Global RNA Structure Analysis of Poliovirus Identifies a Conserved RNA Structure Involved in Viral Replication and Infectivity

The genomes of RNA viruses often contain RNA structures that are crucial for translation and RNA replication and may play additional, uncharacterized roles during the viral replication cycle. For the picornavirus family member poliovirus, a number of functional RNA structures have been identified, but much of its genome, especially the open reading frame, has remained uncharacterized. We have now generated a global RNA structure map of the poliovirus genome using a chemical probing approach that interrogates RNA structure with single-nucleotide resolution. In combination with orthogonal evolutionary analyses, we uncover several conserved RNA structures in the open reading frame of the viral genome. To validate the ability of our global analyses to identify functionally important RNA structures, we further characterized one of the newly identified structures, located in the region encoding the RNA-dependent RNA polymerase, 3D^pol, by site-directed mutagenesis. Our results reveal that the structure is required for viral replication and infectivity, since synonymous mutants are defective in these processes. Furthermore, these defects can be partially suppressed by mutations in the viral protein 3C^pro, which suggests the existence of a novel functional interaction between an RNA structure in the 3D^pol-coding region and the viral protein(s) 3C^pro and/or its precursor 3CD^pro.     

Infectivity of RNA from inactivated poliovirus

During inactivation of poliovirus type 1 (PV-1) by exposure to UV, hypochlorite, and heat (72 degrees C), the infectivity of the virus was compared with that of its RNA. DEAE-dextran (1-mg/ml concentration in Dulbecco's modified Eagle medium buffered with 0.05 M Tris, pH 7.4) was used to facilitate transfecting PV-1 RNA into FRhK-4 host cells. After interaction of PV-1 RNA with cell monolayer at room temperature (21 to 22 degrees C) for 20 min, the monolayers were washed with 5 ml of Hanks balanced salt solution. The remainder of the procedure was the same as that for the conventional plaque technique, which was also used for quantifying the PV-1 whole-particle infectivity. Plaque formation by extracted RNA was approximately 100,000-fold less efficient than that by whole virions. The slopes of best-fit regression lines of inactivation curves for virion infectivity and RNA infectivity were compared to determine the target of inactivation. For UV and hypochlorite inactivation the slopes of inactivation curves of virion infectivity and RNA infectivity were not statistically different. However, the difference of slopes of inactivation curves of virion infectivity and RNA infectivity was statistically significant for thermal inactivation. The results of these experiments indicate that viral RNA is a primary target of UV and hypochlorite inactivations but that the sole target of thermal inactivation is the viral capsid.     

Nonreplicative RNA Recombination in Poliovirus

Current models of recombination between viral RNAs are based on replicative template-switch mechanisms. The existence of nonreplicative RNA recombination in poliovirus is demonstrated in the present study by the rescue of viable viruses after cotransfections with different pairs of genomic RNA fragments with suppressed translatable and replicating capacities. Approximately 100 distinct recombinant genomes have been identified. The majority of crossovers occurred between nonhomologous segments of the partners and might have resulted from transesterification reactions, not necessarily involving an enzymatic activity. Some of the crossover loci are clustered. The origin of some of these "hot spots" could be explained by invoking structures similar to known ribozymes. A significant proportion of recombinant RNAs contained the entire 5' partner, if its 3' end was oxidized or phosphorylated prior to being mixed with the 3' partner. All of these observations are consistent with a mechanism that involves intermediary formation of the 2',3'-cyclic phosphate and 5'-hydroxyl termini. It is proposed that nonreplicative RNA recombination may contribute to evolutionarily significant RNA rearrangements.     

