关键词： ACTING REPLICATION ELEMENT   VPG URIDYLYLATION   REVERSE-TRANSCRIPTASE   POSITIVE-STRAND   STRUCTURAL BASIS   PROTEIN 3AB   CRE   IDENTIFICATION   INITIATION   COMPLEX  

摘要： Protein priming of viral RNA synthesis plays an essential role in the replication of picornavirus RNA. Both poliovirus and coxsackievirus encode a small polypeptide, VPg, which serves as a primer for addition of the first nucleotide during synthesis of both positive and negative strands. This study examined the effects on the VPg uridylylation reaction of the RNA template sequence, the origin of VPg (coxsackievirus or poliovirus), the origin of 3D polymerase (coxsackievirus or poliovirus), the presence and origin of interacting protein 3CD, and the introduction of mutations at specific regions in the poliovirus 3D polymerase. Substantial effects associated with VPg origin were traced to differences in VPg-polymerase interactions. The effects of 3CD proteins and mutations at polymerase-polymerase intermolecular Interface I were most consistent with allosteric effects on the catalytic 3D polymerase molecule. In conclusion, the efficiency and specificity of VPg uridylylation by picornavirus polymerases is greatly influenced by allosteric effects of ligand binding that are likely to be relevant during the viral replicative cycle.

关键词： BMP   CSF   HGF   hippocampal cells   prion gene   TGF   VEGF  

摘要： Transfection of the prion protein gene (Prnp) into prion-deficient mouse cells was shown to reduce the replication of coxsackievirus B3, an enterovirus. Because mice can be susceptible to poliovirus infection by parenteral routes, the authors tested the susceptibility to poliovirus-1 (PV-1) of a panel of murine neuronal cell lines differing in their ability to express Prnp. The investigated cell lines (prionless HpL3.4 cells, HpL3.4 cells transfected with a Prnp vector, HpL3.4 cells transfected with a void vector, wild-type Hw3.5 Prnp(+/+) cells) expressed the murine homologue (Tage4) of human poliovirus receptor (CD155/hPVR). PV-1 infection of Prnp(-/-) HpL3.4 cells resulted in the production of high viral titers, though viral antigens could be detected in only 0.5% to 2% of cells. Wild-type Prnp(+/+) cells and prionless cells transfected with the Prnp gene were not permissive to PV-1. Results of viral titration and immunofluorescence were confirmed by conventional polymerase chain reaction (PCR) and quantitative real-time PCR. Exposure to PV-1 had no influence on the gene expression profile of Prnp(+/+) cells. In contrast, PV-1 infection was associated with upregulation of several genes in permissive Prnp(-/-) cell cultures: type I interferon (IFN) genes, IFN-related developmental regulator 1 (IFNRD1), tumor necrosis factor superfamily member 13b (TNFSF13b), interleukin (IL)-7, granulocyte/macrophage colony-stimulating factors (CSFs), hepatocyte growth factor (HGF), vascular endothelial growth factor-A, transforming growth factors beta1 and beta3 (TGFb1, TGFb3), as well as a variety of bone morphogenetic proteins endowed with neuroprotective activity. Distinction of permissive from nonpermissive neuronal cells on the basis of Prnp expression suggests that prion-deficient mice could represent an extraordinarily sensitive animal model for poliovirus infection.

关键词： poliovirus   OPV   saliva   IgA   ELISA   alpha-capture technique  

摘要： This study describes three ELISA methods for detection of immunoglobulin A (IgA) specific to three types of Sabin strains of poliovirus in saliva taken from 70 children aged 6-7 years vaccinated with a full course of oral poliovirus vaccine (OPV). Of the three ELISA methods (conventional IgA ELISA and two new methods described in this communication, the a-capture ELISA and Inhibition ELISA), a-capture ELISA demonstrated the highest sensitivity, with all saliva samples testing positive for Sabin poliovirus strains specific IgA antibodies of 1-3 types. Of 62 available a-capture ELISA positive saliva samples, all were also positive by the inhibition ELISA, and a significant correlation was found between the results. Fifty-two available saliva samples were screened by the three ELISA tests with positive results, and a significant correlation was found between the a-capture ELISA and the IgA ELISA;the correlation between the IgA ELISA and inhibition ELISA was not significant. The results of this study suggest that determination of Sabin poliovirus-specific IgA in human saliva by the ELISA techniques (especially by the novel α-capture ELISA) can be used reliably for evaluation of mucosal immunity in large groups of people immunized with poliovirus vaccines and for epidemiological studies. © 2005 Elsevier B.V. All rights reserved.

