关键词： FOOT-AND-MOUTH DISEASE VIRUS   POLIOVIRUS   INACTIVATING AGENTS   CROSS-LINKING WITH FORMALDEHYDE   IMMUNOGENICITY  

摘要： The physico-chemical properties and immunogenicity of experimental vaccines against foot-and-mouth disease (FMD) and poliomyelitis, prepared by treatment of the viruses with N-acetylethyleneimine (AEI), formaldehyde or neutral red, have been studied None of these reagents affects the rate of sedimentation of the particles or their reaction with antibody against the major immunogenic sites. FMD vaccines prepared by inactivation with AEI or neutral red behaved like the untreated virus, in that they were disrupted on lowering the pH below 7. The RNA of the AEI-inactivated virus was degraded into slowly sedimenting molecules. Unlike AEI-inactivated virus, from which all the RNA could be extracted with phenol-SDS, the recovery from the neutral red inactivated virus was variable and was sometimes as low as 40%;this RNA gave a heterogenous profile in sucrose gradients. The capsid proteins in the AEI preparation migrated in SDS-PAGE to the sane positions as those of untreated virus, but in the neutral red preparation there was evidence of cross-linking. In contrast, the formaldehyde-inactivated vaccine was stable below pH 7 and the RNA could not be released by extraction with phenol-SDS at pH 5, because the capsid proteins had become cross-linked and/or linked to the RNA. As with foot-and-mouth disease virus (FMD V), poliovirus which had been inactivated with formaldehyde did not release its RNA on extraction with phenol-SDS and the capsid proteins were also cross-linked. Surprisingly, although AEI cleaved the viral RNA slowly in situ, the virus was no longer infectious after 6 h. Neutral red did not reduce the infectivity of the virus. All of the preparations gave similar levels of neutralizing antibody after a single inoculation. The high levels obtained with the formaldehyde-inactivated vaccines have implications for the processing of fixed particles by the antigen-presenting cells.

关键词： WATER   POLIOVIRUS   COXSACKIEVIRUS   REVERSE TRANSCRIPTASE-POLYMERASE CHAIN REACTION (RT-PCR)   CONCENTRATION  

摘要： This study developed a methodology to increase the sensitivity of enteric virus detection in tap water concentrates. Polymerase chain reaction (PCR) detection of virus in reduced volumes of virus-containing water concentrates was successful following removal of PCR inhibitory substances. Poliovirus 1 and coxsackievirus B3 were seeded into 3781 of tap water, concentrated with 1MDS filters, and reconcentrated by organic flocculation. The volume of concentrates was successfully reduced from 25 to 5 ml without loss of virus recovery. PCR detection of virus after treatment of a water concentrate (1.1 X 10(5)-fold concentration) with a Sephadex G-100 plus Chelex-100 column, or Sephadex G-50 plus Chelex-100 column, followed by heat treatment to release viral RNA, was compared with direct phenol-chloroform-isoamyl alcohol (PCI) extraction of viral RNA. The Sephadex G-50 plus Chelex-100 column did not remove inhibitory substances efficiently. The Sephadex G-100 plus Chelex-100 column could remove inhibitory substances, however, 99% of the viruses were also removed by the column. PCI extraction was found to be sufficient to remove inhibitory substances for reverse transcriptase (RT)-seminested PCR with a sensitivity of 0.2 plaque-forming units/10 mu 1 (0.2 PFU/1 tap water).

