A novel peptide inhibits the influenza virus replication by preventing the viral attachment to the host cells

Avian influenza viruses (AIV), the causative agent of avian flu or bird flu, cause widespread morbidity and mortality in poultry. The symptoms of the disease range from mild flu like symptoms to death. These viruses possess two important surface glycoproteins, namely hemagglutinin (HA) and neuraminidase (NA) against which neutralizing antibodies are produced. Due to the highly mutative nature of the genes which encode these proteins, the viruses often confer resistance to the current anti-viral drugs making the prevention and treatment of infection challenging. In our laboratory, we have recently identified a novel anti-viral peptide (P1) against the AIV H9N2 from a phage displayed peptide library. This peptide inhibits the replication of the virus in ovo and in vitro by its binding to the HA glycoprotein. In the current study, we demonstrate that the peptide inhibits the virus replication by preventing the attachment to the host cell but it does not have any effect on the viral fusion. The reduction in the viral nucleoprotein (NP) expression inside the host cell has also been observed during the peptide (P1) treatment. This novel peptide may have the potential to be developed as a therapeutic agent for the treatment and control of avian influenza virus H9N2 infections.     

1-beta-D-arabinofuranosylcytosine inhibits borna disease virus replication and spread

Borna disease virus (BDV) is a nonsegmented, negative-strand RNA virus that causes neurological diseases in a variety of warm-blooded animal species. There is general consensus that BDV can also infect humans, being a possible zoonosis. Although the clinical consequences of human BDV infection are still controversial, experimental BDV infection is a well-described model for human neuropsychiatric diseases. To date, there is no effective treatment against BDV. In this paper, we demonstrate that the nucleoside analog 1-beta-D-arabinofuranosylcytosine (Ara-C), a known inhibitor of DNA polymerases, inhibits BDV replication. Ara-C treatment inhibited BDV RNA and protein synthesis and prevented BDV cell-to-cell spread in vitro. Replication of other negative-strand RNA viruses such as influenza virus or measles virus was not inhibited by Ara-C, underscoring the particularity of the replication machinery of BDV. Strikingly, Ara-C treatment induced nuclear retention of viral ribonucleoparticles. These findings could not be attributed to known effects of Ara-C on the host cell, suggesting that Ara-C directly inhibits the BDV polymerase. Finally, we show that Ara-C inhibits BDV replication in vivo in the brain of infected rats, preventing persistent infection of the central nervous system as well as the development of clinical disease. These findings open the way to the development of effective antiviral therapy against BDV.     

蛋白激酶抑制剂Flavopiridol对流感病毒复制的体外抑制作用

【目的】在细胞水平上研究黄酮类化合物flavopiridol的抗流感病毒效果,初步探索了其抗流感病毒的机制。【方法】首先用Western blot初步检测了在蛋白激酶抑制剂flavopiridol处理下流感病毒NP和M1蛋白的水平,然后通过免疫荧光实验观察了宿主细胞中流感病毒vRNP的合成,又利用噬斑实验检测了flavopiridol对病毒复制的影响,最后通过检测flavopiridol处理的宿主细胞内RNA聚合酶Ⅱ的磷酸化状态和病毒各种RNA的合成量,探究了flavopiridol抑制流感病毒复制的机理。【结果】结果表明,flavopiridol在细胞水平上可以显著抑制流感病毒蛋白质和vRNP的合成及病毒的复制,同时flavopiridol也可以抑制宿主RNA聚合酶Ⅱ大亚基CTD结构域七肽重复序列中的2位丝氨酸的磷酸化来抑制聚合酶的转录延伸活性,显著地减少病毒vRNA的合成。【结论】Flavopiridol可以通过抑制宿主细胞RNA聚合酶Ⅱ的转录延伸活性有效地抑制流感病毒的复制。     

Rabies virus antinucleoprotein antibody protects against rabies virus challenge in vivo and inhibits rabies virus replication in vitro.

