Infectious Viral Quantification of Chikungunya Virus & mdash;Virus Plaque Assay
The plaque assay is an essential method for quantification of infectious virus titer. Cells infected with virus particles are overlaid with a viscous substrate. A suitable incubation period results in the formation of plaques, which can be fixed and stained for visualization. Here, we describe a method for measuring Chikungunya virus (CHIKV) titers via virus plaque assays. (Source: Springer protocols feed by Infectious Diseases)
Source: Springer protocols feed by Infectious Diseases - December 31, 2015 Category: Infectious Diseases Source Type: news

Bioinformatics Based Approaches to Study Virus & ndash;Host Interactions During Chikungunya Virus Infection
The limitations of high-throughput genomic methods used for studying virus & ndash;host interactions make it difficult to directly obtain insights on virus pathogenesis. In this chapter, the central steps of a protein structure similarity based computational approach used to predict the host interactors of Chikungunya virus are explained by highlighting the important aspects that need to be considered. Identification of such conserved set of putative interactions that allow the virus to take control of the host has the potential to deepen our understanding of the virus-specific remodeling processes of the host cell and...
Source: Springer protocols feed by Infectious Diseases - December 31, 2015 Category: Infectious Diseases Source Type: news

Viral & ndash;Host Protein Interaction Studies Using Yeast Two-Hybrid Screening Method
Yeast two-hybrid (Y2H) assay is one of the earliest methods developed to study protein & ndash;protein interactions. In the proteomics era, Y2H has created a niche of its own by providing protein interaction maps for various organisms. Owing to limited coding capacities of their genomes, viruses are dependent on their host cellular machinery for successful infection. Identification of the key players orchestrating the survival of virus in their host is essential for understanding viral life cycle and devising strategies to prevent interactions resulting in pathogenesis. In this chapter, Y2H assay will be explained in d...
Source: Springer protocols feed by Infectious Diseases - December 31, 2015 Category: Infectious Diseases Source Type: news

Neutralization Assay for Chikungunya Virus Infection: Plaque Reduction Neutralization Test
Neutralization assay is a technique that detects and quantifies neutralizing antibody in serum samples by calculating the percentage of reduction of virus activity, as the concentration of virus used is usually constant. Neutralizing antibody titer is conventionally determined by calculating the percentage reduction in total virus infectivity by counting and comparing number of plaques (localized area of infection due to cytopathic effect) with a standard amount of virus. Conventional neutralizing test uses plaque-reduction neutralization test (PRNT) to determine neutralizing antibody titers against Chikungunya virus (CHIK...
Source: Springer protocols feed by Infectious Diseases - December 31, 2015 Category: Infectious Diseases Source Type: news

Using Bicistronic Baculovirus Expression Vector System to Screen the Compounds That Interfere with the Infection of Chikungunya Virus
Chikungunya virus (CHIKV) is the etiologic agent of Chikungunya fever and has emerged in many countries over the past decade. There are no effective drugs for controlling the disease. A bicistronic baculovirus expression system was utilized to co-express CHIKV structural proteins C (capsid), E2 and E1 and the enhanced green fluorescence protein (EGFP) in Spodoptera frugiperda insect cells (Sf21). The EGFP-positive Sf21 cells fused with each other and with uninfected cells to form a syncytium is mediated by the CHIKV E1 allowing it to identify chemicals that can prevent syncytium formation. The compounds characterized by th...
Source: Springer protocols feed by Infectious Diseases - December 31, 2015 Category: Infectious Diseases Source Type: news

A Real-Time Cell Analyzing Assay for Identification of Novel Antiviral Compounds against Chikungunya Virus
Screening of viral inhibitors through induction of cytopathic effects (CPE) by conventional method has been applied for various viruses including Chikungunya virus (CHIKV), a significant arbovirus. However, it does not provide the information about cytopathic effect from the beginning and throughout the course of virus replication. Conventionally, most of the approaches are constructed on laborious end-point assays which are not capable for detecting minute and rapid changes in cellular morphology. Therefore, we developed a label-free and dynamical method for monitoring the cellular features that comprises cell attachment,...
Source: Springer protocols feed by Infectious Diseases - December 31, 2015 Category: Infectious Diseases Source Type: news

