Poliovirus Replication Research Articles

Establishment of measles virus receptor-expressing Vero cells lacking functional poliovirus receptors.

Global efforts are underway to eliminate measles and rubella, and active viral surveillance is the key to achieving this goal. In addition, the World Health Organization announced guidelines for handling materials potentially infectious for poliovirus (PV) to minimize the risk of PV reintroduction and to achieve PV eradication. To support global efforts, we established new PV-non-susceptible cell lines that are useful for the isolation of measles virus (MeV) and rubella virus (RuV) (Vero ΔPVR1/2 hSLAM+). In the cell lines, MeV and RuV replicated efficiently, with no concern regarding PV replication.

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.

Experimental and mathematical insights on the interactions between poliovirus and a defective interfering genome.

During replication, RNA viruses accumulate genome alterations, such as mutations and deletions. The interactions between individual variants can determine the fitness of the virus population and, thus, the outcome of infection. To investigate the effects of defective interfering genomes (DI) on wild-type (WT) poliovirus replication, we developed an ordinary differential equation model, which enables exploring the parameter space of the WT and DI competition. We also experimentally examined virus and DI replication kinetics during co-infection, and used these data to infer model parameters. Our model identifies, and our experimental measurements confirm, that the efficiencies of DI genome replication and encapsidation are two most critical parameters determining the outcome of WT replication. However, an equilibrium can be established which enables WT to replicate, albeit to reduced levels.

Modeling poliovirus replication dynamics from live time-lapse single-cell imaging data

Viruses experience selective pressure on the timing and order of events during infection to maximize the number of viable offspring they produce. Additionally, they may experience variability in cellular environments encountered, as individual eukaryotic cells can display variation in gene expression among cells. This leads to a dynamic phenotypic landscape that viruses must face to replicate. To examine replication dynamics displayed by viruses faced with this variable landscape, we have developed a method for fitting a stochastic mechanistic model of viral infection to time-lapse imaging data from high-throughput single-cell poliovirus infection experiments. The model’s mechanistic parameters provide estimates of several aspects associated with the virus’s intracellular dynamics. We examine distributions of parameter estimates and assess their variability to gain insight into the root causes of variability in viral growth dynamics. We also fit our model to experiments performed under various drug treatments and examine which parameters differ under these conditions. We find that parameters associated with translation and early stage viral replication processes are essential for the model to capture experimentally observed dynamics. In aggregate, our results suggest that differences in viral growth data generated under different treatments can largely be captured by steps that occur early in the replication process.

Temperature controlled high-throughput magnetic tweezers show striking difference in activation energies of replicating viral RNA-dependent RNA polymerases.

RNA virus survival depends on efficient viral genome replication, which is performed by the viral RNA dependent RNA polymerase (RdRp). The recent development of high throughput magnetic tweezers has enabled the simultaneous observation of dozens of viral RdRp elongation traces on kilobases long templates, and this has shown that RdRp nucleotide addition kinetics is stochastically interrupted by rare pauses of 1–1000 s duration, of which the short-lived ones (1–10 s) are the temporal signature of a low fidelity catalytic pathway. We present a simple and precise temperature controlled system for magnetic tweezers to characterize the replication kinetics temperature dependence between 25°C and 45°C of RdRps from three RNA viruses, i.e. the double-stranded RNA bacteriophage Φ6, and the positive-sense single-stranded RNA poliovirus (PV) and human rhinovirus C (HRV-C). We found that Φ6 RdRp is largely temperature insensitive, while PV and HRV-C RdRps replication kinetics are activated by temperature. Furthermore, the activation energies we measured for PV RdRp catalytic state corroborate previous estimations from ensemble pre-steady state kinetic studies, further confirming the catalytic origin of the short pauses and their link to temperature independent RdRp fidelity. This work will enable future temperature controlled study of biomolecular complex at the single molecule level.

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.

Related Enteric Viruses Have Different Requirements for Host Microbiota in Mice.

