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

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

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

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

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

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

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

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

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

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

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

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

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

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

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