METHODS FOR DETECTING ANTIVIRAL-DRUG RESISTANT VIRUS
20210230710 · 2021-07-29
Assignee
Inventors
Cpc classification
C12Q2600/106
CHEMISTRY; METALLURGY
G01N33/56994
PHYSICS
C12Q1/705
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention is directed to a method for detecting the presence or absence of an antiviral drug-resistant HSV, comprising: (a) identifying one or more HSV mutation selected from: (i) a thymidine kinase (TK) mutation selected from 250G>A (HSV-2), 0C>T (HSV-1), 268C>T (HSV-2), 373C>T (HSV-1), 146T>G (HSV-1), 363G>A (HSV-), 497T>A (HSV-1), 558G>T (HSV-2), 641A>G (HSV-2), 715T>C (HSV-1), 938T>C(HSV-2), 437_438insA (HSV-1), 169delC (HSV-1), 170delC (HSV-1), 171delC (HSV-1), 1072delC (HSV-1), 458delC (HSV-2), 459delC (HSV-2), 460delC (HSV-2), 461delC (HSV-2), 881delC (HSV-1), 882delC (HSV-1), 883delC (HSV-1), 884delC (HSV-1), and 885delC (HSV-1); and (ii) a DNA polymerase (DNA pol) mutation selected from 1882C>G(HSV-2), 2405T>G (HSV-1), 2500G>T (HSV-1), 2515A>G (HSV-1), 2892_2893insT(HSV-1), 2893_2894insT (HSV-1), 2894_2895insT (HSV-1), and 2895_2896insT (HSV-1); wherein the presence of said one or more HSV mutation confirms the presence of an antiviral drug-resistant HSV, and wherein the absence of said one or more HSV mutation is indicative of the absence of an antiviral drug-resistant HSV.
Claims
1. A method for detecting the presence or absence of an antiviral drug-resistant HSV, comprising: a. identifying the presence or absence of one or more HSV mutation in a database comprising: i. thymidine kinase (TK) mutations 250G>A (HSV-2), 100C>T (HSV-1), 268C>T (HSV-2), 373C>T (HSV-1), 146T>G (HSV-1), 363G>A (HSV-1), 497T>A (HSV-1), 558G>T (HSV-2), 641A>G (HSV-2), 715T>C (HSV-1), 938T>C (HSV-2), 437_438 insA (HSV-1), 169delC (HSV-1), 170delC (HSV-1), 171delC (HSV-1), 172delC (HSV-1), 458delC (HSV-2), 459delC (HSV-2), 460delC (HSV-2), 461delC (HSV-2), 881delC (HSV-1), 882delC (HSV-1), 883delC (HSV-1), 884delC (HSV-1), and 885delC (HSV-1); and ii. a DNA polymerase (DNA pol) mutations 2405T>G (HSV-1), 2500G>T (HSV-1), 2515A>G (HSV-1), 2892_2893 insT (HSV-1), 2893_2894 insT (HSV-1), 2894_2895 insT (HSV-1), and 2895_2896 insT (HSV-1); wherein the presence of said one or more HSV mutation(s) confirms the presence of an antiviral drug-resistant HSV, and wherein the absence of said one or more HSV mutation(s) is indicative of the absence of an antiviral drug-resistant HSV.
2. The method according to claim 1, wherein the database comprises thymidine kinase (TK) mutations 250G>A (HSV-2), 100C>T (HSV-1), 268C>T (HSV-2), 373C>T (HSV-1), 146T>G (HSV-1), 363G>A (HSV-1), 497T>A (HSV-1), 558G>T (HSV-2), 641A>G (HSV-2), 715T>C (HSV-1), 938T>C (HSV-2), 437_438 insA (HSV-1), 169delC (HSV-1), 170delC (HSV-1), 171delC (HSV-1), 172delC (HSV-1), 458delC (HSV-2), 459delC (HSV-2), 460delC (HSV-2), 461delC (HSV-2), 881delC (HSV-1), 882delC (HSV-1), 883delC (HSV-1), 884delC (HSV-1), and 885delC (HSV-1).
