2, varicella-zoster virus, human cytomegalovirus and vaccinia virus. Their cytostatic activities were determined against murine leukemia cells, human T-lymphocyte cells and cervix carcinoma cells. Compounds were also evaluated on a wide panel of RNA viruses, including Vesicular stomatitis virus, influenza viruses type A (H1N1 and H3N2) and B in MDCK cell cultures, parainfluenza-3 virus, reovirus-1, Sindbis virus and Punta Toro virus in Vero cell cultures and Vesicular stomatitis, Coxsackie B4 and respiratory syncytial virus, with no specific antiviral effect.     

Characterization of humoral and cell-mediated immunity induced by mRNA vaccines expressing influenza hemagglutinin stem and nucleoprotein in mice and nonhuman primates

In response to immune pressure, influenza viruses evolve, producing drifted variants capable of escaping immune recognition. One strategy for inducing a broad-spectrum immune response capable of recognizing multiple antigenically diverse strains is to target conserved proteins or protein domains. To that end, we assessed the efficacy and immunogenicity of mRNA vaccines encoding either the conserved stem domain of a group 1 hemagglutinin (HA), a group 2 nucleoprotein (NP), or a combination of the two antigens in mice, as well as evaluated immunogenicity in naïve and influenza seropositive nonhuman primates (NHPs). HA stem-immunized animals developed a robust anti-stem antibody binding titer, and serum antibodies recognized antigenically distinct group 1 HA proteins. These antibodies showed little to no neutralizing activity in vitro but were active in an assay measuring induction of antibody-dependent cellular cytotoxicity. HA-directed cell-mediated immunity was weak following HA stem mRNA vaccination; however, robust CD4 and CD8 T cell responses were detected in both mice and NHPs after immunization with mRNA vaccines encoding NP. Both HA stem and NP mRNA vaccines partially protected mice from morbidity following lethal influenza virus challenge, and superior efficacy against two different H1N1 strains was observed when the antigens were combined. In vivo T cell depletion suggested that anti-NP cell-mediated immunity contributed to protection in the mouse model. Taken together, these data show that mRNA vaccines encoding conserved influenza antigens, like HA stem and NP in combination, induce broadly reactive humoral responses as well as cell-mediated immunity in mice and NHPs, providing protection against homologous and heterologous influenza infection in mice.     

Enhancement of T cell-mediated immune responses to whole inactivated influenza virus by chloroquine treatment in vivo

Current influenza vaccines induce poor cross-reactive CD8+ T cell responses. Cellular immunity is generally specific for epitopes that are remarkably conserved among different subtypes, suggesting that strategies to improve the cross-presentation of viral antigens by dendritic cells (DC) could elicit a broadly protective immune response. Previous studies have shown that limited proteolysis within the endocytic pathway can favorably influence antigen processing and thus immune responses. Herein, we demonstrate that chloroquine improves the cross-presentation of non-replicating influenza virus in vitro and T cell responses in mice following a single administration of inactivated HI-X31 virus. CD8+ T cells were also recruited to lymph nodes draining the site of infection and able to reduce viral load following pulmonary challenge with the heterologous PR8 virus. These findings may have implications for vaccination strategies aimed at improving the cross-presentation capacity of DCs and thus the size of effector and memory CD8+ T cells against influenza vaccines.