实验基础

Studies that involve antigen processing and presentation often require de novo biosynthesis of the antigen both in vitro and in vivo. Additionally, biosynthesis of the antigen or engineered variants within the antigen-presenting cells is usually simpler than providing purified r ...

For the last 30 yr, interest in vaccinia virus immune monitoring has focused on the use of the vaccinia virus as a recombinant vaccine vector and the potential detrimental effect of antivector immunity on subsequent vaccination with a recombinant vaccinia virus. However, interest in this ar ...

Surface plasmon resonance (SPR) biosensors have become an increasingly popular technology for characterizing the protein-protein interactions of virus-host interactions. Various studies have exploited the versatility of SPR to probe the interaction between virus and host ...

The large size of poxvirus virions (approx 250 � 350 nm) makes them ideal candidates for microscopic studies. Recombinant vaccinia viruses that express a viral envelope-specific, green fluorescent protein (GFP) chimera produce enveloped virions that fluoresce green. This fluores ...

This chapter describes the methods for the study of binding and entry of the two different forms of vaccinia virus (VV)—the intracellular mature virus (IMV) and extracellular enveloped virus (EEV)—using immunofluorescent staining and confocal microscopy. After binding to or penetr ...

Cytoplasmic replication of poxviruses dictates the encoding of most, if not all, of the trans-acting factors required for faithful genome duplication. Several of these proteins have been identified through genetic and biochemical evaluation, including the catalytic DNA polyme ...

The study and use of vaccinia virus-derived RNA-modifying proteins has made a significant contribution to molecular biology. Here, the purification and assay of two such proteins, comprising the vaccinia poly(A) polymerase/cap-specific mRNA 2′-O-methyltransferase, is descri ...

This chapter describes a protocol that allows accurate in vitro transcription of vaccinia virus late genes. In this method, extracts are made from vaccinia virus-infected cells and used as enzyme sources to produce mRNAs from plasmid templates containing late gene promoter sequences.

Transcription of the vaccinia virus early genes occurs within the confines of the virion core structure. Therefore, isolated virions are a particularly rich source of proteins that function in early mRNA biosynthesis. Methods are described here for the extraction of purified vaccinia ...

Biologic and antigenic properties are often useful for identifying and differentiating orthopoxviruses (OPV). However, polymerase chain reaction (PCR) amplification, with either restriction cleavage or sequencing of amplicons, has been gaining credibility as a more rapid, ...

This chapter describes the preparation of template DNA from poxvirus-infected cells, plaques, or crude virus stocks for polymerase chain reaction (PCR) amplification. The advantages of this technique are that it is rapid, inexpensive, and, most importantly, reliable, requiring only ...

Poxviruses are cell-associated viruses that can be grown in adherent- or suspension-cell cultures or chorioallantoic membranes of embryonated hen’s eggs. The main principle of isolating the virus is to mechanically lyse infected cells. The virus can then be semipurified by centrifug ...

Modified vaccinia virus Ankara (MVA) is a valuable tool for the expression of recombinant genes used for such purposes as the study of protein functions or characterization of cellular and humoral immune responses. A major advantage of MVA is its clear safety record, and it can be handled under bio ...

Poxvirus expression vectors have gained widespread use for expression of foreign proteins and as delivery vehicles for vaccine antigens. We have developed a novel method using the poxvirus as a library vector for functional selection of specific cDNA. Poxviruses have several unique and ...

Recombinant DNA technology has made it possible to develop molecular cloning vectors that allow the expression of heterologous genes in a variety of animal viruses. This chapter discusses the use of vaccinia virus encoding bacteriophage T7 RNA polymerase as an expression vector system. A ...

Vaccinia virus (VV) has proven to be a very useful tool for the expression and analysis of foreign gene products. The most common method used to produce recombinant viruses involves the insertion of foreign genes into the thymidine kinase (TK) gene of the VV via homologous recombination. This is acc ...

The standard approach for the isolation of vaccinia virus recombinants involves homologous recombination between a transfected plasmid and the replicating viral DNA. In a typical infection/transfection experiment, recombinant viruses only account for a tiny proportion (10 ...

Vaccinia virus, the prototype Orthopoxvirus, is widely used in the laboratory as a model system to study various aspects of viral biology, virus-host interactions, and as a protein expression system and a vaccine vector. The ubiquitous use of vaccinia viruses in the laboratory raises certain ...

Human infection has been reported with groups A, B, and C rotaviruses (RVs). Of these, Group A RVs are the most important, being a major cause of severe gastroenteritis (GE). Each year, Group A RVs are estimated to cause approx 870,000 deaths worldwide in children less than 5 years (yr) of age, mostly in developi ...

Rotaviruses (RVs) are the chief etiologic agent of viral gastroenteritis in infants and young children, and in the young of a large variety of animal species. Since the discovery of RVs in man 25 yr ago, much has been learned about their genome and protein composition; their three-dimensional struc ...