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.Growth of Human Rhinovirus in H1-HeLa Cell Suspension Culture and Purification of Virions
HeLa cell culture is the most widely used system for in vitro studies of the basic biology of human rhinovirus (HRV). It is also useful for making sufficient quantities of virus for experiments that require highly concentrated and purified virus. This chapter describes the protocols for producing a large amount of HeLa cells in suspension culture, using these cells to grow a large quantity of virus of HeLa-adapted HRV-A and -B serotypes, and making highly concentrated virus stock and highly purified virions. These purified HRV virions are free of cellular components and suitable for experiments that are sensitive to cellul...
Source: Springer protocols feed by Infectious Diseases - September 29, 2014 Category: Infectious Diseases Source Type: news
Molecular Genotyping of Human Rhinovirus by Using PCR and Sanger Sequencing
Human rhinovirus (HRV) is the virus most often associated with acute upper respiratory tract infections. Advances in molecular detection have shown that HRV is also the major viral cause of asthma exacerbations. Genotypic assignment and identification of HRV types are of significant value in the investigation of type-associated differences in disease outcomes, transmission, and epidemiology. Here, we describe a genotyping process involving two separate RT-PCR assays, targeted to VP4/VP2 and 5′ UTR regions of HRV genome, respectively. Together with the reference sequences of each HRV species, the generated sequences a...
Source: Springer protocols feed by Infectious Diseases - September 29, 2014 Category: Infectious Diseases Source Type: news
Molecular Identification and Quantification of Human Rhinoviruses in Respiratory Samples
PCR-based molecular assays have become standard diagnostic procedures for the identification and quantification of human rhinoviruses (HRVs) and other respiratory pathogens in most, if not all, clinical microbiology laboratories. Molecular assays are significantly more sensitive than traditional culture-based and serological methods. This advantage has led to the recognition that HRV infections are common causes for not only upper airway symptoms but also more severe lower respiratory illnesses. In addition, molecular assays improve turnaround time, can be performed by technicians with ordinary skills, and can easily be au...
Source: Springer protocols feed by Infectious Diseases - September 29, 2014 Category: Infectious Diseases Source Type: news
Nested-RT-PCR and Multiplex RT-PCR for Diagnosis of Rhinovirus Infection in Clinical Samples
Human rhinoviruses (HRVs) are positive-stranded RNA viruses belonging to the Enterovirus genus in the family of Picornaviridae. Identification of the specific strain in HRV disease has been difficult because the traditional serological method is insensitive, labor intensive, and cumbersome. With the fast progress in molecular biological technique, more sensitive and faster molecular methods have been developed, such as polymerase chain reaction (PCR), reverse transcriptase (RT)-PCR, and real-time RT-PCR. To improve the technique for defining the links between illnesses and specific strains of HRV, we developed RT-PCR speci...
Source: Springer protocols feed by Infectious Diseases - September 29, 2014 Category: Infectious Diseases Source Type: news
Classification and Evolution of Human Rhinoviruses
The historical classification of human rhinoviruses (RV) by serotyping has been replaced by a logical system of comparative sequencing. Given that strains must diverge within their capsid sequenced by a reasonable degree (>12–13 % pairwise base identities) before becoming immunologically distinct, the new nomenclature system makes allowances for the addition of new, future types, without compromising historical designations. Currently, three species, the RV-A, RV-B, and RV-C, are recognized. Of these, the RV-C, discovered in 2006, are the most unusual in terms of capsid structure, receptor use, and association wit...
Source: Springer protocols feed by Infectious Diseases - September 29, 2014 Category: Infectious Diseases Source Type: news
Mouse Models of Rhinovirus Infection and Airways Disease
Mouse models are invaluable tools for gaining insight into host immunity during virus infection. Until recently, no practical mouse model for rhinovirus infection was available. Development of infection models was complicated by the existence of distinct groups of viruses that utilize different host cell surface proteins for binding and entry. Here, we describe mouse infection models, including virus purification and measurement of host immune responses, for representative viruses from two of these groups: (1) infection of unmodified Balb/c mice with minor group rhinovirus serotype 1B (RV-1B) and (2) infection of transgeni...
Source: Springer protocols feed by Infectious Diseases - September 29, 2014 Category: Infectious Diseases Source Type: news
Reverse Genetic Engineering of the Human Rhinovirus Serotype 16 Genome to Introduce an Antibody-Detectable Tag
The ability to accurately detect viral proteins during infection is essential for virology research, and the lack of specific antibodies can make this detection difficult. Reverse genetic engineering of virus genomes to alter the wild-type genome is a powerful technique to introduce a detectable tag onto a viral protein. Here we outline a method to incorporate an influenza hemagglutinin epitope tag onto the 2A protease of HRV16. The method uses site-directed mutagenesis PCR to introduce the sequence for the HA antigen onto either the C or N termini of 2A protease while keeping the relevant internal cleavage sites intact. T...
Source: Springer protocols feed by Infectious Diseases - September 29, 2014 Category: Infectious Diseases Source Type: news
Reverse Genetics System for Studying Human Rhinovirus Infections
Human rhinovirus (HRV) contains a 7.2 kb messenger-sense RNA genome which is the template for reproducing progeny viruses after it enters the cytoplasm of a host cell. Reverse genetics refers to the regeneration of progeny viruses from an artificial cDNA copy of the RNA genome of an RNA virus. It has been a powerful molecular genetic tool for studying HRV and other RNA viruses because the artificial DNA stage makes it practical to introduce specific mutations into the viral RNA genome. This chapter uses HRV-16 as the model virus to illustrate the strategy and methods for constructing and cloning the artificial cDNA copy of...
