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Certification of global eradication of indigenous wild poliovirus type 2 occurred in 2015 and of type 3 in 2019. Since the launch of the Global Polio Eradication Initiative (GPEI) in 1988 and broad use of live, attenuated oral poliovirus vaccine (OPV), the number of wild poliovirus cases has declined >99.99% (1). Genetically divergent vaccine-derived poliovirus* (VDPV) strains can emerge during vaccine use and spread in underimmunized populations, becoming circulating VDPV (cVDPV) strains, and resulting in outbreaks of paralytic poliomyelitis.† In April 2016, all oral polio vaccination switched from trivalent OPV (tOPV; containing vaccine virus types 1, 2, and 3) to bivalent OPV (bOPV; containing types 1 and 3) (2). Monovalent type 2 OPV (mOPV2) is used in response campaigns to control type 2 cVDPV (cVDPV2) outbreaks. This report presents data on cVDPV outbreaks detected during January 2018-June 2019 (as of September 30, 2019). Compared with January 2017-June 2018 (3), the number of reported cVDPV outbreaks more than tripled, from nine to 29; 25 (86%) of the outbreaks were caused by cVDPV2. The increase in the number of outbreaks in 2019 resulted from VDPV2 both inside and outside of mOPV2 response areas. GPEI is planning future use of a novel type 2 OPV, stabilized to decrease the likelihood of reversion to neurovirulence. However, all countries must maintain high population immunity to decrease the risk for cVDPV emergence. Cessation of all OPV use after certification of polio eradication will eliminate the risk for VDPV emergence.

Since the Global Polio Eradication Initiative (GPEI) was launched in 1988, the number of polio cases worldwide has declined approximately 99.99%; only two countries (Afghanistan and Pakistan) have never interrupted wild poliovirus (WPV) transmission (1). The primary means of detecting poliovirus circulation is through surveillance for acute flaccid paralysis (AFP) among children aged

Circulating vaccine-derived polioviruses (cVDPVs) can emerge in areas with low poliovirus immunity and cause outbreaks* of paralytic polio (1-5). Among the three types of wild poliovirus, type 2 was declared eradicated in 2015 (1,2). The use of trivalent oral poliovirus vaccine (tOPV; types 1, 2, and 3 Sabin strains) ceased in April 2016 via a 1-month-long, global synchronized switch to bivalent OPV (bOPV; types 1 and 3 Sabin strains) in immunization activities (1-4). Monovalent type 2 OPV (mOPV2; type 2 Sabin strain) is available for cVDPV type 2 (cVDPV2) outbreak response immunization (1-5). The number and geographic breadth of post-switch cVDPV2 outbreaks have exceeded forecasts that trended toward zero outbreaks 4 years after the switch and assumed rapid and effective control of any that occurred (4). New cVDPV2 outbreaks have been seeded by mOPV2 use, by both suboptimal mOPV2 coverage within response zones and recently mOPV2-vaccinated children or contacts traveling outside of response zones, where children born after the global switch are fully susceptible to poliovirus type 2 transmission (2-4). In addition, new emergences can develop by inadvertent exposure to Sabin OPV2-containing vaccine (i.e., residual response mOPV2 or tOPV) (4). This report updates the January 2018-June 2019 report with information on global cVDPV outbreaks during July 2019-February 2020 (as of March 25, 2020)† (2). Among 33 cVDPV outbreaks reported during July 2019-February 2020, 31 (94%) were cVDPV2; 18 (58%) of these followed new emergences. In mid-2020, the Global Polio Eradication Initiative (GPEI) plans to introduce a genetically stabilized, novel OPV type 2 (nOPV2) that has a lower risk for generating VDPV2 than does Sabin mOPV2; if nOPV2 is successful in limiting new VDPV2 emergences, GPEI foresees the replacement of Sabin mOPV2 with nOPV2 for cVDPV2 outbreak responses during 2021 (2,4,6).

Objective Primary immunodeficiency (PID) patients are prone to developing viral infections and should not be vaccinated with live vaccines. In such patients, prolonged excretion and viral divergence may occur and they may subsequently act as reservoirs in the community introducing mutated virus and jeopardizing polio eradication. One hundred and thirty PID cases were included for poliovirus detection in stool with assessment of divergence of detected polioviruses from oral polio vaccine (OPV) virus. Clinical presentations of PID patients with detectable poliovirus in stool specimens are described. Results Six PID patients (4.5%) had detectable vaccine-derived poliovirus (VDPV) excretion in stool specimens; of these, five patients had severe combined immunodeficiency (two with acute flaccid paralysis, one with meningoencephalitis and two without neurological manifestations), and one patient had X-linked agammaglobulinemia (paralysis developed shortly after diagnosis of immunodeficiency). All six case-patients received trivalent OPV. Five case-patients had type 2 immunodeficiency-related vaccine-derived polioviruses (iVDPV2) excretion; one had concomitant excretion of Sabin like type 3 virus and one was identified as iVDPV1 excretor. Surveillance for poliovirus excretion among PID patients is critical as these patients represent a potential source to reseed polioviruses into populations.