We previously reported that A/WySnJ mice vaccinated via a tail scratch with a recombinant raccoon poxvirus (RCN) expressing the rabies virus internal structural nucleoprotein (N) (RCN-N) were protected against a street rabies virus (D. L. Lodmell, J. W. Sumner, J.J. Esposito, W.J. Bellini, and L. C. Ewalt, J. Virol. 65:3400-3405, 1991). To improve our understanding of the mechanism(s) of this protection, we investigated whether sera of A/WySnJ mice that had been vaccinated with RCN-N but not challenged with street rabies virus had anti-rabies virus activity. In vivo studies illustrated that mice inoculated in the footpad with preincubated mixtures of anti-N sera and virus were protected. In addition, anti-N sera inoculated into the site of virus challenge protected mice. The antiviral activity of anti-N sera was also demonstrated in vitro. Infectious virus was not detected in cultures 24 h following infection with virus that had been preincubated with anti-N sera. At later time points, infectious virus was detected, but inhibition of viral production was consistently > or = 99% compared with control cultures. The protective and antiviral inhibitory activity of the anti-N sera was identified as anti-N antibody by several methods. First, absorption of anti-N sera with goat anti-mouse immunoglobulin serum, but not normal goat serum, removed the activity. Second, radioimmuno-precipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of sucrose density gradient-fractionated anti-N sera showed that antiviral activity was present only in the fraction containing anti-N antibody. Finally, absorption of anti-N sera with insect cells infected with a baculovirus expressing the N protein removed the protective activity. These data indicate that anti-N antibody is a component of the resistance to rabies virus infections.     

Autophagy is involved in influenza A virus replication

Autophagy is involved in the replication of viruses, especially those that perform RNA assembly on the surface of cytoplasmic membrane in host cells. However, little is known about the regulatory role of autophagy in influenza A virus replication. Using fluorescence and electron microscopy, we observed that autophagosomes can be induced and identified upon influenza A virus infection. The virus increased the amount of the autophagosome marker protein microtubule-associated protein light chain 3-II (LC3-II) and enhanced autophagic flux. When autophagy was pharmacologically inhibited by either 3-methylademine or wortmannin, the titers of influenza A virus were remarkably decreased. Viral reduction via autophagy inhibition was further confirmed by RNA interference, through which two different proteins required for autophagy were depleted. Noticeably, the compounds utilized had no marked effect on virus entry or cell viability, either of which might limit viral replication. Furthermore, alteration of cellular autophagy via pharmacological reagents or RNA interference impaired viral protein accumulation. Taken together, these findings indicate that autophagy is actively involved in influenza A virus replication.     

Passively transferred monoclonal antibody to the M2 protein inhibits influenza A virus replication in mice.

The M2 protein of influenza A virus is expressed on the surfaces of infected cells, and a monoclonal antibody to this protein inhibits plaque enlargement of sensitive influenza A viruses without reducing plaque titer (S.L. Zebedee and R.A. Lamb, J. Virol. 62:2762-2772, 1988). In the current study, passively transferred monoclonal antibody to M2 reduced the level of replication of influenza A virus but not of influenza B virus in the lungs of mice. These experiments demonstrated that antibody to a protein conserved among influenza A virus subtypes inhibits virus growth in vivo.     

MDA7/IL-24 is an anti-viral factor that inhibits influenza virus replication

Melanoma differentiation associated gene-7 (mda-7)/interleukin- 24 (IL-24) is a secreted cytokine, which plays an essential role in tumor suppression. Although its role as a multifunctional protein affecting broad types of cancers is well described, functions of IL-24 in host defense against virus infection are yet to be determined. In this study, we explored the anti-viral effect of recombinant IL-24 treatment during influenza infection. Infection of human lung adenocarcinoma cells (A549) with the influenza A virus up-regulated IL-24 mRNA and protein expression in a time-dependent manner. Pre-treatment of A549 cells with recombinant IL-24 protein effectively suppressed viral plaque formation. Furthermore, IL-24 treatment of A549 cells reduced viral non-structural protein 1 (NS1) synthesis, whereas IL-24 knockdown resulted in increased viral replication. Interestingly, IL-24 treatment following influenza A virus infection led to up-regulation of interferon (IFN)-induced antiviral signaling. Taken together, our results suggest that IL-24 exerts a potent suppressive effect on influenza viral replication and can be used in the treatment of influenza infection.     

