Viral variants and methods for detecting same

Abstract

The present invention relates generally to viral variants exhibiting reduced interactivity with immunological reagents. More particularly, the present invention is directed to hepatitis B variants exhibiting complete or partial resistance to nucleoside analogs and/or reduced interactivity with antibodies to viral surface components. The present invention further contemplates assays for detecting such viral variants which assays are useful in monitoring anti-viral therapeutic agents.

1 . (canceled) 
     
     
         2 . An isolated HBV mutant, comprising one or more mutations in the gene encoding the HBV polymerase resulting in decreased sensitivity to a nucleoside analogue compared to a wild-type HBV, wherein said mutation(s) result(s) in at least one amino acid addition, substitution and/or deletion in the A domain corresponding to amino acid residues 421-436 of a wild-type HBV polymerase and/or in the D domain corresponding to amino acid residues 576-588 of a wild-type HBV polymerase and/or in the E domain corresponding to amino acid residues 592-600 of a wild-type HBV polymerase, or in a region proximal thereto. 
     
     
         3 . An isolated HBV mutant according to  claim 2 , comprising one or more mutations in the gene encoding the HBV polymerase resulting in decreased sensitivity to a nucleoside analogue compared to a wild-type HBV, wherein said mutation(s) result(s) in at least one amino acid addition, substitution and/or deletion in the A domain corresponding to amino acid residues 421-436 of a wild-type HBV polymerase. 
     
     
         4 . An isolated HBV mutant according to  claim 2 , comprising one or more mutations in the gene encoding the HBV polymerase resulting in decreased sensitivity to a nucleoside analogue compared to a wild-type HBV, wherein said mutation(s) result(s) in at least one amino acid addition, substitution and/or deletion in the D domain corresponding to amino acid residues 576-588 of a wild-type HBV polymerase. 
     
     
         5 . An isolated HBV mutant according to  claim 2 , comprising one or more mutations in the gene encoding the HBV polymerase resulting in decreased sensitivity to a nucleoside analogue compared to a wild-type HBV, wherein said mutation(s) result(s) in at least one amino acid addition, substitution and/or deletion in the E domain corresponding to amino acid residues 592-600 of a wild-type HBV polymerase. 
     
     
         6 . A method for determining the potential for an HBV to exhibit reduced sensitivity to a nucleoside analogue, said method comprising isolating DNA or corresponding mRNA from said HBV and screening for one or more mutation(s) in the nucleotide sequence encoding HBV DNA polymerase resulting in at least one amino acid substitution, deletion and/or addition in the A domain or D domain or E domain or a region proximal thereto of said DNA polymerase wherein the presence of such a mutation is an indication of the likelihood of resistance to said nucleoside analogue. 
     
     
         7 . A method for determining whether an HBV isolate encodes a variant DNA polymerase, said method comprising determining the amino acid sequence of its DNA polymerase directly or via a nucleotide sequence and comparing same to the amino acid sequence below: 
       
         
           
                 
                 
               
                            DOMAIN A 
                     
                 
                   421        430         440         450 
                 
                     S N   D     LSWLSLD VSAAFYH   I   P PL HPAAMPHLL I   V   GSSGL S   D RYVA 
                 
                     
                 
                   460           470             480           490 
                 
                   RLSS T   N S R   N N I *N  N Y H Q H   Y   G   R *** D   N LH  D   N   S   Y CSR N   Q LYVS L L   M LLY K   Q T Y   F G R   W   
                 
                     
                 
                                   DOMAIN B 
                 
                   500           510          520        530 
                 
                   KLHL Y L   S   A     HPI I   V     LGFRK I   L     PMG V   G     GLSPFLLAQF  TSAI C   L   A   S   V   M   V   T   R   C R 
                 
                     
                 
                                   DOMAIN C 
                 
                   540            550            560 
                 
                   AF F   P HC L   V       A       V       F S   A     Y MDD V     L M     VLGA   K   R   S   T    V   G   Q   E H L S R E S   F L F   Y   T   A   A   S   
                 
                     
                 
                                   DOMAIN D         DOMAIN E 
                 
                   570              580         590          600 
                 
                     V   I   T   C   N   S   F   V LL   S   D   L   V     GI HLNP N   Q     KTKR W GY SLNFMGY V   I     I G   
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
         where the presence of a different amino acid from the consensus sequence indicates a putative HBV mutant. 
       