Antiviral Strategies Against Chikungunya Virus
In the last few decades the Chikungunya virus (CHIKV) has evolved from a geographically isolated pathogen to a virus that is widespread in many parts of Africa, Asia and recently also in Central- and South-America. Although CHIKV infections are rarely fatal, the disease can evolve into a chronic stage, which is characterized by persisting polyarthralgia and joint stiffness. This chronic CHIKV infection can severely incapacitate patients for weeks up to several years after the initial infection. Despite the burden of CHIKV infections, no vaccine or antivirals are available yet. The current therapy is therefore only symptoma...
Source: Springer protocols feed by Infectious Diseases - December 31, 2015 Category: Infectious Diseases Source Type: news

Immunohistochemical Detection of Chikungunya Virus Antigens in Formalin-Fixed and Paraffin-Embedded Tissues
Immunohistochemistry is a histological technique that allows detection of one or more proteins of interest within a cell using specific antibody binding, followed by microscopic visualization of a chromogenic substrate catalyzed by peroxidase and/or alkaline phosphatase. Here, we describe a method to localize Chikungunya virus (CHIKV) antigens in formalin-fixed and paraffin-embedded infected mouse brain. (Source: Springer protocols feed by Infectious Diseases)
Source: Springer protocols feed by Infectious Diseases - December 31, 2015 Category: Infectious Diseases Source Type: news

Generation of Mouse Monoclonal Antibodies Specific to Chikungunya Virus Using ClonaCell-HY Hybridoma Cloning Kit
Monoclonal antibodies offer high specificity and this makes it an important tool for molecular biology, biochemistry and medicine. Typically, monoclonal antibodies are generated by fusing mouse spleen cells that have been immunized with the desired antigen with myeloma cells to create immortalized hybridomas. Here, we describe the generation of monoclonal antibodies that are specific to Chikungunya virus using ClonaCell-HY system. (Source: Springer protocols feed by Infectious Diseases)
Source: Springer protocols feed by Infectious Diseases - December 31, 2015 Category: Infectious Diseases Source Type: news

Mouse Models of Chikungunya Virus
The majority of medical advances have been made using animals. Studies using mouse models of chikungunya-induced disease have proven invaluable for dissecting the intricate nature of the immune response to this viral infection and identifying potential targets for the development of treatment strategies. Herein we describe the common mouse models used to research the pathobiology of chikungunya virus infection to date. (Source: Springer protocols feed by Infectious Diseases)
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Infectious Viral Quantification of Chikungunya Virus—Virus Plaque Assay
The plaque assay is an essential method for quantification of infectious virus titer. Cells infected with virus particles are overlaid with a viscous substrate. A suitable incubation period results in the formation of plaques, which can be fixed and stained for visualization. Here, we describe a method for measuring Chikungunya virus (CHIKV) titers via virus plaque assays. (Source: Springer protocols feed by Infectious Diseases)
Source: Springer protocols feed by Infectious Diseases - December 31, 2015 Category: Infectious Diseases Source Type: news

Propagation of Chikungunya Virus Using Mosquito Cells
Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that transmits in between a mosquito host vector to a primate host and then back to the mosquito host vector to complete its life cycle. Hence, CHIKV must be able to replicate in both host cellular systems that are genetically and biochemically distinct. The ability to grow and propagate the virus in high titers in the laboratory is fundamentally crucial in order to understand virus replication in different host cellular systems and many other CHIKV research areas. Here, we describe a method on CHIKV propagation using C6/36, a mosquito cell line derived from Aedes al...
Source: Springer protocols feed by Infectious Diseases - December 31, 2015 Category: Infectious Diseases Source Type: news

