Recovery Of Poliovirus Research Articles

Recovery of enteroviruses and poliovirus in Harare sewage using the bag-mediated filtration system at the introduction of the inactivated polio vaccine in Zimbabwe

Environmental surveillance is a sensitive method for detecting circulating virus in the absence of clinical cases and is important for monitoring progress for poliovirus (PV) eradication. This study used the bag-mediated filtration system (BMFS) to determine PV and enterovirus (EV) prevalence in sewage at the transition from oral polio vaccine type 2 (OPV2) use to inactivated polio vaccine (IPV) use in Zimbabwe, and examined the correlation between environmental surveillance results and vaccination coverage of OPV. A total of 18 BMFS samples from 6 sampling sites were analysed for the presence of EV and PV via direct RT-qPCR, direct ITD (intratypic differentiation), and the WHO algorithm. EV prevalence in Harare wastewater was 88.9% (16/18) using direct RT-PCR, 61.1% (11/18) using direct ITD, and 77.8% (14/18) using the WHO algorithm. Of the 18 samples analysed using the WHO algorithm, 10 samples (55.6%) were positive for Sabin-like PV type 3 (SL3). Of these 10 samples, 2 were also positive for non-polio enteroviruses (NPEV), resulting in a total of 6 (33.3%) samples positive for NPEV and 4 negative. The sensitivity of isolation in detecting EVs in sewage was 92.9% when comparing direct RT-qPCR results to the WHO algorithm. Using direct ITD, two high-density, low-income sampling sites were negative for SL3 and one low-density, high-income sampling point was negative for SL3 using the WHO algorithm. There was a strong association between relative EV concentration and the number of OPV3 vaccine recipients (r = 0.8590; p = 0.0284) in sampled areas. This study demonstrated the ability of BMFS to detect PVs circulating in Harare wastewater at the beginning of the OPV–IPV switch and can be used to monitor potential reintroduction of wild PV or vaccine-derived PVs from endemic areas.

Direct detection of polioviruses using a recombinant poliovirus receptor.

Polioviruses are positive-sense, single-stranded RNA picornaviruses and the principal cause of poliomyelitis. Global poliovirus surveillance has relied on poliovirus isolation in cells, which may take a minimum of 10 days, involves maintaining two cell lines, and propagates virus in high titers. With eradication underway, a major objective of the Global Polio Eradication Initiative (GPEI) is to develop culture-independent detection of polioviruses as an alternative method to complement the current virus isolation technique. A culture-independent method on poliovirus-positive stool suspensions was assessed with commercially available recombinant soluble poliovirus receptor (PVR) coupled to Histidine (His) tags. Viral RNA was screened by quantitative real-time reverse transcription PCR using the poliovirus intratypic differentiation kit. Poliovirus recovery was optimized with PVR-His–tagged protein and buffers supplemented with polyethylene glycol. To validate the poliovirus-PVR–His tag purification assay, 182 poliovirus-positive stools of programmatic importance were parallel tested against the GPLN-accepted virus isolation method. The PVR-His tag enrichment method detected poliovirus in 164 of 171 poliovirus-positive stools, whereas the virus isolation method misidentified 38 stools as poliovirus-negative (McNemar χ2 p<0.0001). Using this method in combination with RNA extraction, viral RNA recovery increased and showed similar (WPV1) or higher (Sabin 1) sensitivity than the World Health Organization accredited variation of the virus isolation method. The PVR-His enrichment method could be a viable addition to poliovirus surveillance; similar methods have the potential to capture other human pathogens such as EV71 using an appropriate soluble His tag receptor.

