Rapid and sensitive on-site detection of SARS-CoV-2 RNA from environmental surfaces using portable laboratory devices.

IMPORTANCE: This study presents the development of a highly sensitive on-site method for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA on various surfaces, including doorknobs and tables. Identifying SARS-CoV-2 RNA on these surfaces can be crucial in guiding decision-making for implementing non-pharmaceutical interventions, such as zoning strategies, improving ventilation, maintaining physical distancing, and promoting increased hand hygiene practices. Moreover, the on-site detection system can facilitate the swift initiation of mitigation responses in non-laboratory settings, including long-term care facilities and schools. The protocols established in this study offer a comprehensive approach for achieving both sensitivity and rapidity in on-site SARS-CoV-2 RNA detection. Furthermore, since the RT-qPCR assay serves as the gold standard for detecting viral RNAs, the developed protocol holds potential for application to other viruses, including enteroviruses and noroviruses.

Monitoring Enteroviruses and SARS-CoV-2 in Wastewater Using the Polio Environmental Surveillance System in Japan.

In the global strategy for polio eradication, environmental surveillance (ES) has been established worldwide to monitor polioviruses. In addition, nonpolio enteroviruses are simultaneously isolated from wastewater under this ES program. Hence, ES can be used to monitor enteroviruses in sewage to supplement clinical surveillance. In response to the coronavirus disease 2019 (COVID-19) pandemic, we also monitored severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in sewage using the polio ES system in Japan. Enterovirus and SARS-CoV-2 were detected in sewage from January 2019 to December 2021 and from August 2020 to November 2021, respectively. Enterovirus species such as echoviruses and coxsackieviruses were frequently detected by ES in 2019, indicating the circulation of these viruses. After the onset of the COVID-19 pandemic, sewage enterovirus detection and related patient reports were notably reduced in 2020 to 2021, suggesting changes in the hygiene behaviors of the human population in response to the pandemic. Our comparative experiment with a total of 520 reverse transcription-quantitative PCR (RT-qPCR) assays for SARS-CoV-2 detection demonstrated that the solid-based method had a significantly higher detection rate than that of the liquid-based method (24.6% and 15.9%, respectively). Moreover, the resulting RNA concentrations were correlated with the number of new COVID-19 cases (Spearman's r = 0.61). These findings indicate that the existing polio ES system can be effectively used for enterovirus and SARS-CoV-2 sewage monitoring using different procedures such as virus isolation and molecular-based detection. IMPORTANCE Long-term efforts are required to implement surveillance programs for the ongoing COVID-19 pandemic, and they will be required even in the postpandemic era. We adopted the existing polio environmental surveillance (ES) system for SARS-CoV-2 sewage monitoring in Japan as a practical and cost-effective approach. Moreover, the ES system routinely detects enteroviruses from wastewater and, therefore, can be used for enterovirus monitoring. The liquid fraction of the sewage sample is used for poliovirus and enterovirus detection, and the solid fraction can be used for SARS-CoV-2 RNA detection. The present study demonstrates how the existing ES system can be used for monitoring enteroviruses and SARS-CoV-2 in sewage.

Dynamic changes in polioviruses identified by environmental surveillance in Guangzhou, 2009-2021.

Polio cases can be missed by acute flaccid paralysis (AFP) case surveillance alone, emphasizing the importance of environmental surveillance (ES). In this study, to investigate the serotype distribution and epidemiological trends of poliovirus (PV), we characterized PV isolated from domestic sewage in Guangzhou City, Guangdong Province, China from 2009 to 2021. A total of 624 sewage samples were collected from the Liede Sewage Treatment Plant, and the positive rates of PV and non-polio enteroviruses were 66.67% (416/624) and 78.37% (489/624), respectively. After sewage sample treatment, each sewage sample was inoculated in six replicate tubes of three cell lines, and 3370 viruses were isolated during the 13-year surveillance period. Among these, 1086 isolates were identified as PV, including type 1 PV (21.36%), type 2 PV (29.19%), and type 3 PV (49.48%). Based on VP1 sequences, 1057 strains were identified as Sabin-like, 21 strains were high-mutant vaccines, and eight strains were vaccine-derived poliovirus (VDPV). The numbers and serotypes of PV isolates in sewage were influenced by the vaccine switch strategy. After type 2 OPV was removed from the trivalent oral PV (OPV) vaccine and a bivalent OPV (bOPV) was adopted in May 2016, the last type 2 PV strain was isolated from sewage, with no detection thereafter. Type 3 PV isolates increased significantly and became the dominant serotype. Before and after the second vaccine switch in January 2020, that is, from the first dose of IPV and second-fourth doses of bOPV to the first two doses of IPV and third-fourth doses of bOPV, there was also a statistical difference in PV positivity rates in sewage samples. Seven type 2 VDPVs and one type 3 VDPV were identified in sewage samples in 2009-2021, and phylogenetic analysis indicated that all VDPVs isolated from ES in Guangdong are newly discovered VDPVs, different from VDPV previously discovered in China, and were classified as ambiguous VDPV. It is noteworthy that no VDPV cases were reported in AFP case surveillance in the same period. In conclusion, continued PV ES in Guangzhou since April 2008 has been a useful supplement to AFP case surveillance, providing an important basis for evaluating the effectiveness of vaccine immunization strategies. ES improves early detection, prevention, and control; accordingly, this strategy can curb the circulation of VDPVs and provide a strong laboratory basis for maintaining a polio-free status.