Since the 1988 inception of the Global Polio Eradication Initiative (GPEI), progress toward interruption of wild poliovirus (WPV) transmission has occurred mostly through extensive use of oral poliovirus vaccine (OPV) in mass vaccination campaigns and through routine immunization services (1,2). However, because OPV contains live, attenuated virus, it carries the rare risk for reversion to neurovirulence. In areas with very low OPV coverage, prolonged transmission of vaccine-associated viruses can lead to the emergence of vaccine-derived polioviruses (VDPVs), which can cause outbreaks of paralytic poliomyelitis. Although WPV type 2 has not been detected since 1999, and was declared eradicated in 2015,* most VDPV outbreaks have been attributable to VDPV serotype 2 (VDPV2) (3,4). After the synchronized global switch from trivalent OPV (tOPV) (containing vaccine virus types 1, 2, and 3) to bivalent OPV (bOPV) (types 1 and 3) in April 2016 (5), GPEI regards any VDPV2 emergence as a public health emergency (6,7). During May-June 2017, VDPV2 was isolated from stool specimens from two children with acute flaccid paralysis (AFP) in Deir-ez-Zor governorate, Syria. The first isolate differed from Sabin vaccine virus by 22 nucleotides in the VP1 coding region (903 nucleotides). Genetic sequence analysis linked the two cases, confirming an outbreak of circulating VDPV2 (cVDPV2). Poliovirus surveillance activities were intensified, and three rounds of vaccination campaigns, aimed at children aged

The last confirmed wild poliovirus (WPV) case in Democratic Republic of the Congo (DRC) had paralysis onset in December 2011 (1). DRC has had cases of vaccine-derived polioviruses (VDPVs) documented since 2004 (Table 1) (1-6). After an outbreak of 30 circulating VDPV type 2 (cVDPV2) cases during 2011-2012, only five VDPV2 cases were reported during 2013-2016 (Table 1) (1-6). VDPVs can emerge from oral poliovirus vaccine (OPV types 1, 2, or 3; Sabin) polioviruses that have genetically mutated resulting in reversion to neurovirulence. This process occurs during extensive person-to-person transmission in populations with low immunity or after extended replication in the intestines of immune-deficient persons following vaccination (1-6). During 2017 (as of March 8, 2018), 25 VDPV cases were reported in three provinces in DRC: in Tanganyika province, an emergence with one VDPV2 case (pending final classification) in Kabalo health zone and an emergence with one ambiguous VDPV type 1 (aVDPV1) case in Ankoro health zone; in Maniema province, an emergence with two cVDPV2 cases; and in Haut Lomami province, an emergence with 20 cVDPV2 cases that originated in Haut Lomami province and later spread to Tanganyika province (hereafter referred to as the Haut Lomami outbreak area) and an emergence with one aVDPV type 2 (aVDPV2) case in Lwamba health zone (Table 1) (Figure) (6). Outbreak response supplementary immunization activities (SIAs) were conducted during June-December 2017 (Table 2) (6). Because of limitations in surveillance and suboptimal SIA quality and geographic scope, cVDPV2 circulation is likely continuing in 2018, requiring additional SIAs. DRC health officials and Global Polio Eradication Initiative (GPEI) partners are increasing human and financial resources to improve all aspects of outbreak response.

In 1988, the World Health Assembly launched the Global Polio Eradication Initiative (GPEI) (1). Among the three wild poliovirus (WPV) serotypes, only type 1 (WPV1) has been detected since 2012. Since 2014, detection of WPV1 has been limited to three countries, with 37 cases in 2016 and 11 cases in 2017 as of September 27. The99.99% decline worldwide in polio cases since the launch of the GPEI is attributable to the extensive use of the live, attenuated oral poliovirus vaccine (OPV) in mass vaccination campaigns and comprehensive national routine immunization programs. Despite its well-established safety record, OPV use can be associated with rare emergence of genetically divergent vaccine-derived polioviruses (VDPVs) whose genetic drift from the parental OPV strains indicates prolonged replication or circulation (2). VDPVs can also emerge among persons with primary immunodeficiencies (PIDs). Immunodeficiency-associated VDPVs (iVDPVs) can replicate for years in some persons with PIDs. In addition, circulating vaccine-derived polioviruses (cVDPVs) can emerge very rarely among immunologically normal vaccine recipients and their contacts in areas with inadequate OPV coverage and can cause outbreaks of paralytic polio. This report updates previous summaries regarding VDPVs (3). During January 2016-June 2017, new cVDPV outbreaks were identified, including two in the Democratic Republic of the Congo (DRC) (eight cases), and another in Syria (35 cases), whereas the circulation of cVDPV type 2 (cVDPV2) in Nigeria resulted in cVDPV2 detection linked to a previous emergence. The last confirmed case from the 2015-2016 cVDPV type 1 (cVDPV1) outbreak in Laos occurred in January 2016. Fourteen newly identified persons in 10 countries were found to excrete iVDPVs, and three previously reported patients in the United Kingdom and Iran (3) were still excreting type 2 iVDPV (iVDPV2) during the reporting period. Ambiguous VDPVs (aVDPVs), isolates that cannot be classified definitively, were found among immunocompetent persons and environmental samples in 10 countries. Cessation of all OPV use after certification of polio eradication will eliminate the risk for new VDPV infections.