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. II. Total Culturable Virus Assay

A standardized method is required when national studies on virus occurrence in environmental and drinking waters utilize multiple analytical laboratories. The U.S Environmental Protection Agency’s (USEPA) Method 1615 was developed with the goal of providing such a standard for measuring Enterovirus and Norovirus in these waters. Virus is concentrated from water using an electropositive filter, eluted from the filter surface with beef extract, and then concentrated further using organic flocculation. Herein we present the protocol from Method 1615 for filter elution, secondary concentration, and measurement of total culturable viruses. A portion of the concentrated eluate from each sample is inoculated onto ten replicate flasks of Buffalo Green Monkey kidney cells. The number of flasks demonstrating cytopathic effects is used to quantify the most probable number (MPN) of infectious units per liter. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. Laboratories must meet defined performance standards. Method 1615 was evaluated by examining virus recovery from reagent-grade and ground waters seeded with Sabin poliovirus type 3. Mean poliovirus recoveries with the total culturable assay were 111% in reagent grade water and 58% in groundwaters.

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. II. Total Culturable Virus Assay.

A standardized method is required when national studies on virus occurrence in environmental and drinking waters utilize multiple analytical laboratories. The U.S Environmental Protection Agency’s (USEPA) Method 1615 was developed with the goal of providing such a standard for measuring Enterovirus and Norovirus in these waters. Virus is concentrated from water using an electropositive filter, eluted from the filter surface with beef extract, and then concentrated further using organic flocculation. Herein we present the protocol from Method 1615 for filter elution, secondary concentration, and measurement of total culturable viruses. A portion of the concentrated eluate from each sample is inoculated onto ten replicate flasks of Buffalo Green Monkey kidney cells. The number of flasks demonstrating cytopathic effects is used to quantify the most probable number (MPN) of infectious units per liter. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. Laboratories must meet defined performance standards. Method 1615 was evaluated by examining virus recovery from reagent-grade and ground waters seeded with Sabin poliovirus type 3. Mean poliovirus recoveries with the total culturable assay were 111% in reagent grade water and 58% in groundwaters.

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a standardized method for use in multiple analytical laboratories during monitoring period 3 of the Unregulated Contaminant Monitoring Rule. Herein we present the protocol for extraction of viral ribonucleic acid (RNA) from water sample concentrates and for quantitatively measuring enterovirus and norovirus concentrations using reverse transcription-quantitative PCR (RT-qPCR). Virus concentrations for the molecular assay are calculated in terms of genomic copies of viral RNA per liter based upon a standard curve. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. The method has been evaluated by examining virus recovery from ground and reagent grade waters seeded with poliovirus type 3 and murine norovirus as a surrogate for human noroviruses. Mean poliovirus recoveries were 20% in groundwaters and 44% in reagent grade water. Mean murine norovirus recoveries with the RT-qPCR assay were 30% in groundwaters and 4% in reagent grade water.

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR.

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a standardized method for use in multiple analytical laboratories during monitoring period 3 of the Unregulated Contaminant Monitoring Rule. Herein we present the protocol for extraction of viral ribonucleic acid (RNA) from water sample concentrates and for quantitatively measuring enterovirus and norovirus concentrations using reverse transcription-quantitative PCR (RT-qPCR). Virus concentrations for the molecular assay are calculated in terms of genomic copies of viral RNA per liter based upon a standard curve. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. The method has been evaluated by examining virus recovery from ground and reagent grade waters seeded with poliovirus type 3 and murine norovirus as a surrogate for human noroviruses. Mean poliovirus recoveries were 20% in groundwaters and 44% in reagent grade water. Mean murine norovirus recoveries with the RT-qPCR assay were 30% in groundwaters and 4% in reagent grade water.

Effectiveness of Poliovirus Concentration and Recovery from Treated Wastewater by Two Electropositive Filter Methods

