Springer protocols feed by Microbiology 
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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.Transient Dominant Selection for the Modification and Generation of Recombinant Infectious Bronchitis Coronaviruses
We have developed a reverse genetics system for the avian coronavirus infectious bronchitis virus (IBV) in which a full-length cDNA corresponding to the IBV genome is inserted into the vaccinia virus genome under the control of a T7 promoter sequence. Vaccinia virus as a vector for the full-length IBV cDNA has the advantage that modifications can be introduced into the IBV cDNA using homologous recombination, a method frequently used to insert and delete sequences from the vaccinia virus genome. Here, we describe the use of transient dominant selection as a method for introducing modifications into the IBV cDNA; this has b...
Source: Springer protocols feed by Microbiology - February 28, 2015 Category: Microbiology Source Type: news
Quantification of Infectious Bronchitis Coronavirus by Titration In Vitro and In Ovo
Quantification of the number of infectious viruses in a sample is a basic virological technique. In this chapter we provide a detailed description of three techniques to estimate the number of viable infectious avian coronaviruses in a sample. All three techniques are serial dilution assays, better known as titrations. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - February 28, 2015 Category: Microbiology Source Type: news
Characterization of Human Coronaviruses on Well-Differentiated Human Airway Epithelial Cell Cultures
The human airway serves as the entry point of human respiratory viruses, including human coronaviruses. In this chapter we outline the methods by which we establish fully differentiated airway epithelium and its use for human coronavirus propagation. Additionally, we outline methods for immunofluorescence staining of these cultures for virus detection, characterization of cell tropism, and how to perform antiviral assays and quantify viral replication. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - February 28, 2015 Category: Microbiology Source Type: news
Isolation and Propagation of Coronaviruses in Embryonated Eggs
The embryonated egg is a complex structure comprised of an embryo and its supporting membranes (chorioallantoic, amniotic, yolk). The developing embryo and its membranes provide the diversity of cell types that are needed for successful replication of a wide variety of different viruses. Within the family Coronaviridae the embryonated egg has been used as a host system primarily for two avian coronaviruses within the genus Gammacoronavirus, infectious bronchitis virus (IBV) and turkey coronavirus (TCoV). The embryonated egg also has been shown to be suitable for isolation and propagation of pheasant coronavirus, a proposed...
Source: Springer protocols feed by Microbiology - February 28, 2015 Category: Microbiology Source Type: news
The Preparation of Chicken Kidney Cell Cultures for Virus Propagation
Chicken kidney (CK) cell cultures have historically proved useful for the assay of a number of viruses including coronaviruses. A technique for the preparation of such cell cultures, using a combination of manual and trypsin disaggregation of kidneys dissected from 2- to 3-week-old birds is described. This technique routinely gives high cell yield together with high viability and the resultant adherent primary cultures can be used for virus growth and plaque formation. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - February 28, 2015 Category: Microbiology Source Type: news
The Preparation of Chicken Tracheal Organ Cultures for Virus Isolation, Propagation, and Titration
Chicken tracheal organ cultures (TOCs), comprising transverse sections of chick embryo trachea with beating cilia, have proved useful in the isolation of several respiratory viruses and as a viral assay system, using ciliostasis as the criterion for infection. A simple technique for the preparation of chicken tracheal organ cultures in glass test tubes, in which virus growth and ciliostasis can be readily observed, is described. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - February 28, 2015 Category: Microbiology Source Type: news
Partial Purification of IBV and Subsequent Isolation of Viral RNA for Next-Generation Sequencing
RNA viruses are known for a high mutation rate and rapid genomic evolution. As such an RNA virus population does not consist of a single genotype but is rather a collection of individual viruses with closely related genotypes—a quasispecies, which can be analyzed by next-generation sequencing (NGS). This diversity of genotypes provides a mechanism in which a virus population can evolve and adapt to a changing environment. Sample preparation is vital for successful sequencing. The following protocol describes the process of generating a high-quality RNA preparation from IBV grown in embryonated eggs and then partially...
Source: Springer protocols feed by Microbiology - February 28, 2015 Category: Microbiology Source Type: news
Purification of Coronavirus Virions for Cryo-EM and Proteomic Analysis
Purification of intact enveloped virus particles can be useful as a first step in understanding the structure and function of both viral and host proteins that are incorporated into the virion. Purified preparations of virions can be used to address these questions using techniques such as mass spectrometry proteomics. Recent studies on the proteome of coronavirus virions have shown that in addition to the structural proteins, accessory and non-structural virus proteins and a wide variety of host cell proteins associate with virus particles. To further study the presence of virion proteins, high-quality sample preparation ...
