HCV variants

A method for identifying an adaptive Hepatitis C Virus (HCV) variant polynucleotide with increased transfection comprising the steps of:

• (A) providing a first HCV polynucleotide that comprises a non-naturally occurring HCV sequence capable of replication, said first HCV polynucleotide comprising an HCV 5′ non-translated region (5′ NTR), an HCV polyprotein coding region, and a 3′ non-translated region (3′ NTR), wherein a selection marker gene is operably linked to said first HCV polynucleotide;
• (B) transfecting an Huh7 cell line permissive for HCV RNA replication with the HCV polynucleotide of (A);
• (C) selecting a transfected cell line from (B) containing an HCV polynucleotide based on the presence or absence of said selection marker gene function;
• (D) subpassaging said selected cell line of (C) on fresh media that provides for the identification of cell lines containing an HCV polynucleotide comprising an HCV 5′ NTR, an HCV polyprotein coding region, an HCV 3′ non-translated region (3′ NTR), and said selection marker gene of (A);
• (E) identifying a subpassaged cell line from (D) that contains said HCV polynucleotide of (D);
• (F) isolating said subpassaged cell line of (E);
• (G) isolating an HCV cDNA by reverse transcription of RNA from said isolated cell line of (F);
• (H) (i) transfecting an Huh7 cell line permissive for HCV RNA replication with said first HCV polynucleotide of (A) and measuring the transfection efficiency thereof and
• (ii) transfecting an Huh7 cell line permissive for HCV RNA replication with a second HCV polynucleotide obtained from said HCV cDNA of (G) and measuring the transfection efficiency thereof;
• and
• (I) comparing the transfection efficiencies from (H)(i) and (H)(ii) to identify an adaptive HCV variant polynucleotide that transfects sensitive cell lines with increased efficiency.