T-Cell Epitope Prediction of Chikungunya Virus
There has been a growing demand for vaccines against Chikungunya virus (CHIKV), and epitope-based vaccine is a promising solution. Identification of CHIKV T-cell epitopes is critical to ensure successful trigger of immune response for epitope-based vaccine design. Bioinformatics tools are able to significantly reduce time and effort in this process by systematically scanning for immunogenic peptides in CHIKV proteins. This chapter provides the steps in utilizing machine learning algorithms to train on major histocompatibility complex (MHC) class I peptide binding data and build prediction models for the classification of b...
Source: Springer protocols feed by Infectious Diseases - December 31, 2015 Category: Infectious Diseases Source Type: news

Bioinformatics Based Approaches to Study Virus–Host Interactions During Chikungunya Virus Infection
The limitations of high-throughput genomic methods used for studying virus–host interactions make it difficult to directly obtain insights on virus pathogenesis. In this chapter, the central steps of a protein structure similarity based computational approach used to predict the host interactors of Chikungunya virus are explained by highlighting the important aspects that need to be considered. Identification of such conserved set of putative interactions that allow the virus to take control of the host has the potential to deepen our understanding of the virus-specific remodeling processes of the host cell and illum...
Source: Springer protocols feed by Infectious Diseases - December 31, 2015 Category: Infectious Diseases Source Type: news

Application of GelC-MS/MS to Proteomic Profiling of Chikungunya Virus Infection: Preparation of Peptides for Analysis
Gel-enhanced liquid chromatography coupled with tandem mass spectrometry (GeLC-MS/MS) is a labor intensive, but relatively straightforward methodology that generates high proteome coverage which can be applied to the proteome analysis of a range of starting materials such as cells or patient specimens. Sample proteins are resolved electrophoretically in one dimension through a sodium dodecyl sulfate (SDS) polyacrylamide gel after which the lanes are sliced into sections. The sections are further diced and the gel cubes generated are subjected to in-gel tryptic digestion. The resultant peptides can then be analyzed by tande...
Source: Springer protocols feed by Infectious Diseases - December 31, 2015 Category: Infectious Diseases Source Type: news

Viral–Host Protein Interaction Studies Using Yeast Two-Hybrid Screening Method
Yeast two-hybrid (Y2H) assay is one of the earliest methods developed to study protein–protein interactions. In the proteomics era, Y2H has created a niche of its own by providing protein interaction maps for various organisms. Owing to limited coding capacities of their genomes, viruses are dependent on their host cellular machinery for successful infection. Identification of the key players orchestrating the survival of virus in their host is essential for understanding viral life cycle and devising strategies to prevent interactions resulting in pathogenesis. In this chapter, Y2H assay will be explained in detail ...
Source: Springer protocols feed by Infectious Diseases - December 31, 2015 Category: Infectious Diseases Source Type: news