Prevalence of Non-Polio Enteroviruses in the Sewage of Guangzhou City, China, from 2013 to 2021.

Continuous surveillance of enteroviruses (EVs) in urban domestic sewage can timely reflect the circulation of EVs in the environment and crowds, and play a predictive and early warning role in EV-related diseases. To better understand the long-term epidemiological trends of circulating EVs and EV-related diseases, we conducted a 9-year (2013 to 2021) surveillance study of non-polio EVs (NPEVs) in urban sewage in Guangzhou city, China. After concentrating and isolating the viruses from the sewage samples, NPEVs were detected and molecular typing was performed. Twenty-one different NPEV serotypes were identified. The most isolated EVs were echovirus 11 (E11), followed by coxsackievirus (CV) B5, E6, and CVB3. EV species B prevailed in sewage samples, but variations in the annual frequency of different serotypes were also observed in different seasons, due to spatial and temporal factors. E11 and E6 were detected continuously before 2017, and the number of isolates was relatively stable during the surveillance period. However, after their explosive growth in 2018 and 2019, their numbers suddenly decreased significantly. CVB3 and CVB5 had alternating trends; CVB5 was most frequently detected in 2013 to 2014 and 2017 to 2018, while CVB3 was most frequently detected in 2015 to 2016 and 2020 to 2021. Phylogenetic analysis showed that at least two different transmission chains of CVB3 and CVB5 were prevalent in Guangzhou City. Our results show that in the absence of a comprehensive and systematic EV-related disease surveillance system in China, environmental surveillance is a powerful and effective tool to strengthen and further investigate the invisible transmission of EVs in the population. IMPORTANCE This study surveilled urban sewage samples from north China for 9 years to monitor enteroviruses. Samples were collected, processed, and viral identification and molecular typing were performed. We detected 21 different non-polio enteroviruses (NPEVs) with yearly variations in prevalence and peak seasons. In addition, this study is very important for understanding the epidemiology of EVs during the COVID-19 pandemic, as the detection frequency and serotypes of EVs in sewage changed considerably around 2020. We believe that our study makes a significant contribution to the literature because our results strongly suggest that environmental surveillance is an exceptionally important tool, which can be employed to detect and monitor organisms of public health concern, which would otherwise be missed and under-reported by case-based surveillance systems alone.

[Polio Environmental Surveillance and Its Application to SARS-CoV-2 Detection].

The polio eradication program, launched in 1988, has successfully decreased the number of poliomyelitis patients worldwide. However, in areas with immunization gaps where oral polio vaccine coverage has dropped, outbreaks of more virulent vaccine-derived polioviruses (VDPVs) have become a threat to public health. In Japan, inactivated polio vaccine replaced oral polio vaccine as the routine immunization in 2012. Polio environmental surveillance (ES) has been conducted nationwide since 2013 to efficiently monitor the wild type poliovirus or VDPV, which may be imported from overseas. ES may also be utilized to detect other viruses in stool samples. We propose a method of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection based on the polio ES network, and establish a procedure to detect fragments of SARS-CoV-2 genome in wastewater solids. Our findings suggest that polio ES can be used to simultaneously monitor SARS-CoV-2 RNA fragments in sewage waters.

Efficient detection of SARS-CoV-2 RNA in the solid fraction of wastewater.

