scholarly journals Chemical Mutagenesis of Dengue Virus Type 4 Yields Mutant Viruses Which Are Temperature Sensitive in Vero Cells or Human Liver Cells and Attenuated in Mice

2001 ◽  
Vol 75 (20) ◽  
pp. 9731-9740 ◽  
Author(s):  
Joseph E. Blaney ◽  
Daniel H. Johnson ◽  
Cai-Yen Firestone ◽  
Christopher T. Hanson ◽  
Brian R. Murphy ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Virus Type ◽  
Human Liver ◽  
Vero Cells ◽  
Chemical Mutagenesis ◽  
Temperature Sensitive ◽  
Nucleotide Position ◽  
Nucleotide Substitutions ◽  
Ns3 Protein ◽  
Old Mice

ABSTRACT A recombinant live attenuated dengue virus type 4 (DEN4) vaccine candidate, 2AΔ30, was found previously to be generally well tolerated in humans, but a rash and an elevation of liver enzymes in the serum occurred in some vaccinees. 2AΔ30, a non-temperature-sensitive (non-ts) virus, contains a 30-nucleotide deletion (Δ30) in the 3′ untranslated region (UTR) of the viral genome. In the present study, chemical mutagenesis of DEN4 was utilized to generate attenuating mutations which may be useful in further attenuation of the 2AΔ30 candidate vaccine. Wild-type DEN4 2A virus was grown in Vero cells in the presence of 5-fluorouracil, and a panel of 1,248 clones were isolated. Twenty ts mutant viruses were identified that were ts in both simian Vero and human liver HuH-7 cells (n = 13) or only in HuH-7 cells (n = 7). Each of the 20 ts mutant viruses possessed an attenuation phenotype, as indicated by restricted replication in the brains of 7-day-old mice. The complete nucleotide sequence of the 20 ts mutant viruses identified nucleotide substitutions in structural and nonstructural genes as well as in the 5′ and 3′ UTRs, with more than one change occurring, in general, per mutant virus. A ts mutation in the NS3 protein (nucleotide position 4995) was introduced into a recombinant DEN4 virus possessing the Δ30 deletion, thereby creating rDEN4Δ30-4995, a recombinant virus which is ts and more attenuated than rDEN4Δ30 virus in the brains of mice. We are assembling a menu of attenuating mutations that should be useful in generating satisfactorily attenuated recombinant dengue vaccine viruses and in increasing our understanding of the pathogenesis of dengue virus.

scholarly journals Paired Charge-to-Alanine Mutagenesis of Dengue Virus Type 4 NS5 Generates Mutants with Temperature-Sensitive, Host Range, and Mouse Attenuation Phenotypes

2002 ◽  
Vol 76 (2) ◽  
pp. 525-531 ◽  
Author(s):  
Kathryn A. Hanley ◽  
Jay J. Lee ◽  
Joseph E. Blaney ◽  
Brian R. Murphy ◽  
Stephen S. Whitehead
Keyword(s):  
Dengue Virus ◽  
Host Range ◽  
Virus Type ◽  
Temperature Sensitivity ◽  
Vero Cells ◽  
Fine Tuning ◽  
Temperature Sensitive ◽  
Large Set ◽  
Human Hepatoma Cells ◽  
Wide Range

ABSTRACT Charge-to-alanine mutagenesis of dengue virus type 4 (DEN4) NS5 gene generated a collection of attenuating mutations for potential use in a recombinant live attenuated DEN vaccine. Codons for 80 contiguous pairs of charged amino acids in NS5 were individually mutagenized to create uncharged pairs of alanine residues, and 32 recombinant mutant viruses were recovered from the 80 full-length mutant DEN4 cDNA constructs. These mutant viruses were tested for temperature-sensitive (ts) replication in both Vero cells and HuH-7 human hepatoma cells. Of the 32 mutants, 13 were temperature sensitive (ts) in both cell lines, 11 were not ts in either cell line, and 8 exhibited a host range (tshr) phenotype. One tshr mutant was ts only in Vero cells, and seven were ts only in HuH-7 cells. Nineteen of the 32 mutants were 10-fold or more restricted in replication in the brains of suckling mice compared to that of wild-type DEN4, and three mutants were approximately 10,000-fold restricted in replication. The level of temperature sensitivity of replication in vitro did not correlate with attenuation in vivo. A virus bearing two pairs of charge-to-alanine mutations was constructed and demonstrated increased temperature sensitivity and attenuation relative to either parent virus. This large set of charge-to-alanine mutations specifying a wide range of attenuation for mouse brain should prove useful in fine-tuning recombinant live attenuated DEN vaccines.