Systematic Review of Mucosal Immunity Induced by Oral and Inactivated Poliovirus Vaccines against Virus Shedding following Oral Poliovirus Challenge

Inactivated poliovirus vaccine (IPV) may be used in mass vaccination campaigns during the final stages of polio eradication. It is also likely to be adopted by many countries following the coordinated global cessation of vaccination with oral poliovirus vaccine (OPV) after eradication. The success of IPV in the control of poliomyelitis outbreaks will depend on the degree of nasopharyngeal and intestinal mucosal immunity induced against poliovirus infection. We performed a systematic review of studies published through May 2011 that recorded the prevalence of poliovirus shedding in stool samples or nasopharyngeal secretions collected 5–30 days after a “challenge” dose of OPV. Studies were combined in a meta-analysis of the odds of shedding among children vaccinated according to IPV, OPV, and combination schedules. We identified 31 studies of shedding in stool and four in nasopharyngeal samples that met the inclusion criteria. Individuals vaccinated with OPV were protected against infection and shedding of poliovirus in stool samples collected after challenge compared with unvaccinated individuals (summary odds ratio [OR] for shedding 0.13 (95% confidence interval [CI] 0.08–0.24)). In contrast, IPV provided no protection against shedding compared with unvaccinated individuals (summary OR 0.81 [95% CI 0.59–1.11]) or when given in addition to OPV, compared with individuals given OPV alone (summary OR 1.14 [95% CI 0.82–1.58]). There were insufficient studies of nasopharyngeal shedding to draw a conclusion. IPV does not induce sufficient intestinal mucosal immunity to reduce the prevalence of fecal poliovirus shedding after challenge, although there was some evidence that it can reduce the quantity of virus shed. The impact of IPV on poliovirus transmission in countries where fecal-oral spread is common is unknown but is likely to be limited compared with OPV.     

Differential Effect of Phleomycin on the Infectivity of Poliovirus and Poliovirus-Induced Ribonucleic Acids

The infectivity of intact poliovirus was not affected by exposure to the antibiotic phleomycin at concentrations as high as 200 mug/ml, whereas that of the singlestranded poliovirus ribonucleic acid (RNA) was inactivated to 99% by pretreatment of the RNA with phleomycin at a concentration of 2 mug/ml. The infectivity of double and multistranded RNA was 10 times less sensitive than that of singlestranded RNA to the action of this antibiotic. Preincubation of HeLa cells for 30 min with 10 to 50 mug of phleomycin reduced the sensitivity of the cells to infection by viral RNA and intact virus, indicating that phleomycin interferes with cellular functions necessary for virus replication. When phleomycin was added to cells at different times after infection with single- or double-stranded RNA, the highest inactivation of infective centers was observed immediately after infection. With time of incubation at 37 C, the infective centers became more resistant to the action of phleomycin.     

Factors Influencing the Enhancement of the Infectivity of Poliovirus Ribonucleic Acid by Diethylaminoethyl-Dextran

The enhancement by diethylaminoethyl-dextran (DEAE-D) of the infectivity of poliovirus ribonucleic acid (RNA) for cell cultures was demonstrated by infective-center as well as by plaque assays, both in nonprimate (L) and primate cell systems (MK, HeLa, LLC-MK(2)). The sensitivity of plaque assays was greatly improved by using a tris (hydroxymethyl)aminomethane-buffered synthetic medium (basal medium Eagle) and freshly confluent cell monolayers. Enhancement of nucleic acid infectivity was directly dependent on the molecular weight of the DEAE-D. Two observations bearing on the action of DEAE-D appeared important: ribonuclease activity was reduced by DEAE-D, and cells pretreated with DEAE-D remained susceptible to infection with RNA in isotonic medium. Appreciable susceptibility of the treated cells persisted for at least 2 hr; the susceptible state could be reversed at will by an application of heparin. Enhancement of nucleic acid infectivity was independent of an effect of DEAE-D on intact virus and agar inhibitors.     

Improved ELISA test for determination of potency of Inactivated Poliovirus Vaccine (IPV).

