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A Charpilienne

Publications and source records attributed to A Charpilienne.

At least 19 recordsLinked to original sources
PubMed2006

Complete genomic characterization and antigenic relatedness of genogroup III, genotype 2 bovine noroviruses.

Bovine enteric noroviruses form a genogroup, III, distinct from the 2 human norovirus genogroups, I and II. Two genogroup III genotypes were suggested by partial genomic analyses. In the present study, analysis of the full-length genome sequence of Bo/Newbury2/76/UK and the more contemporary Newbury2-like virus, Bo/Dumfries/1994/UK, showed that both were 7311 nucleotides in length and had three open reading frames (ORFs), amino acids motifs typical of noroviruses, and 95% or greater amino acid identities to each other in all regions of their genome. Apart from the ORF1 NTPase region, their ORF1 regions had less than 90% identity to the genogroup III genotype 1 Bo/Jena/80/DE virus, confirming two genogroup III genotypes. A close antigenic relationship was demonstrated by ELISA between the genotype 2 viruses, which will allow their serological diagnosis.

5' Untranslated Regions↗
PubMed2006

Genotype 1 and genotype 2 bovine noroviruses are antigenically distinct but share a cross-reactive epitope with human noroviruses.

The bovine enteric caliciviruses Bo/Jena/1980/DE and Bo/Newbury2/1976/UK represent two distinct genotypes within a new genogroup, genogroup III, in the genus Norovirus of the family Caliciviridae. In the present study, the antigenic relatedness of these two genotypes was determined for the first time to enable the development of tests to detect and differentiate between both genotypes. Two approaches were used. First, cross-reactivity was examined by enzyme-linked immunosorbent assay (ELISA) using recombinant virus-like particles (VLPs) and convalescent-phase sera from calves infected with either Jena (genotype 1) or Newbury2 (genotype 2). Second, cross-reactivity was examined between the two genotypes with a monoclonal antibody, CM39, derived using Jena VLPs. The two genotypes, Jena and Newbury2, were antigenically distinct with little or no cross-reactivity by ELISA to the heterologous VLPs using convalescent calf sera that had homologous immunoglobulin G titers of log10 3.1 to 3.3. CM39 reacted with both Jena and heterologous Newbury2 VLPs. The CM39 epitope was mapped to nine amino acids (31PTAGAQIAA39) in the Jena capsid protein, which was not fully conserved for Newbury2 (31PTAGAPVAA39). Molecular modeling showed that the CM39 epitope was located within the NH2-terminal arm inside the virus capsid. Surprisingly, CM39 also reacted with VLPs from two genogroup II/3 human noroviruses by ELISA and Western blotting. Thus, although the bovine noroviruses Jena and Newbury2 corresponded to two distinct antigenic types or serotypes, they shared at least one cross-reactive epitope. These findings have relevance for epidemiological studies to determine the prevalence of bovine norovirus serotypes and to develop vaccines to bovine noroviruses.

Amino Acid Sequence↗
PubMed2001

Individual rotavirus-like particles containing 120 molecules of fluorescent protein are visible in living cells.

Rotaviruses are large, complex icosahedral particles consisting of three concentric capsid layers. When the innermost capsid protein VP2 is expressed in the baculovirus-insect cell system it assembles as core-like particles. The amino terminus region of VP2 is dispensable for assembly of virus-like particles (VLP). Coexpression of VP2 and VP6 produces double layered VLP. We hypothesized that the amino end of VP2 could be extended without altering the auto assembly properties of VP2. Using the green fluorescent protein (GFP) or the DsRed protein as model inserts we have shown that the chimeric protein GFP (or DsRed)-VP2 auto assembles perfectly well and forms fluorescent VLP (GFP-VLP2/6 or DsRed-VLP2/6) when coexpressed with VP6. The presence of GFP inside the core does not prevent the assembly of the outer capsid layer proteins VP7 and VP4 to give VLP2/6/7/4. Cryo-electron microscopy of purified GFP-VLP2/6 showed that GFP molecules are located at the 5-fold vertices of the core. It is possible to visualize a single fluorescent VLP in living cells by confocal fluorescent microscopy. In vitro VLP2/6 did not enter into permissive cells or in dendritic cells. In contrast, fluorescent VLP2/6/7/4 entered the cells and then the fluorescence signal disappear rapidly. Presented data indicate that fluorescent VLP are interesting tools to follow in real time the entry process of rotavirus and that chimeric VLP could be envisaged as "nanoboxes" carrying macromolecules to living cells.

