Enterovirus A71 does not meet the uncoating receptor SCARB2 at the cell surface.

Enterovirus A71 (EV-A71) infection involves a variety of receptors. Among them, two transmembrane protein receptors have been investigated in detail and shown to be critical for infection: P-selectin glycoprotein ligand-1 (PSGL-1) in lymphocytes (Jurkat cells), and scavenger receptor class B member 2 (SCARB2) in rhabdomyosarcoma (RD) cells. PSGL-1 and SCARB2 have been reported to be expressed on the surface of Jurkat and RD cells, respectively. In the work reported here, we investigated the roles of PSGL-1 and SCARB2 in the process of EV-A71 entry. We first examined the expression of SCARB2 in Jurkat cells, and detected it within the cytoplasm, but not on the cell surface. Further, using PSGL-1 and SCARB2 knockout cells, we found that although both PSGL-1 and SCARB2 are essential for virus infection of Jurkat cells, virus attachment to these cells requires only PSGL-1. These results led us to evaluate the cell surface expression and the roles of SCARB2 in other EV-A71-susceptible cell lines. Surprisingly, in contrast to the results of previous studies, we found that SCARB2 is absent from the surface of RD cells and other susceptible cell lines we examined, and that although SCARB2 is essential for infection of these cells, it is dispensable for virus attachment. These results indicate that a receptor other than SCARB2 is responsible for virus attachment to the cell and probably for internalization of virions, not only in Jurkat cells but also in RD cells and other EV-A71-susceptible cells. SCARB2 is highly concentrated in lysosomes and late endosomes, where it is likely to trigger acid-dependent uncoating of virions, the critical final step of the entry process. Our results suggest that the essential interactions between EV-A71 and SCARB2 occur, not at the cell surface, but within the cell.

Evaluation of Direct Detection Protocols for Poliovirus from Stool Samples of Acute Flaccid Paralysis Patients.

Polio surveillance in the Global Polio Eradication Initiative has been conducted with virus isolation from stool samples of acute flaccid paralysis (AFP) cases. Under the current biorisk management/regulations, challenges arise in the timelines of the report, sensitivity of the test and containment of poliovirus (PV) isolates. In the present study, we evaluated protocols of previously reported direct detection (DD) methods targeting the VP1 or VP4-VP2 regions of the PV genome in terms of sensitivity and sequencability. An optimized protocol targeting the entire-capsid region for the VP1 sequencing showed a high sensitivity (limit of detection = 82 copies of PV genome) with a simpler and faster reaction than reported ones (i.e., with the addition of all the primers at the start of the reaction, the RT-PCR reaction finishes within 2.5 h). The DD methods targeting the VP1 region detected PV in 60 to 80% of PV-positive stool samples from AFP cases; however, minor populations of PV strains in the samples with virus mixtures were missed by the methods. Sequencability of the DD methods was primarily determined by the efficiency of the PCRs for both Sanger and nanopore sequencing. The DD method targeting the VP4-VP2 region showed higher sensitivity than that targeting the VP1 region (limit of detection = 25 copies of PV genome) and successfully detected PV from all the stool samples examined. These results suggest that DD methods are effective for the detection of PV and that further improvement of the sensitivity is essential to serve as an alternative to the current polio surveillance algorithm.

Concurrent outbreaks of circulating vaccine-derived poliovirus types 1 and 2 affecting the Republic of the Philippines and Malaysia, 2019-2021.

Concurrent outbreaks of circulating vaccine-derived poliovirus serotypes 1 and 2 (cVDPV1, cVDPV2) were confirmed in the Republic of the Philippines in September 2019 and were subsequently confirmed in Malaysia by early 2020. There is continuous population subgroup movement in specific geographies between the two countries. Outbreak response efforts focused on sequential supplemental immunization activities with monovalent Sabin strain oral poliovirus vaccine type 2 (mOPV2) and bivalent oral poliovirus vaccines (bOPV, containing Sabin strain types 1 and 3) as well as activities to enhance poliovirus surveillance sensitivity to detect virus circulation. A total of six cVDPV1 cases, 13 cVDPV2 cases, and one immunodeficiency-associated vaccine-derived poliovirus type 2 case were detected, and there were 35 cVDPV1 and 31 cVDPV2 isolates from environmental surveillance sewage collection sites. No further cVDPV1 or cVDPV2 have been detected in either country since March 2020. Response efforts in both countries encountered challenges, particularly those caused by the global COVID-19 pandemic. Important lessons were identified and could be useful for other countries that experience outbreaks of concurrent cVDPV serotypes.

