-
Food and Environmental Virology Mar 2020The bag-mediated filtration system (BMFS) was developed to facilitate poliovirus (PV) environmental surveillance, a supplement to acute flaccid paralysis surveillance in...
The bag-mediated filtration system (BMFS) was developed to facilitate poliovirus (PV) environmental surveillance, a supplement to acute flaccid paralysis surveillance in PV eradication efforts. From April to September 2015, environmental samples were collected from four sites in Nairobi, Kenya, and processed using two collection/concentration methodologies: BMFS (> 3 L filtered) and grab sample (1 L collected; 0.5 L concentrated) with two-phase separation. BMFS and two-phase samples were analyzed for PV by the standard World Health Organization poliovirus isolation algorithm followed by intratypic differentiation. BMFS samples were also analyzed by a cell culture independent real-time reverse transcription polymerase chain reaction (rRT-PCR) and an alternative cell culture method (integrated cell culture-rRT-PCR with PLC/PRF/5, L20B, and BGM cell lines). Sabin polioviruses were detected in a majority of samples using BMFS (37/42) and two-phase separation (32/42). There was statistically more frequent detection of Sabin-like PV type 3 in samples concentrated with BMFS (22/42) than by two-phase separation (14/42, p = 0.035), possibly due to greater effective volume assayed (870 mL vs. 150 mL). Despite this effective volume assayed, there was no statistical difference in Sabin-like PV type 1 and Sabin-like PV type 2 detection between these methods (9/42 vs. 8/42, p = 0.80 and 27/42 vs. 32/42, p = 0.18, respectively). This study demonstrated that BMFS can be used for PV environmental surveillance and established a feasible study design for future research.
Topics: Environmental Monitoring; Feasibility Studies; Filtration; Fresh Water; Humans; Kenya; Poliomyelitis; Poliovirus
PubMed: 31679104
DOI: 10.1007/s12560-019-09412-1 -
Journal of Applied Microbiology Dec 2020A continuous quench-flow (CQF) reactor was developed to collect samples at the reaction times of less than one second. The reactor is applied to determine ozone...
AIMS
A continuous quench-flow (CQF) reactor was developed to collect samples at the reaction times of less than one second. The reactor is applied to determine ozone disinfection kinetics of poliovirus and to study whether EMA-qPCR can assess the viral infectivity after ozone disinfection.
METHODS
Ozone disinfection of poliovirus was conducted in the developed CQF, and the disinfection kinetics were tested in the range of 0·7-5·0 s at ozone concentration of 0·08 and 0·25 mg l . Inactivation, damage on viral genome and damage on capsid integrity were determined by plaque assay, quantitative reverse transcription polymerase chain reaction (RT-qPCR) and ethidium monoazide treatment coupled with RT-qPCR (EMA-qPCR), respectively.
RESULTS
By using CQF, 2·18 and 2·76 log reductions were observed at the reaction time of 0·7 s and ozone concentration of 0·08 and 0·25 mg l , respectively, followed by tailing. Ozone disinfection kinetics of poliovirus 1 were better fit by the efficiency factor Hom model than by the Chick-Watson model, or the modified Chick-Watson model. Kinetics observed were similar between RT-qPCR and EMA-qPCR assays at the reaction times of <2·0 s and ozone concentrations of 0·08 and 0·25 mg l . At reaction times > 5 s, viral concentration evaluated by EMA-qPCR was reduced in comparison to stable RT-qPCR results. Both assays still underestimated the virus inactivation.
CONCLUSION
The simple developed reactor can be used to investigate viral ozone disinfection kinetics and to elucidate inactivation characteristics or mechanisms at very short exposure times.
SIGNIFICANCE AND IMPACT OF THE STUDY
The developed CQF reactor is beneficial for better understanding of virus inactivation by ozone, and the reactor can be used to better elucidate disinfection kinetics and mechanisms for future research. This work constitutes an important contribution to the existing knowledge of the application and limitation of the EMA/PMA-qPCR to assess virus infectivity after ozone disinfection.
Topics: Azides; Capsid; Disinfection; Genome, Viral; Kinetics; Ozone; Poliovirus; Real-Time Polymerase Chain Reaction; Viral Plaque Assay; Virus Inactivation
PubMed: 32681543
DOI: 10.1111/jam.14787 -
The Journal of Infectious Diseases Mar 2018Environmental surveillance (ES) is a sensitive method for detecting human enterovirus (HEV) circulation, and it is used worldwide to support global polio eradication. We...