Enteric viruses are often present in low numbers in various water matrices. Virus sampling therefore involves multiple concentration steps to condense large samples down to small volumes for detection by cell culture or molecular assays. The NanoCeram® Virus Sampler has been demonstrated to be effective for the recovery of viruses from tap water, surface waters, and seawater. The goal of this study was to evaluate a new method using NanoCeram® filters for the recovery of poliovirus 1 (PV-1) from treated wastewater. Activated sludge effluent samples were spiked with PV-1 and concentrated in side-by-side tests by two methods: (1) NanoCeram® filtration, elution with sodium polyphosphate buffer, secondary concentration via centrifugal ultrafiltration; and (2) 1MDS filtration, elution with beef extract, secondary concentration via organic flocculation. The virus retention and elution efficiencies did not differ significantly between the two methods. In contrast, the secondary concentrate volume was smaller for the NanoCeram® method (8.4 vs. 30 mL) and the secondary concentration efficiencies were different between the two methods with 98 % for centrifugal ultrafiltration (NanoCeram® and 45 % for organic flocculation (1MDS). The overall method efficiencies were significantly different (P ≤ 0.05) with the NanoCeram® method yielding a 57 % and the 1MDS a 23 % virus recovery. In addition, there appeared to be less interference with viral detection via polymerase chain reaction with the NanoCeram® concentrates. This NanoCeram® method therefore is able to efficiently recover PV-1 from large volumes of wastewater and may serve as an inexpensive alternative to the standard 1MDS filter method for such applications.

Application of Acidic Elution to Virus Concentration Using Electropositive Filters

The effect of the type and pH of an elution solution on the recovery of poliovirus from water by a virus concentration method using an electropositive filter was evaluated. The experimental results obtained indicated the potential usefulness of H2SO4 (pH 1.5-3.5) as a novel solution for virus elution.

Development and Evaluation of EPA Method 1615 for Detection of Enterovirus and Norovirus in Water

The U.S. EPA developed a sample concentration and preparation assay in conjunction with the total culturable virus assay for concentrating and measuring culturable viruses in source and drinking waters as part of the Information Collection Rule (ICR) promulgated in 1996. In an effort to improve upon this method, the U.S. EPA recently developed Method 1615: Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Method 1615 uses a culturable virus assay with reduced equipment and labor costs compared to the costs associated with the ICR virus method and introduces a new molecular assay for the detection of enteroviruses and noroviruses by reverse transcription-quantitative PCR. In this study, we describe the optimization of several new components of the molecular assay and examine virus recovery from ground, reagent-grade, and surface water samples seeded with poliovirus type 3 and murine norovirus. For the culturable virus and molecular assays, mean poliovirus recovery using the complete method was 58% and 20% in groundwater samples, 122% and 39% using low-titer spikes in reagent-grade water, 42% and 48% using high-titer spikes in reagent-grade water, and 11% and 10% in surface water with high turbidity, respectively. Murine norovirus recovery by the molecular assay was 30% in groundwater samples, less than 8% in both low- and high-titer spikes in reagent-grade water, and 6% in surface water with high turbidity. This study demonstrates the effectiveness of Method 1615 for use with groundwater samples and highlights the need for further research into its effectiveness with surface water.

Evaluation of hollow-fiber ultrafiltration primary concentration of pathogens and secondary concentration of viruses from water

Tangential flow hollow-fiber ultrafiltration (HFUF) was evaluated for virus and Cryptosporidium parvum concentration from water. Recovery of viruses at a low filtration rate was found to be significantly greater than at a higher filtration rate, with the recoveries of bacteriophage MS2 at high and low filtration rates shown to be 64.7% and 98.7%, respectively. Poliovirus recoveries from tap water were similar to MS2, with recoveries of 62.9% and 104.5% for high and low filtration rates, respectively. C. parvum, which was only tested at high filtration rates, had an average recovery was 105.1%. In addition to the optimization of the primary concentration technique, this study also compared several secondary concentration procedures. The highest recovery (89.5%) of poliovirus from tap water concentrates was obtained when a beef extract–celite method was used and the virus was eluted from the celite with phosphate buffered saline, pH 9.0. When HFUF primary concentration and the optimal secondary concentration methods were combined, an average recovery of 97.0 ± 35.6% or 89.3 ± 19.3%, depending on spike level, was achieved for poliovirus. This study demonstrated that HFUF primary concentration method is effective at recovering MS2, poliovirus and C. parvum from large volumes of water and that beef extract–celite method is an effective secondary concentration method for the poliovirus tested.