Integrin α3 is involved in non-enveloped hepatitis E virus infection.

Hepatitis E virus (HEV) causes acute and fulminant hepatitis worldwide. Although enveloped (e) and non-enveloped (ne) forms of HEV have been discovered, host factors involved in infection, including receptors, remain to be elucidated. Here, we identified integrin α3 (encoded by ITGA3), a protein that binds and responds to the extracellular matrix, as an essential host factor for HEV infection. Integrin α3 expression was lower in four HEV-non-permissive cell subclones than in an HEV-permissive subclone. ITGA3 knockout cells lost HEV permissibility, suggesting that integrin α3 is critical for HEV infection. Stable expression of integrin α3 in an HEV-non-permissive subclone provided permissibility only to infection by neHEV; expression of integrin α3 lacking the ectodomain did not. Direct interaction between neHEV and the integrin α3 ectodomain was confirmed by co-precipitation using a soluble integrin α3-Fc. These results strongly suggest that integrin α3 is a key molecule for cellular attachment and entry of neHEV.

An Insight into Recombination with Enterovirus Species C and Nucleotide G-480 Reversion from the Viewpoint of Neurovirulence of Vaccine-Derived Polioviruses.

A poliomyelitis outbreak caused by type 1 circulating vaccine-derived polioviruses (cVDPVs) was identified in China in 2004. Six independent cVDPVs (eight isolates) could be grouped into a single cluster with pathways of divergence different from a single cVDPV progenitor, which circulated and evolved into both a highly neurovirulent lineage and a less neurovirulent lineage. They were as neurovirulent as the wild type 1 Mahoney strain, recombination was absent, and their nucleotide 480-G was identical to that of the Sabin strain. The Guizhou/China cVDPV strains shared 4 amino acid replacements in the NAg sites: 3 located at the BC loop, which may underlie the aberrant results of the ELISA intratypic differentiation (ITD) test. The complete ORF tree diverged into two main branches from a common ancestral infection estimated to have occurred in about mid-September 2003, nine months before the appearance of the VDPV case, which indicated recently evolved VDPV. Further, recombination with species C enteroviruses may indicate the presence and density of these enteroviruses in the population and prolonged virus circulation in the community. The aforementioned cVDPVs has important implications in the global initiative to eradicate polio: high quality surveillance permitted earliest detection and response.

The Suramin Derivative NF449 Interacts with the 5-fold Vertex of the Enterovirus A71 Capsid to Prevent Virus Attachment to PSGL-1 and Heparan Sulfate.

NF449, a sulfated compound derived from the antiparasitic drug suramin, was previously reported to inhibit infection by enterovirus A71 (EV-A71). In the current work, we found that NF449 inhibits virus attachment to target cells, and specifically blocks virus interaction with two identified receptors--the P-selectin ligand, PSGL-1, and heparan sulfate glycosaminoglycan--with no effect on virus binding to a third receptor, the scavenger receptor SCARB2. We also examined a number of commercially available suramin analogues, and newly synthesized derivatives of NF449; among these, NF110 and NM16, like NF449, inhibited virus attachment at submicromolar concentrations. PSGL-1 and heparan sulfate, but not SCARB2, are both sulfated molecules, and their interaction with EV-A71 is thought to involve positively charged capsid residues, including a conserved lysine at VP1-244, near the icosahedral 5-fold vertex. We found that mutation of VP1-244 resulted in resistance to NF449, suggesting that this residue is involved in NF449 interaction with the virus capsid. Consistent with this idea, NF449 and NF110 prevented virus interaction with monoclonal antibody MA28-7, which specifically recognizes an epitope overlapping VP1-244 at the 5-fold vertex. Based on these observations we propose that NF449 and related compounds compete with sulfated receptor molecules for a binding site at the 5-fold vertex of the EV-A71 capsid.

