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.

Characterization of Anti-Poliovirus Compounds Isolated from Edible Plants.

Poliovirus (PV) is the causative agent of poliomyelitis and is a target of the global eradication programs of the World Health Organization (WHO). After eradication of type 2 and 3 wild-type PVs, vaccine-derived PV remains a substantial threat against the eradication as well as type 1 wild-type PV. Antivirals could serve as an effective means to suppress the outbreak; however, no anti-PV drugs have been approved at present. Here, we screened for effective anti-PV compounds in a library of edible plant extracts (a total of 6032 extracts). We found anti-PV activity in the extracts of seven different plant species. We isolated chrysophanol and vanicoside B (VCB) as the identities of the anti-PV activities of the extracts of Rheum rhaponticum and Fallopia sachalinensis, respectively. VCB targeted the host PI4KB/OSBP pathway for its anti-PV activity (EC50 = 9.2 µM) with an inhibitory effect on in vitro PI4KB activity (IC50 = 5.0 µM). This work offers new insights into the anti-PV activity in edible plants that may serve as potent antivirals for PV infection.

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.

Essential Domains of Oxysterol-Binding Protein Required for Poliovirus Replication.

Oxysterol-binding protein (OSBP) is a host factor required for enterovirus (EV) replication. OSBP locates at membrane contact site and acts as a lipid exchanger of cholesterol and phosphatidylinositol 4-phosphate (PI4P) between cellular organelles; however, the essential domains required for the viral replication remain unknown. In this study, we define essential domains of OSBP for poliovirus (PV) replication by a functional dominance assay with a series of deletion variants of OSBP. We show that the pleckstrin homology domain (PHD) and the ligand-binding domain, but not the N-terminal intrinsically disordered domain, coiled-coil region, or the FFAT motif, are essential for PV replication. The PHD serves as the primary determinant of OSBP targeting to the replication organelle in the infected cells. These results suggest that not all the domains that support important biological functions of OSBP are essential for the viral replication.

High-throughput analysis of anti-poliovirus neutralization antibody titre in human serum by the pseudovirus neutralization test.

To monitor vulnerability of countries to poliovirus (PV) outbreaks, serosurveillance of anti-PV neutralization antibody is conducted by conventional PV neutralization test (cPNT), which uses live PV strains. We previously developed a pseudovirus PV neutralization test (pPNT) as an alternative to cPNT, which uses PV pseudovirus that expresses luciferase as a reporter in the infection without producing infectious PV. In the present study, we established a high-throughput pPNT (HTpPNT) for a large-scale serosurveillance. The HTpPNT system was evaluated with 600 human serum samples obtained from a broad range of age groups of healthy volunteers (ages of 0-89 years). HTpPNT showed high correlation with cPNT (R2 for anti-type 1, 2, and 3 PV neutralization antibody titres are 0.90, 0.84, and 0.90, respectively). By using HTpPNT, we analyzed relative neutralizing antibody titre of the sera against a type 1 PV wild-type strain (Mahoney strain) to that against the type 1 Sabin strain. As a result, a correlation between the age (≥ 60 years) and the relative neutralizing antibody titre was observed (n = 15-16, P = 0.0000023-0.041), while the types of PV vaccine (i.e., oral PV vaccine and Sabin strain-based IPV) had no effect. HTpPNT would serve as a useful alternative to cPNT in a large-scale serosurveillance.

Ligand Recognition by the Lipid Transfer Domain of Human OSBP Is Important for Enterovirus Replication.

