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1.
J Clin Virol ; 56(3): 238-43, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23219893

ABSTRACT

BACKGROUND: Resequencing DNA microarray (RMA) technology uses probes designed to identify a panel of viral sequences. It can be used for detecting emerging viruses by revealing the nucleotide polymorphisms within the target of interest. OBJECTIVES/STUDY DESIGN: As a new tool for molecular diagnosis of arbovirus infection, high density PathogenID v2.0 RMA (PID2-RMA) was assessed for the detection and genetic analysis of dengue, West Nile, and Chikungunya viruses in spiked blood samples or sera from individuals infected with dengue virus. Viral RNAs extracted from biological samples were retrotranscribed into cDNA and amplified using the Phi 29 polymerase-based method. This amplified cDNA was used for hybridization on PID2-RMA. RESULTS: A good specificity of RMA-based detection was demonstrated using a panel of arboviruses including Dengue, West Nile and Chikungunya viruses. This technology was also efficient for the detection and genetic analysis of the different serotypes of dengue virus in sera of infected patients. Furthermore, the mixing of dengue, West Nile and Chikungunya prototype viruses within a single sample of human blood did not interfere with the sensitivity of PID2-RMA. CONCLUSIONS: Our data show that high density PID2-RMA was suitable for the identification of medically important arboviruses. It appears to be particularly adapted to the genetic analysis of dengue, West Nile, and Chikungunya viruses in urgent clinical situations where the rapid identification and characterization of the pathogen is essential.


Subject(s)
Alphavirus Infections/diagnosis , Arboviruses/isolation & purification , Dengue/diagnosis , Molecular Diagnostic Techniques/methods , Virology/methods , West Nile Fever/diagnosis , Alphavirus Infections/virology , Arboviruses/classification , Arboviruses/genetics , Chikungunya Fever , Dengue/virology , Humans , Microarray Analysis/methods , Sequence Analysis, DNA/methods , West Nile Fever/virology
2.
Virology ; 274(2): 292-308, 2000 Sep 01.
Article in English | MEDLINE | ID: mdl-10964773

ABSTRACT

One mechanism by which dengue (DEN) virus may cause cell death is apoptosis. In this study, we investigated whether the genetic determinants responsible for acquisition by DEN type 1 (DEN-1) virus of mouse neurovirulence interfere with the induction of apoptosis. Neurovirulent variant FGA/NA d1d was generated during the adaptation of the human isolate of DEN-1 virus strain FGA/89 to grow in newborn mouse brains and mosquito cells in vitro [Desprès, P. Frenkiel, M. -P. Ceccaldi, P.-E. Duarte Dos Santos, C. and Deubel, V. (1998) J. Virol., 72: 823-829]. Genetic determinants possibly responsible for mouse neurovirulence were studied by sequencing the entire genomes of both DEN-1 viruses. Three amino acid differences in the envelope E protein and one in the nonstructural NS3 protein were found. The cytotoxicity of the mouse-neurovirulent DEN-1 variant was studied in different target cells in vitro and compared with the parental strain. FGA/NA d1d was more pathogenic for mouse neuroblastoma cells and attenuated for human hepatoma cells. Changes in virus replicative functions and virus assembly may account, in a large part, for the differences in the induction of apoptosis. Our data suggest that identified amino acid substitutions in the envelope E protein and viral RNA helicase NS3 may influence DEN-1 virus pathogenicity by altering viral growth.


Subject(s)
Apoptosis , Dengue Virus/pathogenicity , RNA Helicases/chemistry , RNA Helicases/metabolism , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/metabolism , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Chlorocebus aethiops , Culicidae , Dengue Virus/enzymology , Dengue Virus/genetics , Dengue Virus/growth & development , Epithelial Cells/pathology , Epithelial Cells/virology , Glycoproteins/metabolism , Humans , Kinetics , Membrane Fusion , Models, Molecular , Molecular Sequence Data , Mutation/genetics , Neurons/pathology , Neurons/virology , Protein Conformation , Protein Processing, Post-Translational , RNA Helicases/genetics , RNA, Viral/biosynthesis , Viral Envelope Proteins/genetics , Viral Proteins/biosynthesis , Viral Proteins/metabolism , Virulence , Virus Replication
3.
J Virol ; 74(1): 564-72, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10590151

ABSTRACT

We report that endoplasmic reticulum alpha-glucosidase inhibitors have antiviral effects on dengue (DEN) virus. We found that glucosidase inhibition strongly affects productive folding pathways of the envelope glycoproteins prM (the intracellular glycosylated precursor of M [membrane protein]) and E (envelope protein): the proper folding of prM bearing unprocessed N-linked oligosaccharide is inefficient, and this causes delayed formation of prME heterodimer. The complexes formed between incompletely folded prM and E appear to be unstable, leading to a nonproductive pathway. Inhibition of alpha-glucosidase-mediated N-linked oligosaccharide trimming may thus prevent the assembly of DEN virus by affecting the early stages of envelope glycoprotein processing.