Vaccine-Derived Polioviruses (VDPVs) may emerge during person-to-person transmission (circulating VDPV [cVDPV]) or during prolonged replication in individuals with primary immunodeficiency (iVDPV). To prevent the emergence of type 2 vaccine-derived polioviruses (VDPV2s) which represents >90% of the VDPV during the past two years, WHO implemented a worldwide synchronized switch from trivalent oral poliovirus vaccine (tOPV; types 1, 2, and 3) to bivalent OPV (bOPV; types 1 and 3) in April 2016. When planning the scope of a post-switch outbreak response, it is important to know if the recently identified VDPV is likely to be cVDPV or iVDPV, as the public health responses dramatically differ in the two events: outbreak requires a community immunization response while chronic infection requires careful patient monitoring and potential individual treatment. Here we demonstrate that genetic distinctions between cVDPV and iVDPV sequences can be calculated (to some extent) to inform global poliovirus outbreak response, particularly for VDPV2. We performed a survey of cVDPV and iVDPV nucleotide sequences of the VP1 capsid region (∼900 nucleotides) from GenBank. A non-redundant dataset contains 33 cVDPV2 isolates and 33 iVDPV2 isolates was built. We compared each sequence to its Sabin 2 parental strain to determine the number of genetic nucleotide (NT) and amino acid (AA) substitutions. cVDPV2 and iVDPV2 sequences show similar profiles for NT and AA frequencies, codon usage, and AA conservation. Simple counts of NT, AA, and codon substitutions in comparative analyses between cVDPV2 and iVDPV2 were insufficient for a clear distinction between the two VDPV2 categories. However, the combined measures of NT and AA substitutions (from VP1 and neutralizing antigenic site sequences) were more informative, with more non-synonymous (AA changes) in iVDPV2 than cVDPV2. This work highlights that the genetic variations between iVDPVs and cVDPVs may be reflect the differences of the viral micro-environments, interactions between virus and host, and the selective pressures during person-to-person transmission compared with chronic infections in immune-deficient patients. The dataset was small but the issue is certainly deserving of attentions and suggesting the needs for further data collections from poliovirus surveillance. This is the first attempt using computational techniques to quantify the genetic difference between cVDPVs and iVDPVs.

In September 2015, more than 1 year after reporting its last wild poliovirus (WPV) case in July 2014 (1), Nigeria was removed from the list of countries with endemic poliovirus transmission,* leaving Afghanistan and Pakistan as the only remaining countries with endemic WPV. However, on April 29, 2016, a laboratory-confirmed, circulating vaccine-derived poliovirus type 2 (cVDPV2) isolate was reported from an environmental sample collected in March from a sewage effluent site in Maiduguri Municipal Council, Borno State, a security-compromised area in northeastern Nigeria. VDPVs are genetic variants of the vaccine viruses with the potential to cause paralysis and can circulate in areas with low population immunity. The Nigeria National Polio Emergency Operations Center initiated emergency response activities, including administration of at least 2 doses of oral poliovirus vaccine (OPV) to all children aged

Background Efficient implementation of the global eradication strategies consisting of Acute Flaccid Paralysis (AFP) surveillance and mass immunization campaigns led to interruption of indigenous wild poliovirus transmission in Cameroon in 1999. Objectives This study describes type 1 and type 3 wild poliovirus (WPV) importation, incidence, geographic distribution and control since the original interruption of transmission in Cameroon. Study design Stool samples from AFP patients under the age of 15 years in Cameroon were collected nationwide and subjected to virus isolation on RD and L20B cell cultures. Resulting virus isolates were typed by intratypic differentiation (ITD) and analysis of the VP1 coding sequence of the viral genome. Surveillance data originating from Cameroon between 2000 and 2014 were considered for retrospective descriptive analyses. Results From 2003 to 2009, multiple WPV importation events from neighboring countries affected mainly in the northern regions of Cameroon but did not led to sustained local transmission. Throughout this period, 16 WPV1 and 5 WPV3 were detected and identified as members of multiple clusters within type-specific West Africa B genotypes (WEAF-B). In 2013–2014, a polio outbreak associated to a highly evolved (“orphan”) WPV1 affected four southern regions of Cameroon. Conclusions The appearance of highly evolved lineage of type 1 WPV suggests potential surveillance gap and underscore the need to maintain comprehensive polio immunization activities and sensitive surveillance systems in place as long as any country in the world remains endemic for WPV.

