Current protocols in microbiology

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ISSN 1934-8533

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    ABSTRACT: Human papillomaviruses (HPV) infect skin or mucosal epidermis. The simplistic capsid consists of a major capsid protein L1, a minor capsid protein L2, and a double-stranded circular DNA of about 8kB in size. The development of HPV-based vectors [i.e., pseudovirions (PsV)] as tools to study the initial infection has facilitated our understanding of HPV entry. The covalent coupling of fluorescent molecules to these PsV allows following the viruses en route to the nucleus, i.e., the site of replication. In the first section, we describe a facile method to covalently label HPV PsV that retain their infectivity. In this method, fluorophores coupled to a reactive succinimidyl ester are covalently attached to amine residues in the virion in a one-step chemical reaction. In the second section of this unit, several assays are outlined that use the fluorescently labeled virions for entry studies in live and fixed cells. © 2015 by John Wiley & Sons, Inc.
    Current protocols in microbiology 09/2015; 37:14B.4.1-14B.4.22. DOI:10.1002/9780471729259.mc14b04s37
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    ABSTRACT: The vast majority of surgical biopsy and post-mortem tissue samples are formalin-fixed and paraffin-embedded (FFPE), but this process leads to RNA degradation that limits gene expression analysis. As an example, the viral RNA genome of the 1918 pandemic influenza A virus was previously determined in a 9-year effort by overlapping RT-PCR from post-mortem samples. Using the protocols described here, the full genome of the 1918 virus was determined at high coverage in one high-throughput sequencing run of a cDNA library derived from total RNA of a 1918 FFPE sample after duplex-specific nuclease treatments. This basic methodological approach should assist in the analysis of FFPE tissue samples isolated over the past century from a variety of infectious diseases. © 2015 by John Wiley & Sons, Inc.
    Current protocols in microbiology 09/2015; 33:1E.8.1-1E.8.16. DOI:10.1002/9780471729259.mc01e08s37
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    ABSTRACT: The prognosis from thyroid cancer subtypes in humans covers a spectrum from "cured at almost 90%" to "100% lethal." Invasive and poorly differentiated forms of thyroid cancer are among the most aggressive human cancers, and there are few effective therapeutic options. Genetically engineered mice, based on mutations observed in patients, can accurately recapitulate the human disease and its progression, providing invaluable tools for the preclinical evaluation of novel therapeutic approaches. This overview details models developed to date as well as their uses for identifying novel anticancer agents. © 2015 by John Wiley & Sons, Inc.
    Current protocols in microbiology 09/2015; 33:14.33.1-14.33.14. DOI:10.1002/0471141755.ph1433s69
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    ABSTRACT: Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging highly pathogenic respiratory virus. Although MERS-CoV only emerged in 2012, we and others have developed assays to grow and quantify infectious MERS-CoV and RNA products of replication in vitro. MERS-CoV is able to infect a range of cell types, but replicates to high titers in Vero E6 cells. Protocols for the propagation and quantification of MERS-CoV are presented. © 2015 by John Wiley & Sons, Inc.
    Current protocols in microbiology 09/2015; 37:15E.2.1-15E.2.9. DOI:10.1002/9780471729259.mc15e02s37
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    ABSTRACT: Epichloë species (including former Neotyphodium species) are endophytic fungi that significantly affect fitness of cool-season grass hosts, potentially by increasing nutrient uptake and resistance to drought, parasitism and herbivory. Epichloë species are obligately biotrophic, living in the intercellular spaces of their plant hosts, and spreading systemically throughout host aerial tissues. The reproduction of Epichloë species is versatile; some strains have both sexual and asexual modes of reproduction, but others are restricted to one or the other mode. The reproduction mode determines the dissemination mechanism, and the asexual species most important to agriculture are strictly seed-borne, cause no signs or symptoms, and are undetectable except by specialized microscopic, molecular or antigenic procedures. These procedures can be used to identify endophytes in a variety of plant tissues. Similar protocols can be modified to detect less common symbionts, such as the penicillate "p-endophytes," when they occur by themselves or together with Epichloë species. © 2015 by John Wiley & Sons, Inc.
    Current protocols in microbiology 08/2015; 38:19A.1.1-19A.1.24. DOI:10.1002/9780471729259.mc19a01s38
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    ABSTRACT: Merkel cell polyomavirus (MCPyV) genomes are clonally integrated in tumor cells of ∼95% of all Merkel cell carcinoma (MCC) cases. The virus is highly prevalent; however, where the virus persists and which cell types are permissive for MCPyV replication is still unknown. As a consequence, very little information is available about the life cycle and no fully permissive in vitro replication system has been established. Recently, semi-permissive replication systems based on wild-type MCPyV genomes recovered from the skin of healthy donors or synthetic MCPyV genomes constructed from consensus sequences have been established. The transfection of this intramolecular re-circularized MCPyV DNA into some human cell lines recapitulates efficient DNA replication of the viral genome, viral gene expression as well as moderate levels of virus particle formation. However, serial transmission of infectious virus is still restricted in these cells. © 2015 by John Wiley & Sons, Inc.
