Detection and use of antiviral resistance mutations

A method for determining the potential for an HBV variant to exhibit reduced sensitivity to a nucleoside or nucleotide analog said method comprising:

• isolating DNA or corresponding mRNA from said HBV;
• and
• screening for a mutation in the nucleotide sequence encoding an HBV DNA polymerase resulting in an amino acid substitution in the rt of said DNA polymerase selected from the list consisting of:
• (i) a mutation at codon 239, 240, 241, 242 and/or 243;
• (ii) a mutation resulting in an N236I/D/V substitution;
• (iii) a mutation resulting in an S246H/P/Y/S substitution;
• (iv) a mutation resulting in a P170H substitution;
• (v) a mutation resulting in an I253V substitution;
• (vi) co-mutation rtR242K/R and rtW243G/W substitutions;
• (vii) co-mutation rtS256R/S and rtW257G/W substitutions;
• (viii) co-mutations of two or more substitutions selected from the list consisting of rtL42L/V, rtS78T, rtS85T, rtT128N, rtL180M, rtT184G, rtS202I, rtM204V and rtN236I/D/V;
• (vii) co-mutations of two or more substitutions selected from the list consisting of rtF61L, rtS78T, rtV84M/V, rtV142E, rtQ149Q/R, rtM204I/M, rtI233T/I and rtS246H/P/Y/S;
• and
• (ix) co-mutations of two or more substitutions selected from the list consisting of rtI29L, rtY50S, rtP170H, rtL180M, rtM204V and rtI253V;
• (x) co-mutations of two or more substitutions selected from the listing consisting of rtL180M, rtA181V, rtM207V, rtH216H/P, rtE218D/E, rtH238D, rtR242K/R, rtW243G/W and rtI254I/F.
• wherein the presence of such a mutation is an indication of resistance to a nucleoside or nucleotide analog.

A method for detecting an agent which exhibits inhibitory activity to an HBV variant which exhibits resistance or decreased sensitivity to one or more nucleoside or nucleotide analogs or an immunological reagent said method comprising:

• generating a genetic construct comprising a replication competent-effective amount of the genome from said HBV contained in a plasmid vector and then transfecting said cells with said construct;
• contacting said cells, before, during and/or after transfection, with the agent to be tested;
• culturing said cells for a time and under conditions sufficient for the HBV to replicate, express genetic sequences and/or assemble and/or release virus or virus-like particles if resistant to said agent;
• and
• subjecting the cells, cell lysates or culture supernatant fluid to viral- or viral-component-detection means to determine whether or not the virus has replicated, expressed genetic material and/or assembled and/or been released in the presence of said agent.