Accumulating evidence suggests that intestinal bacteria promote enteric virus infection in mice. For example, previous work demonstrated that antibiotic treatment of mice prior to oral infection with poliovirus reduced viral replication and pathogenesis. Here, we examined the effect of antibiotic treatment on infection with coxsackievirus B3 (CVB3), a picornavirus closely related to poliovirus. We treated mice with a mixture of five antibiotics to deplete host microbiota and examined CVB3 replication and pathogenesis following oral inoculation. We found that, as seen with poliovirus, CVB3 shedding and pathogenesis were reduced in antibiotic-treated mice. While treatment with just two antibiotics, vancomycin and ampicillin, was sufficient to reduce CVB3 replication and pathogenesis, this treatment had no effect on poliovirus. The quantity and composition of bacterial communities were altered by treatment with the five-antibiotic cocktail and by treatment with vancomycin and ampicillin. To determine whether more-subtle changes in bacterial populations impact viral replication, we examined viral infection in mice treated with milder antibiotic regimens. Mice treated with one-tenth the standard concentration of the normal antibiotic cocktail supported replication of poliovirus but not CVB3. Importantly, a single dose of one antibiotic, streptomycin, was sufficient to reduce CVB3 shedding and pathogenesis while having no effect on poliovirus shedding and pathogenesis. Overall, replication and pathogenesis of CVB3 are more sensitive to antibiotic treatment than poliovirus, indicating that closely related viruses may differ with respect to their reliance on microbiota.IMPORTANCE Recent data indicate that intestinal bacteria promote intestinal infection of several enteric viruses. Here, we show that coxsackievirus, an enteric virus in the picornavirus family, also relies on microbiota for intestinal replication and pathogenesis. Relatively minor depletion of the microbiota was sufficient to decrease coxsackievirus infection, while poliovirus infection was unaffected. Surprisingly, a single dose of one antibiotic was sufficient to reduce coxsackievirus infection. Therefore, these data indicate that closely related viruses may differ with respect to their reliance on microbiota.

A Redundant Mechanism of Recruitment Underlies the Remarkable Plasticity of the Requirement of Poliovirus Replication for the Cellular ArfGEF GBF1.

The replication of many positive-strand RNA viruses [(+)RNA viruses] depends on the cellular protein GBF1, but its role in the replication process is not clear. In uninfected cells, GBF1 activates small GTPases of the Arf family and coordinates multiple steps of membrane metabolism, including functioning of the cellular secretory pathway. The nonstructural protein 3A of poliovirus and related viruses has been shown to directly interact with GBF1, likely mediating its recruitment to the replication complexes. Surprisingly, viral mutants with a severely reduced level of 3A-GBF1 interaction demonstrate minimal replication defects in cell culture. Here, we systematically investigated the conserved elements of GBF1 to understand which determinants are important to support poliovirus replication. We demonstrate that multiple GBF1 mutants inactive in cellular metabolism could still be fully functional in the replication complexes. Our results show that the Arf-activating property, but not the primary structure of the Sec7 domain, is indispensable for viral replication. They also suggest a redundant mechanism of recruitment of GBF1 to the replication sites, which is dependent not only on direct interaction of the protein with the viral protein 3A but also on determinants located in the noncatalytic C-terminal domains of GBF1. Such a double-targeting mechanism explains the previous observations of the remarkable tolerance of different levels of GBF1-3A interaction by the virus and likely constitutes an important element of the resilience of viral replication.IMPORTANCE Enteroviruses are a vast group of viruses associated with diverse human diseases, but only two of them could be controlled with vaccines, and effective antiviral therapeutics are lacking. Here, we investigated in detail the contribution of a cellular protein, GBF1, in the replication of poliovirus, a representative enterovirus. GBF1 supports the functioning of cellular membrane metabolism and is recruited to viral replication complexes upon infection. Our results demonstrate that the virus requires a limited subset of the normal GBF1 functions and reveal the elements of GBF1 essential to support viral replication under different conditions. Since diverse viruses often rely on the same cellular proteins for replication, understanding the mechanisms by which these proteins support infection is essential for the development of broad-spectrum antiviral therapeutics.

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

BackgroundOur understanding of the acquisition of intestinal mucosal immunity and the control of poliovirus replication and transmission in later life is still emerging.MethodsAs part of a 2011 randomised, blinded, placebo-controlled...