3. A method for detecting the presence or absence of an antiviral drug-resistant HSV, comprising: a. identifying one or more HSV mutation selected from: i. a thymidine kinase (TK) mutation selected from 250G>A (HSV-2), 1000>T (HSV-1), 268C>T (HSV-2), 373C>T (HSV-1), 146T>G (HSV-1), 363G>A (HSV-1), 497T>A (HSV-1), 558G>T (HSV-2), 641A>G (HSV-2), 715T>C (HSV-1), 938T>C (HSV-2), 437_438 insA (HSV-1), 169delC (HSV-1), 170delC (HSV-1), 171delC (HSV-1), 172delC (HSV-1), 458delC (HSV-2), 459delC (HSV-2), 460delC (HSV-2), 461delC (HSV-2), 881delC (HSV-1), 882delC (HSV-1), 883delC (HSV-1), 884delC (HSV-1), and 885delC (HSV-1); and ii. a DNA polymerase (DNA pol) mutation selected from 2405T>G (HSV-1), 2500G>T (HSV-1), 2515A>G (HSV-1), 2892_2893 insT (HSV-1), 2893_2894 insT (HSV-1), 2894_2895 insT (HSV-1), and 2895_2896 insT (HSV-1); wherein the presence of said one or more HSV mutation confirms the presence of an antiviral drug-resistant HSV, and wherein the absence of said one or more HSV mutation is indicative of the absence of an antiviral drug-resistant HSV.
4. A method for detecting the presence or absence of an antiviral drug-resistant HSV, comprising: a. identifying the presence or absence of one or more HSV polymorphism in a database comprising: i. thymidine kinase (TK) polymorphisms E84K (HSV-2), Q34* (HSV-1), Q90* (HSV-2), Q125* (HSV-1), L49R (HSV-1), M1211 (HSV-1), 1166N (HSV-1), Q186H (HSV-2), H214R (HSV-2), E146fs (HSV-1), D228* (HSV-1), Y239H (HSV-1), L313S (HSV-2), T183* (HSV-1), H58fs (HSV-1), M85* (HSV-1), P154fs (HSV-2), M183* (HSV-2), A294fs (HSV-1), P295fs (HSV-1), and E296fs (HSV-1); and ii. a DNA polymerase (DNA pol) polymorphisms L802R (HSV-1), A834S (HSV-1), T839A (HSV-1), F965_I966insF (HSV-1), and 1966* (HSV-1); wherein the presence of said one or more HSV polymorphism(s) confirms the presence of an antiviral drug-resistant HSV, and wherein the absence of said one or more HSV polymorphism(s) is indicative of the absence of an antiviral drug-resistant HSV.
5. A method for detecting the presence or absence of an antiviral drug-resistant HSV, comprising: a. identifying one or more HSV polymorphism selected from: i. a TK polymorphism selected from E84K (HSV-2), Q34* (HSV-1), Q90* (HSV-2), Q125* (HSV-1), L49R (HSV-1), M1211 (HSV-1), 1166N (HSV-1), Q186H (HSV-2), H214R (HSV-2), E146fs (HSV-1), D228* (HSV-1), Y239H (HSV-1), L313S (HSV-2), T183* (HSV-1), H58fs (HSV-1), M85* (HSV-1), P154fs (HSV-2), M183* (HSV-2), A294fs (HSV-1), P295fs (HSV-1), and E296fs (HSV-1); and ii. a DNA pol polymorphism selected from L802R (HSV-1), A834S (HSV-1), T839A (HSV-1), F965_I966insF (HSV-1), and 1966* (HSV-1); wherein the presence of said one or more HSV polymorphism confirms the presence of an antiviral drug-resistant HSV, and the absence of said one or more HSV polymorphism is indicative of the absence of an antiviral drug-resistant HSV.
6. The method according to any one of the preceding claims, further comprising: a. identifying one or more HSV-1 TK mutation selected from: 110C>T, 205C>A, 1072A>C, 1072A>T, 574G>A, and 766C>T; b. identifying one or more HSV-2 TK mutation selected from: 373G>A, 639A>C or 639A>T, and 1094T>C; c. identifying one or more HSV-1 DNA Pol mutation selected from: 64G>A, 160A>G, 248A>C, 361G>A, 415G>A or 415G>C, 716C>T, 1255C>A, 2039A>C, 2042G>A, 2249A>C, 2548G>C, 2732G>A, 2741C>T, 2915C>T, 2954G>A, 2974G>A, 2977C>T, 3137A>C, 3343G>A, 3359A>C, 3505G>A, and 3595C>A; and/or d. identifying one or more HSV-2 DNA Pol mutation selected from: 520G>T, 1339A>C or 1339A>T, 1481T>C, 2141A>G, 2281G>A, 2323C>T, 2325C>G, and 2326G>C; wherein the presence of said one or more HSV mutation is indicative of the absence of an antiviral drug-resistant HSV.
