Amplification and Spread of Viruses in a Growing Plaque

The two-dimensional propagation of viruses through a "lawn" of receptive hosts, commonly called plaque growth, reflects the dynamics of interactions between viruses and host cells. Here we treat the amplification of viruses during plaque growth as a reaction-diffusion system, where interactions among the virus, uninfected host cells, and virus-producing host-virus complexes are accounted for using rates of viral adsorption to and desorption from the host-cell surface, rates of reproduction and release of progeny viruses by lysis of the host, and by the coupling of these reactions with diffusion of free virus within the agar support. Numerical solution of the system shows the development of a traveling wave of reproducing viruses, where the velocity of the wave is governed by the kinetic and diffusion parameters. The model has been applied to predict the propagation velocity of a bacteriophage plaque. Different mechanisms may account for the dependence of this velocity on the host density during early stages of a growing plaque. The model provides a means to explore how changes in the virus-host interactions may be manifest in a growing plaque.     

Selection of Functional Variants of the NS3-NS4A Protease of Hepatitis C Virus by Using Chimeric Sindbis Viruses

The NS3-NS4A serine protease of hepatitis C virus (HCV) mediates four specific cleavages of the viral polyprotein and its activity is considered essential for the biogenesis of the HCV replication machinery. Despite extensive biochemical and structural characterization, the analysis of natural variants of this enzyme has been limited by the lack of an efficient replication system for HCV in cultured cells. We have recently described the generation of chimeric HCV-Sindbis viruses whose propagation depends on the NS3-NS4A catalytic activity. NS3-NS4A gene sequences were fused to the gene coding for the Sindbis virus structural polyprotein in such a way that processing of the chimeric polyprotein, nucleocapsid assembly, and production of infectious viruses required NS3-NS4A-mediated proteolysis (G. Filocamo, L. Pacini, and G. Migliaccio, J. Virol. 71:1417–1427, 1997). Here we report the use of these chimeric viruses to select and characterize active variants of the NS3-NS4A protease. Our original chimeric viruses displayed a temperature-sensitive phenotype and formed lysis plaques much smaller than those formed by wild-type (wt) Sindbis virus. By serially passaging these chimeric viruses on BHK cells, we have selected virus variants which formed lysis plaques larger than those produced by their progenitors and produced NS3-NS4A proteins different in size and/or sequence from those of the original viruses. Characterization of the selected protease variants revealed that all of the mutated proteases still efficiently processed the chimeric polyprotein in infected cells and also cleaved an HCV substrate in vitro. One of the selected proteases was expressed in a bacterial system and showed a catalytic efficiency comparable to that of the wt recombinant protease.     

Propagation of fluorescent viruses in growing plaques

To study virus propagation, we have developed a method by which the propagation of the Lambda bacteriophage can be observed and quantified. This is done by creating a fusion protein of the capsid protein gpD and the enhanced yellow fluorescent protein (EYFP). We show that this fusion allows capsid formation and that the modified viruses propagate on a surface covered with host bacteria thus forming fluorescent plaques. The intensity of fluorescence in a growing plaque determines the distribution of phages. This provides a new tool to study the propagation of infection at the microscopic level.     

Replication of viruses in a growing plaque: a reaction-diffusion model.

An understanding of the viral replication process commonly referred to as "plaque growth" is developed in the context of a reaction-diffusion model. The interactions among three components: the virus, the healthy host, and the infected host are represented using rates of viral adsorption and desorption to the cell surface, replication and release by host lysis, and diffusion. The solution to the full model reveals a maximum in the dependence of the velocity of viral propagation on its equilibrium adsorption constant, suggesting that conditions can be chosen where viruses which adsorb poorly to their hosts will replicate faster in plaques than those which adsorb well. Analytic expressions for the propagation velocity as a function of the kinetic and diffusion parameters are presented for the limiting cases of equilibrated adsorption, slow adsorption, fast adsorption, and large virus yields. Hindered diffusion at high host concentrations must be included for quantitative agreement with experimental data.     