The Role of VP1 Amino Acid Residue 145 of Enterovirus 71 in Viral Fitness and Pathogenesis in a Cynomolgus Monkey Model.

Enterovirus 71 (EV71), a major causative agent of hand, foot, and mouth disease, occasionally causes severe neurological symptoms. We identified P-selectin glycoprotein ligand-1 (PSGL-1) as an EV71 receptor and found that an amino acid residue 145 in the capsid protein VP1 (VP1-145) defined PSGL-1-binding (PB) and PSGL-1-nonbinding (non-PB) phenotypes of EV71. However, the role of PSGL-1-dependent EV71 replication in neuropathogenesis remains poorly understood. In this study, we investigated viral replication, genetic stability, and the pathogenicity of PB and non-PB strains of EV71 in a cynomolgus monkey model. Monkeys were intravenously inoculated with cDNA-derived PB and non-PB strains of EV71, EV71-02363-EG and EV71-02363-KE strains, respectively, with two amino acid differences at VP1-98 and VP1-145. Mild neurological symptoms, transient lymphocytopenia, and inflammatory cytokine responses, were found predominantly in the 02363-KE-inoculated monkeys. During the early stage of infection, viruses were frequently detected in clinical samples from 02363-KE-inoculated monkeys but rarely in samples from 02363-EG-inoculated monkeys. Histopathological analysis of central nervous system (CNS) tissues at 10 days postinfection revealed that 02363-KE induced neuropathogenesis more efficiently than that induced by 02363-EG. After inoculation with 02363-EG, almost all EV71 variants detected in clinical samples, CNS, and non-CNS tissues, possessed a G to E amino acid substitution at VP1-145, suggesting a strong in vivo selection of VP1-145E variants and CNS spread presumably in a PSGL-1-independent manner. EV71 variants with VP1-145G were identified only in peripheral blood mononuclear cells in two out of four 02363-EG-inoculated monkeys. Thus, VP1-145E variants are mainly responsible for the development of viremia and neuropathogenesis in a non-human primate model, further suggesting the in vivo involvement of amino acid polymorphism at VP1-145 in cell-specific viral replication, in vivo fitness, and pathogenesis in EV71-infected individuals.

Genetic diversity of circulating Saffold viruses in Pakistan and Afghanistan.

Human cardioviruses or Saffold viruses (SAFVs) of the family Picornaviridae are newly emerging viruses whose genetic and phenotypic diversity are poorly understood. We report here the full genome sequence of 11 SAFV genotypes from Pakistan and Afghanistan, along with a re-evaluation of their genetic diversity and recombination. We detected 88 SAFV from stool samples of 943 acute flaccid paralysis cases using reverse transcriptase-PCR targeting the 5' untranslated region (UTR). Further characterization based on complete VP1 analysis revealed 71 SAFVs belonging to 11 genotypes, including three previously unidentified genotypes. SAFV showed high genetic diversity and recombination based on phylogenetic, pairwise distance distributions and recombination mapping analyses performed herein. Phylogenies based on non-structural and UTRs were highly incongruent indicating frequent recombination events among SAFVs. We improved the SAFV genotyping classification criteria by determining new VP1 thresholds based on the principles used for the classification of enteroviruses. For genotype assignment, we propose a threshold of 23 and 10 % divergence for VP1 nucleotide and amino acid sequences, respectively. Other members of the species Theilovirus, such as Thera virus and Theiler's murine encephalomyelitis virus, are difficult to classify in the same species as SAFV, because they are genetically distinct from SAFV, with 41-56 % aa pairwise distances. The new genetic information obtained in this study will improve our understanding of the evolution and classification of SAFV.

Enterovirus 71 binding to PSGL-1 on leukocytes: VP1-145 acts as a molecular switch to control receptor interaction.