7. The method according to any one of the preceding claims, further comprising: a. identifying one or more HSV-1 TK polymorphism selected from: A37V, L69M, 1358L, A192T, and R256W; b. identifying one or more HSV-2 TK polymorphism selected from: A125T, E213D, and L365P; c. identifying one or more HSV-1 DNA Pol polymorphism selected from: G22R, T54A, D83A, G121S, G139R, S239L, L4191, E680A, R681Q, K750T, E850Q, S911N, S914L, A972V, G985E, E992K, R993C, N1046T, A1115T, E1120A, A1169T, and P1199T; and/or d. identifying one or more HSV-2 DNA Pol polymorphism selected from: D174Y, M447L, M494T, H714R, E761K, R775C, R775W, and E776Q; wherein the presence of said one or more HSV polymorphism is indicative of the absence of an antiviral drug-resistant HSV.
8. A method for treating an infection of an antiviral drug-resistant HSV in a subject, comprising: a. identifying the presence or absence of one or more HSV mutation in a database comprising TK mutations 250G>A (HSV-2), 100C>T (HSV-1), 268C>T (HSV-2), 373C>T (HSV-1), 146T>G (HSV-1), 363G>A (HSV-1), 497T>A (HSV-1), 558G>T (HSV-2), 641A>G (HSV-2), 715T>C (HSV-1), 938T>C (HSV-2), 437_438 insA (HSV-1), 169delC (HSV-1), 170delC (HSV-1), 171delC (HSV-1), 172delC (HSV-1), 458delC (HSV-2), 459delC (HSV-2), 460delC (HSV-2), 461delC (HSV-2), 881delC (HSV-1), 882delC (HSV-1), 883delC (HSV-1), 884delC (HSV-1), and 885delC (HSV-1); b. confirming the presence of said one or more HSV mutation(s); and c. administering to said subject a drug selected from a foscarnet drug, a cidofovir drug and a docosanol drug; or other non-penciclovir and/or non-acyclovir drug.
9. A method for treating an infection of an antiviral drug-resistant HSV in a subject, comprising: a. confirming an antiviral drug-resistant HSV comprises a TK mutation selected from 250G>A (HSV-2), 100C>T (HSV-1), 268C>T (HSV-2), 373C>T (HSV-1), 146T>G (HSV-1), 363G>A (HSV-1), 497T>A (HSV-1), 558G>T (HSV-2), 641A>G (HSV-2), 715T>C (HSV-1), 938T>C (HSV-2), 437_438 insA (HSV-1), 169delC (HSV-1), 170delC (HSV-1), 171delC (HSV-1), 172delC (HSV-1), 458delC (HSV-2), 459delC (HSV-2), 460delC (HSV-2), 461delC (HSV-2), 881delC (HSV-1), 882delC (HSV-1), 883delC (HSV-1), 884delC (HSV-1), and 885delC (HSV-1); and b. administering to said subject a drug selected from a foscarnet drug, a cidofovir drug and a docosanol drug; or other non-penciclovir and/or non-acyclovir drug.
10. A method for treating an infection of an antiviral drug-resistant HSV in a subject, comprising: a. identifying the presence or absence of one or more HSV mutation in a database comprising DNA Pol mutations 2405T>G (HSV-1), 2500G>T (HSV-1), 2515A>G (HSV-1), 2892_2893 insT (HSV-1), 2893_2894 insT (HSV-1), 2894_2895 insT (HSV-1), and 2895_2896 insT (HSV-1); b. confirming the presence of said one or more HSV mutation(s); and c. administering to said subject drug selected from a docosanol drug, BAY 54-6322, ASP2151 and BAY 57-1293; or other non-foscarnet drug, non-cidofovir drug, non-penciclovir and/or non-acyclovir drug.
11. A method for treating an infection of an antiviral drug-resistant HSV in a subject, comprising: a. confirming an antiviral drug-resistant HSV comprises a DNA Pol mutation selected from 2405T>G (HSV-1), 2500G>T (HSV-1), 2515A>G (HSV-1), 2892_2893 insT (HSV-1), 2893_2894 insT (HSV-1), 2894_2895 insT (HSV-1), and 2895_2896insT (HSV-1); and b. administering to said subject drug selected from a docosanol drug, BAY 54-6322, ASP2151 and BAY 57-1293; or other non-foscarnet drug, non-cidofovir drug, non-penciclovir and/or non-acyclovir drug.