An improved ELISA test for determination of potency of Inactivated Poliovirus Vaccine (IPV) is proposed. The method is based on the use of IgG purified from immune rabbit serum conjugated with biotin. Optimized and validated materials for the test can be stored for a long time in the form of ready-to-use kits. Optimization included selection of anti-poliovirus rabbit antibody batches with the best specificity to D-antigen as well as finding the most efficient parameters for all steps of ELISA protocol. The assay is based on direct ("sandwich") ELISA scheme, in which antigens are captured on ELISA plates coated with purified rabbit polyclonal D-antigen specific IgG raised against wild polioviruses of three serotypes. D-antigen specificity of the IgG was at least 10 times higher than to H-antigen (heat-inactivated virus). The presence of antigen was detected using biotin-conjugated IgG from the same source. Eight-point dose-response curves were obtained for each sample and the reference vaccine. The protocol ensured low background (less than 0.2 OD), linear response over the entire range of optical density measurements (up to 3.0 OD), and high precision of data (assay variability was about 3%). The quantitative results and the validity of the test were determined by two numerical approaches, linear regression and a new analysis procedure called the local interpolation method. For the first approach we also proposed a new method for testing of parallelism of regression lines. The ELISA protocol for all three types of poliovirus is based on standard off-the-shelf reagents, and is highly reproducible and reliable. An in-house Reference Reagent was formulated and calibrated against the International Reference for IPV.     

