关键词： Animals   Central Nervous System/pathology   Macaca   Observer Variation   Poliovirus/pathogenicity   Poliovirus Vaccine, Inactivated/toxicity   Virulence   World Health Organization  

摘要： Abstract. A collaborative study evaluated the histopathological examination of the WHO neurovirulence test for poliovirus vaccines. Participants scored two sets of slides and the slides were sent to all participants in turn. The results showed an approximately two-fold range in mean lesion scores between readers. As readers scored consistently high or low, however, the range of differences between the two sets of slides was considerably less. From the limited data available, these differences in scoring would not have resulted in different decisions on the acceptability of the particular vaccines included in this study. The study was of value in assisting in developing mutual confidence in the interpretation of histological lesions in the neurovirulence test.

关键词： Animals   Cell Line   Glycosylation   Hela Cells   Humans   Mice   Molecular Weight   Poliovirus/genetics   Poliovirus/metabolism   Precipitin Tests   Precipitin Tests   Receptors, Virus/genetics   Receptors, Virus/metabolism   Viral Envelope Proteins/metabolism  

摘要： The expression of the human poliovirus receptor (hPVR) in several cultured cell lines was studied with the use of different antibodies directed against hPVR proteins. Immunoprecipitations of metabolically labeled cell lysates revealed that membrane-bound glycoforms of hPVR proteins have a molecular weight of about 80 kDa. By applying inhibitors of the glycosylation pathway (deoxymannojirimycin and swainsonine) we were able to monitor the modification of the hPVR glycoproteins when passing through the processing pathway. We show that a 67-kDa hPVR protein identified earlier (using a vaccinia virus expression system) is an intermediate glycoform probably located in the endoplasmic reticulum or cis-Golgi. Further modification of this glycoform is blocked by vaccinia virus infection or by the inhibitor deoxymannojirimycin. We, therefore, conclude that the 80-kDa glycoforms identified here are the fully processed hPVR isoforms. Surface iodination confirms that only the 80-kDa glycoforms are expressed on the cell surface. Treatment with various deglycosylating enzymes (N-Glycanase, O-Glycanase, Endo-H, and neuraminidase) demonstrates that the hPVR proteins bear sialylated complex-type oligosaccharides. Deglycosylation of the hPVR proteins also reveals the presence of both hPVR membrane-bound forms in cultured cells. Their relative expression levels, with respect to each other, vary considerably. The distribution of these hPVR isoforms in tissues may help explain the natural function of the hPVR proteins.

摘要： A picornavirus hybrid genome was constructed in which the internal ribosomal entry site (IRES) of encephalomyocarditis virus was inserted between the 5' nontranslated region and the open reading frame of poliovirus (PV), type 1 (Mahoney). Upon transfection into HeLa cells, the hybrid RNA replicated and yielded a derivative of PV (W1-PNENPO). The PV IRES could be removed from pPNENPO, which resulted in a hybrid picornavirus (W1-P108ENPO) in which the translation of the PV open reading frame normally promoted by the type 1 IRES of PV was promoted by the type 2 IRES of encephalomyocarditis virus. This result indicates that these elements are not likely to contain cis-acting elements necessary for PV replication or encapsidation. A foreign gene (bacterial chloramphenicol acetyltransferase, CAT) was inserted into pPNENPO cDNA between the PV and encephalomyocarditis virus IRES elements. The dicistronic RNA replicated in HeLa cells and yielded a derivative of PV (W1-DICAT) with a genome 17% longer than that of wild-type PV. CAT assays and immunoblot analyses showed that the viral RNA efficiently expressed the foreign gene in cell culture. The CAT activity diminished somewhat with each passage of the dicistronic virus, an observation which suggested that the inserted gene had a deleterious effect on viral replication. However, even after five virus passages, a significant quantity of the foreign gene was still expressed. Insertion of the open reading frame of luciferase (67 kDa) resulted in an RNA species that replicated and expressed luciferase for up to 20 hr after transfection. However, this elongated RNA was not encapsidated.

