Identification of vaccine-derived polioviruses using dual-stage real-time RT-PCR

Kilpatrick, David R.; Ching, Karen; Iber, Jane; Chen, Qi; Yang, Sen; De, Lina; Williams, Alice; Mandelbaum, Mark; Sun, Hong; Oberste, M. Steven; Kew, Olen M.

doi:10.1016/j.jviromet.2013.11.017

Cited by 35 publications

(33 citation statements)

References 25 publications

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“…1). Isolates that were categorized as "Sabin-like" by intratypic differentiation using real-time reverse transcription-PCR (32,33) were included in the study. After samples that contained mixtures of multiple poliovirus strains (about 15% of all available samples) were excluded, the initial sample set was reduced to 241 monotypic samples from 238 unique individuals.…”

Section: Methodsmentioning

confidence: 99%

Sabin Vaccine Reversion in the Field: a Comprehensive Analysis of Sabin-Like Poliovirus Isolates in Nigeria

Famulare

Chang

Iber

et al. 2016

J Virol

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To assess the dynamics of genetic reversion of live poliovirus vaccine in humans, we studied molecular evolution in Sabin-like poliovirus isolates from Nigerian acute flaccid paralysis cases obtained from routine surveillance. We employed a novel modeling approach to infer substitution and recombination rates from whole-genome sequences and information about poliovirus infection dynamics and the individual vaccination history. We confirmed observations from a recent vaccine trial that VP1 substitution rates are increased for Sabin-like isolates relative to the rate for the wild type due to increased nonsynonymous substitution rates. We also inferred substitution rates for attenuating nucleotides and confirmed that reversion can occur in days to weeks after vaccination. We combine our observations for Sabin-like virus evolution with the molecular clock for VP1 of circulating wild-type strains to infer that the mean time from the initiating vaccine dose to the earliest detection of circulating vaccine-derived poliovirus (cVDPV) is 300 days for Sabin-like virus type 1, 210 days for Sabin-like virus type 2, and 390 days for Sabin-like virus type 3. Phylogenetic relationships indicated transient local transmission of Sabin-like virus type 3 and, possibly, Sabin-like virus type 1 during periods of low wild polio incidence. Comparison of Sabin-like virus recombinants with known Nigerian vaccine-derived poliovirus recombinants shows that while recombination with non-Sabin enteroviruses is associated with cVDPV, the recombination rates are similar for Sabin isolate-Sabin isolate and Sabin isolate-non-Sabin enterovirus recombination after accounting for the time from dosing to the time of detection. Our study provides a comprehensive picture of the evolutionary dynamics of the oral polio vaccine in the field. IMPORTANCEThe global polio eradication effort has completed its 26th year. Despite success in eliminating wild poliovirus from most of the world, polio persists in populations where logistical, social, and political factors have not allowed vaccination programs of sustained high quality. One issue of critical importance is eliminating circulating vaccine-derived polioviruses (cVDPVs) that have properties indistinguishable from those of wild poliovirus and can cause paralytic disease. cVDPV emerges due to the genetic instability of the Sabin viruses used in the oral polio vaccine (OPV) in populations that have low levels of immunity to poliovirus. However, the dynamics responsible are incompletely understood because it has historically been difficult to gather and interpret data about evolution of the Sabin viruses used in OPV in regions where cVDPV has occurred. This study is the first to combine whole-genome sequencing of poliovirus isolates collected during routine surveillance with knowledge about the intrahost dynamics of poliovirus to provide quantitative insight into polio vaccine evolution in the field. O ver the last 50 years, mass vaccination campaigns with oral polio vaccine (OPV) have been a key fa...

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Section: Methodsmentioning

confidence: 99%

Sabin Vaccine Reversion in the Field: a Comprehensive Analysis of Sabin-Like Poliovirus Isolates in Nigeria

Famulare

Chang

Iber

et al. 2016

J Virol

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“…Step RT-qPCR ToughMix kit (Quanta) with the same primer/probe sets as provided in the poliovirus diagnostic rRT-PCR kit (US CDC) and dualstage thermal cycling (RT reaction/inactivation at 42°C for 45 min and 95°C for 3 min and then 15 cycles of PCR at 95°C for 24 s, 44°C for 30 s, and 60°C for 24 s, followed by 40 cycles of PCR at 95°C for 24 s, 47°C for 30 s, and 65°C for 24 s) (12).…”

Section: Methodsmentioning

confidence: 99%

“…With nondegenerate primers, highly sensitive rRT-PCR systems to detect PV vaccine strains have been developed (6,11). VDPV strains could also be detected with a recently developed rRT-PCR system (12). However, alternative methodologies remain to be developed to obtain the nucleotide sequence of the entire VP1 coding region from quite small amounts of PV in stool extracts, which could be Ͻ10 0.5 CCID 50 or about 1,000 viral genome copies in 50 l of stool extract (5,6).…”