In the context of the coronavirus disease 2019 (COVID-19) pandemic, environmental surveillance for the detection of SARS-CoV-2 has become increasingly important. Studies have demonstrated that the SARS-CoV-2 RNA is present in the feces of infected individuals; further, its presence in wastewater has been reported. However, an optimized method for its detection in sewage has not yet been adequately investigated. Therefore, in this study, the efficient detection of SARS-CoV-2 RNA in the solid fraction of wastewater was investigated using two quantitative PCR assays. In particular, wastewater samples were collected from a manhole located in the commercial district of a metropolitan region in Japan, where COVID-19 is highly prevalent, and two wastewater treatment plants (WWTPs). The samples were concentrated using four separate methods, namely, electronegative membrane adsorption, polyethylene glycol precipitation, ultrafiltration, and solid precipitation. Each method revealed a significant concentration of pepper mild mottle virus (PMMoV) RNA, which is an indicator virus for wastewater. As expected, non-enveloped PMMoV RNA was enriched in the supernatant fraction such that relatively low concentrations were detected in the solid fraction of the wastewater samples. In contrast, higher SARS-CoV-2 RNA concentrations were consistently detected in the solid fractions compared with the supernatant fractions based on the other methods that were investigated in this study. Spearman's correlation tests showed that the SARS-CoV-2 RNA concentrations in wastewater samples from the WWTP were significantly correlated with the number of COVID-19 cases recorded during the data collection period. These results demonstrate that viral recovery from the solid fraction is an effective method for SARS-CoV-2 RNA surveillance in an aqueous environment.

Environmental Surveillance Can Dynamically Track Ecological Changes in Enteroviruses.

Environmental surveillance can be used to trace enteroviruses shed from human stool using a sewer network that is independent of symptomatic or asymptomatic infection. In this study, the local transmission of enteroviruses was analyzed using two wastewater treatment plants, which were relatively close to each other (15 km), designated as sentinels. Influent was collected at both sentinels once a month from 2013 to 2016, and viruses were isolated. Using neutralizing tests with type-specific polyclonal antisera and molecular typing, 933 isolates were identified as enteroviruses. Our results showed that the frequency of virus isolation varied for each serotype at the two sentinels in a time-dependent manner. Because echovirus 11 (Echo11) and coxsackievirus B5 isolates showed a high frequency and were difficult to distinguish, they were further grouped into various lineages based on the VP1 amino acid sequences. The prevalence of each lineage was visualized using multidimensional scaling. The results showed that Echo11 isolates of the same lineage were isolated continuously, similar to coxsackievirus B5 isolates of three lineages. Conversely, Echo1, Echo13, Echo18, Echo19, Echo20, Echo29, and Echo33 were isolated only once each. Our findings suggested that if an enterovirus is imported into the population, it may result in small-scale transmission, whereas if there are initially many infected individuals, it may be possible for the virus to spread to a wide area, beyond the local community, over time. In addition, our findings could provide insights into risk assessment of transmission for importation of poliovirus in polio-free countries and regions.IMPORTANCE In this study, we showed that environmental enterovirus surveillance can be used to monitor the propagation of nonpolio enteroviruses in addition to poliovirus detection. Since epidemiological studies of virus transmission based on the past were performed using specimens from humans, there were limitations to research design, such as specimen collection for implementation on a large-scale target population. However, environmental monitoring can dynamically track the ecological changes in enteroviruses in the region by monitoring viruses in chronological order and targeting the population within the area by monitoring viruses over time. We observed differences in the transmission of echovirus 11 and coxsackievirus B5 in the region according to lineage in a time-dependent manner and with a multidimensional scaling pattern.

One-year Survey of human enteroviruses from sewage and the factors affecting virus adsorption to the suspended solids.

This study described the results of environmental enterovirus surveillance conducted in Shandong Province of China in 2013. Altogether 39 sewage samples were collected and 873 enterovirus isolates (including 334 polioviruses) belonging to 22 serotypes were obtained. Echovirus (E) -7, coxsackievirus (CV) -B5, E-11, E-6, and E-3 were the most commonly detected non-polio enterovirus serotypes, and phylogeny of E-7 and CV-B5 was described. The numbers of isolates of different serotypes from sewage supernatant were compared with those from the solids. Interestingly, dramatic divergence was observed between the supernatant and solids origin for the serotypes of E-3 and E-6, which were prone to the solids and supernatant, respectively. A following adsorption test with E-3 and E-6 added sewage specimens confirmed the different preference. Furthermore, the adsorption of Sabin poliovirus type 1 to the solids under different conditions was investigated, and the results showed that acid medium, cold temperature, and high solids concentration facilitated the viral adsorption to the solids, whereas change of virus titer did not influence the proportion of adsorption. These results highlighted the importance of combining the enterovirus isolates from the supernatant and solids together in environmental surveillance so as to better understand the local circulation of different serotypes.

