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.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)
Source: Springer protocols feed by Infectious Diseases - December 31, 2015 Category: Infectious Diseases Source Type: news
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...
Source: Springer protocols feed by Infectious Diseases - December 31, 2015 Category: Infectious Diseases Source Type: news
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...
Source: Springer protocols feed by Infectious Diseases - December 31, 2015 Category: Infectious Diseases Source Type: news
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...
Source: Springer protocols feed by Infectious Diseases - December 31, 2015 Category: Infectious Diseases Source Type: news
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...
Source: Springer protocols feed by Infectious Diseases - December 31, 2015 Category: Infectious Diseases Source Type: news
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...
Source: Springer protocols feed by Infectious Diseases - December 31, 2015 Category: Infectious Diseases Source Type: news
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...
Source: Springer protocols feed by Infectious Diseases - December 31, 2015 Category: Infectious Diseases Source Type: news
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 ...
Source: Springer protocols feed by Infectious Diseases - December 31, 2015 Category: Infectious Diseases Source Type: news
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)
Source: Springer protocols feed by Infectious Diseases - December 31, 2015 Category: Infectious Diseases Source Type: news
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)
Source: Springer protocols feed by Infectious Diseases - December 31, 2015 Category: Infectious Diseases Source Type: news
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...
Source: Springer protocols feed by Infectious Diseases - December 31, 2015 Category: Infectious Diseases Source Type: news
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 ...
Source: Springer protocols feed by Infectious Diseases - December 15, 2015 Category: Infectious Diseases Source Type: news
