Natural Spread of Plant Viruses by Birds

Observations made in Mali strongly suggest that Rice yellow mottle virus (RYMV) is spread by weaverbirds (Quelea quelea) below and around baobab trees (Adansonia digitata) in which they nest. Rice leaves in bird nests appeared to be infected. In Spain, an infection of Southern bean mosaic virus (SBMV) in string (climbing) beans (Phaseolus vulgaris) was apparently introduced and spread by sparrows (Passer domesticus) judging from the damage caused on flowers and bean pods. Damaged leaves and pods on SBMV‐infected plants were also found in a screenhouse visited by sparrows and bulbuls (Pycnonotus barbatus) in Morocco. These observations showed that both viruses could be spread by birds when either collecting infected leaves for nesting or feeding on infected plants.     

Human metapneumovirus Induces Reorganization of the Actin Cytoskeleton for Direct Cell-to-Cell Spread

Paramyxovirus spread generally involves assembly of individual viral particles which then infect target cells. We show that infection of human bronchial airway cells with human metapneumovirus (HMPV), a recently identified paramyxovirus which causes significant respiratory disease, results in formation of intercellular extensions and extensive networks of branched cell-associated filaments. Formation of these structures is dependent on actin, but not microtubule, polymerization. Interestingly, using a co-culture assay we show that conditions which block regular infection by HMPV particles, including addition of neutralizing antibodies or removal of cell surface heparan sulfate, did not prevent viral spread from infected to new target cells. In contrast, inhibition of actin polymerization or alterations to Rho GTPase signaling pathways significantly decreased cell-to-cell spread. Furthermore, viral proteins and viral RNA were detected in intercellular extensions, suggesting direct transfer of viral genetic material to new target cells. While roles for paramyxovirus matrix and fusion proteins in membrane deformation have been previously demonstrated, we show that the HMPV phosphoprotein extensively co-localized with actin and induced formation of cellular extensions when transiently expressed, supporting a new model in which a paramyxovirus phosphoprotein is a key player in assembly and spread. Our results reveal a novel mechanism for HMPV direct cell-to-cell spread and provide insights into dissemination of respiratory viruses.     

The Ins and Outs of Nondestructive Cell-to-Cell and Systemic Movement of Plant Viruses

Propagation of viral infection in host plants comprises two distinct and sequential stages: viral transport from the initially infected cell into adjacent neighboring cells, a process termed local or cell-to-cell movement, and a chain of events collectively referred to as systemic movement that consists of entry into the vascular tissue, systemic distribution with the phloem stream, and unloading of the virus into noninfected tissues. To achieve intercellular transport, viruses exploit plasmodesmata, complex cytoplasmic bridges interconnecting plant cells. Viral transport through plasmodesmata is aided by virus-encoded proteins, the movement proteins (MPs), which function by two distinct mechanisms: MPs either bind viral nucleic acids and mediate passage of the resulting movement complexes (M-complexes) between cells, or MPs become a part of pathogenic tubules that penetrate through host cell walls and serve as conduits for transport of viral particles. In the first mechanism, M-complexes pass into neighboring cells without destroying or irreversibly altering plasmodesmata, whereas in the second mechanism plasmodesmata are replaced or significantly modified by the tubules. Here we summarize the current knowledge on both local and systemic movement of viruses that progress from cell to cell as M-complexes in a nondestructive fashion. For local movement, we focus mainly on movement functions of the 30 K superfamily viruses, which encode MPs with structural homology to the 30 kDa MP of Tobacco mosaic virus, one of the most extensively studied plant viruses, whereas systemic movement is primarily described for two well-characterized model systems, Tobacco mosaic virus and Tobacco etch potyvirus. Because local and systemic movement are intimately linked to the molecular infrastructure of the host cell, special emphasis is placed on host factors and cellular structures involved in viral transport.     