关键词： CAP INDEPENDENT   NONCODING   POLIOVIRUS   PROTEASE   TRANSLATION  

摘要： Viral protein synthesis in poliovirus infected cells was found to be influenced by mutations in part of the viral 5'-non-coding region (NCR) in a temperature dependent manner. At elevated temperatures these mutations resulted in virus titre reductions that allowed selection of revertant viruses. Some revertants were found to have retained the 5'-NCR mutations but had compensating mutations in the 2A protease gene that were responsible for the suppression of the temperature sensitive phenotypes. The mutations in 2A enhanced viral protein synthesis at a stage when cap dependent translation was already abolished, suggesting that the virally encoded protein 2A is directly involved in the process of cap independent translation in addition to its role in abolishing cap dependent translation.

摘要： Poliovirus 2C is a nonstructural polypeptide proposed to function in viral RNA replication. Poliovirus 2C is a member of a rapidly expanding family of proteins containing a consensus for nucleotide binding (NTP-B). Site-directed mutagenesis of conserved residues in the consensus A and S sites have suggested a functional role for the NTP-B motif in viral RNA replication and proliferation of poliovirus. We have expressed wildtype 2C and a 2C mutant, carrying a single amino acid exchange in the NTP-B motif A (Lys135Gln) using the baculovirus system. Both wildtype and mutant proteins are membrane associated. Following membrane solubilization, we have purified wildtype and mutant proteins to near homogeneity using conventional chromatography. We present biochemical evidence that wildtype 20 copurifies with an ATPase activity that is absent in the mutant preparation.

摘要： The Saukett/USA/50 strain is the type 3 component of the inactivated poliovirus vaccine. The capsid-coding region of genomic RNA of Saukett strains from five different sources was sequenced and the sequence differences were correlated with antigenic differences measurable with poliovirus type S-specific neutralizing monoclonal antibodies. All strains appeared to have capsid protein genes identical in size to those of the entirely sequenced type 3 poliovirus strains. The nucleotide sequence identity between the strains was 91% on the average and the strains could be divided into three groups. Amino acid differences were seen in 30 positions located throughout the capsid region both within and outside the known antigenic sites. Substitutions at the known antigenic sites explained most of the observed antigenic differences. Use of the atomic coordinates of the crystal structure model of the Sabin 3 virus and prior data based on escape mutants and peptide scanning revealed that most of the exposed substitutions located outside the known antigenic sites are spatially associated with regions found to be antigenic by either or both of these methods.

关键词： Arginine/physiology   Base Sequence   Capsid/genetics   Centrifugation, Density Gradient   DNA, Viral   Genetic Complementation Test   Genome, Viral   Genome, Viral   Hela Cells   Humans   Molecular Sequence Data   Mutagenesis, Site-Directed   Poliovirus/genetics   Protein Precursors/genetics   RNA, Viral/metabolism  

摘要： To begin to identify poliovirus capsid protein determinants required for assembly and RNA encapsidation, we have addressed the functional significance of three arginine residues of the poliovirus capsid in virus assembly and encapsidation of genomic RNA. These studies were conducted by using a recently described system in which recombinant vaccinia viruses are used to supply poliovirus capsid proteins in trans to a poliovirus subgenomic replicon [D. C. Ansardi, D. C. Porter, and C. D. Morrow (1993) J. Virol. 67, 3684-3690]. Two of the arginine residues, located at position 34 of VP4 (VP4-R034) and position 129 of VP1 (VP1-R129), are located within a cavity on the poliovirus capsid interior, whereas the third arginine, residue 223 of VP3 (VP3-R223), is located at a protomer-protomer interface. Five mutants were constructed by site-directed mutagenesis of poliovirus P1 capsid precursor cDNA to separately encode lysine or glutamine substitutions at VP4-R034 (VP4-R034K, VP4-R034Q), lysine or glutamine substitutions at residue 129 of VP1 (VP1-R129K, VP1-R129Q), or a lysine substitution at residue 223 of VP3 (VP3-R223K). Processed capsid proteins derived from the VP3-R223K, VP1-R129K, and VP1-R129Q mutant precursors were unstable and failed to assemble subviral particles or virions at 37°. The assembly defect for cleavage products of the VP3-R223K precursor was partially overcome at 33°, as empty capsids, but not mature virions, assembled from the mutant capsid subunits at the lower temperature. With regard to the third arginine residue analyzed, VP4-R034, processed capsid proteins derived from both the VP4-R034K and the VP4-R034Q mutant precursors assembled 155S virions at 37° however, capsid proteins derived from the VP4-R034Q precursor were temperature-sensitive for virion formation at 39.5°. The reduced virion formation at 39.5° was apparently a reflection of a defect in forming assembly competent subunits which also prevented accumulation of surpl