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

Phosphatidylinositol-4 kinase III β (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.

A New Live-Attenuated Poliovirus Vaccine Constructed by Rational Design Improves Safety by Preventing Reversion to Virulence

The life-attenuated Sabin oral poliovirus vaccine (OPV), created over 60 years ago, replicate efficiently in the vaccinee’s gut and, therefore, elicits robust mucosal and systemic immunity, which limits poliovirus spread. Accordingly, OPV has been used in the context of the Global Polio Eradication Initiative, which aims to end the circulation of all polioviruses. However, the program has met a significant hurdle imposed by the ability of OPV to evolve to regain transmissibility and virulence. This represents a risk of widespread polio re-emergence should immunization cease. We exploited molecular features of poliovirus replication to develop a safer, genetically stable, strain by limiting its ability to increase fitness and regain neurovirulence. This novel attenuated poliovirus strain is immunogenic in preclinical and clinical tests and may enable completion of poliovirus eradication as well as pave the way for the rational design of effective and safe vaccines for polio-related pathogens.

Phospholipid synthesis fueled by lipid droplets drives the structural development of poliovirus replication organelles

Rapid development of complex membranous replication structures is a hallmark of picornavirus infections. However, neither the mechanisms underlying such dramatic reorganization of the cellular membrane architecture, nor the specific role of these membranes in the viral life cycle are sufficiently understood. Here we demonstrate that the cellular enzyme CCTα, responsible for the rate-limiting step in phosphatidylcholine synthesis, translocates from the nuclei to the cytoplasm upon infection and associates with the replication membranes, resulting in the rerouting of lipid synthesis from predominantly neutral lipids to phospholipids. The bulk supply of long chain fatty acids necessary to support the activated phospholipid synthesis in infected cells is provided by the hydrolysis of neutral lipids stored in lipid droplets. Such activation of phospholipid synthesis drives the massive membrane remodeling in infected cells. We also show that complex membranous scaffold of replication organelles is not essential for viral RNA replication but is required for protection of virus propagation from the cellular anti-viral response, especially during multi-cycle replication conditions. Inhibition of infection-specific phospholipid synthesis provides a new paradigm for controlling infection not by suppressing viral replication but by making it more visible to the immune system.

Replicase-mediated shielding of the poliovirus replicative double-stranded RNA to avoid recognition by MDA5.

Replication of the positive-strand RNA viruses generates double-stranded RNAs (dsRNAs) that are recognized by host pattern recognition receptors (PRRs) to trigger innate immune responses. Formation of the viral replication complex (RC) has been thought to shield dsRNA from being recognized by innate sensors. To elucidate the RC-mediated evasion of innate recognition, we selected poliovirus (PV) as a model. We first found that RNAs generated during PV replication were potent interferon (IFN) inducers upon transfection, while there was no obvious IFN production detected in PV-replicating cells. PV replication did not interfere with IFN production when IFN agonists were synchronously introduced with the replicating PV RNAs, and in PV-infected cells, IFN agonist-induced IFN production was only moderately impaired but not completely abolished. When PV-infected cells were in situ permeabilized by digitonin, viral dsRNAs were readily detected by an anti-dsRNA antibody and were resistant to RNase III digestion. When digitonin-permeabilized cells were further solubilized by 1 % triton X-100, the dsRNAs of PV became sensitive to RNase III digestion. A co-localization study showed that PV dsRNA did not co-localize with MDA5 in virally infected cells. Given that the PV replication complex is protruding single-membrane and tubular in form, viral replicative dsRNAs are probably shielded by the replication complex or the viral replicase to avoid being accessed by RNase III and MDA5. We propose that the replication complex- or replicase-mediated shielding of dsRNA may act as a means for innate evasion.