泛素样蛋白ISG15抗病毒机制研究进展

ISG15(Interferon stimulated gene 15,ISG15)蛋白是由干扰素诱导产生的一种泛素样蛋白分子,分子量大小约为15kD。ISG15同泛素分子相类似可以被共价结合于其他蛋白分子上,这种现象称为ISG化(ISGylation)现象。ISG化系统包括ISG15、UBE1L、UBCH8和HERC5四类蛋白分子,协同完成ISG化过程。ISG15及ISG化系统在抗病毒反应中具有重要作用。近几年对于ISG15的抗病毒作用和机制的研究已经有了很大的突破,ISG15的抗病毒作用也越来越受到人们重视,了解清楚ISG15抗病毒机制对于研制新的抗病毒药物及提出新的抗病毒策略具有重要意义。本文对ISG15在不同种病毒中的抗病毒机制研究进展进行了简要综述。     

Combining oncolytic virotherapy and tumour vaccination

The interactions between the immune system, a malignant tumour and an oncolytic virus are complex and poorly understood. For oncolytic viruses to become successful therapeutics we need to better understand these interactions and identify strategies to take advantage of defects in the innate immune response within tumours and avoid cellular anti-viral responses while capitalizing on anti-tumoural immunity. In this review we will discuss the evidence for the induction of tumour-specific immune responses by oncolytic viruses as well as by cancer vaccines. We will then describe some of the barriers to successful cancer immunotherapy, and finally we will outline a strategy for enhancing anti-tumoural immunity while reducing anti-viral immunity by combining tumour vaccination with oncolytic viral therapy.     

Tobacco mosaic virus movement protein enhances the spread of RNA silencing

Eukaryotic cells restrain the activity of foreign genetic elements, including viruses, through RNA silencing. Although viruses encode suppressors of silencing to support their propagation, viruses may also exploit silencing to regulate host gene expression or to control the level of their accumulation and thus to reduce damage to the host. RNA silencing in plants propagates from cell to cell and systemically via a sequence-specific signal. Since the signal spreads between cells through plasmodesmata like the viruses themselves, virus-encoded plasmodesmata-manipulating movement proteins (MP) may have a central role in compatible virus:host interactions by suppressing or enhancing the spread of the signal. Here, we have addressed the propagation of GFP silencing in the presence and absence of MP and MP mutants. We show that the protein enhances the spread of silencing. Small RNA analysis indicates that MP does not enhance the silencing pathway but rather enhances the transport of the signal through plasmodesmata. The ability to enhance the spread of silencing is maintained by certain MP mutants that can move between cells but which have defects in subcellular localization and do not support the spread of viral RNA. Using MP expressing and non-expressing virus mutants with a disabled silencing suppressing function, we provide evidence indicating that viral MP contributes to anti-viral silencing during infection. Our results suggest a role of MP in controlling virus propagation in the infected host by supporting the spread of silencing signal. This activity of MP involves only a subset of its properties implicated in the spread of viral RNA.     

Interplay between the cellular autophagy machinery and positive-stranded RNA viruses

Autophagy is a conserved cellular process that acts as a key regulator in maintaining cellular homeostasis. Recent studies implicate an important role for autophagy in infection and immunity by removing invading pathogens and through modulating innate and adaptive immune responses. However, several pathogens, notably some positive-stranded RNA viruses, have subverted autophagy to their own ends. In this review, we summarize the current understanding of how viruses with a positive-stranded RNA genome interact with the host autophagy machinery to control their replication and spread. We review the mechanisms underlying the induction of autophagy and discuss the pro- and anti-viral functions of autophagy and the potential mechanisms involved.     

Plant RNAi: How a viral silencing suppressor inactivates siRNA

The three-dimensional structure of an siRNA bound to the tombusvirus p19 protein--a suppressor of gene silencing--provides a first glimpse into how plant viruses can defeat their host's anti-viral RNAi defenses.     

Autophagy during Early Virus–Host Cell Interactions

Autophagy refers to the conserved, multi-step mechanism that delivers cytosolic cargoes to vesicles of the endo-lysosomal system for degradation. It maintains cellular homeostasis by ensuring the continuous degradation of misformed/senescent intracellular components and the associated recycling of nutrients. Autophagy also represents an important cell-intrinsic defense mechanism against invasion by intracellular pathogens, including viruses. Autophagy might oppose viral invasion by targeting viral particles or viral components for degradation. It can also promote the interaction of viral constituents with receptors specialized in the activation of innate immunity pathways or facilitate the activation of anti-viral adaptive immunity. In response to such pressures, viruses have evolved various sophisticated strategies to avoid anti-viral autophagic responses or to manipulate the autophagic machinery to promote their own replication. This review focuses on our current knowledge of autophagy-related events that take place at early stages during interaction of viruses with host cells as well as on their associated consequences in terms of virus replication and cell fate.     