Environmental surveillance of poliovirus in sewage water around the introduction period for inactivated polio vaccine in Japan.

Environmental virus surveillance was conducted at two independent sewage plants from urban and rural areas in the northern prefecture of the Kyushu district, Japan, to trace polioviruses (PVs) within communities. Consequently, 83 PVs were isolated over a 34-month period from April 2010 to January 2013. The frequency of PV isolation at the urban plant was 1.5 times higher than that at the rural plant. Molecular sequence analysis of the viral VP1 gene identified all three serotypes among the PV isolates, with the most prevalent serotype being type 2 (46%). Nearly all poliovirus isolates exhibited more than one nucleotide mutation from the Sabin vaccine strains. During this study, inactivated poliovirus vaccine (IPV) was introduced for routine immunization on 1 September 2012, replacing the live oral poliovirus vaccine (OPV). Interestingly, the frequency of PV isolation from sewage waters declined before OPV cessation at both sites. Our study highlights the importance of environmental surveillance for the detection of the excretion of PVs from an OPV-immunized population in a highly sensitive manner, during the OPV-to-IPV transition period.

Elucidation of echovirus 30's origin and transmission during the 2012 aseptic meningitis outbreak in Guangdong, China, through continuing environmental surveillance.

An aseptic meningitis outbreak occurred in Luoding City of Guangdong, China, in 2012, and echovirus type 30 (ECHO30) was identified as the major causative pathogen. Environmental surveillance indicated that ECHO30 was detected in the sewage of a neighboring city, Guangzhou, from 2010 to 2012 and also in Luoding City sewage samples (6/43, 14%) collected after the outbreak. In order to track the potential origin of the outbreak viral strains, we sequenced the VP1 genes of 29 viral strains from clinical patients and environmental samples. Sequence alignments and phylogenetic analyses based on VP1 gene sequences revealed that virus strains isolated from the sewage of Guangzhou and Luoding cities matched well the clinical strains from the outbreak, with high nucleotide sequence similarity (98.5% to 100%) and similar cluster distribution. Five ECHO30 clinical strains were clustered with the Guangdong environmental strains but diverged from strains from other regions, suggesting that this subcluster of viruses most likely originated from the circulating virus in Guangdong rather than having been more recently imported from other regions. These findings underscore the importance of long-term, continuous environmental surveillance and genetic analysis to monitor circulating enteroviruses.

Molecular epidemiology and recombination of human enteroviruses from AFP surveillance in Yunnan, China from 2006 to 2010.

The study represents the genetic overview of non-polio enteroviruses (NPEV) isolated from acute flaccid paralysis (AFP) cases in Yunnan Province from 2006 to 2010. Molecular typing based on VP1 nucleotide sequence was carried out on 98 NPEV isolates, and 33 serotypes were identified. EV-B was detected most frequently with an overall prevalence of 71.4%, followed by EV-A (18.4%) and EV-C (10.2%). No EV-D was identified. NPEV positive rate was higher in children <3 years of age and in summer and autumn months. Clinically, 68.4% patients presented with fever, and 16 cases (16.3%) were classified as Guillain-Barré syndrome, followed by myositis (13.3%). The phylogenetic analysis on the VP1 and 3D regions of prevalent serotypes provided evidence for recombination events among them. EV-A71, an important pathogen previously demonstrated to be associated with paralysis, had also been detected (n = 8) in this study and they all belonged to genotype C4. Great genetic divergence between Yunnan isolates and strains from other regions of the world was revealed. The findings of the study are of great importance for further research on molecular evolution of EV under the circumstance of no specialized EV surveillance system in China.

Environmental surveillance of human enteroviruses in Shandong Province, China, 2008 to 2012: serotypes, temporal fluctuation, and molecular epidemiology.