Oxysterol-binding protein (OSBP), which transports cholesterol and phosphatidylinositol 4-monophosphate (PtdIns[4]P) between different organelles, serves as a conserved host factor for the replication of various viruses, and OSBP inhibitors exhibit antiviral effects. Here, we determined the crystal structure of the lipid transfer domain of human OSBP in complex with endogenous cholesterol. The hydrocarbon tail and tetracyclic ring of cholesterol interact with the hydrophobic tunnel of OSBP, and the hydroxyl group of cholesterol forms a hydrogen bond network at the bottom of the tunnel. Systematic mutagenesis of the ligand-binding region revealed that M446W and L590W substitutions confer functional tolerance to an OSBP inhibitor, T-00127-HEV2. Employing the M446W variant as a functional replacement for the endogenous OSBP in the presence of T-00127-HEV2, we have identified previously unappreciated amino acid residues required for viral replication. The combined use of the inhibitor and the OSBP variant will be useful in elucidating the enigmatic in vivo functions of OSBP.

Intestinal Antibody Responses to 2 Novel Live Attenuated Type 2 Oral Poliovirus Vaccines in Healthy Adults in Belgium.

In a blinded phase 1 trial (EudraCT 2017-0000908-21; NCT03430349) in Belgium, healthy adults (aged 18-50 years) previously immunized exclusively with inactivated poliovirus vaccine were administered a single dose of 1 of 2 novel type 2 oral poliovirus vaccines (nOPV2-c1: S2/cre5/S15domV/rec1/hifi3 (n = 15); nOPV2-c2: S2/S15domV/CpG40 (n = 15)) and isolated for 28 days in a purpose-built containment facility. Using stool samples collected near days 0, 14, 21, and 28, we evaluated intestinal neutralization and immunoglobulin A responses to the nOPV2s and found that nOPV2-c1 and nOPV2-c2 induced detectable poliovirus type 2-specific intestinal neutralizing responses in 40.0% and 46.7% of participants, respectively.

High-Order Epistasis and Functional Coupling of Infection Steps Drive Virus Evolution toward Independence from a Host Pathway.

The phosphatidylinositol-4 kinase IIIß (PI4KB)/oxysterol-binding protein (OSBP) family I pathway serves as an essential host pathway for the formation of viral replication complex for viral plus-strand RNA synthesis; however, poliovirus (PV) could evolve toward substantial independence from this host pathway with four mutations. Recessive epistasis of the two mutations (3A-R54W and 2B-F17L) is essential for viral RNA replication. Quantitative analysis of effects of the other two mutations (2B-Q20H and 2C-M187V) on each step of infection reveals functional couplings between viral replication, growth, and spread conferred by the 2B-Q20H mutation, while no enhancing effect was conferred by the 2C-M187V mutation. The effects of the 2B-Q20H mutation occur only via another recessive epistasis between the 3A-R54W/2B-F17L mutations. These mutations confer enhanced replication in PI4KB/OSBP-independent infection concomitantly with an increased ratio of viral plus-strand RNA to the minus-strand RNA. This work reveals the essential roles of the functional coupling and high-order, multi-tiered recessive epistasis in viral evolution toward independence from an obligatory host pathway. IMPORTANCE Each virus has a different strategy for its replication, which requires different host factors. Enterovirus, a model RNA virus, requires host factors PI4KB and OSBP, which form an obligatory functional axis to support viral replication. In an experimental evolution system in vitro, virus mutants that do not depend on these host factors could arise only with four mutations. The two mutations (3A-R54W and 2B-F17L) are required for the replication but are not sufficient to support efficient infection. Another mutation (2B-Q20H) is essential for efficient spread of the virus. The order of introduction of the mutations in the viral genome is essential (known as "epistasis"), and functional couplings of infection steps (i.e., viral replication, growth, and spread) have substantial roles to show the effects of the 2B-Q20H mutation. These observations would provide novel insights into an evolutionary pathway of the virus to require host factors for infection.

Sphingomyelin Is Essential for the Structure and Function of the Double-Membrane Vesicles in Hepatitis C Virus RNA Replication Factories.