12. A method for treating an infection of an antiviral drug-resistant HSV in a subject, comprising: a. identifying the presence or absence of one or more HSV polymorphism in a database comprising TK polymorphisms E84K (HSV-2), Q34* (HSV-1), Q90* (HSV-2), Q125* (HSV-1), L49R (HSV-1), M1211 (HSV-1), 1166N (HSV-1), Q186H (HSV-2), H214R (HSV-2), E146fs (HSV-1), D228* (HSV-1), Y239H (HSV-1), L313S (HSV-2), T183* (HSV-1), H58fs (HSV-1), M85* (HSV-1), P154fs (HSV-2), M183* (HSV-2), A294fs (HSV-1), P295fs (HSV-1), and E296fs (HSV-1); b. confirming the presence of said one or more HSV polymorphism(s); and c. administering to said subject a drug selected from a foscarnet drug, a cidofovir drug and a docosanol drug; or other non-penciclovir and/or non-acyclovir drug.
13. A method for treating an infection of an antiviral drug-resistant HSV in a subject, comprising: a. confirming an antiviral drug-resistant HSV comprises a TK polymorphism selected from E84K (HSV-2), Q34* (HSV-1), Q90* (HSV-2), Q125* (HSV-1), L49R (HSV-1), M1211 (HSV-1), 1166N (HSV-1), Q186H (HSV-2), H214R (HSV-2), E146fs (HSV-1), D228* (HSV-1), Y239H (HSV-1), L313S (HSV-2), T183* (HSV-1), H58fs (HSV-1), M85* (HSV-1), P154fs (HSV-2), M183* (HSV-2), A294fs (HSV-1), P295fs (HSV-1), and E296fs (HSV-1); and b. administering to said subject a drug selected from a foscarnet drug, a cidofovir drug and a docosanol drug; or other non-penciclovir and/or non-acyclovir drug.
14. A method for treating an infection of an antiviral drug-resistant HSV in a subject, comprising: a. identifying the presence or absence of one or more HSV polymorphism in a database comprising DNA Pol polymorphisms L802R (HSV-1), A834S (HSV-1), T839A (HSV-1), F965_I966insF (HSV-1), and 1966* (HSV-1); b. confirming the presence of said one or more HSV polymorphism(s); and c. administering to said subject a drug selected from a docosanol drug, BAY 54-6322, ASP2151 and BAY 57-1293; or other non-foscarnet drug, non-cidofovir drug, non-penciclovir and/or non-acyclovir drug.
15. A method for treating an infection of an antiviral drug-resistant HSV in a subject, comprising: a. confirming an antiviral drug-resistant HSV comprises a DNA Pol polymorphism selected from L802R (HSV-1), A834S (HSV-1), T839A (HSV-1), F965_I966insF (HSV-1), and 1966* (HSV-1); and b. administering to said subject a drug selected from a docosanol drug, BAY 54-6322, ASP2151 and BAY 57-1293; or other non-foscarnet drug, non-cidofovir drug, non-penciclovir and/or non-acyclovir drug.
16. An antiviral drug for use in a method for treating an infection of an antiviral drug-resistant HSV (e.g. HSV-1 and/or HSV-2), wherein said method comprises: a. identifying the presence or absence of one or more HSV mutation in a database comprising TK mutations 250G>A (HSV-2), 100C>T (HSV-1), 268C>T (HSV-2), 373C>T (HSV-1), 146T>G (HSV-1), 363G>A (HSV-1), 497T>A (HSV-1), 558G>T (HSV-2), 641A>G (HSV-2), 715T>C (HSV-1), 938T>C (HSV-2), 437_438 insA (HSV-1), 169delC (HSV-1), 170delC (HSV-1), 171delC (HSV-1), 172delC (HSV-1), 458delC (HSV-2), 459delC (HSV-2), 460delC (HSV-2), 461delC (HSV-2), 881delC (HSV-1), 882delC (HSV-1), 883delC (HSV-1), 884delC (HSV-1), and 885delC (HSV-1); and b. confirming the presence of said one or more HSV mutation(s); and c. administering to said subject a drug selected from foscarnet, cidofovir drug and docosanol; or other non-penciclovir and/or non-acyclovir drug.
