2'-branched nucleosides and Flaviviridae mutation

• US 20050031588A1
• Filed: 11/17/2003
• Published: 02/10/2005
• Est. Priority Date: 11/15/2002
• Status: Active Grant

• Current Assignee: Idenix Pharmaceuticals (Merck & Co., Inc.)
• Original Assignee: David Standring, Jean-Pierre Sommadossi, Lin Qu, Paolo Lacolla, Vadim Bichko
• Inventors: Qu, Lin, Bichko, Vadim, Sommadossi, Jean-Pierre, Standring, David, LaColla, Paolo
• Granted Patent: US 7,824,851 B2
• Time in Patent Office: Days
• US Class Current: 424/85.700
• CPC Class Codes: A61K 2300/00 Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• CPC Class Codes: A61K 31/7052 having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
• CPC Class Codes: A61K 31/7072 having two oxo groups directly attached to the pyrimidine ring, e.g. uridine, uridylic acid, thymidine, zidovudine
• CPC Class Codes: A61K 38/21 Interferons [IFN]
• CPC Class Codes: A61K 38/212 IFN-alpha
• CPC Class Codes: A61K 38/215 IFN-beta
• CPC Class Codes: A61K 38/217 IFN-gamma
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 31/14 for RNA viruses
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/16 Purine radicals
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C12N 2770/24022 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 2770/24122 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 2770/24222 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12Q 1/04 Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor C12Q1/6897 takes precedence
• CPC Class Codes: C12Q 1/18 : Testing for antimicrobial activity of a material
• CPC Class Codes: C12Q 1/701 : Specific hybridization probes
• CPC Class Codes: C12Q 1/707 : non-A, non-B Hepatitis, excluding hepatitis D
• CPC Class Codes: C12Q 2600/156 : Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/158 : Expression markers
• CPC Class Codes: G01N 2333/186 : Hepatitis C; Hepatitis NANB
• CPC Class Codes: Y02A 50/30 : Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The present invention discloses a method for the treatment of Flaviviridae infection that includes the administration of a 2′-branched nucleoside, or a pharmaceutically acceptable prodrug and/or salt thereof, to a human in need of therapy in combination or alternation with a drug that directly or indirectly induces a mutation in the viral genome at a location other than a mutation of a nucleotide that results in a change from serine to a different amino acid in the highly conserved consensus sequence, XRXSGXXXT, of domain B of the RNA polymerase region, or is associated with such a mutation. The invention also includes a method to detect a mutant strain of Flaviviridae and a method for its treatment.

Claims

1. A pharmaceutical composition effective for the treatment of a Flaviviridae infection in a host, comprising an effective amount of a 2′

-branched nucleoside, or pharmaceutically acceptable prodrug and/or salt thereof, optionally in a pharmaceutically acceptable carrier or diluent, in combination with one or more drugs that directly or indirectly induce a mutation in a Flaviviridae at a location other than a mutation of a nucleotide that results in a change from serine to a different amino acid in the highly conserved consensus sequence, XRXSGXXXT (Sequence ID No.

     63), of domain B of the RNA polymerase region, and/or one or more drugs that are associated with such a mutation.

2. The pharmaceutical composition of claim 1 wherein the drug is drug that directly or indirectly induces or is associated with a mutation in a Flaviviridae at a location other than nucleotide 1214 (G to C) or 405 Ser to Thr of the RNA polymerase region of BVDV or nucleotide 8443 (G to C) of the HCV genome or 282 Ser to Thr of the RNA polymerase region of HCV.

5. The pharmaceutical composition of any one of claims 1-4, wherein the 2′

-branched nucleoside is a 2′

-branched pyrimidine nucleoside.

6. The pharmaceutical composition of claim 5, wherein the 2′

-branched nucleoside is β

-D-2′

-CH₃-riboC.

7. The pharmaceutical composition of claim 5, wherein the 2′

-branched nucleoside is a 3′

-amino acid prodrug of β

-D-2′

-CH₃-riboC.

8. The pharmaceutical composition of claim 7, wherein the 2′

-branched nucleoside is 3′

-L-valinyl prodrug of β

-D-2′

-CH₃-riboC.

9. The pharmaceutical composition of any one of claims 1-4, wherein the 2′

-branched nucleoside is a 2′

-branched purine nucleoside.

10. The pharmaceutical composition of claim 9, wherein the 2′

-branched nucleoside is β

-D-2′

-CH₃-riboA.