Virus Isolation and Preparation of Sucrose-Banded Chikungunya Virus Samples for Transmission Electron Microscopy
Virus isolation and purification is an invaluable technique in virology to detect and characterize viruses. This chapter describes a large-scale Chikungunya virus (CHIKV) propagation and purification methods by using discontinuous sucrose gradient, and sample preparation for transmission electron microscopy. Sucrose-banding yields large quantities of high-titer (1010 pfu/ml) CHIKV stocks. Such stocks are stable for years when stored at −70 °C. (Source: Springer protocols feed by Infectious Diseases)
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Chikungunya Virus Growth and Fluorescent Labeling: Detection of Chikungunya Virus by Immunofluorescence Assay
Immunofluorescence assay (IFA) is a highly versatile and sensitive assay for detection and titration of chikungunya virus (CHIKV). The IFA technique requires virus-infected cells (viral antigen) and antibodies specific to the viral antigens for detection. Suitable antibodies for detection include monoclonal antibodies specific to CHIKV structural and nonstructural proteins, polyclonal antibodies, and convalescent serum samples. Here, the details of virus antigen preparation, detection by IFA method, and applications are described. The described IFA method is potentially useful in a wide range of studies including virus gro...
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Analysis of CHIKV in Mosquitoes Infected via Artificial Blood Meal
Having a mechanism to assess the transmission dynamics of a vector-borne virus is one critical component of understanding the life cycle of these viruses. Laboratory infection systems using artificial blood meals is one valuable approach for monitoring the progress of virus in its mosquito host and evaluating potential points for interruption of the cycle for control purposes. Here, we describe an artificial blood meal system with Chikungunya virus (CHIKV) and the processing of mosquito tissues and saliva to understand the movement and time course of virus infection in the invertebrate host. (Source: Springer protocols fee...
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Chikungunya Virus Infection of Aedes Mosquitoes
In vivo infection of mosquitoes is an important method to study and characterize arthropod-borne viruses. Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that is transmitted primarily by Aedes mosquitoes. In this chapter, we describe a protocol for infection of CHIKV in two species of Aedes mosquitoes, Aedes aegypti and Aedes albopictus, together with the isolation of CHIKV in different parts of the infected mosquito such as midgut, legs, wings, salivary gland, head, and saliva. This allows the study of viral infection, replication and dissemination within the mosquito vector. (Source: Springer protocols feed by Infectious Diseases)
Source: Springer protocols feed by Infectious Diseases - December 31, 2015 Category: Infectious Diseases Source Type: news