Some plus-stranded RNA viruses generate double-membrane vesicles (DMVs), one type of the membrane replication factories, as replication sites. Little is known about the lipid components involved in the biogenesis of these vesicles. Sphingomyelin (SM) is required for hepatitis C virus (HCV) replication, but the mechanism of SM involvement remains poorly understood. SM biosynthesis starts in the endoplasmic reticulum (ER) and gives rise to ceramide, which is transported from the ER to the Golgi by the action of ceramide transfer protein (CERT), where it can be converted to SM. In this study, inhibition of SM biosynthesis, either by using small-molecule inhibitors or by knockout (KO) of CERT, suppressed HCV replication in a genotype-independent manner. This reduction in HCV replication was rescued by exogenous SM or ectopic expression of the CERT protein, but not by ectopic expression of nonfunctional CERT mutants. Observing low numbers of DMVs in stable replicon cells treated with a SM biosynthesis inhibitor or in CERT-KO cells transfected with either HCV replicon or with constructs that drive HCV protein production in a replication-independent system indicated the significant importance of SM to DMVs. The degradation of SM of the in vitro-isolated DMVs affected their morphology and increased the vulnerability of HCV RNA and proteins to RNase and protease treatment, respectively. Poliovirus, known to induce DMVs, showed decreased replication in CERT-KO cells, while dengue virus, known to induce invaginated vesicles, did not. In conclusion, these findings indicated that SM is an essential constituent of DMVs generated by some plus-stranded RNA viruses.IMPORTANCE Previous reports assumed that sphingomyelin (SM) is essential for HCV replication, but the mechanism was unclear. In this study, we showed for the first time that SM and ceramide transfer protein (CERT), which is in the SM biosynthesis pathway, are essential for the biosynthesis of double-membrane vesicles (DMVs), the sites of viral replication. Low numbers of DMVs were observed in CERT-KO cells transfected with replicon RNA or with constructs that drive HCV protein production in a replication-independent system. HCV replication was rescued by ectopic expression of the CERT protein, but not by CERT mutants, that abolishes the binding of CERT to vesicle-associated membrane protein-associated protein (VAP) or phosphatidylinositol 4-phosphate (PI4P), indicating new roles for VAP and PI4P in HCV replication. The biosynthesis of DMVs has great importance to replication by a variety of plus-stranded RNA viruses. Understanding of this process is expected to facilitate the development of diagnosis and antivirus.

Characterization of a New Antienterovirus D68 Compound Purified from Avocado.

One of the major challenges in development of antienterovirus (EV) drugs is in the safety of the drug. Here, we attempted to identify anti-EV compounds from an edible plant extract library and found potent antienterovirus D68 (EV-D68) activity in avocado (Persea americana). The purified identity is determined as 2R,4R-(12Z,15Z)-heneicosa-12,15-diene-1,2,4-triol, named avoenin. Avoenin shows an EC50 of 2.0 µM for EV-D68 (Fermon) infection with CC50 of >150 µM in RD cells by targeting the uncoating step of EV-D68 infection. Resistant mutations of EV-D68 (VP3-V24I, S173P, and S180G) to avoenin confer cross-resistance to pleconaril, an uncoating inhibitor of EV-D68. The inhibitory effect of avoenin is substantially specific to EV-D68 among the EVs. This work reveals avoenin as the identity of anti-EV-D68 activity in avocado and offers insights into development of a novel and effective strategy to overcome EV-D68 infection and its related respiratory diseases.

Surfeit 4 Contributes to the Replication of Hepatitis C Virus Using Double-Membrane Vesicles.