关键词： poliovirus   environmental surveillance   concentration methods   detection methods   virus mixture  

摘要： Examination of sewage specimens for poliovirus (environmental surveillance) was adopted as a supplementary tool in the surveillance of poliomyelitis in Egypt. Sewage samples were concentrated about 50-fold using a simple two-phase separation technique, and inoculated in cell cultures in two collaborating laboratories in parallel. All but 9 of the 293 (97%) samples collected from January 2001 to December 2002 contained poliovirus and/or other enteroviruses, with polioviruses being detected in 84% of the samples. The proportion of specimens containing type 1 wild poliovirus (PV1W, the North-East African (NEAF) genotype) was less in 2002 (16%) than in 2001 (57%), and further decreased in 2003. While the overall sensitivity to detect PV1W was similar in the two collaborating laboratories, the specimens scored positive were not identical. Parallel cultures inoculated with aliquots of a given specimen very frequently resulted in isolation of different viruses. Moreover, partial sequence analysis occasionally revealed representatives of different genetic lineages of PV1W in a given specimen. These results emphasize the need to use intensive laboratory analysis to optimise sample sensitivity in environmental poliovirus surveillance, and the difficulties in reproducing the isolation results by simple re-inoculation of samples containing a mixture of different viruses. (c) 2005 Elsevier B.V. All rights reserved.

关键词： CRYOELECTRON MICROSCOPY   3-DIMENSIONAL STRUCTURE   ELECTRON-MICROSCOPY   RECEPTOR   VIRUS   TRANSITIONS   MICROGRAPHS   MUTANTS   MODEL   IMAGE  

摘要： Poliovirus provides a well-characterized system for understanding how nonenveloped viruses enter and infect cells. Upon binding its receptor, poliovirus undergoes an irreversible conformational change to the 135S cell entry intermediate. This transition involves shifts of the capsid protein 0 barrels, accompanied by the externalization of VP4 and the N terminus of V-P1. Both polypeptides associate with membranes and are postulated to facilitate entry by forming a translocation pore for the viral RNA. We have calculated cryoelectron microscopic reconstructions of 135S particles that permit accurate placement of the 0 barrels, loops, and terminal extensions of the capsid proteins. The reconstructions and resulting models indicate that each N terminus of VP1 exits the capsid though an opening in the interface between VP1 and VP3 at the base of the canyon that surrounds the fivefold axis. Comparison with reconstructions of 135S particles in which the first 31 residues of VP1 were proteolytically removed revealed that the externalized N terminus is located near the tips of propeller-like features surrounding the threefold axes rather than at the fivefold axes, as had been proposed in previous models. These observations have forced a reexamination of current models for the role of the 135S particle in transmembrane pore formation and suggest testable alternatives.

关键词： differential scanning calorimetry   influenza virus   poliovirus   thermal stability   vaccine  

摘要： Vaccines against poliomyelitis and influenza contain inactivated forms of poliovirus and influenza virus. These antigens are generated on an industrial scale from the purified active viruses that have been analysed in this study by DSC (differential scanning calorimetry). Multiple unfolding transitions are seen for influenza virus A/New Caleclonia/20/99 (H IN 1), A/Panama/2007/99 (H3N2) and B/Shangdong/7/97. These data, combined with previously reported data on other influenza viruses, indicates that each influenza virus strain has a characteristic unfolding behaviour. Only minor changes were seen in the thermogram of beta PL (beta-propiolactone)-inactivated influenza virus, which is consistent with the proposition that beta PL reacts mainly with the nucleotide fraction of the virus. We demonstrate that a peak annotation of the thermogram of the native virus is possible using bromelain-treated virus and vircisomes. At pH 1.5-23, poliovirus of type I unfolds in a single unfolding event with respective T, (midpoint of protein unfolding transition) values between 34 and 45 degrees C. At PH 2, policiviruses of type 11 unfold equally in a single event, but, compared with the type I virus, with a T-m value increased by 3.7 degrees C. At neutral pH, the DSC thermogram of type I poliovirus was very 'noisy'. Data obtained offer the possibility of precisely characterizing and identifying different viral strains.

关键词： BREFELDIN-A   PHOSPHOLIPASE-D   GOLGI MEMBRANES   ENDOPLASMIC-RETICULUM   RNAREPLICATION   CELL-FREE   VESICLE FORMATION   BINDING-PROTEIN   COATED VESICLES   IN-VITRO  