Animals↗
PubMed2001

Food supplementation with milk fermented by Lactobacillus casei DN-114 001 protects suckling rats from rotavirus-associated diarrhea.

Group A rotavirus is the leading cause of diarrhea among children aged 3-36 mo worldwide. Introducing fermented milk products into the infant diet has been proposed for the prevention or treatment of rotavirus diarrhea. The preventive effect of milk fermented by the Lactobacillus casei strain DN-114 001 was studied in a model of germfree suckling rats supplemented daily from d 2 of life and infected with SA11 rotavirus at d 5 (RF group). One group was supplemented with nonfermented milk (RM) and two uninfected groups (CM and CF) received either nonfermented or fermented milk. Frequency and severity of diarrhea were observed. Rats were killed at various times from 0 to 120 h postinfection (p.i.). Bacteria were measured in the intestine, and rotavirus antigens were detected by ELISA in fecal samples and in different parts of the intestine. Histologic observations were made, including vacuolation, morphology of intestinal villi and number of mucin cells. RM rats had diarrhea for 6 d; compared with the CM group, they had alterations of the intestinal mucosa characterized by cellular vacuolation 48 and 72 h p.i. and a lower number of sulfated mucin cells 72 and 96 h p.i. (P: < 0.05). Early supplementation with fermented milk significantly decreased the clinical signs of diarrhea from 24 to 144 h p.i. (P: < 0.05) and prevented rotavirus infection in all sections of the intestine. Histologic lesions of the small intestine were greatly reduced (P: < 0.05) and the number of mucin cells remained unchanged. The data are discussed with respect to the possibility of reducing rotavirus diarrhea in young children by consumption of fermented milk.

Animals↗
PubMed1998

The N terminus of rotavirus VP2 is necessary for encapsidation of VP1 and VP3.

The innermost core of rotavirus is composed of VP2, which forms a protein layer that surrounds the two minor proteins VP1 and VP3, and the genome of 11 segments of double-stranded RNA. This inner core layer surrounded by VP6, the major capsid protein, constitutes double-layered particles that are transcriptionally active. Each gene encoding a structural protein of double-layered particles has been cloned into baculovirus recombinants and expressed in insect cells. Previously, we showed that coexpression of different combinations of the structural proteins of rotavirus double-layered particles results in the formation of virus-like particles (VLPs), and each VLP containing VP1, the presumed RNA-dependent RNA polymerase, possesses replicase activity as assayed in an in vitro template-dependent assay system (C. Q.-Y. Zeng, M. J. Wentz, J. Cohen, M. E. Estes, and R. F. Ramig, J. Virol. 70:2736-2742, 1996). This work reports construction and characterization of VLPs containing a truncated VP2 (VPdelta2, containing amino acids [aa] Met-93 to 880). Expression of VPdelta2 alone resulted in the formation of single-layered delta2-VLPs. Coexpression of VPdelta2 with VP6 produced double-layered delta2/6-VLPs. VLPs formed by coexpression of VPdelta2 and VP1 or VP3, or both VP1 and VP3, resulted in the formation of VLPs lacking both VP1 and VP3. The presence of VP6 with VPdelta2 did not result in encapsidation of VP1 and VP3. To determine the domain of VP2 required for binding VP1, far-Western blot analyses using a series of truncated VP2 constructs were performed to test their ability to bind VP1. These analyses showed that (i) full-length VP2 (aa 1 to 880) binds to VP1, (ii) any N-terminal truncation lacking aa 1 to 25 fails to bind VP1, and (iii) a C-terminal 296-aa truncated VP2 construct (aa 1 to 583) maintains the ability to bind VP1. These analyses indicate that the N terminus of rotavirus VP2 is necessary for the encapsidation of VP1 and VP3.

Animals↗
PubMed1998

Crystallization and preliminary X-Ray analysis of rotavirus protein VP6.

As a first step to gain insight into the structure of the rotavirus virion at atomic resolution, we report here the expression, purification, and crystallization of recombinant rotavirus protein VP6. This protein has the property of polymerizing in the form of tubular structures in solution which have hindered crystallization thus far. Using a combination of electron microscopy and small-angle X-ray scattering, we found that addition of Ca2+ at concentrations higher than 100 mM results in depolymerization of the tubes, leading to an essentially monodisperse solution of trimeric VP6 even at high protein concentrations (higher than 10 mg/ml), thereby enabling us to search for crystallization conditions. We have thus obtained crystals of VP6 which diffract to better than 2.4 A resolution and belong to the cubic space group P4132 with a cell dimension a of 160 A. The crystals contain a trimer of VP6 lying along the diagonal of the cubic unit cell, resulting in one VP6 monomer per asymmetric unit and a solvent content of roughly 70%.