Development of an Effective Method for Recovery of Viral Genomic RNA from Environmental Silty Sediments for Quantitative Molecular Detection

Nine approaches to recover viral RNA from environmental silty sediments were newly developed and compared to quantify RNA viruses in sediments using molecular methods. Four of the nine approaches employed direct procedures for extracting RNA from sediments (direct methods), and the remaining five approaches used indirect methods wherein viral particles were recovered before RNA extraction. A direct method using an SDS buffer with EDTA to lyse viral capsids in sediments, phenol-chloroform-isoamyl alcohol to extract RNA, isopropanol to concentrate RNA, and magnetic beads to purify RNA resulted in the highest rate of recovery (geometric mean of 11%, with a geometric standard deviation of 0.02; n = 7) of poliovirus 1 (PV1) inoculated in an environmental sediment sample. The direct method exhibiting the highest rate of PV1 recovery was applied to environmental sediment samples. One hundred eight sediment samples were collected from the Takagi River, Miyagi, Japan, and its estuary from November 2007 to April 2009, and the genomic RNAs of enterovirus and human norovirus in these samples were quantified by reverse transcription (RT)-quantitative PCR (qPCR). The human norovirus genome was detected in one sample collected at the bay, although its concentration was below the quantification limit. Meanwhile, the enterovirus genome was detected in two samples at the river mouth and river at concentrations of 8.6 × 10(2) and 2.4 × 10(2) copies/g (wet weight), respectively. This is the first report to obtain quantitative data for a human pathogenic virus in a river and in estuarine sediments using RT-qPCR.

Polyaluminum chloride-enhanced concentration efficiency of poliovirus and f2phage from sewage water

To enhance the recovery of f(2) bacteriophage and poliovirus by an established method based on the adsorption to and elution from positively-charged Al(OH)(3)-treated silica gel. Polyaluminum Chloride (PAC) was added to water samples to neutralize the negatively charged materials, which can reduce virus recovery by providing a competing adsorption mode on the media surface. Using this improved process (PAC 30 mg l(-1), pH 6.5, temperature 20 approximately 30 degrees C), the recoveries of Poliovirus I and f(2) from small-volume sewage (100 ml) were 110.76 +/- 36.0% and 92.06 +/- 8.65%, respectively (P < 0.05 vs. traditional methods). Recovery from a 20-L volume of sewage averaged 85.65 +/- 4.43% for f(2) and 88.73 +/- 9.76% for poliovirus, significantly higher than the recoveries in the traditional methods (P < 0.05). PAC could enhance concentration efficiency of poliovirus and f(2) phage from sewage water. This method should significantly improve the recovery of viruses from sewage.

Mitochondrial DNA and retroviral RNA analyses of archival oral polio vaccine (OPV CHAT) materials: evidence of macaque nuclear sequences confirms substrate identity

Inoculation of live experimental oral poliovirus vaccines (OPV CHAT) during the 1950s in central Africa has been proposed to account for the introduction of HIV into human populations. For this to have occurred, it would have been necessary for chimpanzee rather than macaque kidney epithelial cells to have been included in the preparation of early OPV materials. Theoretically, this could have led to contamination with a progenitor of HIV-1 derived from a related simian immunodeficiency virus of chimpanzees (SIV CPZ). In this article we present further detailed analyses of two samples of OPV, CHAT 10A-11 and CHAT 6039/Yugo, which were used in early human trials of poliovirus vaccination. Recovery of poliovirus by culture techniques confirmed the biological viability of the vaccines and sequence analysis of poliovirus RNA specifically identified the presence of the CHAT strain. Independent nested sets of oligonucleotide primers specific for HIV-1/SIV CPZ and HIV-2/SIV MAC/SIV SM phylogenetic lineages, respectively, indicated no evidence of HIV/SIV RNA in either vaccine preparation, at a sensitivity of 100 RNA equivalents/ml. Analysis of cellular substrate by the amplification of two distinct regions of mitochondrial DNA (D-loop control region and 12S ribosomal sequences) revealed no evidence of chimpanzee cellular sequences. However, this approach positively identified rhesus and cynomolgus macaque DNA for the CHAT 10A-11 and CHAT 6039/Yugo vaccine preparations, respectively. Analysis of multiple clones of mtDNA 12S rDNA indicated a relatively high number of nuclear mitochondrial DNA sequences (numts) in the CHAT 10A-11 material, but confirmed the macaque origin of cellular substrate used in vaccine preparation. These data re-inforce earlier findings on this topic providing no evidence to support the contention that poliovirus vaccination was responsible for the introduction of HIV into humans and sparking the AIDS pandemic.