Poliovirus RNA is released from the capsid near a twofold symmetry axis

After recognizing and binding to its host cell, poliovirus (like other nonenveloped viruses) faces the challenge of translocating its genome across a cellular membrane and into the cytoplasm. To avoid entanglement with the capsid, the RNA must exit via a single site on the virion surface. However, the mechanism by which a single site is selected (from among 60 equivalents) is unknown; and until now, even its location on the virion surface has been controversial. To help to elucidate the mechanism of infection, we have used single-particle cryo-electron microscopy and tomography to reconstruct conformationally altered intermediates that are formed by the poliovirion at various stages of the poliovirus infection process. Recently, we reported icosahedrally symmetric structures for two forms of the end-state 80S empty capsid particle. Surprisingly, RNA was frequently visible near the capsid; and in a subset of the virions, RNA was seen on both the inside and outside of the capsid, caught in the act of exiting. To visualize RNA exiting, we have now determined asymmetric reconstructions from that subset, using both single-particle cryo-electron microscopy and cryo-electron tomographic methods, producing independent reconstructions at ∼50-Å resolution. Contrary to predictions in the literature, the footprint of RNA on the capsid surface is located close to a viral 2-fold axis, covering a slot-shaped area of reduced density that is present in both of the symmetrized 80S reconstructions and which extends by about 20 Å away from the 2-fold axis toward each neighboring 5-fold axis.In its role as the intermediate that links one round of infection with the next, a virus particle protects the viral genome during passage from cell to cell and from host to host, it specifically recognizes and binds to target cells, and it delivers the viral genome into the appropriate compartment in the target cell. For enveloped viruses, which have their own external membranes, fusion of the viral membrane with a host membrane presents a conceptually simple mechanism for delivery of the genome or nucleoprotein into the cytoplasm. For nonenveloped viruses, the viral particle must provide the machinery necessary for either the entire virion, a nucleoprotein complex, or the viral genome to cross a membrane. This process remains poorly understood. Poliovirus provides an excellent model system for probing the mechanisms used for genome translocation. As the type member of the Picornavirus family and the etiological agent of poliomyelitis, poliovirus has been well characterized biochemically and genetically (42), its cell entry pathways have been well characterized (5, 15, 30, 52), and a number of cell entry intermediates have been identified and are accessible for structural studies (2-4, 7, 8, 18, 34, 38, 42, 55, 56).The capsid of the mature poliovirion (160S particle) consists of 60 copies of each of the four coat proteins VP1, VP2, VP3, and VP4 (which is myristolated at its amino-terminal glycine [13]) and encloses a 7.5-kbp positive-sense RNA genome. The outer surface of the capsid has a number of major features, including star-shaped mesas at its 5-fold axes, 3-fold propeller-like protrusions, canyon-like depressions surrounding each of the 5-fold mesas, and depressions at the 2-fold axes (30, 31).Poliovirus infection is initiated when the virus binds to the host-cell-surface poliovirus receptor (called Pvr or CD155) (41), triggering a conformational change of the native capsid into an altered particle called the A particle or 135S particle (18, 19). The 135S particle has been shown to be expanded by about 4% (2, 7), is infectious (16, 33), and is believed to be a productive intermediate in viral entry (30, 33). This conformational change results in the externalization of the small myristoylated capsid protein, VP4 (18), and of the amino-terminal extension of VP1 (which includes a conserved amphipathic helix) (23). Both of these externalized polypeptides then associate with membranes (17, 23). In subsequent steps, the viral genome is released from the capsid and translocated across a membrane (probably an endosomal membrane [5]) to gain access to the cytoplasm, leaving behind an end-state empty capsid shell (called the 80S particle). The trigger for RNA release and the mechanism of genome translocation are both poorly understood (30, 52).Electrophysiology and mutational experiments have shown that the externalization of VP4 and of the amino terminus of VP1 is associated with the formation of channels in membranes (17, 49, 50) and, furthermore, that point mutations in threonine 28 of VP4 can either eliminate (T28G) or alter (T28V, T28S) the ability to form channels and either eliminate (T28G) or slow (T28V, T28S) the kinetics of productive RNA release (17). These observations have led to the hypothesis that the viral polypeptides insert into host cell membranes during infection and rearrange to form channels that permit the viral genome to pass through the membrane, thereby gaining access to the cytoplasm (7, 17, 49, 50).Speculation about the sites of externalization of the viral peptides and of the viral genome began soon after the structures of mature rhinovirus and poliovirus were determined crystallographically 25 years ago (31, 44). In both structures there is a solvent-filled channel running along each 5-fold axis. This channel is closed off at the outer surface of the capsid by polypeptide loops and on the inner surface by a plug that is formed by five intertwined copies of the amino terminus of VP3, forming a parallel beta tube (31, 44). In poliovirus this tube is flanked on its inner surface by five copies of a three-stranded beta sheet in which the outermost two strands come from a beta hairpin at the amino terminus of VP4 and the innermost strand comes from residues at the extreme amino terminus of VP1 (20). The presence of this channel, together with its proximity to peptide segments that were known to be externalized upon receptor attachment, and analogies with other viruses led to a model in which both the peptides and the viral RNA are externalized via the channel at the 5-fold axis (25, 45). At that time, an alternative model for the egress of polypeptides was proposed, based on an analogy with the externalization of the amino-terminal extensions of capsid proteins in expanded states of the topologically similar T=3 plant viruses (26, 32, 43, 47) and on genetic and biochemical studies of mutations that affect cell entry and capsid stability in poliovirus (14, 39, 54). In the latter model, the peptides were proposed to exit from the base of the canyon and then proceed along the outer surface toward the 5-fold peak (43, 47). Both models suggested that five copies of each of the externalized peptides would interact in some way to form a pore in the membrane