关键词： CD4 Lymphocyte Count   Adult   Antibodies   Bacterial   Antibodies   Viral   Case-Control Studies   Diphtheria Toxoid   Female   HIV Infections   Humans   Male   Middle Aged   Poliovirus Vaccine   Inactivated   Prospective Studies   Tetanus Toxoid  

摘要： A prospective study of antibody production by adults infected with human immunodeficiency virus (HIV) after vaccination with T lymphocyte-dependent diphtheria toroid, tetanus toroid, and inactivated trivalent poliovirus vaccine was conducted. Individuals were divided into three groups according to CD4(+) T-lymphocyte count: group 1 had a count of less than or equal to 100 X 10(6)/L;group 2, 101-300 X 10(6)/L;and group 3, >300 X 10(6)/L. After vaccination, 61%, 70%, and 73% of the individuals in groups 1, 2, and 3, respectively, developed protective titers of antibody to diphtheria toxin;the mean postvaccination antibody titer of HIV-infected individuals was significantly lower than that of healthy controls not infected with HIV. Furthermore, the mean titers of antibodies to tetanus toxin and poliovirus were significantly lower in HIV-infected individuals with CD4(+) lymphocyte counts of <300 X 10(6)/L than in controls. Of the HN-infected vaccinees, 83%-100% were protected against tetanus and 78%-100% against polio. We conclude that HIV-infected individuals with CD4(+) lymphocyte counts of <300 x 10(6)/L have an impaired (secondary) antibody response after receipt of T lymphocyte-dependent vaccines.

关键词： Membrane Proteins   Blotting   Northern   Cell Differentiation   Cell Survival   Comparative Study   Flow Cytometry   Hemin   Hemoglobins   Histocompatibility Antigens Class I   Humans   Leukemia   Erythroblastic   Acute   Major Histocompatibility Complex   Neoplasm Proteins   Polioviruses   RNA   RNA   Viral   Receptors   Virus   Research Support   Non-U.S. Gov't   Research Support   U.S. Gov't   P.H.S   Tumor Cells   Cultured   Viral Proteins   Virus Replication  

摘要： Poliovirus readily establishes a persistent infection in the K562-Mu erythroleukemia cell strain. In this study, three additional K562 cell strains were analyzed for their responses to poliovirus infection and found to be quite variable. K562 cells obtained from the ATCC established a persistent infection, similar to the K562-Mu cell strain, while the majority of cells from two other strains, K562-Kl and K562-We, were killed by 4 or 11 days postinfection (p.i.), respectively. Several characteristics of the uninfected and infected cell strains were examined to determine if differences existed which could explain the dramatically different responses to infection. Since K562 cell strains can differentiate toward several cell lineages, the four strains were analyzed for physical and functional likeness to the original K562 cell line using well-established functional criteria to determine whether gross changes in differentiation state had occurred. Based on the lack of MHC class I antigen expression and a dose-dependent increase in globin synthesis in response to hemin, all three laboratory K562 cell strains were indistinguishable from the ATCC reference strain. Surface poliovirus receptor levels were also similar in all K562 cell strains, although four- to fivefold lower than those in HeLa cells. Most biochemical events in virus replication either were very similar among K562 cells or were slightly variable and did not correlate with the degree of cell killing. These included levels of virus production, levels of viral protein produced, and processing and turnover of viral polypeptides. The key difference between the cell strains which consistently correlated with cell killing was the degree of virus-induced host translation shutoff, which was always greatest in the most virus-sensitive K562-KI cells. In addition, levels of 2A(pro) produced in K562 cell strains did not appear to correlate with the levels of host protein shutoff. A related and novel finding in these stud

关键词： Membrane Proteins   Antigens   CD44   Base Sequence   Capsid   Clone Cells   Cytopathogenic Effect   Viral   Humans   Molecular Sequence Data   Point Mutation   Poliovirus   RNA   Viral   Receptors   Virus   Research Support   Non-U.S. Gov't   Transfection   Virulence  

