scholarly journals Utilization of Chimeras between Human (HM-175) and Simian (AGM-27) Strains of Hepatitis A Virus To Study the Molecular Basis of Virulence

1998 ◽  
Vol 72 (9) ◽  
pp. 7467-7475 ◽  
Author(s):  
Gopa Raychaudhuri ◽  
Sugantha Govindarajan ◽  
Max Shapiro ◽  
Robert H. Purcell ◽  
and Suzanne U. Emerson
Keyword(s):  
Cell Culture ◽  
Hepatitis A ◽  
Cultured Cells ◽  
Hepatitis A Virus ◽  
Kinetic Studies ◽  
Simian Virus ◽  
Saguinus Mystax ◽  
Amino Terminal ◽  
A Cell ◽  
Carboxy Terminal

ABSTRACT Chimeras between human (HM-175) and simian (AGM-27) strains of hepatitis A virus (HAV) were constructed to evaluate the effect of the 2C gene of AGM-27 on HAV replication in cell culture and virulence in tamarins (Saguinus mystax) and chimpanzees (Pan troglodytes). Kinetic studies and radioimmunofocus assays demonstrated that replacement of the 2C gene of HAV/7, a cell culture-adapted strain of HM-175, with that of AGM-27 drastically reduced the ability of the virus to replicate in cultured cells. Intragenic chimeras containing AGM-27 sequences in either the 5′ or 3′ half of the 2C gene replicated in cell culture at an intermediate level. Whereas HAV/7 is attenuated for tamarins, a chimera containing the simian virus 2C gene in the HAV/7 background was virulent in tamarins, demonstrating that the simian virus 2C gene alone can confer the phenotype of virulence to an otherwise attenuated virus. Clusters of AGM-27-specific residues near both ends of the 2C protein were required for virulence since a chimera containing AGM-27 sequences in the carboxy-terminal half of 2C was partially attenuated for tamarins while one containing AGM-27 sequences only in the amino-terminal half of 2C was even more attenuated. Chimeras containing either the entire or only the 3′ half of the simian virus 2C gene in the HAV/7 background were attenuated for chimpanzees.

scholarly journals Replication of Subgenomic Hepatitis A Virus RNAs Expressing Firefly Luciferase Is Enhanced by Mutations Associated with Adaptation of Virus to Growth in Cultured Cells

2002 ◽  
Vol 76 (3) ◽  
pp. 1171-1180 ◽  
Author(s):  
MinKyung Yi ◽  
Stanley M. Lemon
Keyword(s):  
Cell Culture ◽  
Hepatitis A ◽  
Cultured Cells ◽  
Hepatitis A Virus ◽  
Internal Ribosomal Entry Site ◽  
Firefly Luciferase ◽  
Virus Genome ◽  
Wild Type Virus ◽  
Reporter System ◽  
Entry Site

ABSTRACT Replication of hepatitis A virus (HAV) in cultured cells is inefficient and difficult to study due to its protracted and generally noncytopathic cycle. To gain a better understanding of the mechanisms involved, we constructed a subgenomic HAV replicon by replacing most of the P1 capsid-coding sequence from an infectious cDNA copy of the cell culture-adapted HM175/18f virus genome with sequence encoding firefly luciferase. Replication of this RNA in transfected Huh-7 cells (derived from a human hepatocellular carcinoma) led to increased expression of luciferase relative to that in cells transfected with similar RNA transcripts containing a lethal premature termination mutation in 3Dpol (RNA polymerase). However, replication could not be confirmed in either FrhK4 cells or BSC-1 cells, cells that are typically used for propagation of HAV. Replication was substantially slower than that observed with replicons derived from other picornaviruses, as the basal luciferase activity produced by translation of input RNA did not begin to increase until 24 to 48 h after transfection. Replication of the RNA was reversibly inhibited by guanidine. The inclusion of VP4 sequence downstream of the viral internal ribosomal entry site had no effect on the basal level of luciferase or subsequent increases in luciferase related to its amplification. Thus, in this system this sequence does not contribute to viral translation or replication, as suggested previously. Amplification of the replicon RNA was profoundly enhanced by the inclusion of P2 (but not 5′ noncoding sequence or P3) segment mutations associated with adaptation of wild-type virus to growth in cell culture. These results provide a simple reporter system for monitoring the translation and replication of HAV RNA and show that critical mutations that enhance the growth of virus in cultured cells do so by promoting replication of viral RNA in the absence of encapsidation, packaging, and cellular export of the viral genome.