DNA-tumor virus entry—From plasma membrane to the nucleus

DNA-tumor viruses comprise enveloped and non-enveloped agents that cause malignancies in a large variety of cell types and tissues by interfering with cell cycle control and immortalization. Those DNA-tumor viruses that replicate in the nucleus use cellular mechanisms to transport their genome and newly synthesized viral proteins into the nucleus. This requires cytoplasmic transport and nuclear import of their genome. Agents that employ this strategy include adenoviruses, hepadnaviruses, herpesviruses, and likely also papillomaviruses, and polyomaviruses, but not poxviruses which replicate in the cytoplasm. Here, we discuss how DNA-tumor viruses enter cells, take advantage of cytoplasmic transport, and import their DNA genome through the nuclear pore complex into the nucleus. Remarkably, nuclear import of incoming genomes does not necessarily follow the same pathways used by the structural proteins of the viruses during the replication and assembly phases of the viral life cycle. Understanding the mechanisms of DNA nuclear import can identify new pathways of cell regulation and anti-viral therapies.     

siRNA, miRNA and HIV: promises and challenges

Small interfering RNA (siRNA) and microRNA (miRNA) are small RNAs of 18-25 nucleotides (nt) in length that play important roles in regulating gene expression. They are incorporated into an RNA-induced silencing complex (RISC) and serve as guides for silencing their corresponding target mRNAs based on complementary base-pairing. The promise of gene silencing has led many researchers to consider siRNA as an anti-viral tool. However, in long-term settings, many viruses appear to escape from this therapeutical strategy. An example of this may be seen in the case of human immunodeficiency virus type-1 (HIV-1) which is able to evade RNA silencing by either mutating the siRNA-targeted sequence or by encoding for a partial suppressor of RNAi (RNA interference). On the other hand, because miRNA targeting does not require absolute complementarity of base-pairing, mutational escape by viruses from miRNA-specified silencing may be more difficult to achieve. In this review, we discuss stratagems used by various viruses to avoid the cells' antiviral si/mi-RNA defenses and notions of how viruses might control and regulate host cell genes by encoding viral miRNAs (vmiRNAs).     

Strong and regulated expression of Escherichia coli beta-galactosidase in insect cells with a baculovirus vector.

The N-terminal region of the gene encoding polyhedrin, the major occlusion protein of the insect baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV), has been fused to DNA encoding Escherichia coli beta-galactosidase. The fused gene was inserted into the AcNPV DNA genome by cotransfection of insect cells with recombinant plasmid DNA and wild-type AcNPV genomic DNA. Recombinant viruses were selected as blue plaques in the presence of a beta-galactosidase indicator, 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside. Studies of one such virus, L1GP-gal3, indicated that the synthesis of beta-galactosidase is temporally controlled beginning late (20 h) in infection after the release of infectious virus particles from the cell. By 48 h postinfection, a remarkably high level of expression is achieved. On the basis of these results, AcNPV should be a useful vector for the stable propagation and expression of passenger genes in a lepidopteran cell background. A generalized transplacement vector that facilitates the construction and selection of recombinant viruses carrying passenger genes under their own promoter control has also been developed.     

Exploration of the in vitro cytotoxic and antiviral activities of different medicinal plants against infectious bursal disease (IBD) virus

Infectious bursal disease (IBD) caused by non-enveloped double stranded RNA virus is an acute and contagious poultry disease. Outbreak of IBD could result in 10–75% mortality of the birds; hence it has gained socio-economic importance worldwide. Medicinal plants have shown broad spectrum anti-viral activities against RNA and DNA viruses. Moringa oleifera Lam (MOL), Phyllanthus emblicus Linn (PEL), Glycyrrhiza glabra Linn (GGL), and Eugenia jambolana Lam (EJL) are commonly available medicinal plants of the sub-continent and exhibited anti-viral potential against different viruses. Ethanolic extracts of the leaves of MOL and EJL, roots of GGL and dried fruit of PEL were investigated for their cytotoxic and anti-viral potential against IBD virus using MTT colorimetric assay and anti-viral assay. Significant anti-viral potential (P<0.001) was demonstrated at concentrations 12.5, 25, 50 and 100 µg ml^?1 of GGL, PEL, MOL and EJL, respectively, with no cytotoxicity. Data also spotlighted that all tested plant extracts possess significant anti-viral potential and this trend was higher in GGL followed by PEL, MOL, and EJL. The data undoubtedly conclude that these medicinal plants contain several health beneficial phyto-chemicals which got significant anti-viral potential and effectively be utilized against IBD virus. Moreover, the outcomes of this study provide a platform on the way to discover novel anti-viral agents against IBD virus and other viruses from plant origin.     