    Current protocols in microbiology 08/2015; 38:14F.2.1-14F.2.19. DOI:10.1002/9780471729259.mc14f02s38
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    ABSTRACT: Immunization with Human Papillomavirus (HPV) L1 virus-like particles or L2 capsid protein elicits neutralizing antibodies that mediate protection. A high-throughput and sensitive in vitro neutralization assay is therefore valuable for prophylactic HPV vaccine studies. Over several hours during infection of the genital tract, virions take on a distinct intermediate conformation, including a required furin cleavage of L2 at its N-terminus. This intermediate is an important target for neutralization by L2-specific antibody, but it is very transiently exposed during in vitro infection of most cell lines resulting in insensitive measurement for L2, but not L1-specific neutralizing antibodies. To model this intermediate, we describe a protocol to generate furin-cleaved HPV pseudovirions (fc-PsV), which deliver an encapsidated reporter plasmid to facilitate infectivity measurements. We also describe a protocol for use of fc-PsV in a high-throughput in vitro neutralization assay for the sensitive measurement of both L1 and L2-specific neutralizing antibodies. © 2015 by John Wiley & Sons, Inc.
    Current protocols in microbiology 08/2015; 38:14B.5.1-14B.5.26. DOI:10.1002/9780471729259.mc14b05s38
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    ABSTRACT: Vibrio cholerae is the agent of cholera, a potentially lethal diarrheal disease that remains a significant threat to populations in developing nations. The infant rabbit model of cholera is the only non-surgical small animal model system that closely mimics human cholera. Following orogastric inoculation, V. cholerae colonizes the intestines of infant rabbits, and the animals develop severe cholera-like diarrhea. In this unit, we provide a detailed description of the preparation of the V. cholerae inoculum, the inoculation process and the collection and processing of tissue samples. This infection model is useful for studies of V. cholerae factors and mechanisms that promote its intestinal colonization and enterotoxicity, as well as the host response to infection. The infant rabbit model of cholera enables investigations that will further our understanding of the pathophysiology of cholera and provides a platform for testing new therapeutics. © 2015 by John Wiley & Sons, Inc.
    Current protocols in microbiology 08/2015; 38:6A.6.1-6A.6.15. DOI:10.1002/9780471729259.mc06a06s38
  • Current protocols in microbiology 05/2015; 37. DOI:10.1002/9780471729259.mc15c06s37
  • Current protocols in microbiology 05/2015; 6F. 2.1-6F. 2.14. DOI:10.1002/9780471729259.mc06f02s37
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    ABSTRACT: There is extensive genomic diversity among Streptococcus pneumoniae isolates. Approximately half of the comprehensive set of genes in the species (the supragenome or pangenome) is present in all the isolates (core set), and the remaining is unevenly distributed among strains (distributed set). The Streptococcus pneumoniae Supragenome Hybridization (SpSGH) array provides coverage for an extensive set of genes and polymorphisms encountered within this species, capturing this genomic diversity. Further, the capture is quantitative. In this manner, the SpSGH array allows for both genomic and transcriptomic analyses of diverse S. pneumoniae isolates on a single platform. In this unit, we present the SpSGH array, and describe in detail its design and implementation for both genomic and transcriptomic analyses. The methodology can be applied to construction and modification of SpSGH array platforms, as well to other bacterial species as long as multiple whole-genome sequences are available that collectively capture the vast majority of the species supragenome. © 2015 by John Wiley & Sons, Inc. Copyright © 2015 John Wiley & Sons, Inc.
    Current protocols in microbiology 02/2015; 36:9D.4.1-9D.4.20. DOI:10.1002/9780471729259.mc09d04s36
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    ABSTRACT: Vaccination has a proven record as one of the most effective medical approaches to prevent the spread of infectious diseases. Traditional vaccine approaches involve the administration of whole killed or weakened microorganisms to stimulate protective immune responses. Such approaches deliver many microbial components, some of which contribute to protective immunity, and assist in guiding the type of immune response that is elicited. Despite their impeccable record, these approaches have failed to yield vaccines for many important infectious organisms. This has prompted a move towards more defined vaccines ('subunit vaccines'), where individual protective components are administered. This unit provides an overview of the components that are used for the development of modern vaccines including: an introduction to different vaccine types (whole organism, protein/peptide, polysaccharide, conjugate, and DNA vaccines); techniques for identifying subunit antigens; vaccine delivery systems; and immunostimulatory agents ('adjuvants'), which are fundamental for the development of effective subunit vaccines. © 2015 by John Wiley & Sons, Inc. Copyright © 2015 John Wiley & Sons, Inc.