11. The pharmaceutical composition of claim 9, wherein the 2′

-branched nucleoside is a 3′

-amino acid prodrug of β

-D-2′

-CH₃-riboA.

12. The pharmaceutical composition of claim 11, wherein the 2′

-branched nucleoside is a 3′

-L-valinyl prodrug of β

-D-2′

-CH₃-riboA.

13. The pharmaceutical composition of claim 9, wherein the 2′

-branched nucleoside is β

-D-2′

-CH₃-ribo-6-N-methylaminopurine.

14. The pharmaceutical composition of claim 9, wherein the 2′

-branched nucleoside is a 3′

-amino acid prodrug of β

-D-2′

-CH₃-ribo-6-N-methylaminopurine.

15. The pharmaceutical composition of claim 14, wherein the 2′

-branched nucleoside is a 3′

-L-valinyl prodrug of β

-D-2′

-CH₃-ribo-6-N-methylaminopurine.

16. The pharmaceutical compositions of any one of claims 1-4, wherein the 2′

-branched nucleoside is of the formula;

17. The pharmaceutical composition of claim 16, wherein base is selected from the group consisting of adenine, N⁶-alkylpurines, N⁶-acylpurines (wherein acyl is C(O)(alkyl, aryl, alkylaryl, or arylalkyl), N⁶-benzylpurine, N⁶-halopurine, N⁶-vinylpurine, N⁶-acetylenic purine, N⁶-acyl purine, N⁶-hydroxyalkyl purine, N⁶-thioalkyl purine, N²-alkylpurines, N²-alkyl-6-thiopurines, thymine, cytosine, 5-fluorocytosine, 5-methylcytosine, 6-azapyrimidine, including 6-azacytosine, 2- and/or 4-mercaptopyrmidine, uracil, 5-halouracil, including 5-fluorouracil, C⁵-alkylpyrimidines, C⁵-benzylpyrimidines, C⁵-halopyrimidines, C⁵-vinyl-pyrimidine, C⁵-acetylenic pyrimidine, C⁵-acyl pyrimidine, C⁵-hydroxyalkyl purine, C⁵-amidopyrimidine, C⁵-cyanopyrimidine, C⁵-nitropyrimidine, C⁵-aminopyrimidine, N²-alkylpurines, N²-alkyl-6-thiopurines, 5-azacytidinyl, 5-aza-uracilyl, triazolopyridinyl, imidazolopyridinyl, pyrrolopyrimidinyl, and pyrazolopyrimidinyl.

18. The pharmaceutical composition of claim 16, wherein base is of the formula:

19. The pharmaceutical composition of claim 16, wherein base is of the formula:

20. The pharmaceutical composition of claim 16, wherein base is of the formula:

3. A pharmaceutical composition effective for the treatment of a Flaviviridae infection in a host, comprising an effective amount of a 2′

-branched nucleoside, or pharmaceutically acceptable prodrug and/or salt thereof, optionally in a pharmaceutically acceptable carrier or diluent, in combination with interferon.

4. The pharmaceutical composition of claim 3, wherein the interferon is selected from the group consisting of Intron-A (interferon alpha-2b), PEG-INTRON (pegylated interferon alpha-2b), Roferon-A (interferon alfa-2a), PEGASYS (pegylated interferon alfa-2a), INFERGEN (interferon alfacon-1), OMNIFERON (natural interferon), ALBUFERON, REBIF (interferon beta-1a), Omega Interferon, Oral Interferon Alpha, and Interferon gamma-1b.

21. A pharmaceutical composition effective for the treatment of a Flaviviridae infection in a host, comprising:

an effective amount of a 2′

, 3′ and

/or 5′

-prodrug of a 2′

-branched nucleoside, or pharmaceutically acceptable salt thereof, optionally in a pharmaceutically acceptable carrier or diluent;

in combination with one or more drugs that directly or indirectly induce a mutation in a Flaviviridae at a location other than a mutation of a nucleotide that results in a change from serine to a different amino acid in the highly conserved consensus sequence, XRXSGXXXT (Sequence ID No.

     63), of domain B of the RNA polymerase region, and/or one or more drugs that are associated with such a mutation.

22. The pharmaceutical composition of claim 21 wherein the 2′

, 3′ and

/or 5′

-prodrug of a 2′

-branched nucleoside is a 2′

, 3′ and

/or 5′

-prodrug of a 2′

-branched pyrimidine nucleoside.