Animals↗
PubMed1997

Productive penetration of rotavirus in cultured cells induces coentry of the translation inhibitor alpha-sarcin.

Internalization of rotavirus in MA104 cells was found to induce coentry of alpha-sarcin, a toxin that inhibits translation in cell-free systems and to which cells are normally impermeable. Entry of the toxin, measured by inhibition of protein synthesis at early times after infection, correlated with virus penetration leading to expression of infectivity, since toxin entry (1) was induced only by trypsin-treated triple-layered virions, to a degree dependent on the toxin and the virus concentration; (2) correlated with the degree of permissivity of different cell lines to rotavirus infection; (3) was inhibited to a similar extent as infectivity by treatment of cells with neuraminidase; and (4) was inhibited by pre- or postadsorption incubation of the virus with neutralizing monoclonal antibodies to VP7 and VP4 (VP8*). Neither the virus infectivity nor the toxin coentry was significantly affected by treatment of cells with bafilomycin A1, an inhibitor of the vacuolar proton ATPase, indicating that both events are independent of the endosomal acid pH. Virus-like particles (VLP), composed of rotavirus proteins 2/6/7/4, but not 2/6/7 or 2/6, were able to induce toxin entry as efficiently as virions. Use of genetically modified VLP in combination with the toxin coentry assay, which measures entry through a productive pathway, should allow identification of the regions of the outer capsid proteins essential for rotavirus penetration.

Animals↗
PubMed1997

Pattern of metabolism and composition of the fecal microflora in infants 10 to 18 months old from day care centers.

BACKGROUND: From 5 months of age, infants are progressively introduced to a variety of foods which influence the equilibrium of the intestinal microflora. METHODS: Thirty-five children age 10-18 months from 8 day care centers in France were studied. Fecal specimens were examined for their biochemical and microbiological criteria. RESULTS: Bifidobacteria and Bacteroides belong to the predominant populations (9.7 and 8.6 log 10 cfu/g, respectively). The mean level of enterobacteria and enterococci were 8.0 and 7.8 log 10 cfu/g, respectively. Only 10% of the infants presented a lactobacilli amount above 6 log 10 cfu/g. Most feces had similar pH values (mean 6.4), percentage of water (mean 76.4%), and short chain fatty acid, ammonia and lactic acid concentrations (means 77, 6.7 and 2.3 mumol/g, respectively). beta-galactosidase had the highest activity (106 IU/g of protein) and nitroreductase, the lowest (0.1 IU/g of protein). alpha-glucosidase and nitrate reductase showed intermediate values of 17 and 4 IU/g of protein, respectively. With the exception of 4 infants, beta-glucosidase and beta-glucuronidase values were low (4 and 2 IU/g of protein). Age and day care center were not significant factors for most parameters studied, except that rotavirus was related to day care center, with detection in 5 infants from the same center. CONCLUSIONS: Many biochemical parameters were comparable to those found in adults, with the exception of ammonia concentration and beta-galactosidase activity. The fecal bacterial profile was different than in adults, with more Bifidobacteria than Bacteroides and higher levels of facultative anaerobes. One infant suffering from gastroenteritis had distinctive biochemical and bacterial parameters.

Ammonia↗
PubMed1997

Cell lines susceptible to infection are permeabilized by cleaved and solubilized outer layer proteins of rotavirus.

It has previously been shown that trypsinized triple-layered particles of rotavirus induce destabilization of liposomes and membrane vesicles in the absence of Ca2+, a condition which leads to solubilization of the outer capsid proteins of the virus. In this work, we have studied the relationship between outer capsid solubilization and permeabilization of membrane vesicles, monitoring particle and vesicle size simultaneously by changes in light scattering. Permeabilization of intact cells induced by solubilized outer capsid proteins was monitored by following the rate of entry of ethidium bromide into the cells. Solubilized outer capsid proteins separated from double-layered particles induced vesicle permeabilization. Solubilization of the outer capsid preceded and was required for vesicle or cell permeabilization. Membrane damage induced by rotaviral outer proteins was not repaired upon addition of 1 mM Ca2+ to the medium. Rotavirus infection and cell permeabilization were correlated in six different cell lines tested. This phenomenon might be related to the mechanism of virus entry into the cell. We propose a new model for rotavirus internalization based on the permeabilizing ability of outer capsid proteins and the cycling of trapped calcium in the endosomal compartment.