    Current Protocols in Microbiology, Edited by Richard Coico, 02/2015: chapter UNIT 18.1 Progress in vaccine development.: pages 18.1.1-18.1.26; John Wiley & Sons., ISBN: 9780471729259
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    ABSTRACT: Bacterial persisters are cells with an impressive, yet transient, tolerance toward extraordinary concentrations of antibiotics. Persisters are believed to impose a significant burden on the healthcare system because of their role in the proclivity of infections to relapse. During antibiotic challenge, these rare, phenotypic variants enter a dormant state where antibiotic primary targets are rendered inactive, allowing them to survive. Once the antibiotic is removed, persisters reawaken and resume growth, leading to repopulation of the environment. Metabolism plays a pivotal role in coordinating the entry, maintenance, and exit from the persister state. However, the low abundance, transient nature, and similarity of persisters to other cell types have prevented their isolation, which is needed for direct metabolic measurements. In this unit, we describe a technique known as the aminoglycoside (AG) potentiation assay, which can be used to rapidly and specifically measure the breadth of persister metabolism in heterogeneous populations. © 2015 by John Wiley & Sons, Inc. Copyright © 2015 John Wiley & Sons, Inc.
    Current protocols in microbiology 02/2015; 36:17.9.1-17.9.14. DOI:10.1002/9780471729259.mc1709s36
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    ABSTRACT: The lagging annotation of bacterial genomes and the inherent genetic complexity of many phenotypes is hindering the discovery of new drug targets and the development of new antimicrobial agents and vaccines. This unit presents Tn-seq, a method that has made it possible to quantitatively determine fitness for most genes in a microorganism and to screen for quantitative genetic interactions on a genome-wide scale and in a high-throughput fashion. Tn-seq can thus direct studies on the annotation of genes and untangle complex phenotypes. The method is based on the construction of a saturated transposon insertion library. After library selection, changes in the frequency of each insertion mutant are determined by sequencing flanking regions en masse. These changes are used to calculate each mutant's fitness. The method was originally developed for the Gram-positive bacterium Streptococcus pneumoniae, a causative agent of pneumonia and meningitis, but has now been applied to several different microbial species. © 2015 by John Wiley & Sons, Inc.
    Current protocols in microbiology 02/2015; 36:1E.3.1-1E.3.24. DOI:10.1002/9780471729259.mc01e03s36
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    ABSTRACT: Many bacteria can become naturally competent to take up extracellular DNA across their outer and inner membranes by a dedicated competence apparatus. Whereas some studies show that the DNA delivered to the cytoplasm may be used for genome repair or for nutrition, it can also be recombined onto the chromosome by homologous recombination: a process called natural transformation. Along with conjugation and transduction, natural transformation represents a mechanism for horizontal transfer of genetic material, e.g., antibiotic resistance genes, which can confer new beneficial characteristics onto the recipient bacteria. Described here are protocols for quantifying the frequency of transformation for the human pathogen Vibrio cholerae, one of several Vibrio species recently shown to be capable of natural transformation. © 2014 by John Wiley & Sons, Inc.
    Current protocols in microbiology 11/2014; 35:6A.4.1-6A.4.12. DOI:10.1002/9780471729259.mc06a04s35
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    ABSTRACT: As with all Herpesviruses, Herpes simplex virus (HSV) has both a lytic replication phase and a latency reactivation cycle. During lytic replication, there is an ordered cascade of viral gene expression that leads to the synthesis of infectious viral progeny. In contrast, latency is characterized by the lack of significant lytic gene expression and the absence of infectious virus. Reactivation from latency is characterized by the re-entry of the virus into the lytic replication cycle and the production of recurrent disease. This unit describes the establishment of the mouse sensory neuron model of HSV-1 latency-reactivation as a useful in vivo system for the analysis of mechanisms involved in latency and reactivation. Assays including the determination of viral yields, immunohistochemical/immunofluorescent detection of viral antigens, and mRNA quantitation are used in experiments designed to investigate the network of cellular and viral proteins regulating HSV-1 lytic infection, latency, and reactivation.
    Current protocols in microbiology 11/2014; 35(14E.6):14E.6.1 - 14E.6.21. DOI:10.1002/9780471729259.mc14e06s35
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    ABSTRACT: Herpes Simplex Virus (HSV) is a human pathogen that establishes latency and undergoes periodic reactivation, resulting in chronic recurrent lytic infection. HSV lytic infection is characterized by an organized cascade of three gene classes; however, successful transcription and expression of the first, the immediate early class, is critical to the overall success of viral infection. This initial event of lytic infection is also highly dependent on host cell factors. This unit uses RNA interference and small molecule inhibitors to examine the role of host and viral proteins in HSV lytic infection. Methods detailing isolation of viral and host RNA and genomic DNA followed by quantitative real-time PCR allow characterization of impacts on viral transcription and replication, respectively. Western blots can be used to confirm quantitative PCR results. This combination of protocols represents a starting point for researchers interested in virus-host interactions during HSV lytic infection.
    Current protocols in microbiology 11/2014; 35(14E.5):14E.5.1 - 14E.5.27. DOI:10.1002/9780471729259.mc14e05s35