A Novel Human Cytomegalovirus Glycoprotein, gpUS9, Which Promotes Cell-to-Cell Spread in Polarized Epithelial Cells, Colocalizes with the Cytoskeletal Proteins E-Cadherin and F-Actin

Processes by which human herpesviruses penetrate and are released from polarized epithelial cells, which have distinct apical and basolateral membrane domains differing in protein and lipid content, are poorly understood. We recently reported that human cytomegalovirus (CMV) mutants with deletions of the gene US9 formed wild-type plaques in cultures of human fibroblasts but were impaired in the capacity for cell-to-cell spread in polarized human retinal pigment epithelial cells. Unlike the glycoproteins that are required for infection, the protein encoded by CMV US9 plays an accessory role by promoting dissemination of virus across cell-cell junctions of polarized epithelial cells. To identify the product and investigate its specialized functions, we selected Madine-Darby canine kidney II (MDCK) epithelial cells that constitutively express CMV US9 or, as a control, US8. The gene products, designated gpUS9 and gpUS8, were glycosylated proteins of comparable molecular masses but differed considerably in intracellular distribution and solubility. Immunofluorescence laser scanning confocal microscopy indicated that, like gpUS8, gpUS9 was present in the endoplasmic reticulum and Golgi compartments of nonpolarized cells. In polarized epithelial cells, gpUS9 also accumulated along lateral membranes, colocalizing with cadherin and actin, and was insoluble in Triton X-100, a property shared with proteins that associate with the cytoskeleton. We hypothesize that gpUS9 may enhance the dissemination of CMV in infected epithelial tissues by associating with the cytoskeletal matrix.     

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.     

Hsf1 Phosphorylation Generates Cell-to-Cell Variation in Hsp90 Levels and Promotes Phenotypic Plasticity

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Increase in Plasmodesmatal Permeability during Cell-to-Cell Spread of Tobacco Rattle Virus from Individually Inoculated Cells

A microinjection technique was devised for inoculation of single Nicotiana clevelandii leaf trichome cells with virus particles. By removing inoculated trichomes at various times after microinjection, it was shown that at least 4 hr were required for tobacco rattle virus (TRV; tobravirus group) to move out of primarily inoculated cells. Effects of the early stages of TRV infection on plasmodesmatal permeability were examined by microinjection of fluorochrome-labeled molecules. Fluorescein-labeled insulin A chain (Mr, 2921) and fluorescein-labeled dextran (Mr, 4400) were observed to pass out of individual N. clevelandii trichome cells that had been inoculated with TRV by microinjection 5 hr previously. By contrast, Lucifer Yellow CH-labeled dextran (Mr, 10,000) was restricted to the inoculated cell. None of these fluorescent probes were able to move out of uninoculated cells or out of cells that had been inoculated with TRV only 2 hr previously. The movement of macromolecules through plasmodesmata, therefore, coincided with and probably resulted from cell-to-cell movement of TRV. The results are discussed with reference to the interaction of viruses and plasmodesmata and mechanisms of intercellular virus movement.     

Animal Viruses Probe Dataset (AVPDS) for Microarray-Based Diagnosis and Identification of Viruses

AVPDS (Animal Viruses Probe dataset) is a dataset of virus-specific and conserve oligonucleotides for identification and diagnosis of viruses infecting animals. The current dataset contain 20,619 virus specific probes for 833 viruses and their subtypes and 3,988 conserved probes for 146 viral genera. Dataset of virus specific probe has been divided into two fields namely virus name and probe sequence. Similarly conserved probes for virus genera table have genus, and subgroup within genus name and probe sequence. The subgroup within genus is artificially divided subgroups with no taxonomic significance and contains probes which identifies viruses in that specific subgroup of the genus. Using this dataset we have successfully diagnosed the first case of Newcastle disease virus in sheep and reported a mixed infection of Bovine viral diarrhea and Bovine herpesvirus in cattle. These dataset also contains probes which cross reacts across species experimentally though computationally they meet specifications. These probes have been marked. We hope that this dataset will be useful in microarray-based detection of viruses. The dataset can be accessed through the link https://dl.dropboxusercontent.com/u/94060831/avpds/HOME.html.     