摘要： We report the isolation and characterization of HEp-2c cell clones obtained after two successive persistent poliovirus (PV) infections. Once cured, some of the cell clones displayed selective permissivity toward the wild-type Mahoney strain and partial resistance to particular mutants of this strain, including the Sabin 1 strain. Two cell clones, CI 4 and CI 10, were studied in greater detail. The cytopathic effects of Mahoney infection were comparable in the cell clones and in HEp-2c cells. The cytopathic effects of infection by Sabin 1 or Mahoney mutants were greatly delayed in CI 4 and CI 10. In the genomic region encoding the capsid proteins, determinants involved in the resistance of the cell clones to the Mahoney mutants were localized in the amino-terminal part of VPI (amino acids 22 and 43), the B-C loop of VP1 (amino acids 94-102), and the loop of VP3 connecting its amino-terminal to beta strand B (amino acid 60). These genomic regions are thought to be involved in the early steps of viral infection. Virus adsorption was slower and less efficient on CI 10 cells than on parental HEp-2c cells. Virus adsorption was faster on CI 4 than on HEp-2c cells, and at least as efficient, but there was less receptor-induced structural modification of the capsid, a step that is required for decapsidation. Furthermore, infection of CI 4 by a Mahoney mutant in which the B-C loop of VP1 has been deleted was affected in the later steps of infection. These results indicate that, in cells cured of persistent PV infection, poliovirus multiplication was restricted at several stages and particularly at two steps of virus entry: adsorption and/or the uncoating transitions following adsorption onto the receptor. (C) 1995 Academic Press, Inc.

关键词： DETERGENT   VIRAL PARTICLE   CELLULAR MONOLAYER   INTERFACIAL INTERACTION   VIRAL INFECTIVITY  

摘要： Monolayer cultures of Buffalo green monkey (BGM) kidney cells were inoculated with different aqueous solutions of anionic detergents prior to infection with poliomyelitisvirus type 1 (PMV-1). Alteration of inoculated cell culture monolayers was assessed by polarized light microscopy. Based on their visual appearance, altered monolayers were arbitrarily assigned different rating categories (r.c.) ranging from 1 to 8. For each detergent concentration, the rate of monolayer destruction was recorded and graphically integrated into the plots of r.c. against detergent concentration. In addition to a direct detergent-mediated alteration of the cellular monolayers, inoculation of detergent-conditioned BGM cultures with PMV-1 apparently affected virus-mediated alteration as well. The type of alteration as well as the initial rate of cytopathic destruction of the monolayer depended on several factors including the stereochemical properties of the detergent used, the final detergent concentration as well as the organic content and inoculation volume of the preconditioning solution, and the infectious titer of the viral inoculum. Moreover, infectivity kinetics revealed that cytopathic effects may be reactivated after a period of viral inhibition. In this way, at least two cycles of virus generation successively alternating with cell proliferation could be recorded simply by supplementing the system with energy for growth. From the presented results, it became apparent that modifications of cell surface activity contribute to virus infectivity mechanisms. Since treatment of aqueous PMV-1 suspensions has been previously described to largely affect viral infectious titers, viral uptake seems to be triggered by the phase behavior of viral particles upon contact with the plasma membrane. Therefore, aquatic samples that have been treated with anionic detergents in order to optimize virus isolation rates should be extensively washed prior to inoculation on cell culture.

关键词： POLIOVIRUS   CENTRAL AFRICAN REPUBLIC  

关键词： POLIOVIRUS   SACCHAROMYCES CEIEVISIAE   POLIOVIRUS PROTEASE 2A   TOXIC PROTEIN  

摘要： Poliovirus encodes two proteases, 2A(pro) and 3C(pro) that participate in the processing of the viral polyprotein and cleave a number of host proteins, Both proteases have been cloned and expressed in an inducible manner in Saccharomyces cerevisiae cells, The expression of 2A(pro), but not 3C(pro), was highly toxic for yeast cells such that growth,vas arrested after 5 h of induction and cell survival sharply declined, Cellular morphology was profoundly modified by expression of poliovirus 2A(pro), in such a way that electron dense granules and autophagosomic bodies arise in the cytoplasm. Experiments aimed at defining the yeast function affected by 2A(pro) suggested that translation was not the target of protease toxicity, but showed that RNA synthesis was profoundly blocked.