Since the World Health Assembly's 1988 resolution to eradicate poliomyelitis, one of the main tools of the World Health Organization (WHO) Global Polio Eradication Initiative (GPEI) has been the live, attenuated oral poliovirus vaccine (OPV). OPV might require several doses to induce immunity but provides long-term protection against paralytic disease. Through effective use of OPV, GPEI has brought polio to the threshold of eradication. Wild poliovirus type 2 (WPV2) was eliminated in 1999, WPV3 has not been detected since November 2012, and WPV1 circulation appears to be restricted to parts of Pakistan and Afghanistan. However, continued use of OPV carries two key risks. The first, vaccine-associated paralytic poliomyelitis (VAPP) has been recognized since the early 1960s. VAPP is a very rare event that occurs sporadically when an administered dose of OPV reverts to neurovirulence and causes paralysis in the vaccine recipient or a nonimmune contact. VAPP can occur among immunologically normal vaccine recipients and their contacts as well as among persons who have primary immunodeficiencies (PIDs) manifested by defects in antibody production; it is not associated with outbreaks. The second, the emergence of genetically divergent, neurovirulent vaccine-derived polioviruses (VDPVs) was recognized more recently. Circulating VDPVs (cVDPVs) resemble WPVs and, in areas with low OPV coverage, can cause polio outbreaks. Immunodeficiency-associated VDPVs (iVDPVs) can replicate and be excreted for years in some persons with PIDs; GPEI maintains a registry of iVDPV cases. Ambiguous VDPVs (aVDPVs) are isolates that cannot be classified definitively. This report updates previous surveillance summaries and describes VDPVs detected worldwide during January 2014-March 2015. Those include new cVDPV outbreaks in Madagascar and South Sudan, and sharply reduced type 2 cVDPV (cVDPV2) circulation in Nigeria and Pakistan during the latter half of 2014. Eight newly identified persons in six countries were found to excrete iVDPVs, and a patient in the United Kingdom was still excreting iVDPV2 in 2014 after more than 28 years. Ambiguous VDPVs were found among immunocompetent persons and environmental samples in 16 countries. Because the large majority of VDPV case-isolates are type 2, WHO has developed a plan for coordinated worldwide withdrawal of trivalent (types 1, 2, and 3) OPV (tOPV) and replacement with bivalent (types 1 and 3) OPV (bOPV) in April 2016, preceded by introduction of at least 1 dose of injectable inactivated poliovirus vaccine (IPV) into routine immunization schedules worldwide to maintain immunity to type 2 viruses.

We followed the dynamics of capsid amino acid replacement among 403 Nigerian outbreak isolates of type 2 circulating vaccine-derived poliovirus (cVDPV2) from 2005 through 2011. Four different functional domains were analyzed: (i) neutralizing antigenic (NAg) sites, (ii) residues binding the poliovirus receptor (PVR), (iii) VP1 residues 1 to 32, and (iv) the capsid structural core. Amino acid replacements mapped to 37 of 43 positions across all 4 NAg sites; the most variable and polymorphic residues were in NAg sites 2 and 3b. The most divergent of the 120 NAg variants had no more than 5 replacements in all NAg sites and were still neutralized at titers similar to those of Sabin 2. PVR-binding residues were less variable (25 different variants; 0 to 2 replacements per isolate; 30/44 invariant positions), with the most variable residues also forming parts of NAg sites 2 and 3a. Residues 1 to 32 of VP1 were highly variable (133 different variants; 0 to 6 replacements per isolate; 5/32 invariant positions), with residues 1 to 18 predicted to form a well-conserved amphipathic helix. Replacement events were dated by mapping them onto the branches of time-scaled phylogenies. Rates of amino acid replacement varied widely across positions and followed no simple substitution model. Replacements in the structural core were the most conservative and were fixed at an overall rate ∼20-fold lower than the rates for the NAg sites and VP1 1 to 32 and ∼5-fold lower than the rate for the PVR-binding sites. Only VP1 143-Ile, a non-NAg site surface residue and known attenuation site, appeared to be under strong negative selection. IMPORTANCE The high rate of poliovirus evolution is offset by strong selection against amino acid replacement at most positions of the capsid. Consequently, poliovirus vaccines developed from strains isolated decades ago have been used worldwide to bring wild polioviruses almost to extinction. The apparent antigenic stability of poliovirus obscures a dynamic of continuous change within the neutralizing antigenic (NAg) sites. During 7 years of a large outbreak in Nigeria, the circulating type 2 vaccine-derived polioviruses generated 120 different NAg site variants via multiple independent pathways. Nonetheless, overall antigenic evolution was constrained, as no isolate had fixed more than 5 amino acid differences from the Sabin 2 NAg sites, and the most divergent isolates were efficiently neutralized by human immune sera. Evolution elsewhere in the capsid was also constrained. Amino acids binding the poliovirus receptor were strongly conserved, and extensive variation in the VP1 amino terminus still conserved a predicted amphipathic helix.

Background Israel has used an inactivated polio vaccine (IPV)-only schedule since 2005 (95% coverage). Silent reintroduction of wild type poliovirus 1 (WPV1) into Israel in early 2013 was detected in Southern Israel via routine environmental surveillance without clinical cases. Objectives To estimate the rate of WPV1 excretion by age and residence and inform decision-making regarding supplemental immunization with OPV. Study design A convenience sample of Bedouin and Jewish residential areas in the epicenter of the incident, focusing on under 8 year-olds who not previously given OPV. Fecal samples were directly tested for WPV1 RNA using a novel qRT-PCR assay. Positive samples were confirmed by gold standard cell culture and subject to genotyping. Results Overall, 2196 non-duplicate fecal samples were collected and analyzed. WPV1 was detected in 61 samples (2.8%), 55 of which (90.2%) were from Bedouins. WPV1 excretion rates were 5.4% among Bedouins and 0.6% among Jewish individuals. Respective age-specific rates among Bedouin and Jewish children were 4.9% and 0.2% for 0–2 years and 7.2% and 1.7% for 2–8 years. Molecular testing had 89.5% sensitivity (higher than culture) and 100% specificity. Conclusion The rapid performance of a field study to evaluate WPV1 excretion unequivocally demonstrated substantial WPV1 infection rates among children under 8 years in Southern Israel, thus informing the decision to vaccinate this age group with bOPV and risk communication to both healthcare personnel and the public. Rapid development and implementation of molecular screening can thus underpin risk assessment and management in complex epidemiological situations.

