Increasing detections of vaccine-derived poliovirus (VDPV) globally, including in countries previously declared polio free, is a public health emergency of international concern. Individuals with primary immunodeficiency (PID) can excrete polioviruses for prolonged periods, which could act as a source of cryptic transmission of viruses with potential to cause neurological disease. Here, we report on the detection of immunodeficiency-associated VDPVs (iVDPV) from two asymptomatic male PID children in the UK in 2019. The first child cleared poliovirus with increased doses of intravenous immunoglobulin, the second child following haematopoetic stem cell transplantation. We perform genetic and phenotypic characterisation of the infecting strains, demonstrating intra-host evolution and a neurovirulent phenotype in transgenic mice. Our findings highlight a pressing need to strengthen polio surveillance. Systematic collection of stool from asymptomatic PID patients who are at high risk for poliovirus excretion could improve the ability to detect and contain iVDPVs.

Direct detection by PCR of poliovirus RNA in stool samples provides a rapid diagnostic and surveillance tool that can replace virus isolation by cell culture in global polio surveillance. The sensitivity of direct detection methods is likely to depend on the choice of RNA extraction method and sample volume. We report a comparative analysis of 11 nucleic acid extraction methods (7 manual and 4 semiautomated) for poliovirus molecular detection using stool samples (n = 59) that had been previously identified as poliovirus positive by cell culture. To assess the effect of RNA recovery methods, extracted RNA using each of the 11 methods was tested with a poliovirus-specific reverse transcription-quantitative PCR (RT-qPCR), a pan-poliovirus RT-PCR (near-whole-genome amplification), a pan-enterovirus RT-PCR (entire capsid region), and a nested VP1 PCR that is the basis of a direct detection method based on nanopore sequencing. We also assessed extracted RNA integrity and quantity. The overall effect of extraction method on poliovirus PCR amplification assays tested in this study was found to be statistically significant (P < 0.001), thus indicating that the choice of RNA extraction method is an important component that needs to be carefully considered for any diagnostic based on nucleic acid amplification. Performance of the methods was generally consistent across the different assays used. Of the 11 extraction methods tested, the MagMAX viral RNA isolation kit used manually or automatically was found to be the preferable method for poliovirus molecular direct detection considering performance, cost, and processing time. IMPORTANCE Poliovirus, the causative agent of poliomyelitis, is a target of global eradication led by the World Health Organization since 1988. Direct molecular detection and genomic sequencing without virus propagation in cell culture is arguably a critical tool in the final stages of polio eradication. Efficient recovery of good-quality viral RNA from stool samples is a prerequisite for direct detection by nucleic acid amplification. We tested 11 nucleic acid extraction methods to identify those facilitating sensitive, fast, simple, and cost-effective extraction, with flexibility for manual and automated protocols considered. Several different PCR assays were used to compare the recovered viral RNA to test suitability for poliovirus direct molecular detection. Our findings highlight the importance of choosing a suitable RNA extraction protocol and provide useful information to diagnostic laboratories and researchers facing the choice of RNA extraction method for direct molecular virus detection from stool.

Immunodeficient patients whose gut is chronically infected by vaccine-derived poliovirus (VDPV) may excrete large amounts of virus for years. To investigate how poliovirus (PV) establishes chronic infections in the gut, we tested whether it is possible to establish persistent VDPV infections in human intestinal Caco-2 cells. Four type 3 VDPV mutants, representative of the viral evolution in the gut of a hypogammaglobulinemic patient over almost 2 years [J. Virol. 74 (2000) 3001], were used to infect both undifferentiated, dividing cells, and differentiated, polarized enterocytes. A VDPV mutant excreted 36 days postvaccination by the patient was lytic in both types of intestinal cell cultures, like the parental Sabin 3 (S3) strain. In contrast, three VDPVs excreted 136, 442, and 637 days postvaccination, established persistent infections both in undifferentiated cells and in enterocytes. Thus, viral determinants selected between day 36 and 136 conferred on VDPV mutants the capacity to infect intestinal cells persistently. The percentage of persistently VDPV-infected cultures was higher in enterocytes than in undifferentiated cells, implicating cellular determinants involved in the differentiation of enterocytes in persistent VDPV infections. The establishment of persistent infections in enterocytes was not due to poor replication of VDPVs in these cells, but was associated with reduced viral adsorption to the cell surface.

There are currently huge efforts by the World Health Organization and partners to complete global polio eradication. With the significant decline in poliomyelitis cases due to wild poliovirus in recent years, rare cases related to the use of live-attenuated oral polio vaccine assume greater importance. Poliovirus strains in the oral vaccine are known to quickly revert to neurovirulent phenotype following replication in humans after immunisation. These strains can transmit from person to person leading to poliomyelitis outbreaks and can replicate for long periods of time in immunodeficient individuals leading to paralysis or chronic infection, with currently no effective treatment to stop excretion from these patients. Here, we describe an individual who has been excreting type 2 vaccine-derived poliovirus for twenty eight years as estimated by the molecular clock established with VP1 capsid gene nucleotide sequences of serial isolates. This represents by far the longest period of excretion described from such a patient who is the only identified individual known to be excreting highly evolved vaccine-derived poliovirus at present. Using a range of in vivo and in vitro assays we show that the viruses are very virulent, antigenically drifted and excreted at high titre suggesting that such chronic excreters pose an obvious risk to the eradication programme. Our results in virus neutralization assays with human sera and immunisation-challenge experiments using transgenic mice expressing the human poliovirus receptor indicate that while maintaining high immunisation coverage will likely confer protection against paralytic disease caused by these viruses, significant changes in immunisation strategies might be required to effectively stop their occurrence and potential widespread transmission. Eventually, new stable live-attenuated polio vaccines with no risk of reversion might be required to respond to any poliovirus isolation in the post-eradication era.

