Biomedical subjects

V Deubel

Publications and source records attributed to V Deubel.

At least 19 recordsLinked to original sources

PubMed1992

Expression and secretion of Japanese encephalitis virus nonstructural protein NS1 by insect cells using a recombinant baculovirus.

The nonstructural protein NS1 of Japanese encephalitis virus (JEV) was expressed at a high level under the control of the polyhedrin promoter in Spodoptera frugiperda (Sf9) insect cells using a recombinant baculovirus. Recombinant NS1 was designed to contain its natural signal sequence at its N-terminus and no C-terminal hydrophobic domain that could act as a membrane anchor. This recombinant protein exhibited similar size to native NS1 expressed in Aedes albopictus (C6/36) insect cells infected with wild-type JEV. The signal sequence of NS1 allowed translocation of the protein into the endoplasmic reticulum where it underwent glycosylation. A small fraction of synthesized NS1 was able, in the absence of any other viral protein, to associate as a homodimer, showing similar characteristics to the native dimer. Interestingly, this recombinant dimeric form seemed to be exported and released in the extracellular medium of infected cell culture. During its transport, one of the two N-linked oligosaccharides of the polymannose type was processed to an endoglycosidase H-resistant form, suggesting that the protein had passed through the Golgi compartment before reaching the cell surface. Moreover, Triton X-114 partitioning analysis showed that monomeric NS1 behaved essentially as a hydrophilic protein, whereas both intracellular and extracellular dimeric NS1 were either free of or associated to membraneous components.

Animals↗

PubMed1992

Recent advances and prospective researches on molecular epidemiology of dengue viruses.

The determination of amino acid changes in the envelope protein by direct sequencing of either genomic RNA or PCR-amplified cDNA fragments provides useful informations for assessing the genetic variability and the geographic distribution of the actually most widespread dengue-2 serotype. The possible link of variations in the envelope protein-gene and virus virulence is discussed.

Dengue↗

PubMed1991

Demonstration of concurrent dengue 1 and dengue 3 infection in six patients by the polymerase chain reaction.

A dual viremia resulting from naturally acquired dengue 1 and dengue 3 infections in six patients experiencing a dengue-like syndrome during the epidemic in New Caledonia in 1989 is reported. Serotype identification was first based on virus isolation in mosquito cells and immunofluorescence using type-specific monoclonal antibodies. The double infection was confirmed directly in blood samples by the polymerase chain reaction (PCR) on genomic RNA and hybridization of the amplified cDNA fragments with type-specific DNA probes.

Base Sequence↗

PubMed1991

Processing, secretion, and immunoreactivity of carboxy terminally truncated dengue-2 virus envelope proteins expressed in insect cells by recombinant baculoviruses.

Two recombinant baculoviruses were constructed by inserting via the transfer vector pAcYM1 the genes coding for the structural proteins of dengue (DEN)-2 virus downstream from the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus. The two recombinants differed in truncation of 26 and 71 amino acids, respectively, in the carboxy-terminal sequence of DEN-specific envelope (E) glycoprotein. Recombinant DEN-2 E glycoproteins were processed and transported to the surface of Spodoptera frugiperda Sf9 cells infected with both viruses. We show that about one-third of the E glycoprotein minus its whole C-terminal hydrophobic anchor domain was secreted into an endoglycosidase H-resistant form. The type-specific neutralizing epitopes were conserved in the recombinant proteins as shown with a panel of monoclonal antibodies.

Animals↗

PubMed1991

[Application of the polymerase chain reaction (PCR) to diagnosis in virology].

Recall of the principle of the PCR and of its advantages: rapidity, possible automatization, high sensitivity allowing the detection of minute amounts of infectious agents, specificity depending on that of the primers used for the detection of the amplified product. But this technic has to be used with care to avoid any contamination which might give false positive results and requires controls. This procedure would allow to analyze the evolution and the variability of infectious agents by their sequence determination, thus providing a powerful tool for molecular epidemiology investigations. This method has already been used in many diagnoses of viral infections. The authors show results obtained on dengue viruses: 24 out of 32 sera were found positive (75%) by PCR whereas only 15 (47%) were positive by cell culture. On the other hand, 7 strains of dengue 2 virus of various regions throughout the world and isolated between 1969 and 1982 showed extensive degrees of variability.

Humans↗

PubMed1990

Identification of dengue sequences by genomic amplification: rapid diagnosis of dengue virus serotypes in peripheral blood.