scholarly journals Dengue Virus Type 1 Nonstructural Glycoprotein NS1 Is Secreted from Mammalian Cells as a Soluble Hexamer in a Glycosylation-Dependent Fashion

1999 ◽  
Vol 73 (7) ◽  
pp. 6104-6110 ◽  
Author(s):  
Marie Flamand ◽  
Françoise Megret ◽  
Magali Mathieu ◽  
Jean Lepault ◽  
Félix A. Rey ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Virus Type ◽  
Mammalian Cells ◽  
Culture Fluid ◽  
Vero Cells ◽  
Soluble Form ◽  
Complex Type ◽  
Infected Insect ◽  
Infected Cells

ABSTRACT Nonstructural glycoprotein NS1, specified by dengue virus type 1 (Den-1), is secreted from infected green monkey kidney (Vero) cells in a major soluble form characterized by biochemical and biophysical means as a unique hexameric species. This noncovalently bound oligomer is formed by three dimeric subunits and has a molecular mass of 310 kDa and a Stokes radius of 64.4 Å. During protein export, one of the two oligosaccharides of NS1 is processed into an endo-β-N-acetylglucosaminidase F-resistant complex-type sugar while the other remains of the polymannose type, protected in the dimeric subunit from the action of maturation enzymes. Complete processing of the complex-type sugar appears to be required for efficient release of soluble NS1 into the culture fluid of infected cells, as suggested by the repressive effects of the N-glycan processing inhibitors swainsonine and deoxymannojyrimicin. These results, together with observations related to the absence of secretion of NS1 from Den-infected insect cells, suggest that maturation and secretion of hexameric NS1 depend on the glycosylation status of the host cell.

scholarly journals Generation and Molecular Characterization of New Temperature-Sensitive Plasmids Intended for Genetic Engineering of Pasteurellaceae

2005 ◽  
Vol 71 (11) ◽  
pp. 7187-7195 ◽  
Author(s):  
Robert E. Briggs ◽  
Fred M. Tatum
Keyword(s):  
Base Pair ◽  
Pasteurella Multocida ◽  
Chemical Mutagenesis ◽  
Temperature Sensitive ◽  
Origin Of Replication ◽  
Wild Type ◽  
Recognition Sequence ◽  
Nucleotide Substitutions ◽  
Serotype 1

ABSTRACT Temperature-sensitive (TS) plasmids were generated through chemical mutagenesis of a derivative of the streptomycin resistance parent plasmid pD70, isolated from Mannheimia hemolytica serotype 1. Three TS plasmids which failed to replicate at or above 42°C in M. hemolytica but which were fully functional below 31°C were selected for further analysis. Two of the TS plasmids were shown by sequencing to possess unique single-base-pair mutations. The third TS plasmid contained a unique base pair substitution and a second mutation that had been previously identified. These mutations were clustered within a 200-bp region of the presumed plasmid origin of replication. Site-directed single-nucleotide substitutions were introduced into the wild-type pD70 origin of replication to confirm that mutations identified by sequencing had conferred thermoregulated replication. Deletion analysis on the wild-type pD70 plasmid replicon revealed that approximately 720 bp are necessary for plasmid maintenance. Replication of the TS plasmids was thermoregulated in Pasteurella multocida and Haemophilus somnus as well. To consistently transform H. somnus with TS plasmid, in vitro DNA methylation with commercially available HhaI methyltransferase was necessary to protect against the organism's restriction enzyme HsoI (recognition sequence 5′-GCGC-3′) characterized herein.

scholarly journals Mutagenesis of the Dengue Virus Type 2 NS3 Protein within and outside Helicase Motifs: Effects on Enzyme Activity and Virus Replication

2001 ◽  
Vol 75 (20) ◽  
pp. 9633-9643 ◽  
Author(s):  
Anita E. Matusan ◽  
Melinda J. Pryor ◽  
Andrew D. Davidson ◽  
Peter J. Wright
Keyword(s):  
Dengue Virus ◽  
Virus Replication ◽  
Virus Type ◽  
Nonstructural Protein ◽  
Serine Proteinase ◽  
Temperature Shift ◽  
Temperature Sensitive ◽  
Helicase Activity ◽  
Dengue Virus Type 2