23. The pharmaceutical composition of claim 22 wherein the 2′

, 3′ and

/or 5′

-prodrug of a 2′

-branched nucleoside is 2′

, 3′ and

/or 5′

-prodrug of β

-D-2′

-CH₃-riboC.

24. The pharmaceutical composition of claim 23 wherein the 2′

, 3′ and

/or 5′

-prodrug of a 2′

-branched nucleoside is 3′

-amino acid prodrug of β

-D-2′

-CH₃-riboC.

25. The pharmaceutical composition of claim 24 wherein the 2′

, 3′ and

/or 5′

-prodrug of a 2′

-branched nucleoside is 3′

-L-valinyl prodrug of β

-D-2′

-CH₃-riboC.

26. The pharmaceutical composition of claim 21 wherein the 2′

, 3′ and

/or 5′

-prodrug of a 2′

-branched nucleoside is a 2′

, 3′ and

/or 5′

-prodrug of a 2′

-branched purine nucleoside.

27. The pharmaceutical composition of claim 26 wherein the 2′

, 3′ and

/or 5′

-prodrug of a 2′

-branched nucleoside is 2′

, 3′ and

/or 5′

-prodrug of β

-D-2′

-CH₃-riboA.

28. The pharmaceutical composition of claim 27 wherein the 2′

, 3′ and

/or 5′

-prodrug of a 2′

-branched nucleoside is 3′

-amino acid prodrug of β

-D-2′

-CH₃-riboA.

29. The pharmaceutical composition of claim 28 wherein the 2′

, 3′ and

/or 5′

-prodrug of a 2′

-branched nucleoside is 3′

-L-valinyl prodrug of β

-D-2′

-CH₃-riboA.

30. The pharmaceutical composition of claim 26 wherein the 2′

, 3′ and

/or 5′

-prodrug of a 2′

-branched nucleoside is 2′

, 3′ and

/or 5′

-prodrug of β

-D-2′

-CH₃-ribo-6-N-methylamino-purine.

31. The pharmaceutical composition of claim 30 wherein the 2′

, 3′ and

/or 5′

-prodrug of a 2′

-branched nucleoside is 3′

-amino acid prodrug of β

-D-2′

-CH₃-ribo-6-N-methylamino-purine.

32. The pharmaceutical composition of claim 31 wherein the 2′

, 3′ and

/or 5′

-prodrug of a 2′

-branched nucleoside is 3′

-L-valinyl prodrug of β

-D-2′

-CH₃-ribo-6-N-methylamino-purine.

33. A method for treating a Flaviviridae infection in a host, comprising administering an effective amount of a 2′

-branched nucleoside, or its pharmaceutically acceptable prodrug or salt to the host, optionally in a pharmaceutically acceptable carrier or diluent, in combination and/or alternation with one or more drugs that directly or indirectly induce a mutation in a Flaviviridae at a location other than a mutation of a nucleotide that results in a change from serine to a different amino acid in the highly conserved consensus sequence, XRXSGXXXT (Sequence ID No.

     63), of domain B of the RNA polymerase region, and/or one or more drugs that are associated with such a mutation.

34. The method of claim 33 wherein the drug is drug that directly or indirectly induces or is associated with a mutation in a Flaviviridae at a location other than nucleotide 1214 (G to C) or 405 Ser to Thr of the RNA polymerase region of BVDV or nucleotide 8443 (G to C) of the HCV genome or 282 Ser to Thr of the RNA polymerase region of HCV.

37. The method of any one of claims 33-36, wherein the 2′

-branched nucleoside is a 2′

-branched pyrimidine nucleoside.

38. The method of claim 37, wherein the 2′

-branched nucleoside is β

-D-2′

-CH₃-riboC.

39. The method of claim 37, wherein the 2′

-branched nucleoside is a 3′

-amino acid prodrug of β

-D-2′

-CH₃-riboC.

40. The method of claim 39, wherein the 2′

-branched nucleoside is 3′

-L-valinyl prodrug of β

-D-2′

-CH₃-riboC.

41. The method of any one of claims 33-36, wherein the 2′

-branched nucleoside is a 2′

-branched purine nucleoside.

42. The method of claim 41, wherein the 2′

-branched nucleoside is β

-D-2′

-CH₃-riboA.

43. The method of claim 41, wherein the 2′

-branched nucleoside is a 3′

-amino acid prodrug of β

-D-2′

-CH₃-riboA.

44. The method of claim 43, wherein the 2′

-branched nucleoside is a 3′

-L-valinyl prodrug of β

-D-2′

-CH₃-riboA.