17. An antiviral drug for use in a method for treating an infection of an antiviral drug-resistant HSV (e.g. HSV-1 and/or HSV-2), wherein said method comprises: a. confirming an antiviral drug-resistant HSV comprises a TK mutation selected from 250G>A (HSV-2), 100C>T (HSV-1), 268C>T (HSV-2), 373C>T (HSV-1), 146T>G (HSV-1), 363G>A (HSV-1), 497T>A (HSV-1), 558G>T (HSV-2), 641A>G (HSV-2), 715T>C (HSV-1), 938T>C (HSV-2), 437_438 insA (HSV-1), 169delC (HSV-1), 170delC (HSV-1), 171delC (HSV-1), 172delC (HSV-1), 458delC (HSV-2), 459delC (HSV-2), 460delC (HSV-2), 461delC (HSV-2), 881delC (HSV-1), 882delC (HSV-1), 883delC (HSV-1), 884delC (HSV-1), and 885delC (HSV-1); and b. administering to said subject a drug selected from foscarnet, cidofovir drug and docosanol; or other non-penciclovir and/or non-acyclovir drug.
18. An antiviral drug for use in a method for treating an infection of an antiviral drug-resistant HSV (e.g. HSV-1 and/or HSV-2), wherein said method comprises: a. identifying the presence or absence of one or more HSV mutation in a database comprising DNA Pol mutations 2405T>G (HSV-1), 2500G>T (HSV-1), 2515A>G (HSV-1), 2892_2893 insT (HSV-1), 2893_2894 insT (HSV-1), 2894_2895 insT (HSV-1), and 2895_2896 insT (HSV-1); b. confirming the presence of said one or more HSV mutation(s); and c. administering to said subject a drug selected from a docosanol drug, BAY 54-6322, ASP2151 and BAY 57-1293; or other non-foscarnet drug, non-cidofovir drug, non-penciclovir and/or non-acyclovir drug.
19. An antiviral drug for use in a method for treating an infection of an antiviral drug-resistant HSV (e.g. HSV-1 and/or HSV-2), wherein said method comprises: a. confirming an antiviral drug-resistant HSV comprises a DNA Pol mutation selected from 2405T>G (HSV-1), 2500G>T (HSV-1), 2515A>G (HSV-1), 2892_2893 insT (HSV-1), 2893_2894 insT (HSV-1), 2894_2895 insT (HSV-1), and 2895_2896 insT (HSV-1); and b. administering to said subject a drug selected from a docosanol drug, BAY 54-6322, ASP2151 and BAY 57-1293; or other non-foscarnet drug, non-cidofovir drug, non-penciclovir and/or non-acyclovir drug.
20. An antiviral drug for use in a method for treating an infection of an antiviral drug-resistant HSV (e.g. HSV-1 and/or HSV-2), wherein said method comprises: a. identifying the presence or absence of one or more HSV polymorphism in a database comprising TK polymorphisms E84K (HSV-2), Q34* (HSV-1), Q90* (HSV-2), Q125* (HSV-1), L49R (HSV-1), M1211 (HSV-1), 1166N (HSV-1), Q186H (HSV-2), H214R (HSV-2), E146fs (HSV-1), D228* (HSV-1), Y239H (HSV-1), L313S (HSV-2), T183* (HSV-1), H58fs (HSV-1), M85* (HSV-1), P154fs (HSV-2), M183* (HSV-2), A294fs (HSV-1), P295fs (HSV-1), and E296fs (HSV-1); b. confirming the presence said one or more HSV polymorphism(s); and c. administering to said subject a drug selected from foscarnet, cidofovir drug and docosanol; or other non-penciclovir and/or non-acyclovir drug.
21. An antiviral drug for use in a method for treating an infection of an antiviral drug-resistant HSV (e.g. HSV-1 and/or HSV-2), wherein said method comprises: a. confirming an antiviral drug-resistant HSV comprises a TK polymorphism selected from E84K (HSV-2), Q34* (HSV-1), Q90* (HSV-2), Q125* (HSV-1), L49R (HSV-1), M1211 (HSV-1), 1166N (HSV-1), Q186H (HSV-2), H214R (HSV-2), E146fs (HSV-1), D228* (HSV-1), Y239H (HSV-1), L313S (HSV-2), T183* (HSV-1), H58fs (HSV-1), M85* (HSV-1), P154fs (HSV-2), M183* (HSV-2), A294fs (HSV-1), P295fs (HSV-1), and E296fs (HSV-1); and b. administering to said subject a drug selected from foscarnet, cidofovir drug and docosanol; or other non-penciclovir and/or non-acyclovir drug.