Subject(s)
Dengue Virus/drug effects , Endoplasmic Reticulum/virology , Enzyme Inhibitors/pharmacology , Virion/growth & development , Virus Replication/drug effects , alpha-Glucosidases/pharmacology , 1-Deoxynojirimycin/pharmacology , Amino Acid Sequence , Animals , Dengue/virology , Dengue Virus/physiology , Indolizines/pharmacology , Mice , Molecular Sequence Data , Protein Processing, Post-Translational , Viral Structural Proteins/chemistry , Viral Structural Proteins/metabolism
4.
J Virol ; 72(1): 823-9, 1998 Jan.
Article in English | MEDLINE | ID: mdl-9420294

ABSTRACT

Apoptosis has been suggested as a mechanism by which dengue (DEN) virus infection may cause neuronal cell death (P. Desprès, M. Flamand, P.-E. Ceccaldi, and V. Deubel, J. Virol. 70:4090-4096, 1996). In this study, we investigated whether apoptotic cell death occurred in the central nervous system (CNS) of neonatal mice inoculated intracerebrally with DEN virus. We showed that serial passage of a wild-type human isolate of DEN virus in mouse brains selected highly neurovirulent variants which replicated more efficiently in the CNS. Infection of newborn mice with these neurovirulent variants produced fatal encephalitis within 10 days after inoculation. Virus-induced cell death and oligonucleosomal DNA fragmentation were observed in mouse brain tissue by day 9. Infected mouse brain tissue was assayed for apoptosis by in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and for virus replication by immunostaining of viral antigens and in situ hybridization. Apoptotic cell death and DEN virus replication were restricted to the neurons of the cortical and hippocampal regions. Thus, DEN virus-induced apoptosis in the CNS was a direct result of virus infection. In the murine neuronal cell line Neuro 2a, neuroadapted DEN virus variants showed infection patterns similar to those of the parental strain. However, DEN virus-induced apoptosis in these cells was more pronounced after infection with the neurovirulent variants than after infection with the parental strain.


Subject(s)
Apoptosis , Brain/pathology , Brain/virology , Dengue Virus/pathogenicity , Dengue/pathology , Dengue/virology , Encephalitis, Viral/pathology , Encephalitis, Viral/virology , Animals , Animals, Newborn , Cerebral Cortex/pathology , Cerebral Cortex/virology , DNA Fragmentation , Dengue Virus/genetics , Dengue Virus/physiology , Hippocampus/pathology , Hippocampus/virology , Humans , In Situ Hybridization , Mice , RNA, Viral/genetics , RNA, Viral/metabolism , Virulence , Virus Replication
5.
Vaccine ; 15(17-18): 1946-54, 1997 Dec.
Article in English | MEDLINE | ID: mdl-9413106

ABSTRACT

We constructed a recombinant baculovirus which produces a dengue (DEN)-2 virus envelope (E) protein containing a six-histidine (H6) tag in place of the last 100 amino acids at its C-terminus. The recombinant protein was purified from the supernatant of baculovirus-infected Spodoptera frugiperda insect cell cultures to apparent homogeneity by cation-chelation chromatography (TALON) in which the H6-tagged E-protein was eluted under non-denaturing conditions with 100 mM imidazole at pH 8.0. Mice vaccinated with the purified E mixed with aluminium hydroxide adjuvant showed an immune response of IgM and IgG1, IgG2a and IgG2b isotypes, and neutralizing antibodies, similar to that following immunization with purified inactivated DEN-2 virus. Moreover, mice that received the purified recombinant protein were significantly protected against 200 50% lethal dose of DEN-2 virus. This combination of affinity purified H6-tagged protein and adsorption onto a cationic carrier seems promising for the production of immunogenic particulate proteins, especially DEN protein for the four serotypes, and the development of a new generation of vaccines.