MDCK cell-cultured influenza virus vaccine protects mice from lethal challenge with different influenza viruses

Influenza epidemics are major health concern worldwide. Vaccination is the major strategy to protect the general population from a pandemic. Currently, most influenza vaccines are manufactured using chicken embroynated eggs, but this manufacturing method has potential limitations, and cell-based vaccines offer a number of advantages over the traditional method. We reported here using the scalable bioreactor to produce pandemic influenza virus vaccine in a Madin-Darby canine kidney cell culture system. In the 7.5-L bioreactor, the cell concentration reached to 3.2 × 10(6) cells/mL and the highest virus titers of 256 HAU/50 μL and 1 × 10(7) TCID50/mL. The HA concentration was found to be 11.2 μg/mL. The vaccines produced by the cell-cultured system induced neutralization antibodies, cross-reactive T-cell responses, and were protective in a mouse model against different lethal influenza virus challenge. These data indicate that microcarrier-based cell-cultured influenza virus vaccine manufacture system in scalable bioreactor could be used to produce effective pandemic influenza virus vaccines.     

ANXA1促进Ⅰ型干扰素表达抑制口蹄疫病毒的复制

【目的】研究膜联蛋白A1(Annexin A1,ANXA1)对Ⅰ型干扰素(I-IFN)表达及口蹄疫病毒(FMDV)复制的影响。【方法】开展过表达及Knockdown实验,检测ANXA1对FMDV复制的影响。利用双荧光素酶报告系统检测ANXA1对ISRE和IFN-β启动子元件活化的影响。双荧光素酶报告系统鉴定ANXA1调控Ⅰ-IFN通路活化靶分子。Western blotting检测ANXA1对干扰素调解因子3(IRF3)磷酸化的影响。Real-time PCR检测ANXA1对干扰素刺激基因(ISGs)的影响。【结果】过表达ANXA1显著抑制FMDV的复制;下调ANXA1表达促进FMDV复制(P0.01或P0.05);ANXA1促Ⅰ型干扰素通路活化,呈现剂量依赖性(P0.01)。ANXA1显著增强IRF3的磷酸化,促进ISGs的表达(P0.01或P0.05)。【结论】ANXA1促进Ⅰ-IFN表达,抑制FMDV的复制。     

Sulfatide is required for efficient replication of influenza A virus

Sulfatide is abundantly expressed in various mammalian organs, including the intestines and trachea, in which influenza A viruses (IAVs) replicate. However, the function of sulfatide in IAV infection remains unknown. Sulfatide is synthesized by two transferases, ceramide galactosyltransferase (CGT) and cerebroside sulfotransferase (CST), and is degraded by arylsulfatase A (ASA). In this study, we demonstrated that sulfatide enhanced IAV replication through efficient translocation of the newly synthesized IAV nucleoprotein (NP) from the nucleus to the cytoplasm, by using genetically produced cells in which sulfatide expression was down-regulated by RNA interference against CST mRNA or overexpression of the ASA gene and in which sulfatide expression was up-regulated by overexpression of both the CST and CGT genes. Treatment of IAV-infected cells with an antisulfatide monoclonal antibody (MAb) or an anti-hemagglutinin (HA) MAb, which blocks the binding of IAV and sulfatide, resulted in a significant reduction in IAV replication and accumulation of the viral NP in the nucleus. Furthermore, antisulfatide MAb protected mice against lethal challenge with pathogenic influenza A/WSN/33 (H1N1) virus. These results indicate that association of sulfatide with HA delivered to the cell surface induces translocation of the newly synthesized IAV ribonucleoprotein complexes from the nucleus to the cytoplasm. Our findings provide new insights into IAV replication and suggest new therapeutic strategies.     