Background More than 99% of poliovirus infections are non-paralytic and therefore, not detected by acute flaccid paralysis (AFP) surveillance. Environmental surveillance (ES) can detect circulating polioviruses from sewage without relying on clinical presentation. With extensive ES and continued circulation of polioviruses, Pakistan presents a unique opportunity to quantify the impact of ES as a supplement to AFP surveillance on overall completeness and timeliness of poliovirus detection. Methods Genetic, geographic and temporal data were obtained for all wild poliovirus (WPV) isolates detected in Pakistan from January 2011 through December 2013. We used viral genetics to assess gaps in AFP surveillance and ES as measured by detection of ‘orphan viruses’ (≥1.5% different in VP1 capsid nucleotide sequence). We compared preceding detection of closely related circulating isolates (≥99% identity) detected by AFP surveillance or ES to determine which surveillance system first detected circulation before the presentation of each polio case. Findings A total of 1,127 WPV isolates were detected by AFP surveillance and ES in Pakistan from 2011–2013. AFP surveillance and ES combined exhibited fewer gaps (i.e., % orphan viruses) in detection than AFP surveillance alone (3.3% vs. 7.7%, respectively). ES detected circulation before AFP surveillance in nearly 60% of polio cases (200 of 346). For polio cases reported from provinces conducting ES, ES detected circulation nearly four months sooner on average (117.6 days) than did AFP surveillance. Interpretation Our findings suggest ES in Pakistan is providing earlier, more sensitive detection of wild polioviruses than AFP surveillance alone. Overall, targeted ES through strategic selection of sites has important implications in the eradication endgame strategy.

The poliovirus (PV) is currently targeted for worldwide eradication and containment. Sanger-based sequencing of the viral protein 1 (VP1) capsid region is currently the standard method for PV surveillance. However, the whole-genome sequence is sometimes needed for higher resolution global surveillance. In this study, we optimized whole-genome sequencing protocols for poliovirus isolates and FTA cards using next-generation sequencing (NGS), aiming for high sequence coverage, efficiency, and throughput. We found that DNase treatment of poliovirus RNA followed by random reverse transcription (RT), amplification, and the use of the Nextera XT DNA library preparation kit produced significantly better results than other preparations. The average viral reads per total reads, a measurement of efficiency, was as high as 84.2% ± 15.6%. PV genomes covering >99 to 100% of the reference length were obtained and validated with Sanger sequencing. A total of 52 PV genomes were generated, multiplexing as many as 64 samples in a single Illumina MiSeq run. This high-throughput, sequence-independent NGS approach facilitated the detection of a diverse range of PVs, especially for those in vaccine-derived polioviruses (VDPV), circulating VDPV, or immunodeficiency-related VDPV. In contrast to results from previous studies on other viruses, our results showed that filtration and nuclease treatment did not discernibly increase the sequencing efficiency of PV isolates. However, DNase treatment after nucleic acid extraction to remove host DNA significantly improved the sequencing results. This NGS method has been successfully implemented to generate PV genomes for molecular epidemiology of the most recent PV isolates. Additionally, the ability to obtain full PV genomes from FTA cards will aid in facilitating global poliovirus surveillance.

In 1988, the World Health Assembly resolved to eradicate poliomyelitis worldwide (1). One of the main tools used in polio eradication efforts has been the live, attenuated, oral poliovirus vaccine (OPV) (2), an inexpensive vaccine easily administered by trained volunteers. OPV might require several doses to induce immunity, but provides long-term protection against paralytic disease. Through effective use of OPV, the Global Polio Eradication Initiative (GPEI) has brought wild polioviruses to the threshold of eradication (1). However, OPV use, particularly in areas with low routine vaccination coverage, is associated with the emergence of genetically divergent vaccine-derived polioviruses (VDPVs) whose genetic drift from the parental OPV strains indicates prolonged replication or circulation (3). VDPVs can emerge among immunologically normal vaccine recipients and their contacts as well as among persons with primary immunodeficiencies (PIDs). Immunodeficiency-associated VDPVs (iVDPVs) can replicate for years in some persons with PIDs. In addition, circulating vaccine-derived polioviruses (cVDPVs) (3) can emerge in areas with low OPV coverage and can cause outbreaks of paralytic polio. This report updates previous summaries regarding VDPVs (4).

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.