45. The method of claim 41, wherein the 2′

-branched nucleoside is a 3-D-2′

-CH₃-ribo-6-N-methylaminopurine.

46. The method of claim 41, wherein the 2′

-branched nucleoside is a 3′

-amino acid prodrug of β

-D-2′

-CH₃-ribo-6-N-methylaminopurine.

47. The method of claim 46, wherein the 2′

-branched nucleoside is a 3′

-L-valinyl prodrug of β

-D-2′

-CH₃-ribo-6-N-methylaminopurine.

48. The method of any one of claims 33-36, wherein the 2′

-branched nucleoside is of the formula;

49. The method of claim 48, wherein base is selected from the group consisting of adenine, N⁶-alkylpurines, N⁶-acylpurines (wherein acyl is C(O)(alkyl, aryl, alkylaryl, or arylalkyl), N⁶-benzylpurine, N⁶-halopurine, N⁶-vinylpurine, N⁶-acetylenic purine, N⁶-acyl purine, N⁶-hydroxyalkyl purine, N⁶-thioalkyl purine, N²-alkylpurines, N²-alkyl-6-thiopurines, thymine, cytosine, 5-fluorocytosine, 5-methylcytosine, 6-azapyrimidine, including 6-azacytosine, 2- and/or 4-mercaptopyrmidine, uracil, 5-halouracil, including 5-fluorouracil, C⁵-alkylpyrimidines, C⁵-benzylpyrimidines, C⁵-halopyrimidines, C⁵-vinyl-pyrimidine, C⁵-acetylenic pyrimidine, C⁵-acyl pyrimidine, C⁵-hydroxyalkyl purine, C⁵-amidopyrimidine, C⁵-cyanopyrimidine, C⁵-nitropyrimidine, C⁵-amino-pyrimidine, N²-alkylpurines, N²-alkyl-6-thiopurines, 5-azacytidinyl, 5-aza-uracilyl, triazolopyridinyl, imidazolopyridinyl, pyrrolopyrimidinyl, and pyrazolopyrimidinyl.

50. The method of claim 49, wherein base is of the formula:

51. The method of claim 49, wherein base is of the formula:

52. The method of claim 49, wherein base is of the formula:

35. A method for treating a Flaviviridae infection in a host comprising administering an effective amount of a 2′

-branched nucleoside, or pharmaceutically acceptable prodrug and/or salt thereof to the host, optionally in a pharmaceutically acceptable carrier or diluent, in combination and/or alternation with interferon.

36. The method of claim 35, wherein the interferon is selected from the group consisting of Intron-A (interferon alpha-2b), PEG-INTRON (pegylated interferon alpha-2b), Roferon-A (interferon alfa-2a), PEGASYS (pegylated interferon alfa-2a), INFERGEN (interferon alfacon-1), OMNIFERON (natural interferon), ALBUFERON, REBIF (interferon beta-1a), Omega Interferon, Oral Interferon Alpha, and Interferon gamma-1b.

53. A method for treating a patient infected with a Flaviviridae virus that is resistant to a 2′

-branched nucleoside comprising administering an effective amount of interferon, optionally in a pharmaceutically acceptable carrier or diluent, optionally in a manner that substantially eliminates the viral load.

54. A method for treating a patient infected with Flaviviridae comprising:

administering an effective amount of a 2′

, 3′ and

/or 5′

-prodrug of a 2′

-branched nucleoside, or pharmaceutically acceptable salt thereof, optionally in a pharmaceutically acceptable carrier or diluent;

in combination and/or alternation with one or more drugs that directly or indirectly induce a mutation in a Flaviviridae at a location other than a mutation of a nucleotide that results in a change from serine to a different amino acid in the highly conserved consensus sequence, XRXSGXXXT (Sequence ID No.

     63), of domain B of the RNA polymerase region, and/or one or more drugs that are associated with such a mutation.

55. The method of claim 54, wherein the 2′

, 3′ and

/or 5′

-prodrug of a 2′

-branched nucleoside is a 2′

, 3′ and

/or 5′

-prodrug of a 2′

-branched pyrimidine nucleoside.

56. The method of claim 55, wherein the 2′

, 3′ and

/or 5′

-prodrug of a 2′

-branched nucleoside is 2′

, 3′ and

/or 5′

-prodrug of β

-D-2′

-CH₃-riboC.

57. The method of claim 56, wherein the 2′

, 3′ and

/or 5′

-prodrug of a 2′

-branched nucleoside is 3′

-amino acid prodrug of β

-D-2′

-CH₃-riboC.