MicroRNA-134 regulates poliovirus replication by IRES targeting

Global poliovirus eradication efforts include high vaccination coverage with live oral polio vaccine (OPV), surveillance for acute flaccid paralysis, and OPV “mop-up” campaigns. An important objective involves host-directed strategies to reduce PV replication to diminish viral shedding in OPV recipients. In this study, we show that microRNA-134-5p (miR-134) can regulate Sabin-1 replication but not Sabin-2 or Sabin-3 via direct interaction with the PV 5′UTR. Hypochromicity data showed miR-134 binding to Sabin-1 and 3 but not Sabin-2 IRES. Transfection of a miR-134 mimic repressed translation of Sabin-1 5′UTR driven luciferase validating the mechanism of miR-134-mediated repression of Sabin-1. Further, site directed mutagenesis of the miR-134 binding site in Sabin-1 IRES relieved miR-134-mediated repression indicating that these regulatory molecules have an important role in regulating the host gene response to PV. Binding of miR-134 to Sabin-1 IRES caused degradation of the IRES transcript in a miR-134 and sequence specific manner. The miR-134 binding site was found to be highly conserved in wild type PV-1 as well as EV71 strains indicating that miR-134 may regulate function of these IRES sequences in circulation.

Pathogenic Events in a Nonhuman Primate Model of Oral Poliovirus Infection Leading to Paralytic Poliomyelitis.

Despite a great deal of prior research, the early pathogenic events in natural oral poliovirus infection remain poorly defined. To establish a model for study, we infected 39 macaques by feeding them single high doses of the virulent Mahoney strain of wild type 1 poliovirus. Doses ranging from 107 to 109 50% tissue culture infective doses (TCID50) consistently infected all the animals, and many monkeys receiving 108 or 109 TCID50 developed paralysis. There was no apparent difference in the susceptibilities of the three macaque species (rhesus, cynomolgus, and bonnet) used. Virus excretion in stool and nasopharynges was consistently observed, with occasional viremia, and virus was isolated from tonsils, gut mucosa, and draining lymph nodes. Viral replication proteins were detected in both epithelial and lymphoid cell populations expressing CD155 in the tonsil and intestine, as well as in spinal cord neurons. Necrosis was observed in these three cell types, and viral replication in the tonsil/gut was associated with histopathologic destruction and inflammation. The sustained response of neutralizing antibody correlated temporally with resolution of viremia and termination of virus shedding in oropharynges and feces. For the first time, this model demonstrates that early in the infectious process, poliovirus replication occurs in both epithelial cells (explaining virus shedding in the gastrointestinal tract) and lymphoid/monocytic cells in tonsils and Peyer's patches (explaining viremia), extending previous studies of poliovirus pathogenesis in humans. Because the model recapitulates human poliovirus infection and poliomyelitis, it can be used to study polio pathogenesis and to assess the efficacy of candidate antiviral drugs and new vaccines.IMPORTANCE Early pathogenic events of poliovirus infection remain largely undefined, and there is a lack of animal models mimicking natural oral human infection leading to paralytic poliomyelitis. All 39 macaques fed with single high doses ranging from 107 to 109 TCID50 Mahoney type 1 virus were infected, and many of the monkeys developed paralysis. Virus excretion in stool and nasopharynges was consistently observed, with occasional viremia; tonsil, mesentery lymph nodes, and intestinal mucosa served as major target sites of viral replication. For the first time, this model demonstrates that early in the infectious process, poliovirus replication occurs in both epithelial cells (explaining virus shedding in the gastrointestinal tract) and lymphoid/monocytic cells in tonsils and Peyer's patches (explaining viremia), thereby supplementing historical reconstructions of poliovirus pathogenesis. Because the model recapitulates human poliovirus infection and poliomyelitis, it can be used to study polio pathogenesis, candidate antiviral drugs, and the efficacy of new vaccines.

Investigation of the Role of Protein Kinase D in Human Rhinovirus Replication.