that was contiguous with one of the 5-fold channels, thus providing a way for RNA to be released from the virion at a 5-fold axis of symmetry. No data yet exist to specify what specific structural roles VP4 and the amino terminus of VP1 might play in forming pores and serving as membrane anchors. However, both the electrophysiology data (cited above) and the greater sequence conservation of VP4 suggest that its role in pore formation may be the more central (17, 49, 50).To further elucidate various steps along the infection pathway, cryo-electron microscopy (cryo-EM) reconstructions have been determined for a number of cell entry intermediates of poliovirus and rhinoviruses, and their resolutions have been improved over time (2, 3, 7, 28, 38). Structures of the complexes of polioviruses and major-group rhinoviruses with the ectodomains of their respective receptors have confirmed earlier models that suggested that the canyon is the receptor-binding site and have begun to suggest how receptor binding might lead to receptor-induced conformational rearrangements (3, 56). Cryo-EM and cryo-electron tomography structures (cryo-ET) of a poliovirus-receptor-membrane complex (using a novel receptor-decorated liposome model [51]) confirmed that initial receptor binding brings the surface of the 5-fold mesa into close proximity with the membrane and appears to produce an outward distortion of the outer leaflet of the membrane in its area of closest approach to the virus particle (4, 8).Structures have also been determined for the soluble 135S and 80S particles of poliovirus, formed by heating the virus at 50°C (135S) or 56°C (80S) in hypotonic buffers, and for the 80S particles of rhinovirus 14 and 16, formed by exposing virus to acidic pH. All of the biological and immunological evidence that is currently available indicates that the particles prepared in vitro and used for structural studies are indistinguishable from the particles that are released from the cell surface during infection (6, 53). These structures have allowed the models for peptide release and genome release to be extended and refined (7, 38) and indeed have confirmed that VP1 exits from the particle surface at the base of the canyon and climbs up the side of the 5-fold mesa. However, contrary to the assumptions of the earlier models, the 10-Å structures of the poliovirus 135S and 80S particles show that the amino end of the amino-terminal extension of VP1 does not remain associated with the mesa. Instead, it forms an alpha-helical bridge that stretches across the canyon and binds to the large EF loop of VP2, a surface projection that appears as a 3-fold propeller blade (7, 38).Until recently, the mechanism of RNA release (during the 135S-to-80S transition) has been largely a matter of conjecture. We can infer that the RNA must exit via a single site on the virion surface, to avoid entanglement with the capsid (particularly as entanglement has never been observed in electron micrographs), though the mechanism by which a single site is selected (from among 60 equivalents) is unknown. All models presented to date have assumed that the RNA is released from the channel at the 5-fold axes (2, 3, 7, 8, 25, 27, 28, 30, 42, 45). However, in the icosahedrally constrained 10-Å structures of both the poliovirus 135S and 80S particles (7, 38), the apparent intactness and stability of the 5-fold mesa argues against the 5-fold axis being the site of RNA egress, given that the diameter of the opening, as seen in those structures, would be insufficient to accommodate RNA, even if the “plug” formed by the intertwined amino termini of VP3 was displaced. Moreover, both structures revealed significant thinning between 2-fold-related pentamers in the vicinity of the 2-fold axes. Most convincingly, large holes (easily sufficient to accommodate RNA) were seen at and near the 2-fold axes in the atomic model of the late-80S structure. This coincided with an open hole in the reconstruction, when viewed at a contour level that left most of the remainder of the capsid intact. This evidence was suggestive, but not definitive, as a number of other openings were present, particularly in the interfaces between protomers. Furthermore, the behavior of the capsid structure in the immediate vicinity of the unique site of RNA exit is likely to be different from what we see in the icosahedral average, which is dominated by the remainder of the capsid.In the course of solving icosahedrally symmetric cryo-EM structures for the poliovirus end-state 80S empty capsid particle (7, 38), we were surprised to find that RNA was frequently visible near the capsid and that in a subset of about 5% of the sampled virions, RNA was seen on both the inside and outside of the capsid, apparently caught in the act of exiting. This was an exciting development, as images of viral RNA release had never previously been reported. We were able to improve the resolution to ∼10 Å by classifying the projected images into two groups: an early 80Se particle that was more prevalent in the population after a shorter heating time and a late 80Sl particle that was seen more often when the heating time was increased. The amount of RNA density remaining in the interior appears to be continuously variable in both classes, suggesting that release is gradual. Of the 5% subset of particles clearly caught in the act, almost all belonged to the 80Se class. Our interpretation was that the 80Se class may represent particles in which exiting RNA is still engaged with the capsid machinery and traversing the capsid, while the 80Sl class (in which much of the capsid resembles the 135S form more closely in structure) represents particles with the RNA disengaged, possibly after nuclease cleavage. More than two structural classes may be present, but at the current resolution, we could not distinguish them.The present report addresses the question of what we can learn about the details of RNA release from an asymmetric cryo-EM reconstruction, based on the 540-particle caught-in-the-act subset, and independently from cryo-electron tomographic reconstructions of a similarly prepared sample. In each projected particle image or subtomogram, preliminary orientation parameters are first determined from an icosahedrally symmetric calculation, and in a second stage, the symmetry is broken by choosing 1 of the 60 symmetry-equivalent orientations. Both methods have yielded similar information, at about 50-Å resolution, concerning the footprint of the RNA on the virion surface, which demonstrates that RNA is released from an asymmetric site at the base of the canyon near a particle 2-fold axis and not at the channel at the 5-fold axes, as suggested by previous models. Additionally, the demonstrated success of the methodology provides us with a blueprint for resolving the molecular details of the RNA-capsid interaction in future experiments.     