Some strains of enterovirus 71 (EV71), but not others, infect leukocytes by binding to a specific receptor molecule: the P-selectin glycoprotein ligand-1 (PSGL-1). We find that a single amino acid residue within the capsid protein VP1 determines whether EV71 binds to PSGL-1. Examination of capsid sequences of representative EV71 strains revealed that the PSGL-1-binding viruses had either a G or a Q at residue 145 within the capsid protein VP1 (VP1-145G or Q), whereas PSGL-1-nonbinding viruses had VP1-145E. Using site-directed mutagenesis we found that PSGL-1-binding strains lost their capacity to bind when VP1-145G/Q was replaced by E; conversely, nonbinding strains gained the capacity to bind PSGL-1 when VP1-145E was replaced with either G or Q. Viruses with G/Q at VP1-145 productively infected a leukocyte cell line, Jurkat T-cells, whereas viruses with E at this position did not. We previously reported that EV71 binds to the N-terminal region of PSGL-1, and that binding depends on sulfated tyrosine residues within this region. We speculated that binding depends on interaction between negatively charged sulfate groups and positively charged basic residues in the virus capsid. VP1-145 on the virus surface is in close proximity to conserved lysine residues at VP1-242 and VP1-244. Comparison of recently published crystal structures of EV71 isolates with either Q or E at VP1-145 revealed that VP1-145 controls the orientation of the lysine side-chain of VP1-244: with VP1-145Q the lysine side chain faces outward, but with VP1-145E, the lysine side chain is turned toward the virus surface. Mutation of VP1-244 abolished virus binding to PSGL-1, and mutation of VP1-242 greatly reduced binding. We propose that conserved lysine residues on the virus surface are responsible for interaction with sulfated tyrosine residues at the PSGL-1 N-terminus, and that VP1-145 acts as a switch, controlling PSGL-1 binding by modulating the exposure of VP1-244K.

A strain-specific epitope of enterovirus 71 identified by cryo-electron microscopy of the complex with fab from neutralizing antibody.

Enterovirus 71 (EV71) is a picornavirus that causes outbreaks of hand, foot, and mouth disease (HFMD), primarily in the Asia-Pacific area. Unlike coxsackievirus A16, which also causes HFMD, EV71 induces severe neuropathology leading to high fatalities, especially among children under the age of 6 years. Currently, no established vaccines or treatments are available against EV71 infection. The monoclonal antibody MA28-7 neutralizes only specific strains of EV71 that have a conserved glycine at amino acid VP1-145, a surface-exposed residue that maps to the 5-fold vertex and that has been implicated in receptor binding. The cryo-electron microscopy structure of a complex between EV71 and the Fab fragment of MA28-7 shows that only one Fab fragment occupies each 5-fold vertex. A positively charged patch, which has also been implicated in receptor binding, lies within the Fab footprint. We identify the strain-specific epitope of EV71 and discuss the possible neutralization mechanisms of the antibody.

Understanding the biological context of NS5A-host interactions in HCV infection: a network-based approach.

Hepatitis C virus (HCV) is a major cause of chronic liver disease. HCV NS5A protein plays an important role in HCV infection through its interactions with other HCV proteins and host factors. In an attempt to further our understanding of the biological context of protein interactions between NS5A and host factors in HCV pathogenesis, we generated an extensive physical interaction map between NS5A and cellular factors. By combining a yeast two-hybrid assay with comprehensive literature mining, we built the NS5A interactome composed of 132 human proteins that interact with NS5A. These interactions were integrated into a high-confidence human protein interactome (HPI) with the help of the TargetMine data warehouse system to infer an overall protein interaction map linking NS5A with the components of the host cellular networks. The NS5A-host interactions that were integrated with the HPI were shown to participate in compact and well-connected cellular networks. Functional analysis of the NS5A "infection" network using TargetMine highlighted cellular pathways associated with immune system, cellular signaling, cell adhesion, cellular growth and death among others, which were significantly targeted by NS5A-host interactions. In addition, cellular assays with in vitro HCV cell culture systems identified two ER-localized host proteins RTN1 and RTN3 as novel regulators of HCV propagation. Our analysis builds upon the present understanding of the role of NS5A protein in HCV pathogenesis and provides potential targets for more effective anti-HCV therapeutic intervention.

Multiple independent emergences of type 2 vaccine-derived polioviruses during a large outbreak in northern Nigeria.

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 (A481 in the 5'-untranslated region [5'-UTR] and VP1-Ile143) had been replaced in all VDPV2 isolates; most A481 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.