Source: Springer protocols feed by Infectious Diseases - September 29, 2014 Category: Infectious Diseases Source Type: news
A Protocol to Express and Isolate HRV16 3C Protease for Use in Protease Assays
We describe a simple method for isolating HRV16 3C protease from a bacterial expression system, including transformation of bacterial cells with a commercially available cDNA plasmid which can be adapted to use for 3C proteases from any other HRV serotypes. The extracted, active 3C protease can then be used for in vitro protease assays. (Source: Springer protocols feed by Infectious Diseases)
Source: Springer protocols feed by Infectious Diseases - September 29, 2014 Category: Infectious Diseases Source Type: news
Proteases of Human Rhinovirus: Role in Infection
Human rhinoviruses (HRV) are the major etiological agents of the common cold and asthma exacerbations, with significant worldwide health and economic impact. Although large-scale population vaccination has proved successful in limiting or even eradicating many viruses, the more than 100 distinct serotypes mean that conventional vaccination is not a feasible strategy to combat HRV. An alternative strategy is to target conserved viral proteins such as the HRV proteases, 2Apro and 3Cpro, the focus of this review. Necessary for host cell shutoff, virus replication, and pathogenesis, 2Apro and 3Cpro are clearly viable drug targ...
Source: Springer protocols feed by Infectious Diseases - September 29, 2014 Category: Infectious Diseases Source Type: news
Study of the Stn Protein in Salmonella; A Regulator of Membrane Composition and Integrity
Our studies were undertaken to develop new insights into the function of the Salmonella Stn protein. An analysis of total cell membrane protein fraction suggested the possibility that Stn associates with OmpA. This possibility was confirmed by immunogold labeling using anti-OmpA antibody and far-western blotting. From these results, we conclude that Stn regulates membrane composition and integrity in Salmonella. (Source: Springer protocols feed by Infectious Diseases)
Source: Springer protocols feed by Infectious Diseases - September 25, 2014 Category: Infectious Diseases Source Type: news
Detecting Non-typhoid Salmonella in Humans by Enzyme-Linked Immunosorbent Assays (ELISAs): Practical and Epidemiological Aspects
Salmonellosis caused by non-typhoid Salmonella serotypes is one of the most common causes of food-borne illness throughout the world. The diagnosis is primarily by culture and more recently molecular methods, whereas the use of serological methods for diagnosis of Salmonella infections is limited by high running costs as well as low sensitivity and specificity. Fast and reliable immunoassays for detection of S. typhi subunit antigens are commercially available, but there is no international consensus of similar tests for non-typhoid salmonellosis. Most immunoassays for non-typhoid human Salmonella diagnosis are developed i...
Source: Springer protocols feed by Infectious Diseases - September 25, 2014 Category: Infectious Diseases Source Type: news
Detection of Antimicrobial (Poly)Peptides with Acid Urea Polyacrylamide Gel Electrophoresis Followed by Western Immunoblot
Antimicrobial (poly)peptides (AMPs) are ancient key effector molecules of innate host defense and have been identified in mammals, insects, plants, and even fungi (Nakatsuji and Gallo, J Invest Dermatol, 132: 887–895, 2012). They exhibit a cationic net charge at physiological pH and are rich in hydrophobic amino acids (Dufourc et al., Curr Protein Pept Sci, 13: 620–631, 2012). Their mode of action has been best investigated in bacteria. When assuming secondary structure the cationic and hydrophobic amino acids are sequestered creating a bipartitioned molecule in which the cationic amino acids mediate initial el...
Source: Springer protocols feed by Infectious Diseases - September 25, 2014 Category: Infectious Diseases Source Type: news
Generation and Use of Site-Directed Chromosomal cyaA′ Translational Fusions in Salmonella enterica
CyaA from Bordetella pertussis is a calmodulin-dependent adenylate cyclase. Fusions to the catalytic domain of CyaA (CyaA′) are useful tools to detect translocation of type III secretion system effectors from gram-negative pathogens like Salmonella enterica. These fusions are usually generated using plasmids with strong promoters. Here, we describe a protocol to insert the CyaA′-encoding sequence in a specific site in the bacterial chromosome in order to get a monocopy fusion whose expression is driven by the native promoter. We also describe the procedure to detect translocation of a CyaA′ fusion into ma...
Source: Springer protocols feed by Infectious Diseases - September 25, 2014 Category: Infectious Diseases Source Type: news
A Method to Introduce an Internal Tag Sequence into a Salmonella Chromosomal Gene
Epitope tags are short peptide sequences that are particularly useful for the characterization of proteins against which no antibody has been developed. Influenza hemagglutinin (HA) tag is one of the most widely used epitope tags as several valuable monoclonal and polyclonal antibodies that can be used in various techniques are commercially available. Therefore, adding a HA tag to a protein of interest is quite helpful to get rapid and cost less information regarding its localization, its expression or its biological function. In this chapter, we describe a process, derived from the Datsenko and Wanner procedure, which all...
Source: Springer protocols feed by Infectious Diseases - September 25, 2014 Category: Infectious Diseases Source Type: news