Interaction of Poliovirus with Its Receptor Affords a High Level of Infectivity to the Virion in Poliovirus Infections Mediated by the Fc Receptor

Poliovirus infects susceptible cells through the poliovirus receptor (PVR), which functions to bind virus and to change its conformation. These two activities are thought to be necessary for efficient poliovirus infection. How binding and conformation conversion activities contribute to the establishment of poliovirus infection was investigated. Mouse L cells expressing mouse high-affinity Fcγ receptor molecules were established and used to study poliovirus infection mediated by mouse antipoliovirus monoclonal antibodies (MAbs) (immunoglobulin G2a [IgG2a] subtypes) or PVR-IgG2a, a chimeric molecule consisting of the extracellular moiety of PVR and the hinge and Fc portion of mouse IgG2a. The antibodies and PVR-IgG2a showed the same degree of affinity for poliovirus, but the infectivities mediated by these molecules were different. Among the molecules tested, PVR-IgG2a mediated the infection most efficiently, showing 50- to 100-fold-higher efficiency than that attained with the different MAbs. A conformational change of poliovirus was induced only by PVR-IgG2a. These results strongly suggested that some specific interaction(s) between poliovirus and the PVR is required for high-level infectivity of poliovirus in this system.     

Requirements for RNA Replication of a Poliovirus Replicon by Coxsackievirus B3 RNA Polymerase

A chimeric poliovirus type 1 (PV1) genome was constructed in which the 3D RNA polymerase (3D(pol)) coding sequences were replaced with those from coxsackievirus B3 (CVB3). No infectious virus was produced from HeLa cells transfected with the chimeric RNA. Processing of the PV1 capsid protein precursor was incomplete, presumably due to inefficient recognition of the P1 protein substrate by the chimeric 3CD proteinase containing CVB3 3D sequences. The ability of the chimeric RNA to replicate in the absence of capsid formation was measured after replacement of the P1 region with a luciferase reporter gene. No RNA synthesis was detected, despite efficient production of enzymatically active 3D(pol) from the 3D portion of the chimeric 3CD. The chimeric 3CD protein was unable to efficiently bind to the cloverleaf-like structure (CL) at the 5' end of PV1 RNA, which has been demonstrated previously to be required for viral RNA synthesis. The CVB3 3CD protein bound the PV1 CL as well as PV1 3CD. An additional chimeric PV1 RNA that contained CVB3 3CD sequences also failed to produce virus after transfection. Since processing of PV1 capsid protein precursors by the CVB3 3CD was again incomplete, a luciferase-containing replicon was also analyzed for RNA replication. The 3CD chimera replicated at 33 degrees C, but not at 37 degrees C. Replacement of the PV1 5'-terminal CL with that of CVB3 did not rescue the temperature-sensitive phenotype. Thus, there is an essential interaction(s) between 3CD and other viral P2 or P3 protein products required for efficient RNA replication which is not fully achieved between proteins from the two different members of the same virus genus.     

Poliovirus single-stranded RNA and double-stranded RNA: differential infectivity in enucleate cells.

