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1.
J Virol Methods ; 308: 114577, 2022 10.
Article in English | MEDLINE | ID: mdl-35843366

ABSTRACT

Dengue virus (DENV) specific neutralizing and enhancing antibodies play crucial roles in dengue disease prevention and pathogenesis. DENV reporters are gaining popularity in the evaluation of these antibodies; their accessibility and acceptance may improve with more efficient production systems and indications of their antigenic equivalence to the wild-type virus. This study aimed to generate a replication competent luciferase-secreting DENV reporter (LucDENV2) and evaluate its feasibility in neutralizing and infection-enhancing antibody assays in comparison with wild-type DENV2, strain 16681, and a luciferase-secreting, single-round infectious DENV2 reporter (LucSIP). LucDENV2 replicated to similarly high levels as that of the parent 16681 virus in a commonly used mosquito cell line. LucDENV2 was neutralized in an antibody concentration-dependent manner by a monoclonal antibody specific to the flavivirus fusion loop and two antibodies specific to the E domain III, which closely resembled the neutralization patterns employing the LucSIP and wild-type DENV2. Parallel analysis of LucDENV2 and wild-type DENV2 revealed good agreement between the luciferase-based and focus-based neutralization and enhancement assays in a 96-well microplate format when employed against a set of clinical sera, suggesting comparable antigenic properties of LucDENV2 with those of the parent virus. The high-titer, replication competent, luciferase-secreting DENV reporter presented here should be a useful tool for fast and reliable quantitation of neutralizing and infection-enhancing antibodies in populations living in DENV-endemic areas.


Subject(s)
Dengue Virus , Dengue , Animals , Antibodies, Blocking , Antibodies, Neutralizing , Antibodies, Viral , Dengue Virus/genetics , Luciferases/genetics , Viral Envelope Proteins
2.
J Gen Virol ; 102(8)2021 08.
Article in English | MEDLINE | ID: mdl-34410905

ABSTRACT

The capsid protein (C) of dengue virus is required for viral infectivity as it packages viral RNA genome into infectious particles. C exists as a homodimer that forms via hydrophobic interactions between the α2 and α4 helices of monomers. To identify C region(s) important for virus particle production, a complementation system was employed in which single-round infectious particles are generated by trans-encapsidation of a viral C-deleted genome by recombinant C expressed in mosquito cells. Mutants harbouring a complete α3 deletion, or a dual Ile65-/Trp69-to-Ala substitution in the α3 helix, exhibited reduced production of infectious virus. Unexpectedly, higher proportions of oligomeric C were detected in cells expressing both mutated forms as compared with the wild-type counterpart, indicating that the α3 helix, through its internal hydrophobic residues, may down-modulate oligomerization of C during particle formation. Compared with wild-type C, the double Ile65-/Trp69 to Ala mutations appeared to hamper viral infectivity but not C and genomic RNA incorporation into the pseudo-infectious virus particles, suggesting that increased C oligomerization may impair DENV replication at the cell entry step.


Subject(s)
Capsid Proteins , Capsid/metabolism , Dengue Virus/metabolism , Dengue/virology , Aedes , Amino Acid Sequence , Animals , Capsid Proteins/chemistry , Capsid Proteins/metabolism , Chlorocebus aethiops , Humans , Vero Cells , Virus Assembly , Virus Replication
3.
J Virol Methods ; 291: 114119, 2021 05.
Article in English | MEDLINE | ID: mdl-33662412

ABSTRACT

Flavivirus reporters provide a robust tool for viral pathogenesis studies, anti-viral drug screening, disease diagnosis and functional antibody assays. In this study, we generated a luciferase-secreting, single-round reporter virus by replacing the capsid coding region in a DENV-2 genome with the secretory form of Lucia luciferase gene to produce infectious viral particles in a stable capsid-expressing mosquito cell line. Replication of the reporter virus in trans-complementing mosquito cells was sustained for up to two weeks. There were strong correlations between the extracellular luciferase activity and infectious reporter virus inocula upon infection of mosquito and mammalian cell lines with graded quantities of the reporter virus. A set of anti-E and anti-prM monoclonal antibodies affected the infectivity of reporter virus with similar dose-effect relationships as the parent virus. This simplified version of DENV-2 reporter provides a rapid and reliable method for the detection of neutralizing and infection-enhancing antibodies against dengue virus.