BACKGROUND
Environmental surveillance (ES) is a sensitive method for detecting human enterovirus (HEV) circulation, and it is used worldwide to support global polio eradication. We describe a novel ES approach using next-generation sequencing (NGS) to identify HEVs in sewage samples collected in London, United Kingdom, from June 2016 to May 2017.
METHODS
Two different methods were used to process raw sewage specimens: a 2-phase aqueous separation system and size exclusion by filtration and centrifugation. HEVs were isolated using cell cultures and analyzed using NGS.
RESULTS
Type 1 and 3 vaccine-like poliovirus (PV) strains were detected in samples collected from September 2016 through January 2017. NGS analysis allowed us to rapidly obtain whole-genome sequences of PV and non-PV HEV strains. As many as 6 virus strains from different HEV serotypes were identified in a single cell culture flask. PV isolates contained only a small number of mutations from vaccine strains commonly seen in early isolates from vaccinees.
CONCLUSIONS
Our ES setup has high sensitivity for polio and non-PV HEV detection, generating nearly whole-genome sequence information. Such ES systems provide critical information to assist the polio eradication endgame and contribute to the improvement of our understanding of HEV circulation patterns in humans.
Topics: Environmental Monitoring; Genome, Viral; Humans; Nucleic Acid Amplification Techniques; Poliovirus; Poliovirus Vaccines; Sewage; United Kingdom
PubMed: 29309594
DOI: 10.1093/infdis/jix667 -
Advances in Virus Research 2008Poliomyelitis has long served as a model for studies of viral pathogenesis, but there remain many important gaps in our understanding of this disease. It is the intent... (Comparative Study)
Comparative Study Review
Poliomyelitis has long served as a model for studies of viral pathogenesis, but there remain many important gaps in our understanding of this disease. It is the intent of this review to highlight these residual but important questions, in light of a possible future moratorium on research with polioviruses. Salient questions include: (1) What cells in the gastrointestinal tract are initially infected and act as the source of excreted virus? (2) What is the receptor used by mouse-adapted strains of poliovirus and how can some polioviruses use both mouse and primate receptors? (3) What determines species differences in susceptibility of the gastrointestinal tract to polioviruses? Why cannot PVR transgenic mice be infected by the natural enteric route? (4) Why are neuroadapted polioviruses unable to infect nonneural cells? (5) What is the role of postentry blocks in replication as determinants of neurovirulence? (6) What route(s) does poliovirus take to enter the central nervous system and how does it cross the blood-brain barrier? (7) Why does poliovirus preferentially attack lower motor neurons in contrast to many other neuronal types within the central nervous system? (8) Does cellular immunity play any role in recovery from acute infection or in vaccine-induced protection? (9) In which cells does poliovirus persist in patients with gamma-globulin deficiencies? (10) Is there any evidence that poliovirus genomes can persist in immunocompetent hosts? (11) Why has type 2 poliovirus been eradicated while types 1 and 3 have not? (12) Can transmission of vaccine-derived polioviruses be prevented with inactivated poliovirus vaccine? (13) What is the best strategy to control and eliminate vaccine-derived polioviruses?
Topics: Animals; Antibodies, Viral; Central Nervous System; Global Health; Humans; Immunization Programs; Mice; Models, Animal; Poliomyelitis; Poliovirus; Poliovirus Vaccines; Receptors, Virus; Tropism; Viral Vaccines; Virus Replication
PubMed: 18585526
DOI: 10.1016/S0065-3527(08)00001-8 -
Journal of Clinical Microbiology Aug 2020Global poliovirus surveillance involves virus isolation from stool and environmental samples, intratypic differential (ITD) by PCR, and sequencing of the VP1 region to...