22. An antiviral drug for use in a method for treating an infection of an antiviral drug-resistant HSV (e.g. HSV-1 and/or HSV-2), wherein said method comprises: a. identifying the presence or absence of one or more HSV polymorphism in a database comprising DNA Pol polymorphisms L802R (HSV-1), A834S (HSV-1), T839A (HSV-1), F965_I966insF (HSV-1), and 1966* (HSV-1); b. confirming the presence of said one or more HSV polymorphism(s); and c. administering to said subject a drug selected from a docosanol drug, BAY 54-6322, ASP2151 and BAY 57-1293; or other non-foscarnet drug, non-cidofovir drug, non-penciclovir and/or non-acyclovir drug.
23. An antiviral drug for use in a method for treating an infection of an antiviral drug-resistant HSV (e.g. HSV-1 and/or HSV-2), wherein said method comprises: a. confirming an antiviral drug-resistant HSV comprises a DNA Pol polymorphism selected from L802R (HSV-1), A834S (HSV-1), T839A (HSV-1), F965_I966insF (HSV-1), and 1966* (HSV-1); and b. administering to said subject a drug selected from a docosanol drug, BAY 54-6322, ASP2151 and BAY 57-1293; or other non-foscarnet drug, non-cidofovir drug, non-penciclovir and/or non-acyclovir drug.
24. The method or use according to any one of the preceding claims, wherein the TK and/or DNA pol mutation, and/or TK and/or DNA pol polymorphism, is comprised within an HSV virus.
25. The method or use according to any one of the preceding claims, wherein the HSV is HSV-1.
26. The method or use according to any one of claims 1-24, wherein the HSV is HSV-2.
27. The method or use according to any one of the preceding claims, wherein said TK and/or DNA pol mutation is absent from a reference (e.g. wild-type) TK and/or DNA pol sequence; or wherein said TK and/or DNA pol polymorphism is absent from a reference (wild-type) TK and/or DNA pol sequence; preferably wherein a TK and/or DNA pol comprising said reference sequence is inhibited by one or more antiviral drug.
28. The method or use according any one of claim 1-25 or 27, wherein the TK mutation and/or TK polymorphism is relative to a reference (wild-type) HSV TK sequence, and wherein the reference HSV-1 TK sequence comprises: a. the nucleic acid sequence of SEQ ID NO: 1, or a sequence having at least 90% homology thereto; and/or b. the polypeptide sequence of SEQ ID NO: 2, or a sequence having at least 90% homology thereto.
29. The method or use according any one of claim 1-25, or 27, wherein the DNA Pol mutation and/or DNA Pol polymorphism is relative to a reference (wild-type) HSV DNA Pol sequence, and wherein the reference HSV-1 DNA Pol sequence comprises: a. the nucleic acid sequence of SEQ ID NO: 3, or a sequence having at least 90% homology thereto; and/or b. the polypeptide sequence of SEQ ID NO: 4, or a sequence having at least 90% homology thereto.
30. The method or use according to any one of claim 1-24, 26 or 27, wherein the TK mutation and/or TK polymorphism is relative to a reference (wild-type) HSV TK sequence, and wherein the reference HSV-2 TK sequence comprises: a. the nucleic acid sequence of SEQ ID NO: 5, or a sequence having at least 90% homology thereto; and/or b. the polypeptide sequence of SEQ ID NO: 6, or a sequence having at least 90% homology thereto.
31. The method or use according to any one of claim 1-24, 26 or 27, wherein the DNA Pol mutation and/or DNA Pol polymorphism is relative to a reference (wild-type) HSV DNA Pol sequence, and wherein the reference HSV-2 DNA pol sequence comprises: a. the nucleic acid sequence of SEQ ID NO: 7, or a sequence having at least 90% homology thereto; and/or b. the polypeptide sequence of SEQ ID NO: 8, or a sequence having at least 90% homology thereto.
32. The method or use according to any one of the preceding claims, wherein the antiviral drug-resistant HSV is resistant to one or more antiviral drug selected from acyclovir, penciclovir, foscarnet and cidofovir; or wherein the antiviral drug-resistant HSV is resistant to acyclovir and penciclovir.
33. The method or use according to any one the preceding claims, further comprising correlating one or more of said mutation with a corresponding resistance phenotype associated with said mutation to determine the level of antiviral drug resistance of the antiviral drug-resistant HSV comprising said mutation, wherein the level of antiviral drug resistance is selected from weak (e.g. intermediate) resistance and strong resistance.
34. The method or use according to claim 33, wherein said correlating step is conducted with/using an algorithm, preferably wherein the algorithm is trained on the corresponding resistance phenotype for one or more of said mutation.
35. The method or use according to claim 33 or claim 34, wherein said corresponding resistance phenotype is a resistance phenotype determined via a plaque reduction assay.