A number of positive-strand RNA viruses, such as hepatitis C virus (HCV) and poliovirus, use double-membrane vesicles (DMVs) as replication sites. However, the role of cellular proteins in DMV formation during virus replication is poorly understood. HCV NS4B protein induces the formation of a "membranous web" structure that provides a platform for the assembly of viral replication complexes. Our previous screen of NS4B-associated host membrane proteins by dual-affinity purification, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), and small interfering RNA (siRNA) methods revealed that the Surfeit 4 (Surf4) gene, which encodes an integral membrane protein, is involved in the replication of the JFH1 subgenomic replicon. Here, we investigated in detail the effect of Surf4 on HCV replication. Surf4 affects HCV replication in a genotype-independent manner, whereas HCV replication does not alter Surf4 expression. The influence of Surf4 on HCV replication indicates that while Surf4 regulates replication, it has no effect on entry, translation, assembly, or release. Analysis of the underlying mechanism showed that Surf4 is recruited into HCV RNA replication complexes by NS4B and is involved in the formation of DMVs and the structural integrity of RNA replication complexes. Surf4 also participates in the replication of poliovirus, which uses DMVs as replication sites, but it has no effect on the replication of dengue virus, which uses invaginated/sphere-type vesicles as replication sites. These findings clearly show that Surf4 is a novel cofactor that is involved in the replication of positive-strand RNA viruses using DMVs as RNA replication sites, which provides valuable clues for DMV formation during positive-strand RNA virus replication.IMPORTANCE Hepatitis C virus (HCV) NS4B protein induces the formation of a membranous web (MW) structure that provides a platform for the assembly of viral replication complexes. The main constituents of the MW are double-membrane vesicles (DMVs). Here, we found that the cellular protein Surf4, which maintains endoplasmic reticulum (ER)-Golgi intermediate compartments and the Golgi compartment, is recruited into HCV RNA replication complexes by NS4B and is involved in the formation of DMVs. Moreover, Surf4 participates in the replication of poliovirus, which uses DMVs as replication sites, but has no effect on the replication of dengue virus, which uses invaginated vesicles as replication sites. These results indicate that the cellular protein Surf4 is involved in the replication of positive-strand RNA viruses that use DMVs as RNA replication sites, providing new insights into DMV formation during virus replication and potential targets for the diagnosis and treatment of positive-strand RNA viruses.

Intestinal antibody responses to a live oral poliovirus vaccine challenge among adults previously immunized with inactivated polio vaccine in Sweden.

BACKGROUND: Our understanding of the acquisition of intestinal mucosal immunity and the control of poliovirus replication and transmission in later life is still emerging. METHODS: As part of a 2011 randomised, blinded, placebo-controlled clinical trial of the experimental antiviral agent pocapavir (EudraCT 2011-004804-38), Swedish adults, aged 18-50 years, who had previously received four doses of inactivated polio vaccine (IPV) in childhood were challenged with a single dose of monovalent oral polio vaccine type 1 (mOPV1). Using faecal samples collected before and serially, over the course of 45 days, after mOPV1 challenge from a subset of placebo-arm participants who did not receive pocapavir (N=12), we investigated the kinetics of the intestinal antibody response to challenge virus by measuring poliovirus type 1-specific neutralising activity and IgA concentrations. RESULTS: In faecal samples collected prior to mOPV1 challenge, we found no evidence of pre-existing intestinal neutralising antibodies to any of the three poliovirus serotypes. Despite persistent high-titered vaccine virus shedding and rising serum neutralisation responses after mOPV1 challenge, intestinal poliovirus type 1-specific neutralisation remained low with a titer of ≤18.4 across all time points and individuals. Poliovirus types 1-specific, 2-specific and 3-specific IgA remained below the limit of detection for all specimens collected postchallenge. INTERPRETATION: In contrast to recent studies demonstrating brisk intestinal antibody responses to oral polio vaccine challenge in young children previously vaccinated with IPV, this investigation finds that adults previously vaccinated with IPV have only modest intestinal poliovirus type 1-specific neutralisation and no IgA responses that are measurable in stool samples following documented mOPV1 infection.

Evaluation of antigenic differences between wild and Sabin vaccine strains of poliovirus using the pseudovirus neutralization test.