     
     
         8 . The method according to one of  claim 6  or  claim 7 , wherein the screening for a mutation comprises sequencing said isolated HBV DNA or corresponding DNA. 
     
     
         9 . The method according to one of  claim 6  or  claim 7 , wherein the screening for a mutation comprises a PCR method or PCR-based method. 
     
     
         10 . The method according to one of  claim 6  or  claim 7 , wherein the screening for a mutation comprises a hybridization method. 
     
     
         11 . A method for determining whether an HBV isolate encodes a variant DNA polymerase, said method comprising determining the amino acid sequence of its DNA polymerase directly or via a nucleotide sequence and comparing same to the amino acid sequence below: 
       
         
           
                 
                 
               
                            DOMAIN A 
                     
                 
                   421        430         440         450 
                 
                     S N   D     LSWLSLD VSAAFYH   I   P PL HPAAMPHLL I   V  GSSGL S   D RYVA 
                 
                     
                 
                   460           470             480           490 
                 
                   RLSS T   N S R   N N I *N  N Y H Q H   Y   G   R *** D   N LH  D   N   S   Y CSR N   Q LYVS L L   M LLY K   Q T Y   F G R   W   
                 
                     
                 
                                   DOMAIN B 
                 
                   500           510          520        530 
                 
                   KLHL Y L   S   A     HPI I   V     LGFRK I   L     PMG V   G     GLSPFLLAQF  TSAI C   L   A   S   V   M   V   T   R   C R 
                 
                     
                 
                                   DOMAIN C 
                 
                   540            550            560 
                 
                   AF F   P HC L   V       A       V       F S   A     Y MDD V     L M     VLGA   K   R   S   T    V   G   Q   E H L S R E S   F L F   Y   T   A   A   S   
                 
                     
                 
                                   DOMAIN D         DOMAIN E 
                 
                   570              580         590          600 
                 
                     V   I   T   C   N   S   F   V LL   S   D   L   V     GI HLNP N   Q     KTKR W GY SLNFMGY V   I     I G   
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
         where the presence of a different amino acid from the consensus sequence indicates a putative HBV mutant. 
       
     
     
         12 . A method for determining whether an HBV isolate encodes a variant DNA polymerase according to  claim 11  said method comprising determining the amino acid sequence of the A domain (corresponding to amino acid residues 421-436) or a region proximal thereto directly or via a nucleotide sequence and comparing same to the A domain or the region proximal thereto of the following amino acid sequence: 
       
         
           
                 
                 
               
                            DOMAIN A 
                     
                 
                   421        430         440         450 
                 
                     S N   D     LSWLSLD VSAAFYH   I   P PL HPAAMPHLL I   V  GSSGL S   D RYVA 
                 
                     
                 
                   460           470             480           490 
                 
                   RLSS T   N S R   N N I *N  N Y H Q H   Y   G   R *** D   N LH  D   N   S   Y CSR N   Q LYVS L L   M LLY K   Q T Y   F G R   W   
                 
                     
                 
                                   DOMAIN B 
                 
                   500           510          520        530 
                 
                   KLHL Y L   S   A     HPI I   V     LGFRK I   L     PMG V   G     GLSPFLLAQF  TSAI C   L   A   S   V   M   V   T   R   C R 
                 
                     
                 
                                   DOMAIN C 
                 
                   540            550            560 
                 
                   AF F   P HC L   V       A       V       F S   A     Y MDD V     L M     VLGA   K   R   S   T    V   G   Q   E H L S R E S   F L F   Y   T   A   A   S   
                 
                     
                 