The Herpes Simplex Virus gE-gI Complex Facilitates Cell-to-Cell Spread and Binds to Components of Cell Junctions

The herpes simplex virus (HSV) glycoprotein complex gE-gI mediates the spread of viruses between adjacent cells, and this property is especially evident for cells that form extensive cell junctions, e.g., epithelial cells, fibroblasts, and neurons. Mutants lacking gE or gI are not compromised in their ability to enter cells as extracellular viruses. Therefore, gE-gI functions specifically in the movement of virus across cell-cell contacts and, as such, provides a molecular handle on this poorly understood process. We expressed gE-gI in human epithelial cells by using replication-defective adenovirus (Ad) vectors. gE-gI accumulated at lateral surfaces of the epithelial cells, colocalizing with the adherens junction protein β-catenin but was not found on either the apical or basal plasma membranes and did not colocalize with ZO-1, a component of tight junctions. In subconfluent monolayers, gE-gI was found at cell junctions but was absent from those lateral surfaces not in contact with another cell, as was the case for β-catenin. Similar localization of gE-gI to cell junctions was observed in HSV-infected epithelial cells. By contrast, HSV glycoprotein gD, expressed using a recombinant Ad vectors, was found primarily along the apical surfaces of cells, with little or no protein found on the basal or lateral surfaces. Expression of gE-gI without other HSV polypeptides did not cause redistribution of either ZO-1 or β-catenin or alter tight-junction functions. Together these results support a model in which gE-gI accumulates at sites of cell-cell contact by interacting with junctional components. We hypothesize that gE-gI mediates transfer of HSV across cell junctions by virtue of these interactions with cell junction components.     

Mechanisms of Arthropod Transmission of Plant and Animal Viruses

A majority of the plant-infecting viruses and many of the animal-infecting viruses are dependent upon arthropod vectors for transmission between hosts and/or as alternative hosts. The viruses have evolved specific associations with their vectors, and we are beginning to understand the underlying mechanisms that regulate the virus transmission process. A majority of plant viruses are carried on the cuticle lining of a vector’s mouthparts or foregut. This initially appeared to be simple mechanical contamination, but it is now known to be a biologically complex interaction between specific virus proteins and as yet unidentified vector cuticle-associated compounds. Numerous other plant viruses and the majority of animal viruses are carried within the body of the vector. These viruses have evolved specific mechanisms to enable them to be transported through multiple tissues and to evade vector defenses. In response, vector species have evolved so that not all individuals within a species are susceptible to virus infection or can serve as a competent vector. Not only are the virus components of the transmission process being identified, but also the genetic and physiological components of the vectors which determine their ability to be used successfully by the virus are being elucidated. The mechanisms of arthropod-virus associations are many and complex, but common themes are beginning to emerge which may allow the development of novel strategies to ultimately control epidemics caused by arthropod-borne viruses.     

Rho-ROCK and Rac-PAK Signaling Pathways Have Opposing Effects on the Cell-to-Cell Spread of Marek's Disease Virus