A novel influenza A virus activating enzyme from porcine lung: purification and characterization

Proteolytic activation of hemagglutinin, an envelope glycoprotein of the influenza virus, by host proteases is essential for infection and proliferation of the virus. However, there is no well-defined, inherent source of host proteases in man or swine, both of which are natural hosts for human influenza viruses. We have recently isolated a 32 kDa protein in a high salt extract from porcine lungs, which possess the hemagglutinin processing activity. In this study, we attempted to purify another hemagglutinin processing enzyme from porcine lung. The purified enzyme, named tryptase TC30, exhibited a molecular mass of about 30 kDa by SDS-PAGE and 28.5 kDa by gel filtration chromatography, suggesting that it is a monomer. Tryptase TC30 cleaved peptide substrates with Arg at the P1 position, and preferentially substrates with the Ser-Ile-Gin-Ser-Arg sequence corresponding to the HA cleavage site sequence of the A/PR/8/34 influenza virus. Among various inhibitors tested, trypsin-type serine protease inhibitors, such as aprotinin, antipain, benzamidine and leupeptin, efficiently inhibited the proteolytic activity of the enzyme. The N-terminal 40 amino acid sequence of tryptase TC30 exhibits more than 60% homology to mast cell tryptases from mice MCP-6 and human tryptase-alpha and -beta. These data indicate that tryptase TC30, the 30 kDa enzyme from porcine lung, is a novel hemagglutinin-cleaving enzyme.     

A novel compound C12 inhibits inflammatory cytokine production and protects from inflammatory injury in vivo

Inflammation is a hallmark of many diseases. Although steroids and cyclooxygenase inhibitors are main anti-inflammatory therapeutical agents, they may cause serious side effects. Therefore, developing non-steroid anti-inflammatory agents is urgently needed. A novel hydrosoluble compound, C12 (2,6-bis(4-(3-(dimethylamino)-propoxy)benzylidene)cyclohexanone), has been designed and synthesized as an anti-inflammatory agent in our previous study. In the present study, we investigated whether C12 can affect inflammatory processes in vitro and in vivo. In mouse primary peritoneal macrophages, C12 potently inhibited the production of the proinflammatory gene expression including TNF-α, IL-1β, IL-6, iNOS, COX-2 and PGE synthase. The activity of C12 was partly dependent on inhibition of ERK/JNK (but p38) phosphorylation and NF-κB activation. In vivo, C12 suppressed proinflammatory cytokine production in plasma and liver, attenuated lung histopathology, and significantly reduced mortality in endotoxemic mice. In addition, the pre-treatment with C12 reduced the inflammatory pain in the acetic acid and formalin models and reduced the carrageenan-induced paw oedema and acetic acid-increased vascular permeability. Taken together, C12 has multiple anti-inflammatory effects. These findings, coupled with the low toxicity and hydrosolubility of C12, suggests that this agent may be useful in the treatment of inflammatory diseases.     

A recombinant foot-and-mouth disease virus antigen inhibits DNA replication and triggers the SOS response in Escherichia coli

Abstract The 3D gene of foot-and-mouth disease virus encodes the viral RNA dependent RNA polymerase, also called virus infection associated (VIA) antigen, which is the most important serological marker of virus infection. This 3D gene from a serotype Cl virus has been cloned and overexpressed in Escherichia coli under the control of the strong lambda lytic promoters. The resulting 51 kDa recombinant protein has been shown to be immunoreactive with sera from infected animals. After induction of gene expression, an immediate and dramatic arrest of cell DNA synthesis occurs, similar to that produced by genotoxic doses of the drug mitomycin C. This effect does not occur during the production of either a truncated VIA antigen or other related and non-related viral proteins. The inhibition of DNA replication results in a subsequent induction of the host SOS DNA-repair response and in an increase of the mutation frequency in the surviving cells.