Source: Springer protocols feed by Microbiology - February 28, 2015 Category: Microbiology Source Type: news
Estimation of Evolutionary Dynamics and Selection Pressure in Coronaviruses
Evolution of coronaviruses is facilitated by the strong selection, large population size, and great genetic diversity within the susceptible hosts. This predisposition is primarily due to high error rate, and limited proofreading capability of the viral polymerase and by recombination. These characteristics make coronaviruses an interesting model system to study the mechanisms involved in viral evolution and the ways viruses adapt to switch host or to gain novel functions. Here we describe the protocol to estimate selection pressures for the spike gene and evolutionary dynamics of bovine coronaviruses. (Source: Springer pr...
Source: Springer protocols feed by Microbiology - February 28, 2015 Category: Microbiology Source Type: news
Serological Diagnosis of Feline Coronavirus Infection by Immunochromatographic Test
The immunochromatographic assay (ICA) is a simple antibody–antigen detection method, the results of which can be rapidly obtained at a low cost. We designed an ICA to detect anti-feline coronavirus (FCoV) antibodies. A colloidal gold-labeled recombinant FCoV nucleocapsid protein (rNP) is used as a conjugate. The Protein A and affinity-purified cat anti-FCoV IgG are blotted on the test line and the control line, respectively, of the nitrocellulose membrane. The specific detection of anti-FCoV antibodies was possible in all heparin-anticoagulated plasma, serum, whole blood, and ascitic fluid samples from anti-FCoV anti...
Source: Springer protocols feed by Microbiology - February 28, 2015 Category: Microbiology Source Type: news
Identification of a Novel Coronavirus from Guinea Fowl Using Metagenomics
While classical virology techniques such as virus culture, electron microscopy, or classical PCR had been unsuccessful in identifying the causative agent responsible for the fulminating disease of guinea fowl, we identified a novel avian gammacoronavirus associated with the disease using metagenomics. Next-generation sequencing is an unbiased approach that allows the sequencing of virtually all the genetic material present in a given sample. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - February 28, 2015 Category: Microbiology Source Type: news
Coronaviruses: An Overview of Their Replication and Pathogenesis
Coronaviruses (CoVs), enveloped positive-sense RNA viruses, are characterized by club-like spikes that project from their surface, an unusually large RNA genome, and a unique replication strategy. Coronaviruses cause a variety of diseases in mammals and birds ranging from enteritis in cows and pigs and upper respiratory disease in chickens to potentially lethal human respiratory infections. Here we provide a brief introduction to coronaviruses discussing their replication and pathogenicity, and current prevention and treatment strategies. We also discuss the outbreaks of the highly pathogenic Severe Acute Respiratory Syndr...
Source: Springer protocols feed by Microbiology - February 28, 2015 Category: Microbiology Source Type: news
Single-Stranded DNA Aptamers for Functional Probing of Bacterial RNA Polymerase
Bacterial RNA polymerase (RNAP) is the main regulatory hub of gene transcription. During transcription, RNAP interacts with the DNA template, RNA product, nucleotide substrates, metal cofactors, and regulatory molecules that bind to distinct RNAP sites to modulate its activity. RNAP is also inhibited by several known antibiotics and is a promising target for development of novel antibacterial compounds. Despite great progress in structural analysis of RNAP in recent years, many details of RNAP interactions with nucleic acids, regulatory molecules and antibiotics remain insufficiently understood. Aptamers that target variou...
Source: Springer protocols feed by Microbiology - February 13, 2015 Category: Microbiology Source Type: news
Direct Competition Assay for Transcription Fidelity
Accurate transcription is essential for faithful information flow from DNA to RNA and to the protein. Mechanisms of cognate substrate selection by RNA polymerases are currently elucidated by structural, genetic, and biochemical approaches. Here, we describe a fast and reliable approach to quantitative analyses of transcription fidelity, applicable to analyses of RNA polymerase selectivity against misincorporation, incorporation of dNMPs, and chemically modified rNMP analogues. The method is based on different electrophoretic mobility of RNA oligomers of the same length but differing in sequence. (Source: Springer protocols...
Source: Springer protocols feed by Microbiology - February 13, 2015 Category: Microbiology Source Type: news
Reconstitution of Factor-Dependent, Promoter Proximal Pausing in Drosophila Nuclear Extracts
Genomic analyses reveal that RNA polymerase II initiates transcription but pauses shortly downstream on thousands of promoters in Drosophila and mammalian cells. Here, we describe the reconstitution of this promoter proximal pausing in nuclear extracts from Drosophila embryos. This approach is useful for dissecting the role(s) of transcription factors in promoter proximal pausing. Most of our studies employ the hsp70 heat shock gene promoter; however, this technique has successfully reconstituted RNA polymerase II pausing downstream of several other Drosophila promoters. A pulse/chase method is employed to restrict incorpo...
Source: Springer protocols feed by Microbiology - February 13, 2015 Category: Microbiology Source Type: news