36. The method or use according to any one of the preceding claims, comprising detecting the presence or absence of an antiviral drug-resistant HSV in a sample, preferably wherein said one or more HSV mutation and/or HSV polymorphism is identified in an isolated sample obtained from a subject.
37. The method or use according to claim 36, wherein the sample is selected from one or more of a lesion or part thereof, a viral suspension, cerebrospinal fluid, skin, mucous, blood, blood serum, sputum, urine, and/or synovial fluid.
38. The method or use according to claim 36 or claim 37, wherein the sample is obtained from an immunocompromised subject.
39. The method or use according to any one of the preceding claims, wherein said one or more HSV mutation and/or HSV polymorphism is identified by DNA sequencing, sequence capture, mass spectrometry, Western Blot, Enzyme activity assay and/or Enzyme-Linked Immunosorbent Assay (ELISA).
40. The method or use according to any one of the preceding claims, wherein said one or more HSV mutation is identified by DNA sequencing.
41. The method or use according to any one of claims 1-40, wherein said one or more HSV mutation and/or HSV polymorphism is identified by mass spectrometry.
42. The method or use according to any one of the preceding claims, further comprising the step of recording on a suitable data carrier, the data obtained in the step of identifying one or more HSV mutation and/or HSV polymorphism.
43. A data carrier comprising the data obtained in the step of identifying one or more HSV mutation and/or HSV polymorphism according to any one of claims 1-42.
44. A data carrier according to claim 43 for use in a method for diagnosing an infection with an antiviral drug-resistant HSV.
45. A recombinant HSV or fragment thereof comprising a mutation selected from: (i) a TK mutation selected from 100C>T (HSV-1), 268C>T (HSV-2), 373C>T (HSV-1), 146T>G (HSV-1), 250G>A (HSV-2), 363G>A (HSV-1), 497T>A (HSV-1), 558G>T (HSV-2), 641A>G (HSV-2), 715T>C (HSV-1), 938T>C (HSV-2), 437_438 insA (HSV-1), 169delC (HSV-1), 170delC (HSV-1), 171delC (HSV-1), 172delC (HSV-1), 458delC (HSV-2), 459delC (HSV-2), 460delC (HSV-2), 461delC (HSV-2), 881delC (HSV-1), 882delC (HSV-1), 883delC (HSV-1), 884delC (HSV-1), and 885delC (HSV-1); and (ii) a DNA pol mutation selected from 2405T>G (HSV-1), 2500G>T (HSV-1), 2515A>G (HSV-1), 2892_2893 insT (HSV-1), 2893_2894 insT (HSV-1), 2894_2895 insT (HSV-1), and 2895_2896insT (HSV-1).
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0256] Embodiments of the invention will now be described, by way of example only, with reference to the following Figures and Examples.
[0257]
EXAMPLES
Materials & Methods
Subjects
[0258] The samples tested and validated for resistance were obtained from Ireland, London, the midlands, north, south and east of England, Scotland and Wales. Thus, the results provided are representative of HSV strains present over widespread geographic regions. Samples were isolated from patients infected with a HSV and who did not respond (e.g. respond sufficiently) to treatment with one or more antiviral drugs (e.g. acyclovir, penciclovir, foscarnet, cidofovir).
Sample Preparation
[0259] The viruses were typically isolated from clinical swabs taken from patient lesions. The viruses were either cultured directly from the patient samples, or the virus or relevant segments thereof were amplified and cloned into recombinant vectors (e.g. using unique restriction sites or by homologous recombination). The recombinant vectors are then typically introduced into cells by transfection to produce recombinant and/or pseudotyped viruses.
[0260] Isolated viruses are typically titrated e.g. using monolayers of African green monkey kidney cells (Vero cells).
[0261] The samples were anonymized e.g. by removal of any patient identifiable information and assignment of a non-specific project number.
Detection of Mutation
[0262] Mutations were typically detected by DNA sequencing.
[0263] To prepare viral DNA for sequencing, confluent monolayers of cells (e.g. Vero cells) were infected at 5 PFUs/cell for 24 to 48 hours, until cytopathic effect became apparent. Viral supernatants were harvested by freeze-thawing and passed through a 0.45 μM filter. Four ml of the virus supernatant was incubated with 20 U/ml DNase (Promega) for 3 hours at 37° C. before loading onto a 1.5 ml 20% sucrose cushion and centrifuged at 100,000 g for 1 hour. Viral pellets were re-suspended in TE containing DNase chelator and extracted with the Pure-Link Viral RNA/DNA Mini Kit (Invitrogen) following manufacturer's instructions. Alternatively, viral DNA was extracted directly from a clinical sample using QIAamp DNA mini kit (Qiagen) or Pure-Link RNA/DNA mini kit (Invitrogen).