In 1984, a wild type 3 poliovirus (PV3/FIN84) spread all over Finland causing nine cases of paralytic poliomyelitis and one case of aseptic meningitis. The outbreak was ended in 1985 with an intensive vaccination campaign. By limited sequence comparison with previously isolated PV3 strains, closest relatives of PV3/FIN84 were found among strains circulating in the Mediterranean region. Now we wanted to reanalyse the relationships using approaches currently exploited in poliovirus surveillance. Cell lysates of 22 strains isolated during the outbreak and stored frozen were subjected to RT-PCR amplification in three genomic regions without prior subculture. Sequences of the entire VP1 coding region, 150 nucleotides in the VP1-2A junction, most of the 5' non-coding region, partial sequences of the 3D RNA polymerase coding region and partial 3' non-coding region were compared within the outbreak and with sequences available in data banks. In addition, complete nucleotide sequences were obtained for 2 strains isolated from two different cases of disease during the outbreak. The results confirmed the previously described wide intraepidemic variation of the strains, including amino acid substitutions in antigenic sites, as well as the likely Mediterranean region origin of the strains. Simplot and bootscanning analyses of the complete genomes indicated complicated evolutionary history of the non-capsid coding regions of the genome suggesting several recombinations with different HEV-C viruses in the past.

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. IMPORTANCE The 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.

Background The World Health Organization (WHO) poliovirus eradication program includes careful surveillance of acute-flaccid paralysis (AFP) and mass and routine immunization with oral polio vaccine (OPV). In populations with low vaccine coverage, the live-attenuated Sabin strains, OPV types 1, 2 and 3, can evolve into virulent vaccine-derived polioviruses (VDPVs) and circulate in the community. Until recently, circulating VDPVs (cVDPVs) had not been reported in Cameroon despite the fact that VDPV2 outbreaks have occurred in nearby countries. Objectives This study aimed to characterize virus isolates from four AFP patients infected with cVDPV2 in the Extreme North region of Cameroon in 2013. Study design The complete VP1 region of the four VDPV strains was sequenced and the relationships with cVDPVs from neighboring countries were investigated. Results All four patients were infected by cVDPV2 strains showing 1.2–2.0% nucleotide difference compared to the reference Sabin 2 VP1 sequence. Phylogenetic analysis indicated that the VDPV strains were genetically linked to cVDPV2 lineages of the recent Chad cVDPV2 outbreak. Conclusions The circulation of pathogenic VDPVs suggests that there are localized immunization gaps in some districts like Makary, Mada and Kolofata in Cameroon. To avoid poliomyelitis outbreaks in Cameroon, especially in the districts close to neighboring countries with ongoing cVDPV outbreaks, high polio vaccine coverage is essential.

Since 2005, a large poliomyelitis outbreak associated with type 2 circulating vaccine-derived poliovirus (cVDPV2) has occurred in northern Nigeria, where immunization coverage with trivalent oral poliovirus vaccine (tOPV) has been low. Phylogenetic analysis of P1/capsid region sequences of isolates from each of the 403 cases reported in 2005 to 2011 resolved the outbreak into 23 independent type 2 vaccine-derived poliovirus (VDPV2) emergences, at least 7 of which established circulating lineage groups. Virus from one emergence (lineage group 2005-8; 361 isolates) was estimated to have circulated for over 6 years. The population of the major cVDPV2 lineage group expanded rapidly in early 2009, fell sharply after two tOPV rounds in mid-2009, and gradually expanded again through 2011. The two major determinants of attenuation of the Sabin 2 oral poliovirus vaccine strain (A 481 in the 5′-untranslated region [5′-UTR] and VP1-Ile 143 ) had been replaced in all VDPV2 isolates; most A 481 5′-UTR replacements occurred by recombination with other enteroviruses. cVDPV2 isolates representing different lineage groups had biological properties indistinguishable from those of wild polioviruses, including efficient growth in neuron-derived HEK293 cells, the capacity to cause paralytic disease in both humans and PVR-Tg21 transgenic mice, loss of the temperature-sensitive phenotype, and the capacity for sustained person-to-person transmission. We estimate from the poliomyelitis case count and the paralytic case-to-infection ratio for type 2 wild poliovirus infections that ∼700,000 cVDPV2 infections have occurred during the outbreak. The detection of multiple concurrent cVDPV2 outbreaks in northern Nigeria highlights the risks of cVDPV emergence accompanying tOPV use at low rates of coverage in developing countries.

Wild poliovirus has remained endemic in northern Nigeria because of low coverage achieved in the routine immunization program and in supplementary immunization activities (SIAs). An outbreak of infection involving 315 cases of type 2 circulating vaccine-derived poliovirus (cVDPV2; >1% divergent from Sabin 2) occurred during July 2005–June 2010, a period when 23 of 34 SIAs used monovalent or bivalent oral poliovirus vaccine (OPV) lacking Sabin 2. In addition, 21 “pre-VDPV2” (0.5%–1.0% divergent) cases occurred during this period. Both cVDPV and pre-VDPV cases were clinically indistinguishable from cases due to wild poliovirus. The monthly incidence of cases increased sharply in early 2009, as more children aged without trivalent OPV SIAs. Cumulative state incidence of pre-VDPV2/cVDPV2 was correlated with low childhood immunization against poliovirus type 2 assessed by various means. Strengthened routine immunization programs in countries with suboptimal coverage and balanced use of OPV formulations in SIAs are necessary to minimize risks of VDPV emergence and circulation.