Detection and Quantification of Chikungunya Virus by Real-Time RT-PCR Assay
Real-time PCR assay has many advantages over conventional PCR methods, including rapidity, quantitative measurement, low risk of contamination, high sensitivity, high specificity, and ease of standardization (Mackay et al., Nucleic Acids Res 30:1292–1305, 2002). The real-time PCR system relies upon the measurement of a fluorescent reporter during PCR, in which the amount of emitted fluorescence is directly proportional to the amount of the PCR product in a reaction (Gibsons et al., Genome Res 6:995–1001, 1996). Here, we describe the use of SYBR Green I-based and TaqMan® real-time reverse transcription polym...
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Utilization and Assessment of Throat Swab and Urine Specimens for Diagnosis of Chikungunya Virus Infection
Chikungunya is a mosquito-borne infection with clinical presentation of fever, arthralgia, and rash. The etiological agent Chikungunya virus (CHIKV) is generally transmitted from primates to humans through the bites of infected Aedes aegypti and Aedes albopictus mosquitoes. Outbreaks of Chikungunya occur commonly with varied morbidity, mortality, and sequele according to the epidemiological, ecological, seasonal, and geographical impact. Investigations are required to be conducted as a part of the public health service to understand and report the suspected cases as confirmed by laboratory diagnosis. Holistic sampling at a...
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Diagnostic Methods for CHIKV Based on Serological Tools
This chapter presents the most commonly used serological methods for the diagnosis of Chikungunya virus (CHIKV) infection in humans. CHIKV is a mosquito-borne Alphavirus widely distributed in the tropical and subtropical regions of Africa, Asia, and America. CHIKV infection in human causes acute febrile illness frequently accompanied by severe joint pain. Most of the infected patients may develop chronic arthralgia that may persist for several months or years. Laboratory diagnosis of CHIKV infection is mainly based on molecular and serological tests. The serological tests represent a valuable tool for diagnosis and epidemi...
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Expression and Purification of E2 Glycoprotein from Insect Cells (Sf9) for Use in Serology
Chikungunya virus (CHIKV) is a mosquito-borne arbovirus which poses a major threat to global public health. Definitive CHIKV diagnosis is crucial, especially in distinguishing the disease from dengue virus, which co-circulates in endemic areas and shares the same mosquito vectors. Laboratory diagnosis is mainly based on serological or molecular approaches. The E2 glycoprotein is a good candidate for serological diagnosis since it is the immunodominant antigen during the course of infection, and reacts with seropositive CHIKV sera. In this chapter, we describe the generation of stable clone Sf9 (Spodoptera frugiperda) cells...
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Synthetic Peptide-Based Antibody Detection for Diagnosis of Chikungunya Infection with and without Neurological Complications
Synthetic peptide-based diagnosis of Chikungunya can be an efficient and more accessible approach in immunodiagnostics. Here, we describe the identification of Chikungunya-specific 40 kD protein for development of synthetic peptide-based enzyme-linked immunosorbent assay for the detection of Chikungunya virus-specific antibodies in the patient’s sample. The total sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profile of the patient’s sample can be done to identify specific protein bands. The identified proteins can be subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) for ...
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Advanced Genetic Methodologies in Tracking Evolution and Spread of Chikungunya Virus
Recent advances in genetic methodologies have substantially expanded our ability to track evolution and spatio-temporal distribution of rapidly evolving pathogens. The information gathered from such analyses can be used to decipher host adaptations that shape disease epidemiology. In this chapter, we demonstrate the utilization of freely available resources to track the evolution and spread of Chikungunya virus. (Source: Springer protocols feed by Infectious Diseases)
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Molecular Epidemiology of Chikungunya Virus by Sequencing
Molecular surveillance of Chikungunya virus (CHIKV) is important as it provides data on the circulating CHIKV genotypes in endemic countries and enabling activation of measures to be taken in the event of a pending outbreak. Molecular surveillance is carried out by first detecting CHIKV in susceptible humans or among field-caught mosquitoes. This is followed by sequencing a selected region of the virus which will provide evidence on the source of the virus and possible association of the virus to increased cases of Chikungunya infections. (Source: Springer protocols feed by Infectious Diseases)
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Evolution and Epidemiology of Chikungunya Virus
Chikungunya is a mosquito-borne Alphavirus that is spreading worldwide in the tropical areas and that has a 11.8 kb RNA genome. The most relevant vectors belong to the genus Aedes and contribute to the diffusion of the three different genotypes of the virus from the original site of first identification in East Africa. Recently, an additional site of origin has been identified in Asia. The epidemiology of Chikungunya has been extensively evaluated from 2004 when the virus initiated its travel eastbound from the coast of Africa to the Indian Ocean. It is noteworthy that this diffusion has been mainly sustained by Ae. albopi...
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Molecular Properties of Poliovirus Isolates: Nucleotide Sequence Analysis, Typing by PCR and Real-Time RT-PCR
Virologic surveillance is essential to the success of the World Health Organization initiative to eradicate poliomyelitis. Molecular methods have been used to detect polioviruses in tissue culture isolates derived from stool samples obtained through surveillance for acute flaccid paralysis. This chapter describes the use of realtime PCR assays to identify and serotype polioviruses. In particular, a degenerate, inosine-containing, panpoliovirus (panPV) PCR primer set is used to distinguish polioviruses from NPEVs. The high degree of nucleotide sequence diversity among polioviruses presents a challenge to the systematic desi...
Source: Springer protocols feed by Infectious Diseases - December 15, 2015 Category: Infectious Diseases Source Type: news