Quantification of the dynamics of enterovirus 71 infection by experimental-mathematical investigation.

Enterovirus 71 (EV71) is the causative agent of hand-foot-and-mouth disease and can trigger neurological disorders. EV71 outbreaks are a major public health concern in Asia-Pacific countries. By performing experimental-mathematical investigation, we demonstrate here that viral productivity and transmissibility but not viral cytotoxicity are drastically different among EV71 strains and can be associated with their epidemiological backgrounds. This is the first report demonstrating the dynamics of nonenveloped virus replication in cell culture using mathematical modeling.

Anti-adhesive property of P-selectin glycoprotein ligand-1 (PSGL-1) due to steric hindrance effect.

P-selectin glycoprotein ligand-1 (PSGL-1) is an adhesive molecule that is known to be a ligand for P-selectin. An anti-adhesive property of PSGL-1 has not been previously reported. In this study, we show that PSGL-1 expression is anti-adhesive for adherent cells and we have elucidated the underlying mechanism. Overexpression of PSGL-1 induced cell rounding and floating in HEK293T cells. Similar phenomena were demonstrated in other adherent cell lines with overexpression of PSGL-1. PSGL-1 overexpression inhibits access of antibodies to cell surface molecules such as integrins, HLA and CD25. Cells transfected with PSGL-1 deletion mutants that lack a large part of the extracellular domain and chimeric construct expressing extracellular CD86 and intracellular PSGL-1 only showed rounded morphology, but there are no floating cells. These results indicated that PSGL-1 causes steric hindrance due to the extended structure of its extracellular domain that is highly O-glycosylated, but intracellular domain also has some effect on cell rounding. This study implies that PSGL-1 has Janus-faced functions, being both adhesive and anti-adhesive.

[Infection mechanism of enterovirus 71].

Identification of a specific viral receptor is important for understanding the virus infection mechanism. I identified P-selectin glycoprotein ligand-1 (PSGL-1) as one of the functional receptors for enterovirus 71 (EV71), a pathogen that causes hand, foot, and mouth disease. PSGL-1, which belongs to the sialomucin family, is a transmembrane protein mainly expressed on leukocytes. Tyrosine sulfation in the N-terminal region of PSGL-1 is critical for PSGL-1's capacity to bind EV71. The identification of EV71 receptors provides important mechanistic information about viral entry into cells and helps us understand viral pathogenesis and develop new anti-viral strategies.

Cellular receptors for human enterovirus species a.

Human enterovirus species A (HEV-A) is one of the four species of HEV in the genus Enterovirus in the family Picornaviridae. Among HEV-A, coxsackievirus A16 (CVA16) and enterovirus 71 (EV71) are the major causative agents of hand, foot, and mouth disease (HFMD). Some other types of HEV-A are commonly associated with herpangina. Although HFMD and herpangina due to HEV-A are common febrile diseases among infants and children, EV71 can cause various neurological diseases, such as aseptic meningitis and fatal encephalitis. Recently, two human transmembrane proteins, P-selectin glycoprotein ligand-1 (PSGL-1) and scavenger receptor class B, member 2 (SCARB2), were identified as functional receptors for EV71 and CVA16. In in vitro infection experiments using the prototype HEV-A strains, PSGL-1 and SCARB2 could be responsible for the specific receptors for EV71 and CVA16. However, the involvement of both receptors in the in vitro and in vivo infections of clinical isolates of HEV-A has not been clarified yet. To elucidate a diverse array of the clinical outcome of HEV-A-associated diseases, the identification and characterization of HEV-A receptors may provide useful information in understanding the HEV-A pathogenesis at a molecular level.

Adaptive mutations in the genomes of enterovirus 71 strains following infection of mouse cells expressing human P-selectin glycoprotein ligand-1.