Capsid↗
PubMed1997

Solubilized and cleaved VP7, the outer glycoprotein of rotavirus, induces permeabilization of cell membrane vesicles.

It has been previously shown that rotavirus triple-layered particles induce permeabilization of liposomes and membrane vesicles. These effects were mediated by one or both of the solubilized outer-capsid proteins, VP4 and VP7. Permeabilization was dependent on trypsin treatment of the viral particles, suggesting that VP4 was involved. To analyse the respective roles of the outer-capsid proteins in this permeabilization process, we have used membrane vesicles loaded with carboxyfluorescein and virus-like particles derived from insect cells co-expressing various sets of capsid proteins. Virus-like particles containing VP2, VP6 and VP7 (VLP2/6/7) are as efficient in permeabilizing vesicles as triple-layered particles. As with double-layered particles, virus-like particles made of VP2 and VP6 had no effect on vesicle permeabilization. Permeabilization of membrane vesicles required trypsinization of the VP7 solubilized from VLP2/6/7. These results show that solubilized and trypsinized VP7 is able to induce membrane permeabilization, independently of the presence of VP4.

Antigens, Viral↗
PubMed1996

The concentration of Ca2+ that solubilizes outer capsid proteins from rotavirus particles is dependent on the strain.

It has been previously shown that rotavirus maturation and stability of the outer capsid are calcium-dependent processes. More recently, it has been hypothesized that penetration of the cell membrane is also affected by conformational changes of the capsid induced by Ca2+. In this study, we determined quantitatively the critical concentration of calcium ion that leads to solubilization of the outer capsid proteins VP4 and VP7. Since this critical concentration is below or close to trace levels of Ca2+, we have used buffered solutions based on ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and Ca-EGTA. This method allowed us to show a very high variability of the free [Ca2+] needed to stabilize, at room temperature, the outer capsid of several rotavirus strains. This concentration is about 600 nM for the two bovine strains tested (RF and UK), 100 nM for the porcine strain OSU, and only 10 to 20 nM for the simian strain SA11. Titration of viral infectivity after incubation in buffer of defined [Ca2+] confirmed that the loss of infectivity occurs at different [Ca2+] for these three strains. For the bovine strain, the cleavage of VP4 by trypsin has no significant effect on the [Ca2+] that solubilizes outer shell proteins. The outer layer (VP7) of virus-like particles (VLP) made of recombinant proteins VP2, VP6, and VP7 (VLP2/6/7) was also solubilized by lowering the [Ca2+]. The critical concentration of Ca2+ needed to solubilize VP7 from VLP2/6/7 made of protein from the bovine strain is close to the concentration needed for the corresponding virus. Genetic analysis of this phenotype in a set of reassortant viruses from two parental strains having the phenotypes of strains OSU (porcine) and UK (bovine) confirmed that this property of viral particles is probably associated with the gene coding for VP7. The analysis of VLP by reverse genetics might allow the identification of the region(s) essential for calcium binding.

Animals↗
PubMed1994

Identification of the nucleic acid binding domain of the rotavirus VP2 protein.

The bovine rotavirus VP2 protein is the major component of the core and forms the most internal layer surrounding the dsRNA genome. We have constructed recombinant baculoviruses expressing truncated VP2 proteins. The nucleic acid binding activity of these truncated proteins was tested by North-Western blotting experiments with single-stranded and double-stranded probes. The nucleic acid binding domain in VP2 was localized between amino acids 1 to 132. Recombinant proteins bound single-stranded and double-stranded nucleic acids, but showed less affinity for double-stranded RNA and DNA. Interactions of VP2 with the genome were investigated in viral single-shelled particles by u.v.-cross-linking. In these experiments, only VP2 protein bound the genomic RNA in purified single-shelled particles.

Animals↗
PubMed1994

Rotavirus interaction with isolated membrane vesicles.