Subject(s)
Antibodies, Viral/biosynthesis , Severe Dengue/prevention & control , Viral Envelope Proteins/immunology , Viral Envelope Proteins/therapeutic use , Animals , Baculoviridae/genetics , Baculoviridae/metabolism , Chlorocebus aethiops , Chromatography, Affinity , Dimerization , Male , Mice , Mice, Inbred BALB C , Neutralization Tests , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification , Recombinant Proteins/therapeutic use , Severe Dengue/immunology , Spodoptera/virology , Vero Cells , Viral Envelope Proteins/isolation & purification
6.
J Virol Methods ; 56(2): 179-89, 1996 Feb.
Article in English | MEDLINE | ID: mdl-8882648

ABSTRACT

The maltose-binding protein (MalE) contains a signal sequence which allows its translocation in the periplasm of prokaryotic microorganisms. In this study, MalE was produced in Spodoptera frugiperda (Sf9) lepidopterian cells using the baculovirus expression system. The secretion of MalE, following cleavage of its signal sequence, to the supernatant fluid of recombinant baculovirus-infected Sf9 cells and its affinity for maltodextrin polymers allowed recovery of significant amounts (> or = 10 micrograms per 10(6) cells) of highly purified protein. The gene encoding the envelope glycoprotein E of the dengue (DEN) type 2 virus deleted of its C-terminal 102 amino acids (D2E delta 102) was fused to the MalE gene. The resulting hybrid MalE-D2E delta 102 glycoprotein was processed through the Golgi network of Sf9 cells and was secreted. It was retained on a maltodextrin column and was eluted with maltose. Antigenic and immunogenic properties dependent on the three-dimensional structure in the native E protein were preserved in the recombinant MalE-D2E delta 102 protein. Thus MalE with its signal sequence may be used as a carrier protein for production in the baculovirus system and purification of proteins which require transportation through intracellular compartments for correct folding and processing.


Subject(s)
Antigens, Viral/immunology , Carrier Proteins/immunology , Dengue Virus/immunology , Dengue/prevention & control , Viral Envelope Proteins/immunology , Animals , Antigens, Viral/chemistry , Antigens, Viral/isolation & purification , Cell Line , Cloning, Molecular , Dengue/virology , Dengue Virus/chemistry , Maltose-Binding Proteins , Mice , Mice, Inbred BALB C , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Spodoptera/cytology , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/isolation & purification , Viral Envelope Proteins/metabolism
7.
J Gen Virol ; 75 ( Pt 7): 1569-78, 1994 Jul.
Article in English | MEDLINE | ID: mdl-8021588

ABSTRACT

We constructed two recombinant Autographa californica nuclear polyhedrosis baculoviruses. Spodoptera frugiperda (Sf9) cells containing these constructs produce carboxy-terminally truncated envelope E proteins representing dengue (DEN) virus serotypes 2 and 3. The two recombinant proteins contained their homologous signal sequences at the N terminus and were truncated by 71 and 74 amino acids at the C terminus, respectively. This allowed the translocation of the recombinant proteins to the endoplasmic reticulum followed by glycosylation processing and secretion into the extracellular medium. An additional unglycosylated form which was not secreted was detected inside the infected Sf9 cells. Sera from Swiss mice immunized with the infected Sf9 cell lysates gave a DEN cross-reactive response in ELISA and substantial amounts of neutralizing antibodies to the homologous virus. Similar antibody titres were obtained when the two recombinant proteins were inoculated concomitantly. BALB/c mice were vaccinated with three doses of the recombinant E proteins, taken as monovalent or bivalent immunogens, and challenged with mouse-adapted DEN-2 virus. DEN-2 E protein induced a good protection (90%) against lethal encephalitis and recombinant DEN-3 E protein gave a substantial cross-protection (54%). Eighty-two percent of the mice immunized with a mixture of both recombinant E proteins survived the DEN-2 virus challenge.


Subject(s)
Antibodies, Viral/biosynthesis , Dengue Virus/immunology , Dengue/immunology , Gene Expression Regulation, Viral/genetics , Viral Envelope Proteins/immunology , Viral Proteins/immunology , Animals , Base Sequence , Cell Line , Dengue/microbiology , Dengue Virus/genetics , Female , Immunity, Active , Lethal Dose 50 , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Moths , Neutralization Tests , Nucleopolyhedroviruses/genetics , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Viral Envelope Proteins/biosynthesis , Viral Envelope Proteins/genetics , Viral Proteins/biosynthesis , Viral Proteins/genetics
8.
Arch Virol ; 139(1-2): 197-207, 1994.
Article in English | MEDLINE | ID: mdl-7826210

ABSTRACT

We constructed a recombinant baculovirus encoding a dengue (DEN)-2 virus envelope glycoprotein truncated of 102 amino acids (aa) at its C-terminus (D2E delta 102). The production, processing and transportation of the recombinant protein in baculovirus-infected Spodoptera frugiperda (Sf9) cells and its immunogenic properties in mice were compared to those of a previously characterized recombinant DEN-2 E-protein with a 71aa C-terminal truncation (D2E delta 71). Both proteins were transported through the Golgi complex and their N-oligosaccharides of the high mannose type were processed to the complex mannose type. D2E delta 102 transited to the plasma membrane and was secreted whereas D2E delta 71 presumably remained associated with the plasma membrane. The reactivities of the recombinant proteins with neutralizing monoclonal antibodies were similar. Both intracellular and extracellular D2E delta 102 induced neutralizing antibodies in mice and were thus immunogenic. The level of protective immunity to DEN-2 virus encephalitis challenge in mice vaccinated with intracellular D2E delta 102 (80%, p < 0.01) was lower than that induced with D2E delta 71 (90%, P < 0.001). Sixty-eight percent (P < 0.001) of mice vaccinated with 5 micrograms of extracellular D2E delta 102 protein were protected against lethal challenge.


