Springer protocols feed by Infectious Diseases 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Immunohistochemical Staining of Influenza Virus in Tissues
Immunohistochemical methods are commonly used for studying the pathogenesis of influenza A virus by allowing the identification of sites of replication of the virus in infected tissues and the correlation with the histopathological changes observed. In this chapter, the materials and methods for performing immunohistochemical detection of influenza virus antigens in tissues are provided. The technique involves the following steps: heat-induced antigen retrieval; binding of a primary antibody to the virus antigen; antibody–antigen complex binding by a biotinylated secondary antibody; and binding of an enzyme–str...
Source: Springer protocols feed by Infectious Diseases - June 6, 2014 Category: Infectious Diseases Source Type: news
Reverse Genetics of Influenza Virus
Reverse genetics is the creation of a virus from a full-length cDNA copy of the viral genome, referred to as an “infectious clone,” and is one of the most powerful genetic tools in modern virology. Since its development in 1999, plasmid-based reverse genetics has been effectively applied to numerous aspects of influenza studies which include revolutionizing the production of seasonal and pandemic influenza vaccine seed strains. Although continual improvement in reverse genetics system is being made in different laboratories for the efficient rescue of the influenza virus, the basic concept of synthesizing viral...
Source: Springer protocols feed by Infectious Diseases - June 6, 2014 Category: Infectious Diseases Source Type: news
Neuraminidase-Inhibition Assay for the Identification of Influenza A Virus Neuraminidase Virus Subtype or Neuraminidase Antibody Specificity
The neuraminidase-inhibition (NI) assay is a laboratory procedure for the identification of the neuraminidase (NA) glycoprotein subtype in influenza viruses or the NA subtype specificity of antibodies to influenza virus. A serological procedure for subtyping the NA glycoprotein is critical for the identification and classification of avian influenza (AI) viruses. The macroprocedure was first described in 1961 by Aminoff and was later modified to a microtiter plate procedure (micro-NI) by Van Deusen et al. (Avian Dis 27:745–750, 1983). The micro-NI procedure reduces the quantity of reagents required, permits the antig...
Source: Springer protocols feed by Infectious Diseases - June 6, 2014 Category: Infectious Diseases Source Type: news
Hemagglutination-Inhibition Assay for Influenza Virus Subtype Identification and the Detection and Quantitation of Serum Antibodies to Influenza Virus
Hemagglutination-inhibition (HI) assay is a classical laboratory procedure for the classification or subtyping of hemagglutinating viruses. For influenza virus, HI assay is used to identify the hemagglutinin (HA) subtype of an unknown isolate or the HA subtype specificity of antibodies to influenza virus. Since the HI assay is quantitative it is frequently applied to evaluate the antigenic relationships between different influenza virus isolates of the same subtype. The basis of the HI test is inhibition of hemagglutination with subtype-specific antibodies. The HI assay is a relatively inexpensive procedure utilizing stand...
Source: Springer protocols feed by Infectious Diseases - June 6, 2014 Category: Infectious Diseases Source Type: news
Hemagglutination Assay for Influenza Virus
The hemagglutination assay (HA) is a tool used to screen cell culture isolates or amnioallantoic fluid harvested from embryonated chicken eggs for hemagglutinating agents, such as type A influenza. The HA assay is not an identification assay, as other agents also have hemagglutinating properties. Live and inactivated viruses are detected by the HA test. Amplification by virus isolation in embryonated chicken eggs or cell culture is typically required before HA activity can be detected from a clinical sample. The test is, to some extent, quantitative as 1 hemagglutinating unit (HAU) is equal to approximately 5–6 logs ...
Source: Springer protocols feed by Infectious Diseases - June 6, 2014 Category: Infectious Diseases Source Type: news
Herpes Simplex Virus Mutant Generation and Dual-Detection Methods for Gaining Insight into Latent/Lytic Cycles In Vivo
Two important components to a useful strategy to examine viral gene regulation in vivo are (1) a highly efficient protocol to generate viral mutants that limits undesired mutation and retains full replication competency in vivo and (2) an efficient system to detect and quantify viral promoter activity in rare cells in vivo. Our strategy and protocols for generating, characterizing, and employing HSV viral promoter/reporter mutants in vivo are provided in this two-part chapter. (Source: Springer protocols feed by Infectious Diseases)
Source: Springer protocols feed by Infectious Diseases - April 3, 2014 Category: Infectious Diseases Source Type: news
Modification of HSV-1 to an Oncolytic Virus
Cancer-permissive viruses or oncolytic viruses consist of either genetically engineered or naturally occurring strains that possess relatively selective replicative and/or infection abilities for cancer vs. normal cells (Chiocca, Nat Rev Cancer 2: 938–950, 2002). They can also be armed with additional anticancer cDNAs (e.g., cytokines, prodrug-activating, anti-angiogenesis genes, and others) to extend therapeutic effects (Kaur et al., Curr Gene Ther 9: 341–355, 2009). Herpes simplex virus type 1 (HSV-1) possesses several advantages as an oncolytic virus such as a rapid lytic cycle and a large capacity for inser...
