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Virological Methods for the Generation and Characterization of Influenza Viruses with Reduced Sensitivity to 4-Guanidino-Neu5Ac2en

The compound 4-guanidino-Neu5Ac2en (zanamivir) has been described as a selective inhibitor of the influenza virus neuraminidase (NA) (1). Viruses that are less sensitive to this inhibitor can be isolated after several passages in MDCK cells in the presence of the inhibitor. Variants iso ...

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Crystallization of Macromolecules for Three-Dimensional Structure Determination

The last decade has seen a remarkable flourishing of the biological structure field. This blossoming has brought an explosion of stereochemical information, and has been made possible by the combined improvement in techniques of X-ray crystallography, nuclear magnetic resonance ( ...

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Analysis of HSV-1 Transcripts by RNA-PCR

Since the herpes simplex virus type 1 (HSV-1) genome has been sequenced and most HSV-1 RNAs are not spliced (1), detailed information about the structure of many HSV-1 RNAs can be obtained without the considerable time and effort that is required to construct and analyze cDNA libraries. Once the 5′ and 3′ e ...

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In Vitro Systems to Analyze HSV Transcript Processing

During lytic virus replication, herpes simplex virus (HSV) exhibits a closely regulated pattern of viral gene expression and of DNA replication, resulting in virion production (1). Broadly, HSV genes can be divided into immediate early, early, and late categories based on the kinetics of the ...

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Transient Assays for HSV Origin and Replication Protein Function

Investigations of genome replication in DNA viruses involve several facets. These include characterizing the sites at which synthesis is initiated (origins of DNA replication), identifying the viral and host proteins that participate, understanding the enzymatic activities ...

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HSV Amplicons in Gene Therapy

Herpes simplex virus (HSV) amplicons are defective virus vectors capable of introducing amplified foreign genes into variable types of eukaryotic cells, such as fibroblasts, macrophages, glia, and neurons in different organisms including rodents, monkeys, and human (refs. 1–3; rev ...

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HSV-Cellular Protein Interactions

The herpes simplex virus (HSV) lytic cycle is dependent on a precise temporal pattern of viral gene expression with the initial expression of the immediate-early (IE) genes, followed by the early genes, and finally late gene expression (1). Although such a temporal cascade of viral gene express ...

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Analyses of HSV Proteins for Posttranslational Modifications and Enzyme Functions

In order to identify the nature of posttranslational modifications and enzyme functions of herpes simplex virus 1 and 2 (HSV-1 and HSV-2) proteins, it is necessary to apply both biochemical and genetic analyses. The experimental methods described in this chapter have been applied to cells cu ...

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Models of Recurrent Infection with HSV in the Skin and Eye of the Mouse

Animal models remain essential for studies of many aspects of the biology of herpes simplex virus (HSV). Such studies include basic experiments on pathogenesis (including characterization of viral mutants), tests of antiviral drugs, and methods of immunization. With reference to mod ...

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HSV Growth, Preparation, and Assay

Whether herpes simplex virus (HSV) is viewed as a pathogen or as a model eukaryotic system, it is virtually certain that any experimental work will require the virus to be grown and assayed. The following chapter is therefore seen as the fundamental first step before embarking on more intellectua ...

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Pathogenesis and Molecular Biology of HSV Latency and Ocular Reactivation in the Rabbit

Research on herpesvirus infections has commanded the attention of a diverse group of scientists for over a century. Until the advent of the human immunodeficiency virus, the herpes simplex viruses (HSV) were the most intensively studied of all viruses. During the early part of the nineteenth c ...

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HSV Latency In Vitro: In Situ Hybridization Methods

We have developed an in vitro model of herpes simplex virus (HSV) latency in primary neurons that mimics many aspects of HSV latency in animal models and the human disease (1–3). Using this model, we demonstrated that HSV-1 and HSV-2 establish latent infections in vitro in the same neuronal cell types th ...

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Assays for HSV Gene Expression During Establishment and Maintenance of Latent Infection

Assays in use for the analysis of herpes simplex virus (HSV) gene expression during the establishment and maintenance phases of infection in the nervous system include: 1 The use of reporter genes, for example, the lacZ gene from Escher

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Assessing Cell-Mediated Immune Responses to HSV in Murine Systems

Protective immunity against a eajority of viral infections is mediated by a combination of both humoral and cell-mediated immune responses. However, in the case of herpesvirus infections, where viral spread is largely cell-to-cell, cell-mediated immune mechanisms (which facilit ...

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Analysis of HSV-DNA and RNA Using the Polymerase Chain Reaction

The polymerase chain reaction (PCR) technique is a sensitive method for detection of nucleic acids that can be used to detect herpes simplex virus (HSV)-DNA and RNA in tissue samples with greater sensitivity than hybridization with specific probes (1,2). In its most basic form, PCR involves mul ...

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Investigation of the Anti-HSV Activity of Candidate Antiviral Agents

Herpes simplex virus (HSV) is a human pathogen that causes diseases ranging in medical importance from herpes labialis, through genital herpes and herpes keratitis, to herpes encephalitis-a life-threatening disease. HSV types 1 and 2 have the ability to enter a latent phase during in vivo inf ...

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HSV Vectors for Gene Therapy

A number of aspects of the natural biology of herpes simplex virus (HSV) make it an attractive candidate for a vector to express foreign genes within the nervous system. Some of the advantages of an HSV vector are 1 Establishment of a l

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HSV Entry and Spread

This chapter deals with assays commonly used to follow herpes simplex virus type 1 (HSV-1) entry into and spread between cells in tissue culture These are complex processes, known to involve several of the 20 or more HSV-encoded membrane proteins (see refs. 1 and 2 for recent reviews). HSV entry is media ...

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Preparation of HSV-DNA and Production of Infectious Virus

This chapter deals with (1) the preparation of herpes simplex virus (HSV) virion DNA of a quality and purity suitable to be used for the generation of infectious virus, and (2) its use in the preparation of infectious virus. An important development in the understanding of virus genetics and gene prod ...

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Saturating Mutagenesis and Characterization of a Herpesvirus Genome Using In Vivo Reconstitution of Virus from Cloned Subgenomic Regions

The study of genome structure and gene function is pivotal in understanding the mechanisms of replication, pathogenesis, and virulence of herpesviruses. In this respect, mutagenesis and sequence analysis of genes encoded by the virus are of great importance. However, the herpesvirus g ...

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