Detection of noroviruses in tap water in Japan by means of a new method for concentrating enteric viruses in large volumes of freshwater.

A virus concentration method using a cation-coated filter was developed for large-volume freshwater applications. Poliovirus type 1 (LSc 2ab Sabin strain) inoculated into 40 ml of MilliQ (ultrapure) water was adsorbed effectively to a negatively charged filter (Millipore HA, 0.45- micro m pore size) coated with aluminum ions, 99% (range, 81 to 114%) of which were recovered by elution with 1.0 mM NaOH (pH 10.8) following an acid rinse with 0.5 mM H(2)SO(4) (pH 3.0). More than 80% poliovirus recovery yields were obtained from 500-ml, 1,000-ml, and 10-liter MilliQ water samples and from tap water samples. This method, followed by TaqMan PCR detection, was applied to determine the presence of noroviruses in tap water in Tokyo, Japan. In a 14-month survey, 4 (4.1%) and 7 (7.1%) of 98 tap water samples (100 to 532 liters) contained a detectable amount of noroviruses of genotype 1 and genotype 2, respectively. This method was proved to be useful for surveying the occurrence of enteric viruses, including noroviruses, in large volumes of freshwater.

Detection of human enteric viruses in stream water with RT-PCR and cell culture

A multiplex RT-PCR method was used to measure virus occurrence at five stream water sites that span a range of hydroclimatic, water-quality, and land-use characteristics. The performance of the molecular method was evaluated in comparison with traditional cell culture and Escherichia coli membrane filtration assays. The study incorporated multiple quality controls and included a control for virus recovery during the sampling procedure as well as controls to detect potentially false-negative and false-positive data. Poliovirus recovery ranged from 16 to 65% and was variable, even in samples collected within the same stream. All five sites were positive for viruses by both molecular and cell culture-based virus assays. Enteroviruses, reoviruses, rotaviruses, and hepatitis A viruses were detected, but the use of the quality controls proved critical for interpretation of the molecular data. All sites showed evidence of faecal contamination, and culturable viruses were detected in four samples that would have met the US Environmental Protection Agency's recommended E. coli guideline for safe recreational water.

Comparative study of poliovirus recovery techniques from mussel tissues

Four poliovirus extraction and three poliovirus concentration techniques designed for the study of mussel tissues were compared. The recovered infectious viral particles were confirmed by inoculation to cell cultures. The protocol that provided optimum results involved extraction with a solution of NaCl 0.3 M/beef extract 7%/freon at pH 7.5, followed by a concentration with polyethylene glycol 6000. The resulting concentration rate ranged from 80 to 136%. The amount of infectious viral particles thus recovered was 1×10 5 MPNCU g −1 of tissue (approximately) for an initial concentration of seawater of 1.5×10 3 MPNCU ml −1.

Round robin investigation of glass wool method for poliovirus recovery from drinking water and sea water

This study was initiated by the AFNOR water microbiology working group to evaluate the performance of the glass wool method for virus recovery. Its reliability was tested with drinking and sea water by respectively nine and thirteen laboratories. In both trials, six were actively involved in water virology research, one was designated as a central laboratory, the others had no experience in virological practices. Analysis of reproducibility and repeatability according to NF-ISO 5725-2 were realized. For drinking waters (24 assays), the average recovery efficiency was 72%, mean standard deviations: repeatability 12.4%; reproducibility 33.6%; inter-laboratories 21%. For sea waters (39 assays), the average recovery efficiency was 75% and the mean standard deviations 6.9%, 17.9% and 11% respectively.