The ability of poliovirus virion RNA and double-stranded RNA (replicative form) to replicate in enucleate mouse L cells was investigated. Virion RNA replicated successfully in the absence of the cell nucleus, whereas replicative form infection did not produce any detectable progeny in enucleate cells. The results provide direct evidence of a nuclear requirement early in the infection initiated by replicative form RNA.     

Functional Coupling between Replication and Packaging of Poliovirus Replicon RNA

Poliovirus RNA genomes that contained deletions in the capsid-coding regions were synthesized in monkey kidney cells that constitutively expressed T7 RNA polymerase. These replication-competent subgenomic RNAs, or replicons (G. Kaplan and V. R. Racaniello, J. Virol. 62:1687–1696, 1988), were encapsidated in trans by superinfecting polioviruses. When superinfecting poliovirus resistant to the antiviral compound guanidine was used, the RNA replication of the replicon RNAs could be inhibited independently of the RNA replication of the guanidine-resistant helper virus. Inhibiting the replication of the replicon RNA also profoundly inhibited its trans-encapsidation, even though the capsid proteins present in the cells could efficiently encapsidate the helper virus. The observed coupling between RNA replication and RNA packaging could account for the specificity of poliovirus RNA packaging in infected cells and the observed effects of mutations in the coding regions of nonstructural proteins on virion morphogenesis. It is suggested that this coupling results from direct interactions between the RNA replication machinery and the capsid proteins. The coupling of RNA packaging to RNA replication and of RNA replication to translation (J. E. Novak and K. Kirkegaard, Genes Dev. 8:1726–1737, 1994) could serve as mechanisms for late proofreading of poliovirus RNA, allowing only those RNA genomes capable of translating a full complement of functional RNA replication proteins to be propagated.     

Wistar大鼠皮内免疫Sabin株脊髓灰质炎灭活疫苗的免疫持久性及加强免疫效果研究

脊髓灰质炎野毒株消灭后,口服脊髓灰质炎减毒活疫苗(Oral polio vaccine,OPV)将被停止使用,脊髓灰质炎灭活疫苗(Inactivated poliovirus vaccine,IPV)将全面替代OPV,但IPV成本过高,难以满足全球需要。皮内免疫可以降低Sabin株脊髓灰质炎灭活疫苗(Inactivated poliovirus vaccine derived from Sabin strain,sIPV)的免疫剂量,本研究将观察sIPV疫苗皮内免疫大鼠后的免疫持久性及加强免疫效果。本研究采用sIPV,设皮内免疫组、全剂量肌肉免疫组和皮内免疫阴性对照组,接种Wistar大鼠,于3剂基础免疫程序完成后第1个月、12个月采血;第12个月采血后加强免疫1剂,并于加强免疫1个月后采血。中和试验检测各血清抗脊灰病毒中和抗体效价,评价皮内免疫sIPV的免疫持久性及加强免疫效果。Wistar大鼠3剂基础免疫后1个月,1/5、1/3剂量皮内免疫组与全剂量肌肉免疫组Ⅰ、Ⅱ、Ⅲ型抗体阳转率均达到了100%,各型别中和抗体几何平均滴度(Geometric mean titer,GMT)均远高于1∶8保护水平。基础免疫后12个月,sIPV全剂量组各型阳转率均维持在80%以上,1/10剂量皮内免疫组在50%以上,1/5剂量皮内免疫组维持在70%以上,1/3剂量皮内免疫组维持在80%以上,除1/10剂量组Ⅱ型外其余各组各型别GMT均维持在1∶8以上。加强免疫后1个月,1/5剂量皮内免疫组、1/3剂量皮内免疫组及全剂量组的Ⅰ型、Ⅱ型、Ⅲ型各组中和抗体阳转率均达到100%,并能够诱导产生远高于1∶8的抗体水平。本研究结果显示sIPV疫苗皮内免疫具有良好的免疫持久性及加强免疫效果。     

Poliovirus escape from RNA interference: short interfering RNA-target recognition and implications for therapeutic approaches