Marek's Disease Virus (MDV) is an avian alpha-herpesvirus that only spreads from cell-to-cell in cell culture. While its cell-to-cell spread has been shown to be dependent on actin filament dynamics, the mechanisms regulating this spread remain largely unknown. Using a recombinant BAC20 virus expressing an EGFPVP22 tegument protein, we found that the actin cytoskeleton arrangements and cell-cell contacts differ in the center and periphery of MDV infection plaques, with cells in the latter areas showing stress fibers and rare cellular projections. Using specific inhibitors and activators, we determined that Rho-ROCK pathway, known to regulate stress fiber formation, and Rac-PAK, known to promote lamellipodia formation and destabilize stress fibers, had strong contrasting effects on MDV cell-to-cell spread in primary chicken embryo skin cells (CESCs). Inhibition of Rho and its ROCKs effectors led to reduced plaque sizes whereas inhibition of Rac or its group I-PAKs effectors had the adverse effect. Importantly, we observed that the shape of MDV plaques is related to the semi-ordered arrangement of the elongated cells, at the monolayer level in the vicinity of the plaques. Inhibition of Rho-ROCK signaling also resulted in a perturbation of the cell arrangement and a rounding of plaques. These opposing effects of Rho and Rac pathways in MDV cell-to-cell spread were validated for two parental MDV recombinant viruses with different ex vivo spread efficiencies. Finally, we demonstrated that Rho/Rac pathways have opposing effects on the accumulation of N-cadherin at cell-cell contact regions between CESCs, and defined these contacts as adherens junctions. Considering the importance of adherens junctions in HSV-1 cell-to-cell spread in some cell types, this result makes of adherens junctions maintenance one potential and attractive hypothesis to explain the Rho/Rac effects on MDV cell-to-cell spread. Our study provides the first evidence that MDV cell-to-cell spread is regulated by Rho/Rac signaling.     

Impact of Directed Movement on Invasive Spread in Periodic Patchy Environments

To address the effect of taxis of invasive animals on their spreading speed in heterogeneous environments, we deal with an advection-diffusion-reaction equation (ADR) in a periodic patchy environment. Two-types of advection that spatially vary depending on environmental heterogeneity are taken into consideration: a stepwise taxis function and a saw-like taxis function. We first analyze the ADR with the stepwise taxis advection, and derive an invasion criterion. When the invasion criterion holds, an initially localized population evolves to a traveling periodic wave (TPW). The asymptotic speed of the TPW is found to be equal to the minimal speed of the TPW analytically derived. Thus, we examine how the minimal speed is influenced by the taxis. The major results are: (1)?As the magnitude of the taxis toward favorable patches increases, invasion becomes more feasible. However, the spreading speed increases at first, and then decreases to show a one-humped curve against the magnitude of the taxis; (2)?As the scale of fragmentation in the patchy environment is increased, the spreading speed increases when the magnitude of the taxis is small, while it decreases when the magnitude of the taxis becomes sufficiently large. These characteristic features qualitatively apply to the ADR model with the saw-like taxis function.     

动物病毒传染性克隆技术研究进展

综述了6种动物病毒传染性克隆技术:重组质粒转染子挽救系统,PolI PoⅡ转录挽救系统,Bac操作系统,Cosmid操作系统,YAC操作系统以及Bacmid操作系统;涵盖了动物RNA病毒,DNA病毒以及昆虫杆状病毒;比较了各系统的优缺点和适应范围,指出了动物传染性克隆技术的应用前景.     

Detection of Animal Viruses in Coastal Seawater and Sediments

Animal viruses, predominantly enteroviruses, were detected in shallow water at bottom depths and in clastic marine sediments. Viruses accumulated in sandy and slimy deposits of the sea bottom near the shore and could be easily released into water by means of simple mechanical shaking.     

Acquired Resistance in Hypersensitive Tobacco Operates by Limiting Cell-to-Cell Spread of Virus Infection without Affecting Virus Multiplication

Localized and systemic acquired resistance against tobacco mosaic virus (TMV) or tobacco necrosis virus was induced when local lesions were produced in leaves of Xanthi-nc tobacco by mechanical inoculation with TMV. Both types of resistance were characterized by reduction in the size of lesions produced by the challenging viruses, whereas accumulation of viral antigen in lesions was slightly increased. These results, confirming previous findings relative to other hypersensitive plant virus combinations, do not support the view that an inhibitor of virus replication operates in the resistant tissues, but indicate that both types of resistance operate only against cell-to-cell spread of virus.