Subject(s)
Dengue Virus , Dengue , Flavivirus , Animals , Antibodies, Blocking , Antibodies, Viral , Dengue/diagnosis , Dengue Virus/genetics , Luciferases/genetics
4.
J Gen Virol ; 101(1): 59-72, 2020 01.
Article in English | MEDLINE | ID: mdl-31682220

ABSTRACT

Dengue virus assembly involves the encapsidation of genomic RNA by the capsid protein (C) and the acquisition of an envelope comprising the premembrane (prM) and envelope (E) glycoproteins. This rapid process, lacking in detectable nucleocapsid intermediates, may impose authentic C-prM-E arrangement as a prerequisite for efficient particle assembly. A mosquito cell-based complementation system was employed in this study to investigate the possibility that expression of the three structural proteins in trans allows the efficient production of a partially C-deleted dengue virus as compared to the presence of C alone. Following the transfection of ΔC56-capped RNA transcripts into C6/36 cells transiently expressing C or CprME, the production of the single-cycle virus was comparable. Subsequent propagation in the stable CprME-expressing clone, however, supported virus adaptation leading to acquisition of the L29P and S101F (PF) dual mutations in the C protein. The triple mutant, ΔC56(PF), exhibited enhanced levels of virus replication, specific infectivity and frequent increases of intracellular C dimer, as compared with ΔC56 in the CprME-clone. The PF mutations were associated with the accumulation of truncated CprM in ΔC56(PF)-infected cells, and uncleaved CprM as well as reduced intracellular C-dimer when the dual mutations were introduced into the wild-type dengue virus genetic background. These results indicate that the PF mutations may exert a replication-enhancing effect for the triple mutant virus by relieving the interference of trans-complementing structural proteins during viral assembly and suggest that the C-prM-E arrangement may be advantageous for pseudoinfectious virus production.


Subject(s)
Dengue Virus/genetics , Nucleocapsid/genetics , Viral Envelope Proteins/genetics , Viral Nonstructural Proteins/genetics , Virus Assembly/genetics , Amino Acid Sequence , Animals , Capsid Proteins/genetics , Cell Line , Chlorocebus aethiops , Culicidae/virology , Dengue/virology , RNA, Viral/genetics , Vero Cells , Virus Replication/genetics
5.
J Virol ; 87(23): 12667-74, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24049164

ABSTRACT

Here we present an approach that advances the throughput of a genetic analysis of a positive-sense RNA virus by simplifying virus construction. It enabled comprehensive dissection of a complex, multigene phenotype through rapid derivation of a large number of chimeric viruses and construction of a mutant library directly from a virus pool. The versatility of the approach described here expands the applicability of diverse genetic approaches to study these viruses.


Subject(s)
Genetic Engineering/methods , RNA Viruses/genetics , RNA, Viral/genetics , Gene Library
6.
Virus Res ; 174(1-2): 37-46, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23466247

ABSTRACT

A simple system for the generation of pseudoinfectious particles of dengue virus was developed to facilitate studies of virus replication and vaccine development. Selected clones of the C6/36 mosquito cell line expressing an anchored form of the dengue virus capsid protein served as host cells for the trans-complementation of partially capsid-deleted viral RNA generated in vitro. Transfection of the partially capsid-deleted viral RNA into the anchored capsid-expressing C6/36 cells resulted in moderate titers of infectious virus. Progeny viruses multiplied in the capsid trans-complementing C6/36 cells for up to three weeks, but only initiated single rounds of replication in Vero cells lacking the capsid protein. Employing this trans-complementation system, it was found that nearly all of the capsid-coding sequence in the viral RNA was dispensable for the generation of pseudoinfectious dengue virus particles in mosquito cells.