The Global Polio Laboratory Network routinely uses poliovirus-specific PCR primers and probes to determine the serotype and genotype of poliovirus isolates obtained as part of global poliovirus surveillance. To provide detailed molecular epidemiologic information, poliovirus isolates are further characterized by sequencing the ∼900-nucleotide region encoding the major capsid protein, VP1. It is difficult to obtain quality sequence information when clinical or environmental samples contain poliovirus mixtures. As an alternative to conventional methods for resolving poliovirus mixtures, sets of serotype-specific primers were developed for amplifying and sequencing the VP1 regions of individual components of mixed populations of vaccine–vaccine, vaccine–wild, and wild–wild polioviruses.

We have adapted our previously described poliovirus diagnostic reverse transcription-PCR (RT-PCR) assays to a real-time RT-PCR (rRT-PCR) format. Our highly specific assays and rRT-PCR reagents are designed for use in the WHO Global Polio Laboratory Network for rapid and large-scale identification of poliovirus field isolates.

A type 2 vaccine-derived poliovirus (VDPV), differing from Sabin 2 at 2.5% (22/903) of VP1 nucleotide (nt) positions, was isolated from an incompletely immunized 21-month-old Nigerian child who developed acute flaccid paralysis in 2002. Sequences upstream of nt position 620 (within the 5'-untranslated region [5'-UTR]) and downstream of nt position 5840 (in the 3C(pro) region) were derived from species C enteroviruses unrelated to the oral poliovirus vaccine (OPV) strains. The two substitutions associated with the attenuated phenotype had either recombined out (A(481)-->G in the 5'-UTR) or reverted (Ile(143)-->Thr in VP1). The VDPV isolate had lost the temperature sensitive phenotype of Sabin 2 and it was antigenically distinct from the parental OPV strain, having amino acid substitutions in or near neutralizing antigenic sites 1 and 3. The date of the initiating OPV dose, calculated from the number of synonymous substitutions in the capsid region, was estimated to be approximately 16 to 18 months before onset of paralysis, a finding inconsistent with the most recent mass OPV campaign (conducted 12 days before onset of paralysis) as being the source of infection. Although no related type 2 VDPVs were detected in Nigeria or elsewhere, the VDPV was found in an area where conditions favor VDPV emergence and spread.

Erratum to “Field study of fecal excretion as a decision support tool in response to silent reintroduction of wild-type poliovirus 1 into Israel” [J. Clin. Virol. 66 (2015) 51–55] Jacob Moran-Gilada,b,∗, Ella Mendelsonc,d, Cara C. Burnse, Ravit Bassal f, Michael Gdalevichg, Danit Soferc, M. Steven Oberstee, Lester M. Shulmanc,d, Ehud Kalinera, Musa Hindiyehc, Orna Morc, Liora Shaharg, Jane Ibere, Ruth Yishayh, Joseph Manorc, Boaz Lev i, Ronni Gamzud,i, Itamar Grottoa,b

The recent discovery of recombinant circulating vaccine-derived poliovirus (recombinant cVDPV) has highlighted the need for enhanced global poliovirus surveillance to assure timely detection of any future cVDPV outbreaks. Six pairs of Sabin strain-specific recombinant primers were designed to permit rapid screening for VDPV recombinants by PCR.

A total of 34 sabin strains of the poliovirus isolated from 22 children with 60-day follow-up residual acute flaccid paralysis (AFP) were genetically characterized and screened for any form of recombination. Sequence analysis of the 906-nucleotide capsid showed that all the isolates were similar to their original sabin serotypes, however two of the viruses had drifted in their 3D noncapsid regions toward a sabin-sabin and sabin-nonpolio entero combination. Routine immunization in Nigeria is low and in spite of the increase in the frequency of supplemental immunizations, a lot of children are still inadequately immunized, which may be the reason for our observation in this study. Although we are not dealing with a case of circulating vaccine derived poliovirus (cVDPV) yet, if the above condition persists, the advent of cVDVP may not be too far. There is therefore the need to maintain a high quality mass immunization and sustained routine immunization. Key words : Poliovirus, sequence, crossover, non polio enterovirus, recombination, genome, Sabin-like, vaccine, Nigeria. African Journal of Biotechnology Vol.2(11) 2003: 460-464

Sialic acids are important cell-surface molecules of animals in the deuterostome lineage. Although humans do not express easily detectable amounts ofN-glycolylneuraminic acid (Neu5Gc, a hydroxylated form of the common sialic acidN-acetylneuraminic acid, Neu5Ac), it is a major component in great ape tissues, except in the brain. This difference correlates with lack of the hydroxylase activity that converts CMP-Neu5Ac to CMP-Neu5Gc. Here we report cloning of human and chimpanzee hydroxylase cDNAs. Although this chimpanzee cDNA is similar to the murine homologue, the human cDNA contains a 92-bp deletion resulting in a frameshift mutation. The isolated human gene also shows evidence for this deletion. Genomic PCR analysis indicates that this deletion does not occur in any of the African great apes. The gene is localized to 6p22–p23 in both humans and great apes, which does not correspond to known chromosomal rearrangements that occurred during hominoid evolution. Thus, the lineage leading to modern humans suffered a mutation sometime after the common ancestor with the chimpanzee and bonobo, potentially affecting recognition by a variety of endogenous and exogenous sialic acid-binding lectins. Also, the expression of Neu5Gc previously reported in human fetuses and tumors as well as the traces detected in some normal adult humans must be mediated by an alternate pathway.