In the endgame of global polio eradication, serosurveillance is essential to monitor each country's vulnerability to poliomyelitis outbreaks. Previously, we developed pseudovirus poliovirus (PV) neutralization test (pPNT) with type 1, 2, and 3 PV pseudovirus (PVpv), which possess a luciferase-encoding PV replicon in the capsids of wild-type strains (PVpv[WT]), showing that pPNT with type 2 and 3 PVpv(WT) but not type 1 shows high correlation with the conventional PV neutralization test (cPNT) performed with vaccine strains. Here, we analyse the antigenicity of PVpv(WT) and PVpv with capsid proteins of Sabin vaccine strains (PVpv[Sabin]) in human serum. Type 2 and 3 PVpv(WT) and PVpv(Sabin) show similar antigenicity in the analysed set of human sera in contrast to type 1 PVpv. The levels of PVpv(Sabin) infection (%), including about 70% of PVpv infection (%) measured in the presence of human serum diluted to the cPNT titre, serve as the optimal threshold values for pPNT (5% for type 1 and 2, 10% for type 3) to show high correlation with cPNT results. Our results suggest that pPNT with PVpv(Sabin) could serve as an alternative to cPNT and provide a rationale for pPNT threshold values.

Essential domains of phosphatidylinositol-4 kinase III ß required for enterovirus replication.

Phosphatidylinositol-4 kinase III ß (PI4KB) is a host factor that is required for enterovirus (EV) replication. In this study, the importance of host proteins that interact with PI4KB in EV replication was analyzed by trans complementation with PI4KB mutants in a PI4KB-knockout cell line. Ectopically expressed PI4KB mutants, which lack binding regions for ACBD3, RAB11, and 14-3-3 proteins, rescued replication of poliovirus and enterovirus 71. These findings suggest that interaction of PI4KB with these host proteins is not essential for EV replication once PI4KB has been expressed and that PI4KB is functionally independent from these host proteins regarding EV replication.

Poliovirus Evolution toward Independence from the Phosphatidylinositol-4 Kinase III ß/Oxysterol-Binding Protein Family I Pathway.

Phosphatidylinositol-4 kinase III ß (PI4KB) and oxysterol-binding protein (OSBP) family I provide a conserved host pathway required for enterovirus replication. Here, we analyze the role and essentiality of this pathway in enterovirus replication. Phosphatidylinositol 4-phosphate (PI4P) production and cholesterol accumulation in the replication organelle (RO) are severely suppressed in cells infected with a poliovirus (PV) mutant isolated from a PI4KB-knockout cell line (RD[Δ PI4KB]). Major determinants of the mutant for infectivity in RD(Δ PI4KB) cells map to the A5270U(3A-R54W) and U3881C(2B-F17L) mutations. The 3A mutation is required for PI4KB-independent development of RO. The 2B mutation rather sensitizes PV to PI4KB/OSBP inhibitors by itself but confers substantially complete resistance to the inhibitors with the 3A mutation. The 2B mutation also confers hypersensitivity to interferon alpha treatment on PV. These suggest that the PI4KB/OSBP pathway is not necessarily essential for enterovirus replication in vitro. This work supports a two-step resistance model of enterovirus to PI4KB/OSBP inhibitors involving unique recessive epistasis of 3A and 2B and offers insights into a potential evolutionary pathway of enterovirus toward independence from the PI4KB/OSBP pathway.

Intestinal Immunity to Poliovirus Following Sequential Trivalent Inactivated Polio Vaccine/Bivalent Oral Polio Vaccine and Trivalent Inactivated Polio Vaccine-only Immunization Schedules: Analysis of an Open-label, Randomized, Controlled Trial in Chilean Infants.