Global poliovirus surveillance involves virus isolation from stool and environmental samples, intratypic differential (ITD) by PCR, and sequencing of the VP1 region to distinguish vaccine (Sabin), vaccine-derived, and wild-type polioviruses and to ensure an appropriate response. This cell culture algorithm takes 2 to 3 weeks on average between sample receipt and sequencing. Direct detection of viral RNA using PCR allows faster detection but has traditionally faced challenges related to poor sensitivity and difficulties in sequencing common samples containing poliovirus and enterovirus mixtures. We present a nested PCR and nanopore sequencing protocol that allows rapid (<3 days) and sensitive direct detection and sequencing of polioviruses in stool and environmental samples. We developed barcoded primers and a real-time analysis platform that generate accurate VP1 consensus sequences from multiplexed samples. The sensitivity and specificity of our protocol compared with those of cell culture were 90.9% (95% confidence interval, 75.7% to 98.1%) and 99.2% (95.5% to 100.0%) for wild-type 1 poliovirus, 92.5% (79.6% to 98.4%) and 98.7% (95.4% to 99.8%) for vaccine and vaccine-derived serotype 2 poliovirus, and 88.3% (81.2% to 93.5%) and 93.2% (88.6% to 96.3%) for Sabin 1 and 3 poliovirus alone or in mixtures when tested on 155 stool samples in Pakistan. Variant analysis of sequencing reads also allowed the identification of polioviruses and enteroviruses in artificial mixtures and was able to distinguish complex mixtures of polioviruses in environmental samples. The median identity of consensus nanopore sequences with Sanger or Illumina sequences from the same samples was >99.9%. This novel method shows promise as a faster and safer alternative to cell culture for the detection and real-time sequencing of polioviruses in stool and environmental samples.
Topics: Environmental Monitoring; Feces; Humans; Nanopore Sequencing; Poliomyelitis; Poliovirus; Poliovirus Vaccine, Oral
PubMed: 32611795
DOI: 10.1128/JCM.00920-20 -
Proceedings of the National Academy of... Dec 1997Genetic recombination of plus-strand RNA viruses is an important process for promoting genetic variation. By using genetically marked poliovirus RNAs, we have...
Genetic recombination of plus-strand RNA viruses is an important process for promoting genetic variation. By using genetically marked poliovirus RNAs, we have demonstrated that genetic recombination can occur in a cell-free system that generates infective virus from added poliovirus RNA. Recombinant polioviruses were isolated, and the region of crossing over was roughly mapped. Recombinants could be isolated even under conditions where the yield of viruses from one of the parental RNAs was depressed to levels comparable to or less than the yield of recombinant viruses, an observation suggesting that only one of the recombining RNAs needs to be replication-competent. The generation of poliovirus recombinants in a cell-free system offers new possibilities for studying recombination and evolution of RNA viruses.
Topics: Biological Evolution; Cell-Free System; Crossing Over, Genetic; DNA, Viral; Genes, Viral; Genetic Markers; Genetic Variation; Humans; In Vitro Techniques; Phenotype; Poliovirus; Polymerase Chain Reaction; RNA, Viral; Recombination, Genetic; Virus Replication
PubMed: 9391105
DOI: 10.1073/pnas.94.25.13786 -
Antimicrobial Agents and Chemotherapy Nov 2012V-073, a small-molecule capsid inhibitor originally developed for nonpolio enterovirus indications is considerably more potent against polioviruses. All poliovirus...
V-073, a small-molecule capsid inhibitor originally developed for nonpolio enterovirus indications is considerably more potent against polioviruses. All poliovirus isolates tested to date (n = 45), including wild, vaccine, vaccine-derived, and laboratory strains, are susceptible to the antiviral capsid inhibitor V-073. We grew poliovirus in the presence of V-073 to allow for the identification of variants with reduced susceptibility to the drug. Sequence analysis of 160 independent resistant variants (80 isolates of poliovirus type 1, 40 isolates each of types 2 and 3) established that V-073 resistance involved a single amino acid change in either of two virus capsid proteins, VP1 (67 of 160 [42%]) or VP3 (93 of 160 [58%]). In resistant variants with a VP1 change, the majority (53 of 67 [79%]) exhibited a substitution of isoleucine at position 194 (equivalent position 192 in type 3) with either methionine or phenylalanine. Of those with a VP3 change, alanine at position 24 was replaced with valine in all variants (n = 93). The resistance phenotype was relatively stable upon passage of viruses in cell culture in the absence of drug. Single-step growth studies showed no substantial differences between drug-resistant variants and the virus stocks from which they were derived, while the resistant viruses were generally more thermally labile than the corresponding drug-susceptible parental viruses. These studies provide a foundation from which to build a greater understanding of resistance to antiviral compound V-073.
Topics: Amino Acid Substitution; Amino Acids; Animals; Antiviral Agents; Capsid; Capsid Proteins; Cell Line; Drug Resistance, Viral; Halogenated Diphenyl Ethers; Humans; Macaca mulatta; Mutation; Phenyl Ethers; Poliovirus; Viral Plaque Assay
PubMed: 22890765
DOI: 10.1128/AAC.00539-12 -
Methods in Molecular Biology (Clifton,... 2016Virologic surveillance is essential to the success of the World Health Organization initiative to eradicate poliomyelitis. Molecular methods have been used to detect...