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confidence: 99%

Development of an Efficient Entire-Capsid-Coding-Region Amplification Method for Direct Detection of Poliovirus from Stool Extracts

et al. 2015

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Laboratory diagnosis has played a critical role in the Global Polio Eradication Initiative since 1988, by isolating and identifying poliovirus (PV) from stool specimens by using cell culture as a highly sensitive system to detect PV. In the present study, we aimed to develop a molecular method to detect PV directly from stool extracts, with a high efficiency comparable to that of cell culture. We developed a method to efficiently amplify the entire capsid coding region of human enteroviruses (EVs) including PV. cDNAs of the entire capsid coding region (3.9 kb) were obtained from as few as 50 copies of PV genomes. PV was detected from the cDNAs with an improved PV-specific real-time reverse transcription-PCR system and nucleotide sequence analysis of the VP1 coding region. For assay validation, we analyzed 84 stool extracts that were positive for PV in cell culture and detected PV genomes from 100% of the extracts (84/84 samples) with this method in combination with a PV-specific extraction method. PV could be detected in 2/4 stool extract samples that were negative for PV in cell culture. In PV-positive samples, EV species C viruses were also detected with high frequency (27% [23/86 samples]). This method would be useful for direct detection of PV from stool extracts without using cell culture. Laboratory diagnosis has played a critical role in the Global Polio Eradication Initiative since 1988, by detecting and identifying poliovirus (PV). To date, PV has been detected by isolating the virus, using cell culture systems, from stool specimens from acute flaccid paralysis (AFP) cases, including polio and other paralysis cases, in the World Health Organization (WHO) Global Polio Laboratory Network (1, 2). The advantages of the cell culture-based procedure are (i) minimal equipment requirements, (ii) high sensitivity (detection limit of 1 infectious unit containing 50 to 1,000 virions) (3), and, importantly, (iii) biological amplification of PV to high titers (about 10 6 50% cell culture infectious dose [CCID 50 ] or 10 8 to 10 9 viral genome copies per l of cell lysate) for subsequent nucleotide sequence analysis of the capsid protein VP1 coding region, which is required for final identification and molecular epidemiological analysis of PV strains. Sequencing is essential for classifying PV isolates into vaccine strains, wild-type strains, and circulating vaccine-derived PV (VDPV) strains and for determining the necessity of additional vaccination plans by identifying virus reservoirs, along with molecular epidemiological methods (4). A major disadvantage of the cell culture system is the timeliness of reporting; it takes 10 days to confirm that a sample is negative for PV (2). To improve the efficiency of PV detection and identification, including the timeliness, the development of methods for direct detection of PV has been encouraged by WHO (http://www.polioeradication.org /Research/Grantsandcollaboration.aspx). Such direct detection methods must provide efficient PV detection, comparable to that of cell cultur...

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“…Purified PCR products were subjected to direct sequencing using the Big Dye terminator v3.1 kit (Applied Biosystems) and an ABI Prism 3140 automated sequencer (Applied Biosystems). The sequencing of each amplicon was performed in both directions using PCR primers as reported [7,14,15].…”

Section: Rna Extraction and Genomic Amplification And Sequencing For mentioning

confidence: 99%

“…cVDPVs differ from the majority of vaccine-related isolates by having genetic properties consistent with prolonged replication or transmission [4]. Based on molecular characteristics, it has been reported that there was evidence of multiple genetic recombination events between poliovirus and other non-polio enteroviruses, particularly coxsackievirus [7]. cVDPVs are operationally defined as OPVrelated isolates having >1% nucleotide (nt) sequence divergence from the parental OPV strain (usually determined by sequencing the genome region encoding the major viral surface protein, VP1) [6].…”

Section: Introductionmentioning

confidence: 99%

Disease Trends and Characteristics of Circulating Vaccine derived Poliovirus Isolated in the National Reference Polio Laboratory from the Democratic Republic of Congo

Kavunga¹,

Simulundu²,

Ahuka³

et al. 2017

Arch Med

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Background: Despite the fact that wild poliovirus (WPV) circulation has not been detected for the past six years, cases of poliomyelitis caused by pathogenic circulating vaccine-derived polioviruses (cVDPVs) have been identified in the Democratic Republic of Congo (DRC). Most cVDPVs implicated in polio outbreaks are recombinants between mutated strains of the oral poliovaccine (OPV) and other human enteroviruses (HEV) of species C (HEV-C).The aim of this study was to determine the disease trend and the genetic makeup of cVDPVs strains isolated in DRC from 2008 to 2016.

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Identification of vaccine-derived polioviruses using dual-stage real-time RT-PCR

Cited by 35 publications

References 25 publications

Sabin Vaccine Reversion in the Field: a Comprehensive Analysis of Sabin-Like Poliovirus Isolates in Nigeria

Sabin Vaccine Reversion in the Field: a Comprehensive Analysis of Sabin-Like Poliovirus Isolates in Nigeria

Development of an Efficient Entire-Capsid-Coding-Region Amplification Method for Direct Detection of Poliovirus from Stool Extracts

Disease Trends and Characteristics of Circulating Vaccine derived Poliovirus Isolated in the National Reference Polio Laboratory from the Democratic Republic of Congo

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