关键词： Amino Acid Sequence   Animals   Base Sequence   Carrier Proteins/chemistry   Carrier Proteins/genetics   Cloning, Molecular   DNA, Viral   Hela Cells   Humans   Molecular Sequence Data   Mutagenesis, Site-Directed   Protein Conformation   Transfection   Viral Nonstructural Proteins/chemistry   Viral Nonstructural Proteins/genetics   Water/chemistry  

摘要： Poliovirus protein 2C contains near its N-terminus a putative amphipathic helix which is well conserved among picornaviruses. Three mutants were constructed within this region by site-directed mutagenesis. In the first mutant (pT7XL2-2C-N1) two glutamic acids were replaced with valines at the boundary of the charged and uncharged faces of the helix. The second mutant (pT7XL2-2C-N2) contains an isoleucine to lysine change in the hydrophobic half; in the third mutant (pT7XL2-2C-N3) two lysines were replaced with threonines in the hydrophilic half of the helix. Upon transfection of HeLa cells with RNA transcripts made from these plasmids only pT7XL2-2C-N1 yielded viable virus (W1-2C-N1) which had a small-plaque phenotype. A large-plaque revertant of this virus, W1-2C-N1R, was found to contain the original glutamic acid at one of the mutated sites (E19). There is no detectable minus-stranded RNA synthesis following transfection of HeLa cells with transcript RNAs of the other two plasmids, pT7XL2-2C-N2 and -N3. In vitro translation of these two mutant RNA transcripts in HeLa extracts revealed processing abnormalities in the P2/P3 region of the polyprotein. This leads to a nearly complete absence of 2C and 3AB, which might be the primary cause of defective viral RNA synthesis. The putative amphipathic helix was found to overlap a consensus binding site for double-stranded RNA.

关键词： 无菌性脑膜炎   病毒感染性疾病   肠道病毒   病毒学检查   脊髓灰质炎病毒   中枢神经系统   诊断标准   病毒培养   咽部分泌物   诊断率  

摘要： 无菌性脑膜炎(Aseptic Meningitis Am)是一种中枢神经系统的急性病毒感染性疾病,常见于婴幼儿,每年7—10月份多发生流行。目前认为无菌性脑膜炎由肠道病毒引起,主要是Cox病毒B和Echo病毒,少见有脊髓灰质炎病毒及Cox病毒A。诊断标准:主要是依据急做腰穿刺术,做脑脊液性质、生化及病毒学检查。

关键词： 脊髓灰质炎   病毒   野毒株   PCR  

摘要： 用聚合酶链反应试验（ＰＣＲ）检测我国脊髓灰质炎病毒Ⅰ型（ＰＶＩ）野毒株，仅需５μｌＰＶＩ细胞培养液，方法简便、快速、敏感、特异，用本法对我国１４个省市７９份ＰＶＩ分离株的检测结果，能与检定ｓａｂｉｎⅠ相关株的ＳＩ／ＰＣＲ法的检测结果相印证，与其中３１份核苷酸测序判断为野毒株的结果相一致。其检测阳性率为８４．４％，基本能检测我国流行过的５个ＰＶＩ基因型野毒株。另外，从检测结果看，除北京市未发现野毒株外，其它１３个省区都有野毒株流行，表明当前我国消灭脊灰的任务还很重。