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 design of nucleic acid-based reagents. To accommodate the wide variability and rapid evolution of poliovirus genomes, degenerate codon positions on the template were matched to mixed-base or deoxyinosine residues on both the primers and the TaqMan™ probes. Additional assays distinguish between Sabin vaccine strains and non-Sabin strains. This chapter also describes the use of generic poliovirus specific primers, along with degenerate and inosine-containing primers, for routine VP1 sequencing of poliovirus isolates. These primers, along with nondegenerate serotype-specific Sabin primers, can also be used to sequence individual polioviruses in mixtures.
Topics: Base Sequence; Humans; Poliomyelitis; Poliovirus; Polymerase Chain Reaction; RNA, Viral; Sequence Analysis, RNA
PubMed: 26983735
DOI: 10.1007/978-1-4939-3292-4_9 -
The Journal of Infectious Diseases Nov 2014A paralytic poliomyelitis outbreak occurred in Namibia in 2006, almost exclusively among adults. Nineteen cases were virologically confirmed as due to wild poliovirus...
A paralytic poliomyelitis outbreak occurred in Namibia in 2006, almost exclusively among adults. Nineteen cases were virologically confirmed as due to wild poliovirus type 1 (WPV1), and 26 were classified as polio compatible. Eleven deaths occurred among confirmed and compatible cases (24%). Of the confirmed cases, 97% were aged 15-45 years, 89% were male, and 71% lived in settlement areas in Windhoek. The virus was genetically related to a virus detected in 2005 in Angola, which had been imported earlier from India. The outbreak is likely due to immunity gaps among adults who were inadequately vaccinated during childhood. This outbreak underscores the ongoing risks posed by poliovirus importations, the importance of maintaining strong acute flaccid paralysis surveillance even in adults, and the need to maintain high population immunity to avoid polio outbreaks in the preeradication period and outbreaks due to vaccine-derived polioviruses in the posteradication era.
Topics: Adolescent; Adult; Age Factors; Case-Control Studies; Child; Child, Preschool; Disease Outbreaks; Female; Humans; Infant; Infant, Newborn; Male; Middle Aged; Namibia; Poliomyelitis; Poliovirus; Sex Distribution; Topography, Medical; Young Adult
PubMed: 25316855
DOI: 10.1093/infdis/jiu069 -
The Pan African Medical Journal 2014Global poliomyelitis eradication initiative relies on (i) laboratory based surveillance of acute flaccid surveillance (AFP) to monitor the circulation of wild poliovirus...
INTRODUCTION
Global poliomyelitis eradication initiative relies on (i) laboratory based surveillance of acute flaccid surveillance (AFP) to monitor the circulation of wild poliovirus in a population, and (ii) vaccination to prevent its diffusion. However, as poliovirus can survive in the environment namely in sewage, environmental surveillance (ES) is of growing importance as the eradication target is close. This study aimed to assess polioviruses and non polio enteroviruses circulation in sewage drains covering a significant population of Dakar.
METHODS
From April 2007 to May 2013, 271 specimens of raw sewage were collected using the grab method in 6 neighborhoods of Dakar. Samples were processed to extract and concentrate viruses using polyethylene glycol and Dextran (two-phase separation method). Isolation of enteroviruses was attempted in RD, L20B and Hep2 cell lines. Polioviruses were identified by RT-PCR and Elisa. Non Polio Enteroviruses (NPEVs) were identified by RT-PCR and microneutralisation tests.
RESULTS
Polioviruses and NPEVs were respectively detected in 34,3% and 42,8% sewage samples. No wild poliovirus neither circulating vaccine-derived Poliovirus (cVDPV) was detected. Neutralization assays have identified 49 non polio enteroviruses that were subsequently classified in 13 serotypes belonging to HEV-A (22, 4%), HEV-B (12, 24%), HEV-C (26, 53%) and HEV-D (6, 12%) species.
CONCLUSION
This study is the first documentation of enteroviruses environmental detection in Senegal. It shows the usefulness of environmental surveillance for indirect monitoring of the circulation and distribution of enteroviruses in the community.
Topics: Cell Line; Enterovirus; Environmental Monitoring; Humans; Poliovirus; Reverse Transcriptase Polymerase Chain Reaction; Senegal; Sewage
PubMed: 25848458
DOI: 10.11604/pamj.2014.19.243.3538