Subject(s)
Dengue Virus/immunology , Dengue/immunology , Vaccines, Synthetic/immunology , Viral Envelope Proteins/immunology , Animals , Antibodies, Monoclonal , Baculoviridae , Base Sequence , Cell Line , Cell Membrane/metabolism , DNA Primers , Dengue/prevention & control , Glycosylation , Golgi Apparatus/metabolism , Mice , Molecular Sequence Data , Oligosaccharides/biosynthesis , Oligosaccharides/metabolism , Polymerase Chain Reaction , Protein Processing, Post-Translational , Recombinant Proteins/biosynthesis , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification , Spodoptera , Vaccines, Synthetic/biosynthesis , Vaccines, Synthetic/isolation & purification , Viral Envelope Proteins/biosynthesis , Viral Envelope Proteins/isolation & purification
9.
Virology ; 196(1): 209-19, 1993 Sep.
Article in English | MEDLINE | ID: mdl-8356794

ABSTRACT

The genetic diversity of dengue (DEN) virus was explored using two South American DEN-1 virus strains isolated from viremic human sera. DEN-1 virus strains BR/90 and FGA/89 were selected on the basis of their membrane fusion properties in mosquito cell cultures. Infection of mosquito cell lines with BR/90 virus strain induced a cytopathic effect characterized by syncytium formation whereas no cytopathic changes were observed with FGA/89. Cell-to-cell fusion experiments indicated that the fusogenic activity of FGA/89 required a lower pH than BR/90. Immunoreactivity analysis of the DEN-1 envelope (E) protein with monoclonal antibodies revealed a minor difference between the antigenic structures of FGA/89 and BR/90 virions. FGA/89 was less neurovirulent than BR/90 for newborn mouse. To determine the genetic origin of these modifications, the amino acid sequences of the structural proteins from these virus strains were compared. One amino acid difference was found within the carboxy-terminal domain of protein C. Five amino acid substitutions were found in the E proteins at positions 96, 180, 297, 379, and 473. Changes at positions 96, 297, and 379 map within two overlapping antigenic domains of protein E. These limited amino acid differences in the E protein could affect the biological properties and the antigenicity of the DEN virion.


Subject(s)
Dengue Virus/pathogenicity , Dengue Virus/ultrastructure , Membrane Fusion , Adult , Aedes , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Antibodies, Viral/immunology , Base Sequence , Biomarkers , Cell Fusion , Cell Line , DNA, Viral , Dengue/microbiology , Dengue Virus/genetics , Dengue Virus/immunology , Female , Genetic Variation , Hemagglutination, Viral , Humans , Hydrogen-Ion Concentration , Male , Mice , Molecular Sequence Data , Viral Envelope Proteins/genetics
10.
Vaccine ; 10(5): 319-21, 1992.
Article in English | MEDLINE | ID: mdl-1533479

ABSTRACT

In most developing countries, hepatitis B virus is endemic and prevention has to be carried out early in life and on a mass scale. In these regions, simultaneous administration of multiple antigens is normal practice. We have therefore investigated the interaction of hepatitis B vaccine with BCG and inactivated polio vaccine. The serological antibody response to poliovirus and HBsAg as well as the cellular immune response to tuberculin post BCG immunization were assessed. The immune responses to HBsAg, BCG and polio vaccines injected simultaneously were comparable to those observed after separate administration of each vaccine. Moreover, no increase of adverse reactions was noted. Results confirmed that HB vaccine could be introduced into the WHO expanded programmes on immunization without impairing the expected protective efficacy against the targeted vaccine-preventable diseases.


Subject(s)
BCG Vaccine/administration & dosage , Poliovirus Vaccine, Inactivated/administration & dosage , Viral Hepatitis Vaccines/administration & dosage , BCG Vaccine/immunology , Hepatitis B Vaccines , Humans , Immunization , Infant, Newborn , Poliovirus Vaccine, Inactivated/immunology , Vaccines, Inactivated/administration & dosage , Vaccines, Inactivated/immunology , Viral Hepatitis Vaccines/immunology
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