Global poliovirus surveillance involves virus isolation from stool and environmental samples, intratypic differential (ITD) by PCR, and sequencing of the VP1 region to distinguish vaccine (Sabin), vaccine-derived, and wild-type polioviruses and to ensure an appropriate response. This cell culture algorithm takes 2 to 3 weeks on average between sample receipt and sequencing. Direct detection of viral RNA using PCR allows faster detection but has traditionally faced challenges related to poor sensitivity and difficulties in sequencing common samples containing poliovirus and enterovirus mixtures. We present a nested PCR and nanopore sequencing protocol that allows rapid (<3 days) and sensitive direct detection and sequencing of polioviruses in stool and environmental samples. We developed barcoded primers and a real-time analysis platform that generate accurate VP1 consensus sequences from multiplexed samples. The sensitivity and specificity of our protocol compared with those of cell culture were 90.9% (95% confidence interval, 75.7% to 98.1%) and 99.2% (95.5% to 100.0%) for wild-type 1 poliovirus, 92.5% (79.6% to 98.4%) and 98.7% (95.4% to 99.8%) for vaccine and vaccine-derived serotype 2 poliovirus, and 88.3% (81.2% to 93.5%) and 93.2% (88.6% to 96.3%) for Sabin 1 and 3 poliovirus alone or in mixtures when tested on 155 stool samples in Pakistan. Variant analysis of sequencing reads also allowed the identification of polioviruses and enteroviruses in artificial mixtures and was able to distinguish complex mixtures of polioviruses in environmental samples. The median identity of consensus nanopore sequences with Sanger or Illumina sequences from the same samples was >99.9%. This novel method shows promise as a faster and safer alternative to cell culture for the detection and real-time sequencing of polioviruses in stool and environmental samples.

Enteroviruses are common human pathogens occasionally associated with severe disease, notoriously paralytic poliomyelitis caused by poliovirus. Other enterovirus serotypes such as enterovirus A71 and D68 have been linked to severe neurological syndromes. New enterovirus serotypes continue to emerge, some believed to be derived from nonhuman primates. However, little is known about the circulation patterns of many enterovirus serotypes and, in particular, the detailed enterovirus composition of sewage samples.

Enterovirus A71 (EVA71) causes widespread disease in young children with occasional fatal consequences. In common with other picornaviruses, both empty capsids (ECs) and infectious virions are produced during the viral lifecycle. While initially antigenically indistinguishable from virions, ECs readily convert to an expanded conformation at moderate temperatures. In the closely related poliovirus, these conformational changes result in loss of antigenic sites required to elicit protective immune responses. Whether this is true for EVA71 remains to be determined and is the subject of this investigation.We previously reported the selection of a thermally resistant EVA71 genogroup B2 population using successive rounds of heating and passage. The mutations found in the structural protein-coding region of the selected population conferred increased thermal stability to both virions and naturally produced ECs. Here, we introduced these mutations into a recombinant expression system to produce stabilized virus-like particles (VLPs) in Pichia pastoris.The stabilized VLPs retain the native virion-like antigenic conformation as determined by reactivity with a specific antibody. Structural studies suggest multiple potential mechanisms of antigenic stabilization, however, unlike poliovirus, both native and expanded EVA71 particles elicited antibodies able to directly neutralize virus in vitro. Therefore, anti-EVA71 neutralizing antibodies are elicited by sites which are not canonically associated with the native conformation, but whether antigenic sites specific to the native conformation provide additional protective responses in vivo remains unclear. VLPs are likely to provide cheaper and safer alternatives for vaccine production and these data show that VLP vaccines are comparable with inactivated virus vaccines at inducing neutralising antibodies.

Enterovirus A71 (EVA71) causes widespread disease in young children with occasional fatal consequences. In common with other picornaviruses, both empty capsids (ECs) and infectious virions are produced during the viral lifecycle. While initially antigenically indistinguishable from virions, ECs readily convert to an expanded conformation at moderate temperatures. In the closely related poliovirus, these conformational changes result in loss of antigenic sites required to elicit protective immune responses. Whether this is true for EVA71 remains to be determined and is the subject of this investigation. We previously reported the selection of a thermally resistant EVA71 genogroup B2 population using successive rounds of heating and passage. The mutations found in the structural protein-coding region of the selected population conferred increased thermal stability to both virions and naturally produced ECs. Here, we introduced these mutations into a recombinant expression system to produce stabilised virus-like particles (VLPs) in Pichia pastoris . The stabilised VLPs retain the native virion-like antigenic conformation as determined by reactivity with a specific antibody. Structural studies suggest multiple potential mechanisms of antigenic stabilisation, however, unlike poliovirus, both native and expanded EVA71 particles elicited antibodies able to directly neutralise virus in vitro . Therefore, the anti-EVA71 neutralising antibodies are elicited by sites which are not canonically associated with the native conformation, but whether antigenic sites specific to the native conformation provide additional protective responses in vivo remains unclear. VLPs are likely to provide cheaper and safer alternatives for vaccine production and these data show that VLP vaccines are comparable with inactivated virus vaccines at inducing neutralising antibodies.

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 3D(pol) 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.