摘要： Poliovirus infection results in the disintegration of intracellular membrane structures and formation of specific vesicles that serve as sites for replication of viral RNA. The mechanism of membrane rearrangement has not been clearly defined. Replication of poliovirus is sensitive to brefeldin A (BFA), a fungal metabolite known to prevent normal function of the ADP-ribosylation factor (ARF) family of small GTPases. During normal membrane trafficking in uninfected cells, ARFs are involved in vesicle formation from different intracellular sites through interaction with numerous regulatory and coat proteins as well as in regulation of phospholipase D activity and cytoskeleton modifications. We demonstrate here that ARFs 3 and 5, but not ARF6, are translocated to membranes in HeLa cell extracts that are engaged in translation of poliovirus RNA. The accumulation of ARFs on membranes correlates with active replication of poliovirus RNA in vitro, whereas ARF translocation to membranes does not occur in the presence of BFA. ARF translocation can be induced independently by synthesis of poliovirus 3A or 3CD proteins, and we describe mutations that abolished this activity. In infected HeLa cells, an ARF1-enhanced green fluorescent protein fusion redistributes from Golgi stacks to the perinuclear region, where poliovirus RNA replication occurs. Taken together, the data suggest an involvement of ARF in poliovirus RNA replication.

关键词： polio   immunity   immigration   seroprevalence  

摘要： Background. An essential part of the progress towards the global eradication of poliomyelitis is the ongoing evaluation of populations immunity. Migration of unregistered individuals poses a challenge to such estimations. Moreover, partially immunized immigrants might enlarge immunity gaps and facilitate polio outbreaks in their destination countries. Objectives. To study the level of immunity to polio in adult foreign workers in Israel. Methods. Foreign workers (n = 145) from the Philippines, India, and Western Africa were tested for neutralizing antibodies against three IPV strains and an outbreak-associated wild virus. Demographic and immunization data were collected by questionnaires. Results. Seropositivity rates and geometric mean titers (GMTs) for the Mahoney (type 1), MEF (type 2), and Saukett (type 3) poliovirus strains and the wild poliovirus type 1 strain were 99.3% (GMT-233.8), 98.6% (GMT-268.5), 99.3% (GMT-89.4), and 99.3% (GMT-139.5), respectively. Higher GMTs were associated with parenthood and older age. African individuals had significantly higher GMTS for the Saukett strain, compared with individuals from the Philippines and India. Conclusions. Our results indicate a high level of immunity among foreign workers. More serosurveys are indicated in the future in order to monitor the level of immunity in residents in Israel. (c) 2004 Elsevier Inc. All rights reserved.

关键词： TRACT-BINDING PROTEIN   AMINO-ACID-SEQUENCE   POLY(A)-BINDING PROTEIN   GENE-EXPRESSION   C-MYC   PROTEOLYTIC CLEAVAGE   3C PROTEASE   CELL STRESS   VIRUS   INHIBITION  

摘要： Eukaryotic translation initiation factor 4GI (eIF4GI) is an essential scaffolding protein required to recruit the 43 S complex to the 5'-end of mRNA during translation initiation. We have previously demonstrated that eIF4GI protein expression is translationally regulated. This regulation is mediated by cis-acting RNA elements, including an upstream open reading frame and an IRES that directs synthesis of five eIF4GI protein isoforms via alternative AUG initiation codon selection. Here, we further characterize eIF4GI IRES function and show that eIF4GI is expressed from several distinct mRNAs that vary via alternate promoter use and alternate splicing. Several mRNA variants contain the IRES element. We found that IRES activity mapped to multiple regions within the eIF4GI RNA sequence, but not within the 5'-UTR per se. However, the 5'-UTR enhanced IRES activity in vivo and played a role in initiation codon selection. The eIF4GI IRES was active when transfected into cells in an RNA form, and thus, does not require nuclear processing events for its function. However, IRES activity was found to be dependent upon the presence, in cis, of a 5' m(7)guanosine-cap. Despite this requirement, the eIF4GI IRES was activated by 2A protease cleavage of eIF4GI, in vitro, and retained the ability to promote translation during poliovirus-mediated inhibition of cap-dependent translation. These data indicate that intact eIF4GI protein is not required for the de novo synthesis of eIF4GI, suggesting its expression can continue under stress or infection conditions where eIF4GI is cleaved.

关键词： 脊髓灰质炎病毒   急性弛缓性麻痹   病毒学监测   河北   2003年   流行病学  

摘要： 目的　充分发挥脊灰病毒学监测作用。方法　按照《脊灰病毒检验手册》操作规程进行病毒分离与鉴定,由国家脊灰实验室用PCR -RFLP法对分离到的脊灰病毒株做型内鉴别。结果2 0 0 3年河北省脊灰实验室各项监测指标仍然保持较高水平。从AFP病例粪便标本中分离到脊灰病毒(PV) 4 3株,分离率为11.6 2 % ;非脊灰肠道病毒(NPEV) 4 9株,分离率为13.2 4 %。所有PV经国家脊灰实验室进行型内特征鉴定,除2株为疫苗变异株外,其余均为疫苗相关株。结论　三种细胞可以提高PV分离率,RD和HEp - 2细胞对提高NPEV的分离率都是很重要的。NPEV的分离率与月份有关。