Environmental surveillance is an effective approach in investigating the circulation of polioviruses (PVs) and other human enteroviruses (EVs) in the population. The present report describes the results of environmental surveillance conducted in Shandong Province, China, from 2008 to 2012. A total of 129 sewage samples were collected, and 168 PVs and 1,007 nonpolio enteroviruses (NPEVs) were isolated. VP1 sequencing and typing were performed on all isolates. All PV strains were Sabin-like, with the numbers of VP1 substitutions ranging from 0 to 7. The NPEVs belonged to 19 serotypes, and echovirus 6 (E6), E11, coxsackievirus B3 (CVB3), E3, E12, and E7 were the six main serotypes, which accounted for 18.3%, 14.8%, 14.5%, 12.9%, 9.0%, and 5.7% of NPEVs isolated, respectively. Typical summer-fall peaks of NPEV were observed in the monthly distribution of isolation, and an epidemic pattern of annual circulation was revealed for the common serotypes. Phylogenetic analysis was performed on environmental CVB3 and E3 strains with global reference strains and local strains from aseptic meningitis patients. Shandong strains formed distinct clusters, and a close relationship was observed between local environmental and clinical strains. As an EV-specific case surveillance system is absent in China and many other countries, continuous environmental surveillance should be encouraged to investigate the temporal circulation and phylogeny of EVs in the population.

Molecular detection of hepatitis E virus in rivers in the Philippines.

To understand the hepatitis E virus (HEV)-pollution status in the environment in the Philippines, a total of 12 water samples were collected from rivers in Manila City for detection of HEV RNA. Three of 12 samples were positive for HEV RNA indicating that HEV is circulating in the Philippines. Phylogenetic analysis classified all of the HEV sequences into genotype 3.

Molecular evolution of VP3, VP1, 3C(pro) and 3D(pol) coding regions in coxsackievirus group A type 24 variant isolates from acute hemorrhagic conjunctivitis in 2011 in Okinawa, Japan.

A large acute hemorrhagic conjunctivitis (AHC) outbreak occurred in 2011 in Okinawa Prefecture in Japan. Ten strains of coxsackievirus group A type 24 variant (CA24v) were isolated from patients with AHC and full sequence analysis of the VP3, VP1, 3C(pro) and 3D(pol) coding regions performed. To assess time-scale evolution, phylogenetic analysis was performed using the Bayesian Markov chain Monte Carlo method. In addition, similarity plots were constructed and pairwise distance (p-distance) and positive pressure analyses performed. A phylogenetic tree based on the VP1 coding region showed that the present strains belong to genotype 4 (G4). In addition, the present strains could have divided in about 2010 from the same lineages detected in other countries such as China, India and Australia. The mean rates of molecular evolution of four coding regions were estimated at about 6.15 to 7.86 × 10(-3) substitutions/site/year. Similarity plot analyses suggested that nucleotide similarities between the present strains and a prototype strain (EH24/70 strain) were 0.77-0.94. The p-distance of the present strains was relatively short (<0.01). Only one positive selected site (L25H) was identified in the VP1 protein. These findings suggest that the present CA24v strains causing AHC are genetically related to other AHC strains with rapid evolution and emerged in around 2010.

Development of real-time PCR to detect oral vaccine-like poliovirus and its application to environmental surveillance.

In order to perform environmental surveillance to track oral poliovirus vaccine-like poliovirus sensitively and conveniently, real-time PCR was developed and applied to a raw sewage concentrate. The real-time PCR method detected 0.01-0.1 TCID50 of 3 serotypes of Sabin strain specifically. The method also detected the corresponding serotypes of oral poliovirus vaccine-like poliovirus specifically, but detected neither wild poliovirus, except Mahoney for type 1 and Saukett for type 3, nor other enteric viruses, as far as examined. When real-time PCR was applied to environmental surveillance, the overall agreement rates between real-time PCR and the cell culture were 83.3% for all serotypes. Since real-time PCR has the advantages of rapid detection of viruses and minimum requirement of sampling volume as compared with ordinary cell culture, it is suitable to monitor oral poliovirus vaccine-like poliovirus in the environment, especially in areas where an oral vaccine is being replaced by an inactivated vaccine.

Prevalence of nonpolio enteroviruses in the sewage of Guangzhou city, China, from 2009 to 2012.

