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

Abstract

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...

Poliovirus Studies during the Endgame of the Polio Eradication Program

“…The lower limit of PV detection in PVpositive stool extract by the cell culture method is about ＜ 10 0.5 CCID 50 (50z cell culture infectious dose) or 850-1,300 copies of PV genomes in 50 mL of stool extracts (62,63). The stool extracts may contain PV genomes derived from`non-viable' PVs (33,63). For detection, intratypic differentiation, nucleotide sequencing of VP1-coding region of PV, amplification of PV genomes is essential by using cell culture method (i.e., virus isolation) or by RT-PCR.…”

“…For detection, intratypic differentiation, nucleotide sequencing of VP1-coding region of PV, amplification of PV genomes is essential by using cell culture method (i.e., virus isolation) or by RT-PCR. The most sensitive direct detection method for PV to date utilizes PVRsensitized beads (partial purification of PV from stool extract) and an efficient whole capsid-coding region amplification method (amplification of the entire capsid-coding region of Enterovirus C species, including PV), which allow detection of PV with sensitivity equal to or higher than that of the cell culture method, along with VP1 sequencing for intratypic differentiation (33,63). The challenges remained in the implementation include optimization of the condition of the method in terms of the effectiveness and cost, and also the discussion on the target cost of the surveillance.…”

“…Enumerating all of the milestones reached in PV studies is far beyond the scope of this review, however, this list would include sequencing of the PV genome (9,10), the development of reverse genetics for the virus (11), elucidation of the crystal structure of the virion (12), identification of the internal ribosomal entry site (IRES) (13), identification of the PV receptor (PVR, CD155) (14,15), development of a transgenic mouse model of poliomyelitis (16,17), identification of attenuation determinants in oral polio vaccine (OPV) strains (18,19), identification of drug candidates (20,21), development of a cell-free replication system (22), identification of cis-acting replication elements (23,24), identification of circulating vaccine-derived PVs (cVDPVs) (25), and identification of host targets for drug candidates (26)(27)(28)(29)(30). PV studies in the post-vaccine era have produced valuable tools and information essential to the eradication program, including nucleotide sequences of PV and other enteroviruses (9,10,31), which have been used to design primers for current reverse transcription (RT)-PCR systems for PV detection (32,33); neurovirulence determinants of PV that are used for quality control of vaccines and evaluations of environmental isolates (18,34,35); PV-sensitive murine cell line (L20B) (14), which is a cell line that expresses the PVR and is currently used for PV surveillance because of its high specificity for PV infection, a PVR-transgenic mouse (TgPVR21) model as an alternative to monkeys used in neurovirulence tests for PV vaccines (36), and candidate compounds for antivirals, including V-073 (37).…”

“…WHO is calling for collaborative studies, mainly for effective implementation of the vaccine campaign, including biological studies that can support polio eradication (50), including regional serosurveillance, environmental surveillance (51), direct detection of PV (33), and OPV/IPV implementation (52). However, the overall picture of projects supported might not be clear, because not all these studies have been made public as of 2016.…”

Rapid and sensitive direct detection and identification of poliovirus from stool and environmental surveillance samples using nanopore sequencing

“…70 71 Direct molecular detection of polioviruses in stool or ES samples using RT-PCR could allow 72 rapid detection of poliovirus without the need for cell-culture. However, quantitative RT-PCR 73 capable of amplifying all polioviruses through use of degenerate (pan-poliovirus) primers has 74 historically reported low sensitivity compared with cell-culture (3). Use of serotype or strain-75 specific primers improves sensitivity, but multiplex assays with many primers would be required 76 to identify all polioviruses and the resulting products are too short for informative sequencing 77 (4).…”