Growth of wild-type hepatitis a virus in cell culture

A recombinant Hepatitis A Virus nucleic acid comprising:

• i) a nucleotide sequence selected from the group consisting of
• a) SEQ ID NO:
• 1,
• b) the nucleotide sequence of SEQ ID NO:
• 1 at nucleotide 3889 in which a codon encodes valine at amino acid 216 of the 2B protein and
• c) SEQ ID NO:
• 1 having a mutation at nucleotide 4087 in which a codon encodes methionine and a mutation at nucleotide 4222 in which a codon encodes serine,
• d) SEQ ID NO:
• 1 having a mutation at nucleotide 3889 in which a codon encodes valine at amino acid 216 of the 2B protein and a mutation at nucleotide 4087 in which a codon encodes methionine, and
• e) SEQ ID NO:
• 1 having a mutation at nucleotide 3889 in which a codon encodes valine at amino acid 216 of the 2B protein and a mutation at nucleotide 4222 in which a codon encodes serine;
• and
• ii) a nucleic acid encoding a protein conferring a selectable or screenable phenotype upon a cell that expresses said protein.

A recombinant Hepatitis A Virus nucleic acid comprising

• i) a nucleotide sequence representing at least one unique restriction enzyme site located between nucleotides encoding protease 3Cpro cleavage sites;
• and
• ii) a nucleotide sequence located between nucleotides encoding protease 3Cpro cleavage sites providing resistance to an antibiotic that is effective against cultured mammalian cells and inhibits protein translation or promotes apoptosis in the cultured mammalian cells;
• wherein cells that replicate the recombinant Hepatitis A Virus nucleic acid can be selected by the antibiotic resistance or apoptotic phenotype.

A recombinant Hepatitis A Virus nucleic acid comprising:

• i) a nucleotide sequence selected from the group consisting of
• a) SEQ ID NO:
• 1,
• b) the nucleotide sequence of SEQ ID NO:
• 1 at nucleotide 3889 in which a codon encodes valine at amino acid 216 of the 2B protein and
• c) SEQ ID NO:
• 1 having a mutation at nucleotide 4087 in which a codon encodes methionine and a mutation at nucleotide 4222 in which a codon encodes serine,
• d) SEQ ID NO:
• 1 having a mutation at nucleotide 3889 in which a codon encodes valine at amino acid 216 of the 2B protein and a mutation at nucleotide 4087 in which a codon encodes methionine, and
• e) SEQ ID NO:
• 1 having a mutation at nucleotide 3889 in which a codon encodes valine at amino acid 216 of the 2B protein and a mutation at nucleotide 4222 in which a codon encodes serine;
• and
• ii) a nucleotide sequence representing at least one heterologous nucleotide sequence located between nucleotides encoding protease 3Cpro cleavage sites in the Hepatitis A Virus genome.

A method for using a recombinant Hepatitis A Virus comprising a selectable marker gene to identify cellular factors that allow growth of wild-type Hepatitis A Virus in cells containing such cellular factors, comprising:

• I) selecting from a collection of cells that are non-permissive for replication and growth of Hepatitis A Virus, said collection of cells having been transformed with a library of nucleic acids made from a hepatoma cell line in an expression vector, one or more cells that express a selectable or screenable phenotype, said selectable or screenable phenotype being conferred by infection with a virus comprising a nucleic acid, or being conferred by transfection with a nucleic acid, said nucleic acid comprising;
• i) a nucleotide sequence of SEQ ID NO:
• 1, and
• ii) a nucleic acid encoding a protein conferring a selectable or screenable phenotype upon a cell that expresses said protein that is located between nucleotides encoding 3Cpro cleavage sites in the genome of the wild type Hepatitis A Virus;
• and
• II) determining the nucleotide sequence of the nucleic acid present in the expression vector of the selected cell(s).