Standardized Methods for Detection of Poliovirus Antibodies
Testing for neutralizing antibodies against polioviruses has been an established gold standard for assessing individual protection from disease, population immunity, vaccine efficacy studies, and other vaccine clinical trials. Detecting poliovirus specific IgM and IgA in sera and mucosal specimens has been proposed for evaluating the status of population mucosal immunity. More recently, there has been a renewed interest in using dried blood spot cards as a medium for sample collection to enhance surveillance of poliovirus immunity. Here, we describe the modified poliovirus microneutralization assay, poliovirus capture IgM ...
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A Transgenic Mouse Model of Poliomyelitis
This report describes the methods used to analyze the pathogenicity and immune responses of poliovirus using the PVR-tg mouse model. (Source: Springer protocols feed by Infectious Diseases)
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Quality Assurance in the Polio Laboratory. Cell Sensitivity and Cell Authentication Assays
The accuracy of poliovirus surveillance is largely dependent on the quality of the cell lines used for virus isolation, which is the foundation of poliovirus diagnostic work. Many cell lines are available for the isolation of enteroviruses, whilst genetically modified L20B cells can be used as a diagnostic tool for the identification of polioviruses. To be confident that cells can consistently isolate the virus of interest, it is necessary to have a quality assurance system in place, which will ensure that the cells in use are not contaminated with other cell lines or microorganisms and that they remain sensitive to the vi...
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Molecular Characterization of Polio from Environmental Samples: ISSP, The Israeli Sewage Surveillance Protocol
We present methods for adapting standard kits and validating the changes for this purpose based on experience gained during the recent introduction and sustained transmission of a wild type 1 poliovirus in Israel in 2013 in a population with an initial IPV vaccine coverage>90 %. (Source: Springer protocols feed by Infectious Diseases)
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Isolation and Characterization of Poliovirus in Cell Culture Systems
The isolation and characterization of enteroviruses by cell culture was accepted as the “gold standard” by clinical virology laboratories. Methods for the direct detection of all enteroviruses by reverse transcription polymerase chain reaction, targeting a conserved region of the genome, have largely supplanted cell culture as the principal diagnostic procedure. However, the World Health Organization’s Global Polio Eradication Initiative continues to rely upon cell culture to isolate poliovirus due to the lack of a reliable sensitive genetic test for direct typing of enteroviruses from clinical specimens....
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Isolation and Characterization of Enteroviruses from Clinical Samples
Enterovirus infections are common in humans worldwide. Enteroviruses are excreted in feces during infection and can be detected from stool specimens by isolation in continuous laboratory cell lines. Characterization of enteroviruses is based on their antigenic and/or genetic properties. (Source: Springer protocols feed by Infectious Diseases)
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Poliovirus Laboratory Based Surveillance: An Overview
World Health Assembly (WHA) in 1988 encouraged the member states to launch Global Polio Eradication Initiative (GPEI) (resolution WHA41.28) against “the Crippler” called poliovirus, through strong routine immunization program and intensified surveillance systems. Since its launch, global incidence of poliomyelitis has been reduced by more than 99 % and the disease squeezed to only three endemic countries (Afghanistan, Pakistan, and Nigeria) out of 125. Today, poliomyelitis is on the verge of eradication, and their etiological agents, the three poliovirus serotypes, are on the brink of extinction from the natura...
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An Introduction to Poliovirus: Pathogenesis, Vaccination, and the Endgame for Global Eradication
Poliomyelitis is caused by poliovirus, which is a positive strand non-enveloped virus that occurs in three distinct serotypes (1, 2, and 3). Infection is mainly by the fecal–oral route and can be confined to the gut by antibodies induced either by vaccine, previous infection or maternally acquired. Vaccines include the live attenuated strains developed by Sabin and the inactivated vaccines developed by Salk; the live attenuated vaccine (Oral Polio Vaccine or OPV) has been the main tool in the Global Program of Polio eradication of the World Health Organisation. Wild type 2 virus has not caused a case since 1999 and t...
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Identification and Analysis of Antiviral Compounds Against Poliovirus
The Global Polio Eradication Initiative, launched in 1988, had as its goal the eradication of polio worldwide by the year 2000 through large-scale vaccinations campaigns with the live attenuated oral PV vaccine (OPV) (Griffiths et al., Biologicals 34:73–74, 2006). Despite substantial progress, polio remains endemic in several countries and new imported cases are reported on a regular basis ( http://www.polioeradication.org/casecount.asp ). (Source: Springer protocols feed by Infectious Diseases)