Polymerase chain reaction (PCR) was developed for the in vitro amplification of dengue virus RNA via cDNA. A fraction of the N-terminus gene of the envelope protein in the four dengue serotypes was amplified using synthetic oligonucleotide primer pairs. Amplified products were cloned and used as dengue type-specific probes in gel electrophoresis and dot-blot hybridization. We detected and characterized dengue virus serotypes in blood samples by the three-step procedure DNA-PAH consisting in cDNA priming (P), DNA amplification (A) and hybridization (H) using specific non-radiolabelled probes. Our findings showed that DNA-PAH was more rapid and sensitive in the identification of the infecting serotype than the mosquito cell cultures. Moreover, the failure of cultures to detect virus particles in sera containing few copies of viral genome or anti-dengue antibodies justified the approach of DNA-PAH to the dengue identification in clinical specimens.

Base Sequence↗

PubMed1990

Passive protection studies in mice with monoclonal antibodies directed against the non-structural protein NS3 of dengue 1 virus.

Antibody-mediated enhancement of dengue virus replication is thought to be a mechanism contributing to the pathogenesis of dengue haemorrhagic fever and dengue shock syndrome. Enhancement is associated with antibodies to structural components of the virus. To circumvent the problem of immune enhancement, studies to identify protective antigens of dengue virus have involved non-structural proteins. Passive and active protection against lethal dengue virus infection in mice have been demonstrated with the non-structural protein NS1. In this study, the dengue virus non-structural protein NS3 was examined in passive protection studies with monoclonal antibodies prepared against NS3 of dengue 1 virus (Hawaiian). Five monoclonal antibodies that were authenticated to be reactive to NS3 were used to immunize 13- to 14-day old mice intraperitoneally. Thereafter, the mice were challenged intracerebrally with 100 LD50 of neurotropic dengue 1 virus and the survival indices of the mice were calculated. Significant decreases in survival indices (P less than 0.05), indicating increases in survival times were observed with four of five monoclonal antibodies tested. Monoclonal antibodies to NS3 of dengue 1 virus are able to increase the survival time of mice challenged with a lethal dose of dengue 1 virus, although the mechanism remains to be defined.

Animals↗

PubMed1988

Nucleotide sequence and deduced amino acid sequence of the nonstructural proteins of dengue type 2 virus, Jamaica genotype: comparative analysis of the full-length genome.

The sequence of the 5'-end of the genome of dengue 2 (Jamaica genotype) virus has been previously reported (V. Deubel, R. M. Kinney, and D. W. Trent, 1986, Virology 155, 365-377). We have now cloned and sequenced the remaining 75% of the genomic RNA that encodes the nonstructural proteins. The complete genome is 10,723 bases in length with a single open reading frame extending from nucleotides 97 to 10,269 encoding 3391 amino acids. The 3'-noncoding extremity presents a stem- and loop-structure and contains a repeated oligonucleotide sequence. Comparisons of the nucleotide sequences of the genomes of dengue 2 viruses of different topotypes reveal 90-95% similarity, with 64-66% similarity evident between dengue viruses of different serotypes. The amino acid sequence of the polyprotein of dengue 2 Jamaica virus shows 97, 68, 50, and 44% similarity with those of other dengue 2, dengue 1, or dengue 4, West Nile, and yellow fever viruses, respectively. Despite amino acid sequence divergence, the hydrophobic profile of the flavivirus proteins is highly conserved. Proteins NS1, NS3, and NS5 are the most conserved. Conserved amino acid stretches present in all flavivirus proteins may be involved in common essential biological functions.

Amino Acid Sequence↗

PubMed1988

Dengue 2 virus envelope protein expressed by a recombinant vaccinia virus fails to protect monkeys against dengue.

A cDNA copy of the dengue (DEN) 2 virus genome region encoding the virion capsid, membrane and envelope structural proteins has been inserted into vaccinia virus (VV) DNA under the control of its 11K late promoter. The DEN-2 envelope protein was expressed and processed in cells infected with the VV recombinant (VV/D2S). No DEN-2 virus antibody response was detected in mice, hamsters or monkeys vaccinated with VV/D2S. Furthermore, a viraemia was observed in recombinant-vaccinated monkeys after challenge with infectious DEN-2 virus.

Animals↗

PubMed1987

Comparative immunochemical and biological analysis of African and South American yellow fever viruses.