ABSTRACT The protein NS3 of Dengue virus type 2 (DEN-2) is the second largest nonstructural protein specified by the virus and is known to possess multiple enzymatic activities, including a serine proteinase located in the N-terminal region and an NTPase-helicase in the remaining 70% of the protein. The latter region has seven conserved helicase motifs found in all members of the family Flaviviridae. DEN-2 NS3 lacking the proteinase region was synthesized as a fusion protein with glutathione S-transferase in Escherichia coli. The effects of 10 mutations on ATPase and RNA helicase activity were examined. Residues at four sites within enzyme motifs I, II, and VI were substituted, and six sites outside motifs were altered by clustered charged-to-alanine mutagenesis. The mutations were also tested for their effects on virus replication by incorporation into genomic-length cDNA. Two mutations, both in motif I (G198A and K199A) abolished both ATPase and helicase activity. Two further mutations, one in motif VI (R457A,R458A) and the other a clustered charged-to-alanine substitution at R376KNGK380, abolished helicase activity only. No virus was detected for any mutation which prevented helicase activity, demonstrating the requirement of this enzyme for virus replication. The remaining six mutations resulted in various levels of enzyme activities, and four permitted virus replication. For the two nonreplicating viruses encoding clustered changes at R184KR186 and D436GEE439, we propose that the substituted residues are surface located and that the viruses are defective through altered interaction of NS3 with other components of the viral replication complex. Two of the replicating viruses displayed a temperature-sensitive phenotype. One contained a clustered mutation at D334EE336 and grew too poorly for further characterization. However, virus with an M283F substitution in motif II was examined in a temperature shift experiment (33 to 37°C) and showed reduced RNA synthesis at the higher temperature.

The study on the immune response induced by expressing recombinant plasmid of dengue virus type 2 NS3 protein

2000 ◽  
Vol 5 (2) ◽  
pp. 245-248
Author(s):  
Li Xiang-qun ◽  
Mao Lin ◽  
Yan Zhan-qiu ◽  
Jiang Li-feng ◽  
Yan Hui-jun ◽  
...  
Keyword(s):  
Immune Response ◽  
Dengue Virus ◽  
Virus Type ◽  
Recombinant Plasmid ◽  
Ns3 Protein ◽  
Dengue Virus Type 2

scholarly journals Zinc accelerates dengue virus type 2-induced apoptosis in Vero cells

FEBS Letters ◽  
2002 ◽  
Vol 524 (1-3) ◽  
pp. 20-24 ◽  
Author(s):  
Norazizah Shafee ◽  
Sazaly AbuBakar
Keyword(s):  
Dengue Virus ◽  
Virus Type ◽  
Vero Cells ◽  
Induced Apoptosis ◽  
Dengue Virus Type 2

scholarly journals Genetic Basis of Attenuation of Dengue Virus Type 4 Small Plaque Mutants with Restricted Replication in Suckling Mice and in SCID Mice Transplanted with Human Liver Cells

Virology ◽  
2002 ◽  
Vol 300 (1) ◽  
pp. 125-139 ◽  
Author(s):  
Joseph E. Blaney ◽  
Daniel H. Johnson ◽  
Gracielle G. Manipon ◽  
Cai-Yen Firestone ◽  
Christopher T. Hanson ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Virus Type ◽  
Human Liver ◽  
Genetic Basis ◽  
Liver Cells ◽  
Scid Mice ◽  
Small Plaque ◽  
Suckling Mice ◽  
Human Liver Cells

scholarly journals Chloroquine Inhibits Dengue Virus Type 2 Replication in Vero Cells but Not in C6/36 Cells

2013 ◽  
Vol 2013 ◽  
pp. 1-5 ◽  
Author(s):  
Kleber Juvenal Silva Farias ◽  
Paula Renata Lima Machado ◽  
Benedito Antônio Lopes da Fonseca
Keyword(s):  
Dengue Virus ◽  
Virus Type ◽  
Mammalian Cells ◽  
Plaque Assay ◽  
Antiviral Drug ◽  
Vero Cells ◽  
Significant Difference ◽  
Dengue Virus Type 2

Dengue viruses are the most important arthropod-borne viruses in terms of morbidity and mortality in the world. Since there is no dengue vaccine available for human use, we have set out to investigate the use of chloroquine as an antiviral drug against dengue. Chloroquine, an amine acidotropic drug known to affect intracellular exocytic pathways by increasing endosomal pH, was used in the in vitro treatment of Vero and C6/36 cells infected with dengue virus type 2 (DENV-2). Real-time RT-PCR and plaque assays were used to quantify the DENV-2 load in infected Vero and C6/36 cells after chloroquine treatment. Our results showed that a dose of 50 μg/ml of chloroquine was not toxic to the cells and induced a statistically significant inhibition of virus production in infected Vero cells when compared to untreated cells. In C6/36 cells, chloroquine does not induce a statistically significant difference in viral replication when compared to untreated cells, showing that this virus uses an unlikely pathway of penetration in these cells, and results were also confirmed by the plaque assay (PFU). These data suggest that the inhibition of virus infection induced by chloroquine is due to interference with acidic vesicles in mammalian cells.

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