To gain information about the mechanism of epithelial cell infection by rotavirus, we studied the interaction of bovine rotavirus, RF strain, with isolated membrane vesicles from apical membrane of pig enterocytes. Vesicles were charged with high (quenching) concentrations of either carboxyfluorescein or calcein, and the rate of fluorophore release (dequenching) was monitored as a function of time after mixing with purified virus particles. Purified single-shelled particles and untrypsinized double-shelled ones had no effect. Trypsinized double-shelled virions induced carboxyfluorescein release according to sigmoid curves whose lag period and amplitude were a function of virus concentration and depended on both temperature and pH. The presence of 100 mM salts (Tris Cl, NaCl, or KCl) was required, since there was no reaction in isoosmotic salt-free sorbitol media. Other membrane vesicle preparations such as apical membranes of piglet enterocyte and rat placenta syncytiotrophoblasts, basolateral membranes of pig enterocytes, and the undifferentiated plasma membrane of cultured MA104 cells all gave qualitatively similar responses. Inhibition by a specific monoclonal antibody suggests that the active species causing carboxyfluorescein release is VP5*. Ca2+ (1 mM), but not Mg2+, inhibited the reaction. In situ solubilization of the outer capsid of trypsinized double-shelled particles changed release kinetics from sigmoidal to hyperbolic and was not inhibited by Ca2+. Our results indicate that membrane destabilization caused by trypsinized outer capsid proteins of rotavirus leads to fluorophore release. From the data presented here, a hypothetical model of the interaction of the various states of the viral particles with the membrane lipid phase is proposed. Membrane permeabilization induced by rotavirus may be related to the mechanism of entry of the virus into the host cell.

Animals↗
PubMed1993

Expression of two bovine rotavirus non-structural proteins (NSP2, NSP3) in the baculovirus system and production of monoclonal antibodies directed against the expressed proteins.

Studies on rotavirus non-structural proteins have been hampered in the past by difficulties in obtaining monospecific reagents. To make such reagents available, we have expressed in the baculovirus system NSP2 and NSP3 (formerly called NS35 and NS34, respectively) of the bovine rotavirus RF and produced hybridomas against these proteins. Full-length DNA copies of RNA segments 7 (coding for NSP3) and 8 (coding for NSP2) of the virus strain RF were cloned and sequenced. Each cDNA was inserted in the transfer vector pVL941 and used to transfect Spodoptera frugiperda cells (Sf9). Recombinant baculoviruses encoding these proteins were obtained. Infection of Sf9 cells with these recombinant viruses resulted in a high level of expression of NSP2 and NSP3 (range of 1 microgram per 10(6) cells). Monoclonal antibodies (MAbs) were elicited by immunization of BALB/c mice with adjuvented, unpurified recombinant proteins in the rear foot pads. Fusion was performed using lymphocytes from popliteal lymph nodes with SP2/O-Ag14 myeloma line. Screening was by differential indirect immunofluorescent staining on monolayers of Sf9 cells infected with each recombinant virus. Two MAbs proved to be reactive against NSP3 and a single one against NSP2. They showed high specificity by immunofluorescence, immunoprecipitation and Western blot. The isotype of these MAbs was IgG1. Oligomeric forms of NSP3 and NSP2 proteins were detected and the existence of intra-chain disulfide bridge in NSP2 protein was suggested. The levels of synthesis and cellular localization of NSP3 and NSP2 proteins were different as shown by immunoprecipitation and immunofluorescence.

Animals↗
PubMed1992

Sequences of the four larger proteins of a porcine group C rotavirus and comparison with the equivalent group A rotavirus proteins.

The sequences of the four larger proteins of rotavirus group C (Cowden strain) are presented and compared with the sequences of the corresponding group A proteins. They exhibit a significant level of homology, allowing gene coding assignment for the group C rotavirus. The coding strategy of the group C virus RNA segment is the same as that for the group A large segments as one long open reading frame is present in each segment. The genome segment 1 encodes the structural protein VP1 which presents the RNA-dependent RNA polymerase consensus motifs. The VP1 protein is the most highly conserved between the rotaviruses of groups A and C. The genome segment 2 encodes the VP2 protein. The deduced protein sequence does not present the putative leucine zippers identified in the group A protein but its amino terminal is hydrophilic and highly charged as previously noted for the group A protein. The genome segment 3 encodes for a protein homologous to the group A outer capsid protein VP4. As observed among the various group A sequences, the amino terminal is the region presenting the fewest similarities. A cleavage region and a putative fusion motif similar to those present in the group A viruses have been identified. For this protein the comparison has been extended to the IDIRV [corrected] VP3 previously sequenced and indicates that groups A and C VP4 proteins are much more related to each other than to the group B equivalent. The genome segment 4 encodes for a protein showing an approximate 40% sequence identity to the minor core protein, VP3, of the group A rotavirus. This remarkable conservation of primary structures argues for severe functional constraint on the evolution of these proteins.

Amino Acid Sequence↗