Four geographic variants (topotypes) of yellow fever (YF) virus from Africa and South America previously determined by RNA oligonucleotide mapping were analyzed for their structural, antigenic and virulence characteristics. The electrophoretic migration mobility and carbohydrate content of the envelope protein E characterized YF virus strains of South America. The NS 1 protein of South American and Central African YF virus strains was not precipitated by anti-NS 1 monoclonal antibodies (MAB) that precipitated NS 1 of West African strains. No distinction could be made among the YF virus strains on the basis of the neutralizing capacity of available MAB. South American, but not African YF virus strains were virulent for 8-day-old mice upon intraperitoneal inoculation. The results permitted characterization of topotypes of Central Africa and South America but failed to differentiate YF strains from West Africa.

Africa↗

PubMed1987

Partial nucleotide sequence of St. Louis encephalitis virus RNA: structural proteins, NS1, ns2a, and ns2b.

cDNA clones of the St. Louis encephalitis (SLE) virus genome have been obtained and the nucleotide sequence of 4.7 kb corresponding to the 5' terminal half of the genome determined. The genome contains a 5' noncoding region of 98 nucleotides followed by a single continuous open reading frame that encodes three structural proteins in the order capsid (C), membrane precursor (prM)-membrane (M), and envelope (E). Immediately following the C-terminus of E are located nonstructural proteins NS1 through NS3. The SLE amino acid sequence homology with yellow fever (YF), Murray Valley encephalitis (MVE), West Nile (WN), and dengue-2 (DEN) viruses over the sequenced region is 39, 66, 64, and 43%, respectively. The start of each SLE protein has been assigned on the basis of N-terminal sequence data and potential proteolytic cleavage sites homologous with YF and MVE viruses. Flaviviruses have conserved glycosylation sites in prM and NS1 proteins, although only one of the two glycosylation sites in the SLE E protein is conserved in MVE and DEN viruses. An evolutionary tree showing relationships of SLE, MVE, WN, YF, and DEN-2 flaviviruses is proposed on the basis of the amino acid sequences of the C proteins.

Base Sequence↗

PubMed1987

High genetic stability of the region coding for the structural proteins of yellow fever virus strain 17D.

The genome of the Pasteur 17D-204 vaccine strain of yellow fever virus has been cloned into pBR327. The inserts of recombinant plasmids were analysed by restriction cleavage pattern and compared with that of the genome of another substrain previously cloned and sequenced. Ten of the overlapping inserts were found to contain the sequence of the complete genome. We have sequenced approximately 3680 bases of the 5' region which codes for the C, M and E structural proteins and the NS1 non-structural protein. This sequence is the same as that reported previously, indicating a remarkable stability of these two vaccine substrains.

Cloning, Molecular↗

PubMed1986

Simultaneous administration of hepatitis B and yellow fever vaccines.

In most developing countries, hepatitis B prevention is carried out early in life. In these countries, mobile immunization teams have a limited number of sessions to devote to each rural community; simultaneous administration of multiple antigens is thus normal practice. We compared the immune responses of Senegalese children to the separate or simultaneous injections of yellow fever and hepatitis B vaccines. Injections were given at the time of booster injection for hepatitis B vaccine. Yellow fever antibodies were detected in similar proportions in infants immunized with either yellow fever vaccine alone or yellow fever and hepatitis B vaccines simultaneously. However, a lower proportion of high yellow fever antibody levels were observed when the two vaccines were injected simultaneously. No reduction in the anamnestic response of antibodies against the surface antigen of hepatitis B virus (anti-HBs) was observed when yellow fever vaccine was injected at the same time as the booster dose of hepatitis B vaccine. Since no untoward reactions were noted, it is concluded that hepatitis B and yellow fever vaccines can be administered at the same time.

Antibodies, Viral↗

PubMed1986

Nucleotide sequence and deduced amino acid sequence of the structural proteins of dengue type 2 virus, Jamaica genotype.

The nucleotide sequence of the 5'-terminal 2469 bases of dengue 2 (Jamaica genotype) virus has been determined and the encoded proteins compared with those of yellow fever and West Nile viruses, which belong to different flavivirus serogroups. The cDNA clone which was sequenced contains a 5'-noncoding region of 96 nucleotides followed by a single open reading frame coding for the structural proteins 5'-C-prM(M)-E-3' and the beginning of the NS1 nonstructural protein. The amino acid sequence homology between the structural polyprotein precursor of dengue 2 virus and those of yellow fever and West Nile viruses is 36.5 and 42%, respectively. The dengue virus structural proteins are similar in size and composition to those of the other flaviviruses. The basic capsid protein and the membrane and envelope proteins have hydrophobic regions at their C termini. The dengue 2 capsid C, membrane M, and envelope E proteins share 13, 36, and 43% homology, respectively, with the cognate proteins of yellow fever virus, and 33, 32, and 47% homology with the cognate proteins of West Nile virus. All 6 cysteine residues in the dengue 2 premembrane protein and all 12 cysteine residues in the dengue 2 envelope protein are conserved in the cognate proteins of yellow fever and West Nile viruses.