关键词： Viral Proteins   Amino Acid Sequence   Biopolymers   Cysteine Endopeptidases   Hela Cells   Humans   Hydrolysis   Microtubule-Associated Proteins   Microtubules   Molecular Sequence Data   Picornaviridae   Research Support   U.S. Gov't   P.H.S  

摘要： Poliovirus infection results in a number of host cell changes, including specific alterations in cellular proteins. This study further characterizes the cleavage of a cytoskeletal protein, microtubule-associated protein 4 (MAP-4) and investigates the identity of the viral protease which mediates its cleavage. MAP-4 cleavage by poliovirus was previously identified using a monoclonal antibody (M. Joachims and D. Etchison, 1992, J. Virol. 66, 5997-5804). In this study, MAP-4 cleavage was found to occur in cells infected by only some picornaviruses, poliovirus and human rhinovirus 14. Infection by other types of viruses, vesicular stomatitis virus and adenovirus, or by other types of picornaviruses, encephalomyocarditis virus, did not result in MAP-4 cleavage. To determine the viral mediator of MAP-4 cleavage, the effects of purified poliovirus proteases on MAP-4 integrity were examined by immunoblot. When MAP-4 substrates were incubated with concentratons of poliovirus 2A that were more than sufficient to induce p220 cleavage, there was no effect on MAP-4. However, when MAP-4 substrates were incubated with purified 3C protease (3C(pro)), cleavage products were detected that were identical in size to those generated in vivo in poliovirus-infected cells;the use of a mutant so protease did not result in MAP-4 cleavage. Cleavage of MAP-4 was also demonstrated with purified 3CD(pro), and the in vitro cleavage kinetics were examined. Indirect immunofluorescence revealed that MAP-4 cleavage also correlated with a marked ''collapse'' of microtubules during late infection, indicating a possible relationship between 3C(pro)-mediated MAP-4 cleavage and changes in the microtubule system of infected cells. (C) 1995 Academic Press, Inc.

关键词： Evolution   Animals   Base Sequence   Cell Line   Cercopithecus aethiops   Genome   Viral   Molecular Sequence Data   Mutation   Poliovirus Vaccine   Oral   Polioviruses   Quality Control   Serial Passage   Species Specificity   Vero Cells  

摘要： Screening for sequence heterogeneities in Sabin Type 3 strains of attenuated poliovirus demonstrated mutations that consistently accumulate to significant levels following 10 passages in cultures of primary African green monkey kidney (AGMK) cells or continuous cultures of Vero cells. Fourteen newly identified mutations were quantified by mutant analysis by PCR and restriction enzyme cleavage in passages and in batches of commercial vaccines made in AGMK and Vero cells from the Sabin original (SO) seed virus and from a seed virus rederived by RNA plaque purification (RSO or ''Pfizer'' seed). Nine of the 14 mutations were reproducibly observed in more than one series of passages. Although 5 other mutations were observed in only one set of passages each, their content gradually increased to a high percentage, suggesting that all the mutations that we found accumulated consistently. SO-derived samples accumulated more mutations than did RSO-derived ones, and the number of mutations and the rates of their accumulation were higher in Vero than in AGMK cells. While the rates of accumulation of most mutations were higher when passaging was performed at 37 degrees, a U --> C transition at nucleotide 5832 occurred faster at 34 degrees, the temperature used for vaccine production. Analysis of Type 3 oral poliovirus vaccine (OPV) monopools made by six manufacturers found only 5 of these newly identified mutations in Vaccine batches (nucleotides 3956, 4935, 5357, 5788, and 5832). Some of the mutations were found in trace amounts (less than 0.1%) while others were present at up to 1.8% levels, The pattern of these mutations was characteristic for the type of seed virus and the cell substrate but demonstrated no correlation with results of the monkey neurovirulence test. Therefore the only mutation occurring in Type 3 OPV which contributed to neurovirulence in monkeys was the previously described reversion at nucleotide 472. Quantitation of reversion at nucleotide 472 can be util