Short interfering RNAs (siRNAs) directed against poliovirus and other viruses effectively inhibit viral replication. Although RNA interference (RNAi) may provide the basis for specific antiviral therapies, the limitations of RNAi antiviral strategies are ill defined. Here, we show that poliovirus readily escapes highly effective siRNAs through unique point mutations within the targeted regions. Competitive analysis of the escape mutants provides insights into the basis of siRNA recognition. The RNAi machinery can tolerate mismatches but is exquisitely sensitive to mutations within the central region and the 3' end of the target sequence. Indeed, specific mutations in the target sequence resulting in G:U mismatches are sufficient for the virus to escape siRNA inhibition. However, using a pool of siRNAs to simultaneously target multiple sites in the viral genome prevents the emergence of resistant viruses. Our study uncovers the elegant precision of target recognition by the RNAi machinery and provides the basis for the development of effective RNAi-based therapies that prevent viral escape.     

Environmental Surveillance of Poliovirus in Sewage Water around the Introduction Period for Inactivated Polio Vaccine in Japan

Environmental virus surveillance was conducted at two independent sewage plants from urban and rural areas in the northern prefecture of the Kyushu district, Japan, to trace polioviruses (PVs) within communities. Consequently, 83 PVs were isolated over a 34-month period from April 2010 to January 2013. The frequency of PV isolation at the urban plant was 1.5 times higher than that at the rural plant. Molecular sequence analysis of the viral VP1 gene identified all three serotypes among the PV isolates, with the most prevalent serotype being type 2 (46%). Nearly all poliovirus isolates exhibited more than one nucleotide mutation from the Sabin vaccine strains. During this study, inactivated poliovirus vaccine (IPV) was introduced for routine immunization on 1 September 2012, replacing the live oral poliovirus vaccine (OPV). Interestingly, the frequency of PV isolation from sewage waters declined before OPV cessation at both sites. Our study highlights the importance of environmental surveillance for the detection of the excretion of PVs from an OPV-immunized population in a highly sensitive manner, during the OPV-to-IPV transition period.     

Poliovirus 2C region functions during encapsidation of viral RNA.

We have been exploring the mechanism of action of 5-(3,4-dichlorophenyl) methylhydantoin (hydantoin), an antiviral drug that inhibits the replication of poliovirus in culture. By varying the time of drug addition to infected cells, we found that the drug acts at a stage which is late in the replication cycle and subsequent to the step inhibited by guanidine. Furthermore, we detected normal levels of full-length plus-strand virion RNA in hydantoin-treated cultures. A new assembly intermediate in addition to the expected assembly intermediates was detected in drug-treated cultures. This intermediate has properties consistent with that of a packaging intermediate. Drug-resistant mutants were readily isolated. Sequence analysis of three independent drug-resistant mutants identified amino acid substitutions in the 2C coding region. Reconstruction by site-directed mutagenesis confirmed that these single mutations were sufficient to confer drug resistance. Taken together, these data suggest that the poliovirus 2C region is involved in virus encapsidation and that hydantoin inhibits this stage of replication.     

Poliovirus genome RNA hybridizes specifically to higher eukaryotic rRNAs.

The RNA genome of poliovirus hybridizes to 28S and 18S rRNAs of higher eukaryotes under stringent conditions. The hybridization detected by Northern blot analyses is specific since little or no signal was detected for yeast or prokaryotic rRNAs or other major cellular RNAs. Southern blot analysis of DNA clones of mouse rRNA genes leads us to conclude that several regions of 28S rRNA, and at least one region in 18S rRNA, are involved in the hybridization to polio RNA, and that G/C regions are not responsible for this phenomenon. We have precisely mapped one of these hybridizing regions in both molecules. Computer analysis confirms that extensive intermolecular base-pairing (81 out of 104 contiguous bases in the rRNA strand) could be responsible for this one particular site of interaction (polio genome, bases 5075-5250; 28S rRNA, bases 1097-1200). We discuss the possible functional and/or evolutionary significance of this novel type of interaction.