Source: Springer protocols feed by Infectious Diseases - April 3, 2014 Category: Infectious Diseases Source Type: news
HSV-1 Amplicon Vectors as Genetic Vaccines
HSV-1 amplicon vectors have been used as platforms for the generation of genetic vaccines against both DNA and RNA viruses. Mice vaccinated with such vectors encoding structural proteins from both foot-and-mouth disease virus and rotavirus were partially protected from challenge with wild-type virus (D’Antuono et al. Vaccine 28: 7363–7372, 2010; Laimbacher et al. Mol Ther 20: 1810–1820, 2012), indicating that HSV-1 amplicon vectors are attractive tools for the development of complex and safe genetic vaccines. This chapter describes the use of HSV-1 amplicon vectors that encode individual or multiple viral...
Source: Springer protocols feed by Infectious Diseases - April 3, 2014 Category: Infectious Diseases Source Type: news
Herpes Simplex Virus Type 1 (HSV-1)-Derived Amplicon Vectors
Amplicons are defective, helper-dependent, herpes simplex virus type 1 (HSV-1)-derived vectors. The main interest of these vectors as gene transfer tools stems from the fact that the amplicon vector genomes do not carry protein-encoding viral sequences. Consequently, they are completely safe for the host and nontoxic for the infected cells. Moreover, the complete absence of virus genes provides space to accommodate very large foreign DNA sequences, up to almost 150-kb, the size of the virus genome. This large transgene capacity can be used to deliver complete gene loci, including introns and exons, as well as long regulato...
Source: Springer protocols feed by Infectious Diseases - April 3, 2014 Category: Infectious Diseases Source Type: news
Engineering HSV-1 Vectors for Gene Therapy
Virus vectors have been employed as gene transfer vehicles for various preclinical and clinical gene therapy applications, and with the approval of Glybera (alipogene tiparvovec) as the first gene therapy product as a standard medical treatment (Yla-Herttuala, Mol Ther 20: 1831–1832, 2013), gene therapy has reached the status of being a part of standard patient care. Replication-competent herpes simplex virus (HSV) vectors that replicate specifically in actively dividing tumor cells have been used in Phase I–III human trials in patients with glioblastoma multiforme, a fatal form of brain cancer, and in malignan...
Source: Springer protocols feed by Infectious Diseases - April 3, 2014 Category: Infectious Diseases Source Type: news
Construction and Characterization of Bacterial Artificial Chromosomes (BACs) Containing Herpes Simplex Virus Full-Length Genomes
Bacterial artificial chromosomes (BACs) are suitable vectors not only to maintain the large genomes of herpesviruses in Escherichia coli but also to enable the traceless introduction of any mutation using modern tools of bacterial genetics. To clone a herpes simplex virus genome, a BAC replication origin is first introduced into the viral genome by homologous recombination in eukaryotic host cells. As part of their nuclear replication cycle, genomes of herpesviruses circularize and these replication intermediates are then used to transform bacteria. After cloning, the integrity of the recombinant viral genomes is confirmed...
Source: Springer protocols feed by Infectious Diseases - April 3, 2014 Category: Infectious Diseases Source Type: news
Isolation of Herpes Simplex Virus Nucleocapsid DNA
As an inanimate virus, herpes simplex virus type 1 (HSV-1) necessarily encodes all of its functions in its DNA. Isolation of pure viral DNA allows multiple downstream applications, including the creation of recombinant HSV strains, cloning of selected regions, and sequencing of viral DNA. The term nucleocapsid refers to the combination of the viral genome with the enclosing capsid; these viral genomes are necessarily linear and have been packaged for egress, even if they are not yet released from the cell. In contrast, viral DNA that is not associated with capsids may include episomal or concatenated forms and may have mod...
Source: Springer protocols feed by Infectious Diseases - April 3, 2014 Category: Infectious Diseases Source Type: news
Herpes Simplex Virus Growth, Preparation, and Assay
In order to study the biology of herpes simplex virus or to use it as a vector in gene therapy, it is necessary to grow the virus and to prepare virus stocks. Many different protocols are available from different research groups working with herpes simplex virus type 1 or 2 (HSV-1 or HSV-2). This chapter describes the procedures used in our laboratory. (Source: Springer protocols feed by Infectious Diseases)
Source: Springer protocols feed by Infectious Diseases - April 3, 2014 Category: Infectious Diseases Source Type: news
The Murine Intravaginal HSV-2 Challenge Model for Investigation of DNA Vaccines
DNA vaccines have been licensed in veterinary medicine and have promise for humans. This format is relatively immunogenic in mice and guinea pigs, the two principle HSV-2 animal models, permitting rapid assessment of vectors, antigens, adjuvants, and delivery systems. Limitations include the relatively poor immunogenicity of naked DNA in humans and the profound differences in HSV-2 pathogenesis between host species. Herein, we detail lessons learned over the last few years investigating candidate DNA vaccines in the progesterone-primed female mouse vaginal model of HSV-2 infection as a guide to investigators in the field. ...
Source: Springer protocols feed by Infectious Diseases - April 3, 2014 Category: Infectious Diseases Source Type: news
HSV-1 Protein Expression Using Recombinant Baculoviruses
The baculovirus expression system is an invaluable method for the expression of Herpes Simplex Virus 1 (HSV-1) proteins. The use of insect cells provides a eukaryotic system for the robust expression of heterologous proteins under control of the baculovirus polyhedrin gene promoter that naturally drives the high expression of the polyhedrin protein. Additionally, insect cells often initiate the necessary posttranslational modifications and/or disulfide-bond formation important for the proper folding of the protein. We and others have successfully expressed and purified several HSV-1 proteins including the polymerase, helic...
Source: Springer protocols feed by Infectious Diseases - April 3, 2014 Category: Infectious Diseases Source Type: news