ABSTRACTPicornavirus replication is known to cause extensive remodeling of Golgi and endoplasmic reticulum membranes, and a number of the host proteins involved in the viral replication complex have been identified, including oxysterol binding protein (OSBP) and phosphatidylinositol 4-kinase III beta (PI4KB). Since both OSBP and PI4KB are substrates for protein kinase D (PKD) and PKD is known to be involved in the control of Golgi membrane vesicular and lipid transport, we hypothesized that PKD played a role in viral replication. We present multiple lines of evidence in support of this hypothesis. First, infection of HeLa cells with human rhinovirus (HRV) induced the phosphorylation of PKD. Second, PKD inhibitors reduced HRV genome replication, protein expression, and titers in a concentration-dependent fashion and also blocked the replication of poliovirus (PV) and foot-and-mouth disease virus (FMDV) in a variety of cells. Third, HRV replication was significantly reduced in HeLa cells overexpressing wild-type and mutant forms of PKD1. Fourth, HRV genome replication was reduced in HAP1 cells in which the PKD1 gene was knocked out by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9. Although we have not identified the molecular mechanism through which PKD regulates viral replication, our data suggest that this is not due to enhanced interferon signaling or an inhibition of clathrin-mediated endocytosis, and PKD inhibitors do not need to be present during viral uptake. Our data show for the first time that targeting PKD with small molecules can inhibit the replication of HRV, PV, and FMDV, and therefore, PKD may represent a novel antiviral target for drug discovery.IMPORTANCE Picornaviruses remain an important family of human and animal pathogens for which we have a very limited arsenal of antiviral agents. HRV is the causative agent of the common cold, which in itself is a relatively trivial infection; however, in asthma and chronic obstructive pulmonary disease (COPD) patients, this virus is a major cause of exacerbations resulting in an increased use of medication, worsening symptoms, and, frequently, hospital admission. Thus, HRV represents a substantial health care and economic burden for which there are no approved therapies. We sought to identify a novel host target as a potential anti-HRV therapy. HRV infection induces the phosphorylation of PKD, and inhibitors of this kinase effectively block HRV replication at an early stage of the viral life cycle. Moreover, PKD inhibitors also block PV and FMDV replication. This is the first description that PKD may represent a target for antiviral drug discovery.

MicroRNA screening identifies miR-134 as a regulator of poliovirus and enterovirus 71 infection

MicroRNAs (miRNAs) regulate virus replication through multiple mechanisms. Poliovirus causes a highly debilitating disease and though global efforts to eradicate polio have sharply decreased polio incidence, unfortunately three countries (Afghanistan, Nigeria and Pakistan) remain polio-endemic. We hypothesize that understanding the host factors involved in polio replication will identify novel prophylactic and therapeutic targets against polio and related viruses. In this data set, employing genome wide screens of miRNA mimics and inhibitors, we identified miRNAs which significantly suppressed polio replication. Specifically, miR-134 regulates poliovirus replication via modulation of ras-related nuclear protein (RAN), an important component of the nuclear transport system. MiR-134 also inhibited other Picornaviridae viruses including EV71, a growing concern and a high priority for vaccination in Asian countries like China. These findings demonstrate a novel mechanism for miRNA regulation of poliovirus and other Picornaviridae viruses in host cells, and thereby may provide a novel approach in combating infection and a potential approach for the development of anti-Picornaviridae strategies.

Transformation and Tumorigenicity Testing of Simian Cell Lines and Evaluation of Poliovirus Replication.

The key role of cell cultures in different scientific fields is worldwide recognized, both as in vitro research models alternative to laboratory animals and substrates for biological production. However, many safety concerns rise from the use of animal/human cell lines that may be tumorigenic, leading to potential adverse contaminations in cell-derived biologicals.In order to evaluate the suitability of 13 different cell lines for Poliovirus vaccine production, safety and quality, in vitro/in vivo tumorigenicity and Poliovirus propagation properties were evaluated.Our results revealed that non-human primate cell lines CYNOM-K1, FRhK-4, 4MBr-5 and 4647 are free of tumorigenic features and represent highly susceptible substrates for attenuated Sabin Poliovirus strains. In particular, FRhK-4 and 4647 cell lines are characterized by a higher in vitro replication, resulting indicated for the use in large-scale production field.