Bioinformatics databases and tools in virology research: an overview

Viruses are major factors of human infectious diseases. Understanding of the structure-function correlation in viruses is important for the identification of potential anti-viral inhibitors and vaccine targets. In virology research, virus-related databases and bioinformatic analysis tools are essential for discerning relationships within complex datasets about viruses and host-virus interactions. Bioinformatic analyses on viruses include the identification of open reading frames, gene prediction, homology searching, sequence alignment, and motif and epitope recognition. The predictions of features such as transmembrane domains, glycosylation sites, and protein secondary and tertiary structure are important for analyzing the structure-function relationship of proteins encoded in viral genomes. Biochemical pathway analysis can help elucidate information at the biological systems level. Microarray analysis provides methods for high throughput screening and gene expression profiling. Virus-related bioinformatics databases include those concerned with viral sequences, taxonomy, homologous protein families, structures, or dedicated to specific viruses such as influenza and herpes simplex virus (HSV). This review provides a guide and overview of computational programs for these analyses as a resource for genomics and proteomics studies in virology research. These resources are useful for understanding viral diseases, as well as for the design and development of anti-viral agents.     

A membrane antigen of rabbit thymus cells

Rabbit cells, bearing a thymus-specific antigen, which we call rabbit thymus lymphocyte antigen (RTLA), could be detected with a suitably absorbed heterologous antiserum (goat). In the presence of complement, the RTLA antiserum lysed more than 95% of thymus cells, 70 ± 6% of lymph node cells, 46 ± 10% of spleen cells and 12 ± 7% of bone marrow cells. The number of direct or indirect hemolytic spleen plaques was not reduced by treatment with RTLA antiserum and complement, but was greatly diminished by an unabsorbed thymus antiserum which killed more than 90% of bone marrow cells. RTLA-bearing subpopulations of spleen cells were characterized by velocity sedimentation analysis and were distinguished from Ig receptor bearing subpopulations. The antiserum concentration could be so adjusted that the cytotoxicity against bone marrow was not manifested, while the cytotoxicity against other cell populations remained unchanged. The latter were identified by thymidine incorporation induced by treatment with antibody directed against rabbit light chain allotype. A small subpopulation of thymus cells did not have RTLA antigen and sedimented with a velocity distinct from that of the peak of RTLA-bearing cells.     

Virus Movements on the Plasma Membrane Support Infection and Transmission between Cells

How viruses are transmitted across the mucosal epithelia of the respiratory, digestive, or excretory tracts, and how they spread from cell to cell and cause systemic infections, is incompletely understood. Recent advances from single virus tracking experiments have revealed conserved patterns of virus movements on the plasma membrane, including diffusive motions, drifting motions depending on retrograde flow of actin filaments or actin tail formation by polymerization, and confinement to submicrometer areas. Here, we discuss how viruses take advantage of cellular mechanisms that normally drive the movements of proteins and lipids on the cell surface. A concept emerges where short periods of fast diffusive motions allow viruses to rapidly move over several micrometers. Coupling to actin flow supports directional transport of virus particles during entry and cell-cell transmission, and local confinement coincides with either nonproductive stalling or infectious endocytic uptake. These conserved features of virus–host interactions upstream of infectious entry offer new perspectives for anti-viral interference.     

Effects of schistosomes on host anti-viral immune response and the acquisition,virulence, and prevention of viral infections: A systematic review

Although a growing number of studies suggest interactions between Schistosoma parasites and viral infections, the effects of schistosome infections on the host response to viruses have not been evaluated comprehensively. In this systematic review, we investigated how schistosomes impact incidence, virulence, and prevention of viral infections in humans and animals. We also evaluated immune effects of schistosomes in those coinfected with viruses. We screened 4,730 studies and included 103. Schistosomes may increase susceptibility to some viruses, including HIV and Kaposi’s sarcoma-associated herpesvirus, and virulence of hepatitis B and C viruses. In contrast, schistosome infection may be protective in chronic HIV, Human T-cell Lymphotropic Virus-Type 1, and respiratory viruses, though further research is needed. Schistosome infections were consistently reported to impair immune responses to hepatitis B and possibly measles vaccines. Understanding the interplay between schistosomes and viruses has ramifications for anti-viral vaccination strategies and global control of viral infections.