Poliovirus serotype-specific VP1 sequencing primers

Kilpatrick, David R.; Iber, Jane; Chen, Qi; Ching, Karen; Yang, Sen; De, Lina; Mandelbaum, Mark; Emery, Brian; Campagnoli, Raymond P.; Burns, Cara C.; Kew, Olen M.

doi:10.1016/j.jviromet.2011.03.020

Cited by 67 publications

(47 citation statements)

References 16 publications

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“…We also performed type-specific PV ITD rRT-PCR and detected each type of PV (90% for the PV-positive samples [79/84 samples] and 50% for the PV-negative samples [2/4 samples]). Next, we performed nucleotide sequence analysis of the VP1 coding region starting with the second PCR with the Y7/Q8 primer set, which is currently used for PV nucleotide sequence analysis in the Global Polio Laboratory Network (10). With second PCR products obtained with the Y7/Q8 primers, PV VP1 sequences were obtained with the Y7 primer for only 68% of the samples (60/88 samples).…”

Section: Resultsmentioning

confidence: 99%

“…cDNA of the VP1 coding region was amplified with 1.0 l of unpurified first RT-PCR product as the template by using a Qiagen OneStep RT-PCR kit (Qiagen) with primers Y7 (5=-GGGTTTGTGTCAGCCTGTAATGA-3=) and Q8 (5=-AAGAGGTCTCTRTTCCACAT-3=) or with primers Y7 and PV1A (5=-TTIAIIGCRTGICCRTTRTT-3=), according to the manufacturer's instructions (total of 50 l of the second PCR solution) (10). PCR conditions consisted of a denaturing step at 95°C for 15 min, 35 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 1 min, and an additional elongation step at 72°C for 7 min.…”

Section: Methodsmentioning

confidence: 99%

“…However, the sensitivity of pan-PV PCR has been a major challenge [e.g., 25,000 copies for detection of PV1(Sabin)]. This low sensitivity might reflect intrinsic properties of degenerate primers and probes that use inosine and modified nucleotides to detect diverse nucleotide sequences conserved among the 3 types of PV in the VP1 coding region (7)(8)(9)(10). With nondegenerate primers, highly sensitive rRT-PCR systems to detect PV vaccine strains have been developed (6,11).…”

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confidence: 99%

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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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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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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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“…Total nucleic acid was extracted from 200 l of each virus isolate using a MagNA Pure compact nucleic acid isolation kit and a MagNA Pure compact automated extractor (Roche Applied Science, Indianapolis, IN). For each isolate, two fragments of the genome were amplified using primers 5= NCR (5=-AAGCA GGCTTTAAAACAGCTCTGGGGTT-3=) and 3= NCR (5=-TCTCCGAAT TAAAGAAAAATTTACCCCTACA-3=) and primer Internal Y7 (5=-GGG TTTGTGTCAGCCTGTAATGA-3=) and Internal Q8(367) (5=-AAGAGG TCTCTRTTCCACAT-3=) (34). Barcoded sequencing libraries were prepared using Nextera DNA sample preparation kits (Epicentre, Madison, WI).…”

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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“…Poliovirus is isolated from the samples by infecting recombinant mouse cell line L20B according to WHO guidelines [6,7]. Individual plaques propagated in tube cultures are then analysed by real-time polymerase chain reaction (PCR) using United States (US) Centers for Disease Control and Prevention (CDC) analytical kits designed for identification of polioviruses and differentiation between vaccine and wild types [8], followed by characterisation by genome sequencing [9].…”

Section: Epidemiological Investigation Environmental Surveillancementioning

confidence: 99%

Insidious reintroduction of wild poliovirus into Israel, 2013

et al. 2013

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Israel was certified as polio-free country in June 2002, along with the rest of the World Health Organization European Region. Some 11 years later, wild-type polio virus 1 (WPV1) was isolated initially from routine sewage samples collected between 7 and 13 April 2013 in two cities in the Southern district. WPV1-specific analysis of samples indicated WPV1 introduction into that area in early February 2013. National supplementary immunisation with oral polio vaccine has been ongoing since August 2013.

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Poliovirus serotype-specific VP1 sequencing primers

Cited by 67 publications

References 16 publications

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

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

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

Insidious reintroduction of wild poliovirus into Israel, 2013

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