                                   DOMAIN D         DOMAIN E 
                 
                   570              580         590          600 
                 
                     V   I   T   C   N   S   F   V LL   S   D   L   V     GI HLNP N   Q     KTKR W GY SLNFMGY V   I     I G   
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         13 . A method for determining whether an HBV isolate encodes a variant DNA polymerase according to  claim 11  said method comprising determining the amino acid sequence of the D domain (corresponding to amino acid residues 576-588) or a region proximal thereto directly or via a nucleotide sequence and comparing same to the D domain or the region proximal thereto of the following amino acid sequence: 
       
         
           
                 
                 
               
                            DOMAIN A 
                     
                 
                   421        430         440         450 
                 
                     S N   D     LSWLSLD VSAAFYH   I   P PL HPAAMPHLL I   V  GSSGL S   D RYVA 
                 
                     
                 
                   460           470             480           490 
                 
                   RLSS T   N S R   N N I *N  N Y H Q H   Y   G   R *** D   N LH  D   N   S   Y CSR N   Q LYVS L L   M LLY K   Q T Y   F G R   W   
                 
                     
                 
                                   DOMAIN B 
                 
                   500           510          520        530 
                 
                   KLHL Y L   S   A     HPI I   V     LGFRK I   L     PMG V   G     GLSPFLLAQF  TSAI C   L   A   S   V   M   V   T   R   C R 
                 
                     
                 
                                   DOMAIN C 
                 
                   540            550            560 
                 
                   AF F   P HC L   V       A       V       F S   A     Y MDD V     L M     VLGA   K   R   S   T    V   G   Q   E H L S R E S   F L F   Y   T   A   A   S   
                 
                     
                 
                                   DOMAIN D         DOMAIN E 
                 
                   570              580         590          600 
                 
                     V   I   T   C   N   S   F   V LL   S   D   L   V     GI HLNP N   Q     KTKR W GY SLNFMGY V   I     I G   
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         14 . A method for determining whether an HBV isolate encodes a variant DNA polymerase according to  claim 11  said method comprising determining the amino acid sequence of the E domain (corresponding to amino acid residues 592-600) or a region proximal thereto directly or via a nucleotide sequence and comparing same to the E domain or the region proximal thereto of the following amino acid sequence 
       
         
           
                 
                 
               
                            DOMAIN A 
                     
                 
                   421        430         440         450 
                 
                     S N   D     LSWLSLD VSAAFYH   I   P PL HPAAMPHLL I   V  GSSGL S   D RYVA 
                 
                     
                 
                   460           470             480           490 
                 
                   RLSS T   N S R   N N I *N  N Y H Q H   Y   G   R *** D   N LH  D   N   S   Y CSR N   Q LYVS L L   M LLY K   Q T Y   F G R   W   
                 
                     
                 
                                   DOMAIN B 
                 
                   500           510          520        530 
                 
                   KLHL Y L   S   A     HPI I   V     LGFRK I   L     PMG V   G     GLSPFLLAQF  TSAI C   L   A   S   V   M   V   T   R   C R 
                 
                     
                 
                                   DOMAIN C 
                 
                   540            550            560 
                 
                   AF F   P HC L   V       A       V       F S   A     Y MDD V     L M     VLGA   K   R   S   T    V   G   Q   E H L S R E S   F L F   Y   T   A   A   S   
                 
                     
                 
                                   DOMAIN D         DOMAIN E 
                 
                   570              580         590          600 
                 
                     V   I   T   C   N   S   F   V LL   S   D   L   V     GI HLNP N   Q     KTKR W GY SLNFMGY V   I     I G   
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         15 . The method according to any of  claims 11 - 14 , wherein the screening for a mutation comprises sequencing said isolated HBV DNA or corresponding DNA. 
     
     
         16 . The method according to any of  claims 11 - 14 , wherein the screening for a mutation comprises a PCR method or PCR-based method. 
     
     
         17 . The method according to any of  claims 11 - 14 , wherein the screening for a mutation comprises a hybridization method. 
     
     
         18 . HBV isolate encoding a variant DNA polymerase as detected by the method of any one of the  claims 11 - 14 . 
     
     
         19 . A kit for assays for detecting a HBV isolate according to  claim 18  comprising one or more reagents chosen from the group consisting of nucleic acid hybridisation technology as PCR and reagents for immunologically based detection techniques.