58. The method of claim 57, wherein the 2′

, 3′ and

/or 5′

-prodrug of a 2′

-branched nucleoside is 3′

-L-valinyl prodrug of β

-D-2′

-CH₃-riboC.

59. The method of claim 54 wherein the 2′

, 3′ and

/or 5′

-prodrug of a 2′

-branched nucleoside is a 2′

, 3′ and

/or 5′

-prodrug of a 2′

-branched purine nucleoside.

60. The method of claim 59 wherein the 2′

, 3′ and

/or 5′

-prodrug of a 2′

-branched nucleoside is 2′

, 3′ and

/or 5′

-prodrug of β

-D-2′

-CH₃-riboA.

61. The method of claim 60 wherein the 2′

, 3′ and

/or 5′

-prodrug of a 2′

-branched nucleoside is 3′

-amino acid prodrug of β

-D-2′

-CH₃-riboA.

62. The method of claim 61 wherein the 2′

, 3′ and

/or 5′

-prodrug of a 2′

-branched nucleoside is 3′

-L-valinyl prodrug of β

-D-2′

-CH₃-riboA.

63. The method of claim 59 wherein the 2′

, 3′ and

/or 5′

-prodrug of a 2′

-branched nucleoside is 2′

, 3′ and

/or 5′

-prodrug of β

-D-2′

-CH₃-ribo-6-N-methylamino-purine.

64. The method of claim 63 wherein the 2′

, 3′ and

/or 5′

-prodrug of a 2′

-branched nucleoside is 3′

-amino acid prodrug of β

-D-2′

-CH₃-ribo-6-N-methylamino-purine.

65. The method of claim 64 wherein the 2′

, 3′ and

/or 5′

-prodrug of a 2′

-branched nucleoside is 3′

-L-valinyl prodrug of β

-D-2′

-CH₃-ribo-6-N-methylamino-purine.

66. A method for treating a patient infected with Flaviviridae comprising:

(a) administering to the patient an effective amount of a 2′

-branched nucleoside, or a pharmaceutically acceptable salt or prodrug thereof;

optionally in a pharmaceutically acceptable carrier or diluent;

(b) assaying the blood of the patient to test for seroconversion from wildtype to mutant virus;

(c) administering an effective amount of interferon;

optionally in a pharmaceutically acceptable carrier or diluent.

69. The method of any one of claims 66-68, wherein the 2′

-branched nucleoside is a 2′

-branched pyrimidine nucleoside.

70. The method of claim 69, wherein the 2′

-branched nucleoside is β

-D-2′

-CH₃-riboC.

71. The method of claim 69, wherein the 2′

-branched nucleoside is a 3′

-amino acid prodrug of β

-D-2′

-CH₃-riboC.

72. The method of claim 71, wherein the 2′

-branched nucleoside is 3′

-L-valinyl prodrug of β

-D-2′

-CH₃-riboC.

73. The method of any one of claims 66-68, wherein the 2′

-branched nucleoside is a 2′

-branched purine nucleoside.

74. The method of claim 73, wherein the 2′

-branched nucleoside is β

-D-2′

-CH₃-riboA.

75. The method of claim 73, wherein the 2′

-branched nucleoside is a 3′

-amino acid prodrug of β

-D-2′

-CH₃-riboA.

76. The method of claim 75, wherein the 2′

-branched nucleoside is a 3′

-L-valinyl prodrug of β

-D-2′

-CH₃-riboA.

77. The method of claim 73, wherein the 2′

-branched nucleoside is β

-D-2′

-CH₃-ribo-6-N-methylaminopurine.

78. The method of claim 73, wherein the 2′

-branched nucleoside is a 3′

-amino acid prodrug of β

-D-2′

-CH₃-ribo-6-N-methylaminopurine.

79. The method of claim 78, wherein the 2′

-branched nucleoside is a 3′

-L-valinyl prodrug of β

-D-2′

-CH₃-ribo-6-N-methylaminopurine.

80. The method of any one of claims 66-68, wherein the 2′

-branched nucleoside is of the formula;

67. A method for treating a patient infected with Flaviviridae comprising:

(a) administering an effective amount of a 2′

-branched nucleoside, or pharmaceutically acceptable salt or prodrug thereof;

optionally in a pharmaceutically acceptable carrier or diluent;

(b) obtaining a viral sample from the patient;

(c) determining the replication fitness of the virus;

(d) determining whether the replication fitness of the virus in the sample is less than the replication fitness of the wild-type virus, which indicates resistance to β

-D-2′

-CH₃-riboC;

(e) administering an effective amount of interferon to those patients that are resistant to β

-D-2′

-CH₃-riboC.