Round robin investigation of glass wool method for poliovirus recovery from drinking water and sea water

This study was initiated by the AFNOR water microbiology working group to evaluate the performance of the glass wool method for virus recovery. Its reliability was tested with drinking and sea water by respectively nine and thirteen laboratories. In both trials, six were actively involved in water virology research, one was designated as a central laboratory, the others had no experience in virological practices. Analysis of reproducibility and repeatability according to NF-ISO 5725-2 were realized. For drinking waters (24 assays), the average recovery efficiency was 72%, mean standard deviations: repeatability 12.4%; reproducibility 33.6%; inter-laboratories 21%. For sea waters (39 assays), the average recovery efficiency was 75% and the mean standard deviations 6.9%, 17.9% and 11% respectively.

Evaluation of an extraction-precipitation method for recovering hepatitis A virus and poliovirus from hardshell clams (mercenaria mercenaria)

Epidemiological evidence of shellfish mediated transmission of enteric viral diseases and the inability of bacterial indicators to reliably predict viral contamination of shellfish highlight the risks associated with shellfish consumption. This problem suggests the need for direct virological examination of shellfish, and a number of methods have been developed to recover enteroviruses from clams and other bivalve molluscs. These methods have been evaluated primarily for recovery of poliovirus, and there has been little or no testing of their ability to recover hepatitis A virus (HAV). The present study was done to evaluate a new extraction-precipitation method for recovery of both poliovirus and HAV from experimentally contaminated hard clams. In the method 50-gram clam samples are homogenized after supplementing with saline and beef extract and then extracted with an equal volume of fluorocarbon. After centrifugation, viruses in the supernatant are precipitated with 12% polyethylene glycol, resuspended in buffered saline and assayed in cell cultures. From a series of replicate trials using clams seeded with test viruses, this method gave poliovirus and HAV recoveries of about 53 and 22%, respectively. Although the recovery of HAV was lower than that of poliovirus, it was considered adequate for further application. In subsequent studies using clams experimentally contaminated by virus uptake from water, the recovery method detected naturally accumulated HAV and poliovirus in clams. The method evaluated in this study is one of the few documented as capable of detecting HAV as well as enteroviruses in hard clams.

Evaluation of an extraction-precipitation method for recovering hepatitis A virus and poliovirus from hardshell clams (Mercenaria Mercenaria)

Epidemiological evidence of shellfish mediated transmission of enteric viral diseases and the inability of bacterial indicators to reliably predict viral contamination of shellfish highlight the risks associated with shellfish consumption. This problem suggests the need for direct virological examination of shellfish, and a number of methods have been developed to recover enteroviruses from clams and other bivalve molluscs. These methods have been evaluated primarily for recovery of poliovirus, and there has been little or no testing of their ability to recover hepatitis A virus (HAV). The present study was done to evaluate a new extraction-precipitation method for recovery of both poliovirus and HAV from experimentally contaminated bard clams. In the method 50-gram clam samples are homogenized after supplementing with saline and beef extract and then extracted with an equal volume of fluorocarbon. After centrifugation, viruses in the supernatant are precipitated with 12% polyethylene glycol, resuspended in buffered saline and assayed in cell cultures. From a series of replicate trials using clams seeded with test viruses, this method gave poliovirus and HAV recoveries of about 53 and 22%, respectively. Although the recovery of HAV was lower than that of poliovirus, it was considered adequate for further application. In subsequent studies using clams experimentally contaminated by virus uptake from water, the recovery method detected naturally accumulated HAV and poliovirus in clams. The method evaluated in this study is one of the few documented as capable of detecting HAV as well as enteroviruses in hard clams.