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Measuring Poliovirus Antigenicity by Surface Plasmon Resonance. Application for Potency Indicating Assays
The D-antigen ELISA is the commonly accepted test for release of inactivated poliovirus containing vaccines. However, this test has a few drawbacks regarding the many variations in the method to quantify the D-unit. The result may depend on method and reagents used which makes standardization of inactivated polio vaccines, based on D-units, to a real challenge. This chapter describes a surface plasmon resonance based method to quantify D-units. The advantage of the calibrated D-antigen assay is the decrease in test variations because no labels, [no incubation times] and no washing steps are necessary. For standardization o...
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Methods for the Quality Control of Inactivated Poliovirus Vaccines
Inactivated poliovirus vaccine (IPV) plays an instrumental role in the Global Poliovirus Eradication Initiative (GPEI). The quality of IPV is controlled by assessment of the potency of vaccine batches. The potency of IPV can be assessed by both in vivo and in vitro methods. In vitro potency assessment is based upon the assessment of the quantity of the D-Antigen (D-Ag) units in an IPV. The D-Ag unit is used as a measure of potency as it is largely expressed on native infectious virions and is the protective immunogen. The most commonly used in vitro test is the indirect ELISA which is used to ensure consistency throughout ...
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Methods to Monitor Molecular Consistency of Oral Polio Vaccine
Replication of viruses leads to emergence of mutations and their content in viral populations can increase by selection depending on growth conditions. Some of these mutations have deleterious effect on vaccine safety, such as neurovirulent reversions in the 5′-UTR of attenuated Sabin strains of poliovirus. Their content in vaccine batches must be tightly controlled during vaccine manufacture to ensure safety of the product. This chapter describes a quantitative molecular procedure called mutant analysis by PCR and restriction enzyme cleavage (MAPREC) that is used to monitor content of neurovirulent revertants in Ora...
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A Rapid Method for Engineering Recombinant Polioviruses or Other Enteroviruses
The cloning of large enterovirus RNA sequences is labor-intensive because of the frequent instability in bacteria of plasmidic vectors containing the corresponding cDNAs. In order to circumvent this issue we have developed a PCR-based method that allows the generation of highly modified or chimeric full-length enterovirus genomes. This method relies on fusion PCR which enables the concatenation of several overlapping cDNA amplicons produced separately. A T7 promoter sequence added upstream the fusion PCR products allows its transcription into infectious genomic RNAs directly in transfected cells constitutively expressing t...
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Generation of Infectious Poliovirus with Altered Genetic Information from Cloned cDNA
The effect of specific genetic alterations on virus biology and phenotype can be studied by a great number of available assays. The following method describes the basic protocol to generate infectious poliovirus with altered genetic information from cloned cDNA in cultured cells. (Source: Springer protocols feed by Infectious Diseases)
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Phylogenetic Analysis of Poliovirus Sequences
Comparative genomic sequencing is a major surveillance tool in the Polio Laboratory Network. Due to the rapid evolution of polioviruses (~1 % per year), pathways of virus transmission can be reconstructed from the pathways of genomic evolution. Here, we describe three main phylogenetic methods; estimation of genetic distances, reconstruction of a maximum-likelihood (ML) tree, and estimation of substitution rates using Bayesian Markov chain Monte Carlo (MCMC). The data set used consists of complete capsid sequences from a survey of poliovirus sequences available in GenBank. (Source: Springer protocols feed by Infectious Diseases)
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Isolation and Characterization of Vaccine-Derived Polioviruses, Relevance for the Global Polio Eradication Initiative
Stool specimens were collected from children with acute flaccid paralysis (AFP) and their contacts, and viral isolation was performed according to standard procedures. If the specimens tested positive for poliovirus, then intratypic differentiation (ITD) methods were performed on the viral isolates to determine whether the poliovirus isolates were wild or of vaccine origin, these include a poliovirus diagnostic ITD real-time PCR method and a vaccine-derived poliovirus (VDPV) screening real-time PCR method. (Source: Springer protocols feed by Infectious Diseases)
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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...
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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...
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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...
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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...
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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...
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