Attenuation Phenotype of a Cell Culture-Adapted Variant of Hepatitis A Virus (HM175/p16) in Susceptible New World Owl Monkeys

1993 ◽  
Vol 168 (3) ◽  
pp. 592-601 ◽  
Author(s):  
K. L. Taylor ◽  
P. C. Murphy ◽  
L. V. S. Asher ◽  
J. W. LeDuc ◽  
S. M. Lemon
Keyword(s):  
Cell Culture ◽  
Hepatitis A ◽  
New World ◽  
Hepatitis A Virus ◽  
A Cell ◽  
Owl Monkeys

Hepatitis a Virus Concentration from Experimentally Contaminated Distilled, Tap, Waste and Seawater

1989 ◽  
Vol 21 (3) ◽  
pp. 255-258 ◽  
Author(s):  
Evangélos Biziagos ◽  
Jacques Passagot ◽  
Jean-Marc Crance ◽  
Robert Deloince
Keyword(s):  
Cell Culture ◽  
Polyethylene Glycol ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Tap Water ◽  
Virus Concentration ◽  
Beef Extract ◽  
Polyethylene Glycol 6000

The concentration of cell-culture-adapted hepatitis A virus (HAV) from experimentally contaminated distilled, drinking, waste and seawater was performed by using a filter adsorption-elu-tion method in the following conditions: HAV seeded in water was adsorbed at pH 4.0 to two nitrocellulose membranes (1.2 and 0.45 µm porosity for distilled and tap water or 8.0 and 3.0 µm porosity for waste and seawater), then eluted by 3% beef-extract at pH 8.5 and further concentrated by polyethylene glycol 6000 precipitation. Thus, HAV in 5 to 50 liters of seeded waters was concentrated approximately 1,700 to 17,000 fold with greater than 70% recovery of the initial virus added to the samples.

Restricted replication of human hepatitis A virus in cell culture: intracellular biochemical studies.

1981 ◽  
Vol 37 (1) ◽  
pp. 216-225 ◽  
Author(s):  
S A Locarnini ◽  
A G Coulepis ◽  
E G Westaway ◽  
I D Gust
Keyword(s):  
Cell Culture ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Human Hepatitis ◽  
Biochemical Studies

Hepatitis A virus cDNA and its RNA transcripts are infectious in cell culture.

1987 ◽  
Vol 61 (10) ◽  
pp. 3035-3039 ◽  
Author(s):  
J I Cohen ◽  
J R Ticehurst ◽  
S M Feinstone ◽  
B Rosenblum ◽  
R H Purcell
Keyword(s):  
Cell Culture ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Rna Transcripts ◽  
Virus Cdna

A New In Vitro Model for Predicting the Toxicity of Cosmetic Products

1992 ◽  
Vol 20 (1) ◽  
pp. 138-143
Author(s):  
Maria Carrara ◽  
Lorenzo Cima ◽  
Roberto Cerini ◽  
Maurizio Dalle Carbonare
Keyword(s):  
Cell Culture ◽  
Cultured Cells ◽  
Neutral Red ◽  
Total Protein Content ◽  
Cosmetic Products ◽  
Cell Culture Insert ◽  
A Cell ◽  
Vitro Model ◽  
Cosmetic Emulsions

A method has been developed whereby cosmetic products which are not soluble in water or in alcohol can be brought into contact with cell cultures by being placed in a cell culture insert, which is then placed in the cell culture well. Preliminary experiments were carried out with L929 cells, and cytotoxicity was evaluated by measuring neutral red uptake and the total protein content of treated cultured cells. Encouraging results were obtained in comparisons of three cosmetic emulsions and of one emulsion containing a range of concentrations of two preservatives, Kathon CG and Bronopol.

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