Vaccine-induced mucosal immunity to poliovirus: analysis of cohorts from an open-label, randomised controlled trial in Latin American infants

SummaryBackgroundIdentification of mechanisms that limit poliovirus replication is crucial for informing decisions aimed at global polio eradication. Studies of mucosal immunity induced by oral poliovirus (OPV) or inactivated poliovirus (IPV) vaccines and mixed schedules thereof will determine the effectiveness of different vaccine strategies to block virus shedding. We used samples from a clinical trial of different vaccination schedules to measure intestinal immunity as judged by neutralisation of virus and virus-specific IgA in stools.MethodsIn the FIDEC trial, Latin American infants were randomly assigned to nine groups to assess the efficacy of two schedules of bivalent OPV (bOPV) and IPV and challenge with monovalent type 2 OPV, and stools samples were collected. We selected three groups of particular interest—the bOPV control group (serotypes 1 and 3 at 6, 10, and 14 weeks), the trivalent attenuated OPV (tOPV) control group (tOPV at 6, 10, and 14 weeks), and the bOPV–IPV group (bOPV at 6, 10, and 14 weeks plus IPV at 14 weeks). Neutralising activity and poliovirus type-specific IgA were measured in stool after a monovalent OPV type 2 challenge at 18 weeks of age. Mucosal immunity was measured by in-vitro neutralisation of a type 2 polio pseudovirus (PV2). Neutralisation titres and total and poliovirus-type-specific IgG and IgA concentrations in stools were assessed in samples collected before challenge and 2 weeks after challenge from all participants.Findings210 infants from Guatemala and Dominican Republic were included in this analysis. Of 38 infants tested for mucosal antibody in the tOPV group, two were shedding virus 1 week after challenge, compared with 59 of 85 infants receiving bOPV (p<0·0001) and 53 of 87 infants receiving bOPV–IPV (p<0·0001). Mucosal type 2 neutralisation and type-specific IgA were noted primarily in response to tOPV. An inverse correlation was noted between virus shedding and both serum type 2 neutralisation at challenge (p<0·0001) and mucosal type 2 neutralisation at challenge (p<0·0001).InterpretationMucosal type-2-specific antibodies can be measured in stool and develop in response to receipt of OPV type 2 either in the primary vaccine series or at challenge. These mucosal antibodies influence the amount of virus that is shed in an established infection.FundingBill & Melinda Gates Foundation.

BPIFB6 Regulates Secretory Pathway Trafficking and Enterovirus Replication.

Bactericidal/permeability-increasing protein (BPI) fold-containing family B, member 3 (BPIFB3) is an endoplasmic reticulum (ER)-localized host factor that negatively regulates coxsackievirus B (CVB) replication through its control of the autophagic pathway. Here, we show that another member of the BPIFB family, BPIFB6, functions as a positive regulator of CVB, and other enterovirus, replication by controlling secretory pathway trafficking and Golgi complex morphology. We show that similar to BPIFB3, BPIFB6 localizes exclusively to the ER, where it associates with other members of the BPIFB family. However, in contrast to our findings that RNA interference (RNAi)-mediated silencing of BPIFB3 greatly enhances CVB replication, we show that silencing of BPIFB6 expression dramatically suppresses enterovirus replication in a pan-viral manner. Mechanistically, we show that loss of BPIFB6 expression induces pronounced alterations in retrograde and anterograde trafficking, which correlate with dramatic fragmentation of the Golgi complex. Taken together, these data implicate BPIFB6 as a key regulator of secretory pathway trafficking and viral replication and suggest that members of the BPIFB family participate in diverse host cell functions to regulate virus infections. Enterovirus infections are associated with a number of severe pathologies, such as aseptic meningitis, dilated cardiomyopathy, type I diabetes, paralysis, and even death. These viruses, which include coxsackievirus B (CVB), poliovirus (PV), and enterovirus 71 (EV71), co-opt the host cell secretory pathway, which controls the transport of proteins from the endoplasmic reticulum to the Golgi complex, to facilitate their replication. Here we report on the identification of a novel regulator of the secretory pathway, bactericidal/permeability-increasing protein (BPI) fold-containing family B, member 6 (BPIFB6), whose expression is required for enterovirus replication. We show that loss of BPIFB6 expression correlates with pronounced defects in the secretory pathway and greatly reduces the replication of CVB, PV, and EV71. Our results thus identify a novel host cell therapeutic target whose function could be targeted to alter enterovirus replication.