68. A method for treating a patient infected with Flaviviridae comprising:

(a) administering an effective amount of a 2′

-branched nucleoside, or pharmaceutically acceptable salt or prodrug thereof;

optionally in a pharmaceutically acceptable carrier or diluent;

(b) obtaining a viral culture sample from the patient;

(c) culturing the sample and comparing the plaque growth between the sample and wild type virus;

(d) determining whether the plaque growth of the sample is smaller than the plaque growth of the wildtype, which indicates resistance to β

-D-2′

-CH₃-riboC;

(e) administering an effective amount of interferon to those patients that are resistant to β

-D-2′

-CH₃-riboC.

81. A method for assaying a sample suspected of containing a Flaviviridae resistant to a 2′

-branched nucleoside comprising;

(a) contacting a sample containing a Flaviviridae nucleic acid sequence with a detectable oligonucleotide probe having a sequence complimentary a codon that encodes a serine in the highly conserved consensus sequence, XRXSGXXXT (Sequence ID No.

     63), of domain B of the RNA polymerase region of Flaviviridae;

(b) allowing the probe to hybridize to the sequence;

(c) detecting the hybridization of the probe the sequence.

82. A method for assaying a sample suspected of containing a Flaviviridae resistant to a 2′

-branched nucleoside comprising;

(a) contacting a sample containing a Flaviviridae nucleic acid sequence with a detectable oligonucleotide probe having a sequence complimentary to the cytidine at nucleotide 1214 of the RNA polymerase region of BVDV or the cytidine at nucleotide 8443 of HCV;

(b) allowing the probe to hybridize to the sequence;

(c) detecting the hybridization of the probe to cytidine at nucleotide 1214 of the RNA polymerase region of BVDV or at nucleotide 8443 of HCV.

83. A kit for assaying a sample suspected of containing a Flaviviridae resistant to a 2′

-branched nucleoside comprising;

(a) a sample containing a Flaviviridae nucleic acid sequence with a detectable oligonucleotide probe having a sequence complimentary a codon that encodes a serine in the highly conserved consensus sequence, XRXSGXXXT (Sequence ID No.

     63), of domain B of the RNA polymerase region of Flaviviridae;

(b) a means for detecting the hybridization of the probe the sequence; and

(c) optionally with instructional material.

84. A kit for assaying a sample suspected of containing a Flaviviridae resistant to a 2′

-branched nucleoside comprising;

(a) a sample containing a Flaviviridae nucleic acid sequence with a detectable oligonucleotide probe having a sequence complimentary to the cytidine at nucleotide 1214 of the RNA polymerase region of BVDV or the cytidine at nucleotide 8443 of HCV;

(b) a means for detecting the hybridization of the probe to cytidine at nucleotide 1214 of the RNA polymerase region of BVDV or at nucleotide 8443 of HCV; and

(c) optionally with instructional material.

85. A method for diagnosing the presence of Flaviviridae resistant to a 2′

-branched nucleoside in a patient comprising;

(a) obtaining a sample suspected of containing a Flaviviridae nucleic acid sequence;

(b) contacting the sample with a detectable oligonucleotide probe having a sequence complementary a codon that encodes a serine in the highly conserved consensus sequence, XRXSGXXXT (Sequence ID No.

     63), of domain B of the RNA polymerase region of Flaviviridae;

(b) allowing the probe to hybridize to the sequence; and

(c) detecting the hybridization of the probe the sequence to determine the presence of a β

-D-2′

-CH₃-riboC-resistant Flaviviridae.

86. A method for diagnosing the presence of a Flaviviridae resistant to a 2′

-branched nucleoside in a patient comprising;

(a) obtaining a sample suspected of containing a Flaviviridae nucleic acid sequence;

(b) contacting the sample with a detectable oligonucleotide probe having a sequence complementary to the cytidine at nucleotide 1214 of the RNA polymerase region of BVDV or the cytidine at nucleotide 8443 of HCV;

(b) allowing the probe to hybridize to the sequence; and

(c) detecting the hybridization of the probe to cytidine at nucleotide 1214 of the RNA polymerase region of BVDV or at nucleotide 8443 of HCV to determine the presence of a β

-D-2′

-CH₃-riboC-resistant Flaviviridae.