Amino Acid Sequence↗

PubMed1986

Genetic heterogeneity of yellow fever virus strains from Africa and the Americas.

Isolates of yellow fever (YF) virus from Africa (Senegambia, Central African Republic, Ivory Coast, Burkina Faso) and from South America (Panama, Ecuador, Trinidad) were examined by oligonucleotide fingerprinting of the 40S genome RNA. Geographically isolated and epidemiologically unrelated viruses were very distinct. On the basis of the T1 oligonucleotide fingerprints of each isolate, four geographical variants (topotypes) of YF virus isolated within the same period of time have been established. The Ivory Coast and Burkina Faso topotypes were similar. In the Central African Republic, two variants could be found exhibiting 70 to 75% homology to one another. In South America, the three analysed strains exhibited only about 70% homology, but could be classified in the same topotype. The oligonucleotide fingerprints of the genome RNA offered a useful tool for the understanding of YF virus variability.

Aedes↗

PubMed1986

Simultaneous administration of hepatitis B and yellow fever vaccines.

In most developing countries, hepatitis prevention has to be carried out early in life and on a mass scale. In such countries, mobile immunization teams have a limited number of sessions to devote to each rural community ans simultaneous administration of multiple antigens is normal practice. We have, therefore, compared the immune response to yellow fever and hepatitis B vaccines when injected simultaneously or not to Senegalese children. Injections were done at time of booster injections for hepatitis B vaccine. Yellow fever antibodies were detected in a similar proportion in infants immunized with yellow fever vaccine. However, a lower proportion of high yellow fever antibody levels was observed when the two vaccines were injected simultaneously. No reduction in the anti-HBs anamnestic response was observed when yellow fever vaccine was injected at the same time as the booster dose of hepatitis B vaccine. As no untowards reactions were noted, it can be concluded from the results that hepatitis B vaccine and yellow fever vaccine could be injected at the same time.

Antibodies, Viral↗

PubMed1985

Homogeneity among Senegalese strains of yellow fever virus.

A series of 16 yellow fever (YF) viruses isolated from mosquitoes, monkeys and humans in different epidemiological contexts in Senegal and The Gambia between 1976 and 1983, was analyzed by T1 RNase oligonucleotide fingerprints of the genomic 32P-labeled RNA, by SDS-polyacrylamide gel electrophoresis of the intracellular virus-specified polypeptides, by peptide mapping of the envelope E glycoprotein and by immunological reactivities with monoclonal antibody fluids (MAF's) against the E glycoprotein. These strains had not been passed in suckling mice and were isolated in Aedes pseudoscutellaris Mos 61 cultured cells. These strains showed no virulence in three-week-old Swiss mice when injected intraperitoneally. Direct comparison of the large T1 RNase-resistant oligonucleotide maps indicated a relative genetic stability (92%-100%). A greater change was observed when these strains were compared with an epidemic YF strain isolated in 1965 with an oligonucleotide fingerprint map sharing 82%-88% similarity. The YF-specified proteins were identical in their molecular weight, and the fragments obtained after limited proteolysis of the envelope protein using protease V8 or alphachymotrypsine indicated that the strains were chemically similar. Only a few differences were observed when the strains were seroneutralized with MAF's, but no relation could be made with genetic or biological data. This suggested that the YF virus strains isolated from the same geographic area and during a short period of time had evolved slowly. Moreover, all the viruses were closely related and no correlation could be established with the apparent variations in virulence in nature.

Aedes↗

PubMed1977

[Irradiated cell cultures applied to group A "chlamydiae" isolation (author's transl)].

The irradiated Mc Coy cell cultures method modified by Darougar et al. has been used to investigate the frequency of Chlamydiae in non-specific genital tract disease, in Reiter's disease and in patients suffering from conjonctivits associated with non-specific urethritis. Isolates were obtained from 104 men of the 660 suffering from acute urethritis, and from 18 men of the 67 suffering from conjonctivitis associated with urethritis. Fourteen female sexual contacts of these men were tested: Chlamydiae was isolated from 9. Seven patients suffering from acute Reiter's disease were tested: Chlamydiae was isolated from 4. These patients were tested by complement fixation and titers of 1/8 or more were only obtained in the 4 isolated positive cases, the three other cases remaining negative. Isolates of Chlamydiae were obtained from 10 women of the 67 women suffering from exocervicitis. No isolate was obtained, from 27 control patients.

Arthritis, Reactive↗