Background: Identifying polio vaccine regimens that can elicit robust intestinal mucosal immunity and interrupt viral transmission is a key priority of the polio endgame. Methods: In a 2013 Chilean clinical trial (NCT01841671) of trivalent inactivated polio vaccine (IPV) and bivalent oral polio vaccine (bOPV; targeting types 1 and 3), infants were randomized to receive IPV-bOPV-bOPV, IPV-IPV-bOPV, or IPV-IPV-IPV at 8, 16, and 24 weeks of age and challenged with monovalent oral polio vaccine type 2 (mOPV2) at 28 weeks. Using fecal samples collected from 152 participants, we investigated the extent to which IPV-bOPV and IPV-only immunization schedules induced intestinal neutralizing activity and immunoglobulin A against polio types 1 and 2. Results: Overall, 37% of infants in the IPV-bOPV groups and 26% in the IPV-only arm had detectable type 2-specific stool neutralization after the primary vaccine series. In contrast, 1 challenge dose of mOPV2 induced brisk intestinal immune responses in all vaccine groups, and significant rises in type 2-specific stool neutralization titers (P < .0001) and immunoglobulin A concentrations (P < 0.0001) were measured 2 weeks after the challenge. In subsidiary analyses, duration of breastfeeding also appeared to be associated with the magnitude of polio-specific mucosal immune parameters measured in infant fecal samples. Conclusions: Taken together, these results underscore the concept that mucosal and systemic immune responses to polio are separate in their induction, functionality, and potential impacts on transmission and, specifically, provide evidence that primary vaccine regimens lacking homologous live vaccine components are likely to induce only modest, type-specific intestinal immunity.

Intestinal Immune Responses to Type 2 Oral Polio Vaccine (OPV) Challenge in Infants Previously Immunized With Bivalent OPV and Either High-Dose or Standard Inactivated Polio Vaccine.

Background: The impact of inactivated polio vaccines (IPVs) on intestinal mucosal immune responses to live poliovirus is poorly understood. Methods: In a 2014 phase 2 clinical trial, Panamanian infants were immunized at 6, 10, and 14 weeks of age with bivalent oral polio vaccine (bOPV) and randomized to receive either a novel monovalent high-dose type 2-specific IPV (mIPV2HD) or a standard trivalent IPV at 14 weeks. Infants were challenged at 18 weeks with a monovalent type 2 oral polio vaccine (mOPV2). Infants' intestinal immune responses during the 3 weeks following challenge were investigated by measuring poliovirus type-specific neutralization and immunoglobulin (Ig) A, IgA1, IgA2, IgD, IgG, and IgM antibodies in stool samples. Results: Despite mIPV2HD's 4-fold higher type 2 polio D-antigen content and heightened serum neutralization profile, mIPV2HD-immunized infants' intestinal immune responses to mOPV2 challenge were largely indistinguishable from those receiving standard IPV. Mucosal responses were tightly linked to evidence of active infection and, in the 79% of participants who shed virus, robust type 2-specific IgA responses and stool neutralization were observed by 2 weeks after challenge. Conclusions: Enhancing IPV-induced serum neutralization does not substantively improve intestinal mucosal immune responses or limit viral shedding on mOPV2 challenge. Clinical Trials Registration: NCT02111135.

Allosteric Regulation of Phosphatidylinositol 4-Kinase III Beta by an Antipicornavirus Compound MDL-860.

MDL-860 is a broad-spectrum antipicornavirus compound discovered in 1982 and one of the few promising candidates effective in in vivo virus infection. Despite the effectiveness, the target and the mechanism of action of MDL-860 remain unknown. Here, we have characterized antipoliovirus activity of MDL-860 and identified host phosphatidylinositol-4 kinase III beta (PI4KB) as the target. MDL-860 treatment caused covalent modification and irreversible inactivation of PI4KB. A cysteine residue at amino acid 646 of PI4KB, which locates at the bottom of a surface pocket apart from the active site, was identified as the target site of MDL-860. This work reveals the mechanism of action of this class of PI4KB inhibitors and offers insights into novel allosteric regulation of PI4KB activity.

Poliovirus Studies during the Endgame of the Polio Eradication Program.

Since the beginning of Global Polio Eradication Initiative in 1988, poliomyelitis cases caused by wild poliovirus (PV) have been drastically reduced, with only 74 cases reported in 2 endemic countries in 2015. The current limited PV transmission suggests that we are in the endgame of the polio eradication program. However, specific challenges have emerged in the endgame, including tight budget, switching of the vaccines, and changes in biorisk management of PV. To overcome these challenges, several PV studies have been implemented in the eradication program. Some of the responses to the emerging challenges in the polio endgame might be valuable in other infectious diseases eradication programs. Here, I will review challenges that confront the polio eradication program and current research to address these challenges.