Emery-Dreifuss muscular dystrophy (EMD) is an X-linked disorder characterized by contractures, progressive muscle weakness and cardiomyopathy1–3. The emerin gene, located in human Xq28, is approximately 2 kb in length, is composed of 6 exons and falls within a 219-kb region that has been completely sequenced4–6. Immediately centromeric to emerin is the 26-kb filamin gene (FLN1), composed of 48 exons and encoding the actin-binding protein 280 (refs 7,8). Flanking this 48-kb FLN1/emerin region are two large inverted repeats, each 11.3 kb, that exhibit >99% sequence identity6. The high level of genomic detail in this region allowed us to characterize the first complete emerin gene deletion mutation that also involved a partial duplication of the nearby FLN1 gene. This rearrangement could be explained by mispairing of the large inverted repeats, followed by double recombination among one set of mispaired repeats and internal sequences. Furthermore, our characterization of this rare DNA rearrangement revealed a more common result of the mispairing of these large inverted repeats7—recombination contained within the inverted repeats leading to the maintenance of repeat sequence homogeneity and inversion of the 48-kb FLN1/emerin region. The presence of this frequent inversion, found in the heterozygous state in 33% of females, helps to explain the discrepancies observed between the genetic and physical map distances in this region of the X chromosome. It also illustrates the biological insights which can be gleaned by sequencing the human genome.

Fragile X syndrome results from the expansion of the CGG repeat in the FMR1 gene. Expansion has been suggested to be a postzygotic event with the germline protected. From an analysis of intact ovaries of full mutation fetuses, we now show that only full expansion alleles can be detected in oocytes (but in the unmethylated state). Similarly, the testes of a 13-week full mutation fetus show no evidence of premutations while a 17-week full mutation fetus exhibits some germ cells with attributes of premutations. These data discount the hypothesis that the germline is protected from full expansion and suggest full mutation contraction in the immature testis. Thus, full expansion may already exist in the maternal oocyte, or postzygotic expansion, if it occurs, arises quite early in development prior to germline segregation.

Between October 2001 and April 2002, five cases of acute flaccid paralysis (AFP) associated with type 2 vaccine-derived polioviruses (VDPVs) were reported in the southern province of the Republic of Madagascar. To determine viral factors that favor the emergence of these pathogenic VDPVs, we analyzed in detail their genomic and phenotypic characteristics and compared them with co-circulating enteroviruses. These VDPVs appeared to belong to two independent recombinant lineages with sequences from the type 2 strain of the oral poliovaccine (OPV) in the 5′-half of the genome and sequences derived from unidentified species C enteroviruses (HEV-C) in the 3′-half. VDPV strains showed characteristics similar to those of wild neurovirulent viruses including neurovirulence in poliovirus-receptor transgenic mice. We looked for other VDPVs and for circulating enteroviruses in 316 stools collected from healthy children living in the small area where most of the AFP cases occurred. We found vaccine PVs, two VDPVs similar to those found in AFP cases, some echoviruses, and above all, many serotypes of coxsackie A viruses belonging to HEV-C, with substantial genetic diversity. Several coxsackie viruses A17 and A13 carried nucleotide sequences closely related to the 2C and the 3Dpol coding regions of the VDPVs, respectively. There was also evidence of multiple genetic recombination events among the HEV-C resulting in numerous recombinant genotypes. This indicates that co-circulation of HEV-C and OPV strains is associated with evolution by recombination, resulting in unexpectedly extensive viral diversity in small human populations in some tropical regions. This probably contributed to the emergence of recombinant VDPVs. These findings give further insight into viral ecosystems and the evolutionary processes that shape viral biodiversity., Author Summary Following extensive vaccination campaigns using the attenuated oral polio vaccine, wild polioviruses remain endemic in only a few countries. Nevertheless, several poliomyelitis outbreaks associated with vaccine-derived polioviruses (VDPVs) were reported in different parts of the world in recent years, particularly in Madagascar in 2002. We analyzed the molecular characteristics of Madagascar VDPVs and compared them with those of co-circulating enteroviruses. These VDPVs appear to be recombinant viruses between vaccine polioviruses and human enteroviruses of species C (HEV-C) and to present phenotypic characteristics similar to those of wild polioviruses including pathogenicity. Similar VDPVs and other enteroviruses, including several HEV-C of different types, were found in the stools of healthy children living in neighboring villages to where most of the poliomyelitis cases occurred. Some HEV-Cs showed sequences closely related to those of VDPVs, indicating genetic recombination between these viruses and vaccine polioviruses. There was also evidence of multiple genetic recombination events among other HEV-C isolates resulting in numerous different genotypes. These findings indicate that co-circulation of HEV-C and vaccine polioviruses and their evolution by recombination results in unexpectedly extensive viral diversity, at least in some small human populations, probably contributing to the emergence of recombinant VDPVs. Results of this study give further insight into the world of viruses and their biodiversity.