The human-pathogenic viruses in urban sewage have been extensively monitored to obtain information on circulating viruses in human communities. Enteroviruses (EVs) excreted by patients who present with diverse clinical syndromes can remain infectious in the environment for several weeks, and limited data on circulating environmental EVs are available. A 4-year (2009 to 2012) surveillance study was conducted to detect nonpolio enteroviruses (NPEVs) in the urban sewage of Guangzhou city, China. After the viruses in the sewage samples were concentrated and isolated, molecular identification was used to detect and type the NPEVs. During the 4-year study, 17 different NPEV serotypes were identified in the sewage of Guangzhou city. The most common serotypes were echovirus 11 (ECHO11), ECHO6, ECHO7, and ECHO12 and coxsackie group B viruses 5 (CVB5) and CVB3. The predominant serotypes were influenced by spatial and temporal factors and differed each year. CVB5 was commonly detected in 2009 and 2010 but was rarely isolated in 2011 and 2012. In contrast, CVB3 was not observed in 2009 and 2010 but was increasingly detected in 2011 and 2012. Our study provides an overview of the serotype distribution and circulation patterns of NPEVs in the sewage of Guangzhou, China. In the absence of a systematic EV disease surveillance system, the detection and characterization of sewage-borne NPEVs will help us better understand the changes in EV disease trends and the epidemic background of circulating EVs, which could help interpret the EV trends and warn of future outbreaks in this area.

Isolation and characterization of a type 2 vaccine-derived poliovirus from environmental surveillance in China, 2012.

Environmental surveillance of poliovirus on sewage has been conducted in Shandong Province, China since 2008. A type 2 vaccine-derived poliovirus (VDPV) with 7 mutations in VP1 coding region was isolated from the sewage collected in the city of Jinan in December 2012. The complete genome sequencing analysis of this isolate revealed 25 nucleotide substitutions, 7 of which resulted in amino acid alteration. No evidence of recombination with other poliovirus serotypes was observed. The virus did not lose temperature sensitive phenotype at 40°C. An estimation based on the evolution rate of the P1 coding region suggested that evolution time of this strain might be 160-176 days. VP1 sequence analysis revealed that this VDPV strain is of no close relationship with other local type 2 polioviruses (n=66) from sewage collected between May 2012 and June 2013, suggesting the lack of its circulation in the local population. The person who excreted the virus was not known and no closely related virus was isolated in local population via acute flaccid paralysis surveillance. By far this is the first report of VDPV isolated from sewage in China, and these results underscore the value of environmental surveillance in the polio surveillance system even in countries with high rates of OPV coverage.

An optimized method for elution of enteroviral RNA from a cellulose-based substrate.

The Flinders Technology Australia (FTA) Elute Card is a commercial product that facilitates the collection, transport, archiving and processing of nucleic acids from a wide variety of biological samples at room temperature. While the cards have been designed so that sterile/deionized water can elute DNA easily, they are not suitable for some less stable RNAs. This study was undertaken to determine the optimal conditions such as the buffer type, buffer pH and incubation temperature for the elution of enteroviral RNA from FTA Elute Cards prior to quantitative analysis using real-time PCR (qPCR) or consensus degenerate hybrid oligonucleotide primer VP1 RT-semi nested PCR (CODEHOP VP1 RT-snPCR). TE-1 (pH 8.0), rather than sterile water, was the best buffer for high efficiency elution of enteroviral RNA at 95°C. However, as the estimated recovery rate of viral RNA eluted from the cards averaged to be only 6.1%, enterovirus assays using FTA elution should be considered qualitative, especially at low virus titers, and therefore the results of the assay should be interpreted carefully.

Estimating the risk of re-emergence after stopping polio vaccination.

Live vaccination against polio has effectively prevented outbreaks in most developed countries for more than 40 years, and there remain only a few countries where outbreaks of poliomyelitis by the wild strain still threaten the community. It is expected that worldwide eradication will be eventually achieved through careful surveillance and a well-managed immunization program. The present paper argues, however, that based on a simple stochastic model the risk of outbreak by a vaccine-derived strain after the cessation of vaccination is quite high, even if many years have passed since the last confirmed case. As vaccinated hosts are natural reservoirs for virulent poliovirus, the source of the risk is the vaccination itself, employed to prevent the outbreaks. The crisis after stopping vaccination will emerge when the following two conditions are met: the susceptible host density exceeds the threshold for epidemics and the vaccinated host density remains large enough to ensure the occurrence of virulent mutants in the population. Our estimates for transmission, recovery, and mutation rates, show that the probability of an outbreak of vaccine-derived virulent viruses easily exceeds 90%. Moreover, if a small fraction of hosts have a longer infectious period, as observed in individuals with innate immunodeficiency, the risk of an outbreak rises significantly. Under such conditions, successful global eradication of polio is restricted to a certain range of parameters even if inactivated polio vaccine (IPV) is extensively used after the termination of live vaccination.