We recently identified human P-selectin glycoprotein ligand-1 (PSGL-1) as a functional enterovirus 71 (EV71) receptor and demonstrated PSGL-1-dependent replication for some EV71 strains in human leukocytes. Here, we report that four out of five PSGL-1-binding strains of EV71 replicated poorly in mouse L929 cells stably expressing human PSGL-1 (L-PSGL-1 cells). Therefore, we compared the replication kinetics and entire genomic sequence of five original EV71 strains and the corresponding EV71 variants (EV71-LPS), which were propagated once in L-PSGL-1 cells. Direct sequence comparison of the entire genome of the original EV71 strains and EV71-LPS variants identified several possible adaptive mutations during the course of replication in L-PSGL-1 cells, including a putative determinant of the adaptive phenotype in L-PSGL-1 cells at VP2-149. The results suggest that an adaptive mutation, along with a PSGL-1-binding phenotype, may facilitate efficient PSGL-1-dependent replication of the EV71 strains in L-PSGL-1 cells.

Tyrosine sulfation of the amino terminus of PSGL-1 is critical for enterovirus 71 infection.

Enterovirus 71 (EV71) is one of the major causative agents of hand, foot, and mouth disease, a common febrile disease in children; however, EV71 has been also associated with various neurological diseases including fatal cases in large EV71 outbreaks particularly in the Asia Pacific region. Recently we identified human P-selectin glycoprotein ligand-1 (PSGL-1) as a cellular receptor for entry and replication of EV71 in leukocytes. PSGL-1 is a sialomucin expressed on the surface of leukocytes, serves as a high affinity counterreceptor for selectins, and mediates leukocyte rolling on the endothelium. The PSGL-1-P-selectin interaction requires sulfation of at least one of three clustered tyrosines and an adjacent O-glycan expressing sialyl Lewis x in an N-terminal region of PSGL-1. To elucidate the molecular basis of the PSGL-1-EV71 interaction, we generated a series of PSGL-1 mutants and identified the post-translational modifications that are critical for binding of PSGL-1 to EV71. We expressed the PSGL-1 mutants in 293T cells and the transfected cells were assayed for their abilities to bind to EV71 by flow cytometry. We found that O-glycosylation on T57, which is critical for PSGL-1-selectin interaction, is not necessary for PSGL-1 binding to EV71. On the other hand, site-directed mutagenesis at one or more potential tyrosine sulfation sites in the N-terminal region of PSGL-1 significantly impaired PSGL-1 binding to EV71. Furthermore, an inhibitor of sulfation, sodium chlorate, blocked the PSGL-1-EV71 interaction and inhibited PSGL-1-mediated viral replication of EV71 in Jurkat T cells in a dose-dependent manner. Thus, the results presented in this study reveal that tyrosine sulfation, but not O-glycosylation, in the N-terminal region of PSGL-1 may facilitate virus entry and replication of EV71 in leukocytes.

Characterization of a rare natural intertypic type 2/type 3 penta-recombinant vaccine-derived poliovirus isolated from a child with acute flaccid paralysis.

A type 2 vaccine-derived poliovirus (VDPV) (strain CHN1025), with a 1.1 % (10/903) difference from Sabin strain in the VP1 coding region, was isolated from a child with poliomyelitis caused by a poliovirus variant infection. The patient was from Shandong Province of China and developed acute flaccid paralysis in 1997. The child was infected with a rare and complicated penta-recombinant poliovirus with the uncommon genomic recombinant organization S2/S3/S1/S3/S1/S3. At least five successive rounds of recombination occurred in the VP1 capsid coding region and in the 2C, 3C (twice) and 3D(pol) non-capsid coding regions, respectively, during virus evolution. Strain CHN1025 had most of the characteristics of the type 2 vaccine strain; it had Sabin-specific epitopes, suggesting that the virus was antigenically indistinguishable from the Sabin 2 reference strain. Typical mutations in the 5'-untranslated region and VP1 associated with reversion to neurovirulence for Sabin 2 poliovirus were found, and the virus showed moderate neurovirulence in transgenic mice. A few nucleotide substitutions were located in the donor sequences, and two donor sequences contained no nucleotide substitutions, suggesting that these sequences were relatively new. The appearance of these mutations within approximately 192 days of at least five successive rounds of recombination events derived from a single ancestral infection illustrates the rapid emergence of new recombinants among VDPVs. This is the first report on the isolation of a type 2/type 3 poliovirus capsid recombinant with one of the five crossover sites located in the VP1 coding region.