Subject(s)
Capsid Proteins/metabolism , Dengue Virus/physiology , Virus Replication , Animals , Capsid Proteins/genetics , Cell Line , Culicidae , Dengue Virus/genetics , Gene Deletion , Genetic Complementation Test , Transfection , Viral Load , Virus Cultivation
7.
Arch Virol ; 154(8): 1211-21, 2009.
Article in English | MEDLINE | ID: mdl-19565324

ABSTRACT

We produced monoclonal and polyclonal antibodies to the capsid (C) protein of dengue serotype 2 virus (DV2 C). First, a maltose-binding protein fused to DV2 C protein (MBP-C) was overproduced in E. coli. The affinity-purified MBP-C protein was cleaved by factor Xa protease to obtain a recombinant DV2 C protein, which was then used for mouse immunizations. Two hybridoma cell lines producing anti-C Mabs as well as anti-C polyclonal antibody were successfully generated and characterized. Interestingly, all of the generated antibodies specifically recognized the first 20 amino acids of the DV2 C protein, as determined by peptide epitope mapping and via a recombinant DV2 C protein in which this region was deleted. The results suggested that this region is predominantly immunogenic in mice.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Viral/immunology , Capsid Proteins/immunology , Dengue Virus/immunology , Dengue/immunology , Immunodominant Epitopes/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/biosynthesis , Antibodies, Viral/biosynthesis , Capsid Proteins/genetics , Capsid Proteins/metabolism , Carrier Proteins/metabolism , Epitope Mapping , Factor Xa/metabolism , Immunization , Immunodominant Epitopes/genetics , Maltose-Binding Proteins , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Peptide Hydrolases/metabolism , Recombinant Proteins/immunology , Recombinant Proteins/metabolism
8.
J Gen Virol ; 89(Pt 5): 1254-1264, 2008 May.
Article in English | MEDLINE | ID: mdl-18420804

ABSTRACT

During infection, the capsid (C) protein of many flaviviruses localizes to the nuclei and nucleoli of several infected cell lines; the underlying basis and significance of C protein nuclear localization remain poorly understood. In this study, double alanine-substitution mutations were introduced into three previously proposed nuclear-localization signals (at positions 6-9, 73-76 and 85-100) of dengue virus C protein, and four viable mutants, c(K6A,K7A), c(K73A,K74A), c(R85A,K86A) and c(R97A,R98A), were generated in a mosquito cell line in which C protein nuclear localization was rarely observed. Indirect immunofluorescence analysis revealed that, whilst C protein was present in the nuclei of PS and Vero cells throughout infection with a dengue serotype 2 parent virus, the substitution mutations in c(K73A,K74A) and c(R85A,K86A) resulted in an elimination of nuclear localization in PS cells and marked reduction in Vero cells. Mutants c(K6A,K7A) and c(R97A,R98A) exhibited reduced nuclear localization at the late period of infection in PS cells only. All four mutants displayed reduced replication in PS, Vero and C6/36 cells, but there was a lack of correlation between nuclear localization and viral growth properties. Distinct dibasic residues within dengue virus C protein, many of which were located on the solvent-exposed side of the C protein homodimer, contribute to its ability to localize to nuclei during virus infection.


Subject(s)
Capsid Proteins/genetics , Capsid Proteins/metabolism , Cell Nucleus/chemistry , Dengue Virus/physiology , Amino Acid Substitution/genetics , Animals , Cell Line , Chlorocebus aethiops , Culicidae , Microscopy, Fluorescence , Models, Molecular , Mutagenesis, Site-Directed , Swine , Virus Replication/genetics
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