[0264] Regions of interest (e.g. the UL23 and/or UL30 genes) were then amplified by PCR. Primers which flank the UL23 and/or UL30 genes were typically used, although primers designed to amplify only a fragment of said gene in which a polymorphism may be expected to occur may also be suitable. The resulting amplicons were then sequenced using typical DNA sequencing technology available to the skilled person. The DNA sequences are then used to determine the corresponding polypeptide sequence.
[0265] The nucleotide and/or amino acid changes in the amplified sequence are detected by comparison with wild-type reference strains (or pretreatment sequences). Suitable wild-type reference strains include GenBank accession number JN555585.1 and JQ673480.1 as reference for HSV-1; and Z86099.2 and JN561323.2 as reference for HSV-2.
Plaque Reduction Assay
[0266] Viral sample were typically phenotypically characterised using the plaque reduction assay.
[0267] Viral isolates were used to infect a sub-confluent monolayer of Vero cells at a concentration of 75 or 50 plaque forming units (PFU) per well for HSV-1 and HSV-2, respectively. After 1 hour incubation at 37° C. cells were overlaid with CMC medium (4% Carboxymethyl cellulose in PBS) containing a serial dilution of the antiviral drugs acyclovir (ACV), pencyclovir (PCV), cidofovir (CDV) and foscarnet (FOS) or CMC alone, as a no drug control, and incubated for a further 72 hours until plaques became apparent. CMC medium typically used to create a semisolid interface and prevents indiscrimate viral spreading. Other materials (e.g. agar) may also be used to create this interface.
[0268] Cells were fixed with 10% formalin and stained with crystal violet before enumeration of the plaques (microscopic observation or use of fluorescent antibodies for detection is also suitable). The data obtained from these experiments were then expressed as percent inhibition of viral infectivity relative to the no drug control. The data was then used to determine IC.sub.50 values for all four drugs using linear regression—i.e. dose-response curves were constructed from which the drug concentrations required to inhibit virus replication by 50% (IC.sub.50) were determined.
[0269] Definitions of phenotypic drug susceptibility classification as sensitive or resistant were as follows: ACV, <3 μM or >40 μM; PCV, <10 μM or >40 μM; CDV, <24 μM or >30 μM; FOS, <250 μM or >400 μM; and ACV, <6.5 μM or >40 μM; PCV, <38 μM or >40 μM; FOS, <250 μM or >400 μM for HSV-1 and HSV-2, respectively. Any IC.sub.50 values falling in between these sensitive and resistant cut-offs were reported as intermediate resistance.
[0270] The ratio of the IC.sub.50 of patient-derived virus may also be divided by that of a wild-type reference virus (e.g. a virus known to be susceptible to the drug under investigation) to provide a fold change and determine the susceptibility of the patient-derived virus. A fold change greater than one means that the patient-derived virus is less susceptible to that particular drug compared with wild-type virus and vice versa.
[0271] A fold change greater than one does not necessarily mean that the patient will not respond to the treatment; therefore, IC.sub.50 or fold change cutoff values have to be determined for each drug at which a patient-derived virus is considered to be susceptible or resistant. Different types of cutoff values can be used but the most pertinent is the ‘clinical cutoff’ which takes into consideration the relationship between IC.sub.50 or fold change values and virological response or clinical outcome.
Example 1
Detecting Mutations
[0272] Viral samples were isolated from patients suspected of being infected with an antiviral-drug resistant HSV. The mutation (where relevant) resulting in resistance was detected by DNA sequencing as described above. Aligning the TK and/or DNA pol sequence of the antiviral-resistant virus with that of a wild-type reference sequence demonstrated the relevant mutation.
Example 2
Phenotypes of Detected TK and DNA Pol Mutations/Polymorphisms
[0273] The phenotype (i.e. reactivity to a drug) of the virus was characterised by the plaque reduction assay as described above. The present inventors have characterised the resistance-causing phenotype of a large number of TK and DNA pol mutations. Numerous natural polymorphisms (which do not cause drug resistance) have also been detected and characterised. The mutations, polymorphisms and associated phenotypes are demonstrated in Tables 4-7.
[0274] Definitions of phenotypic drug susceptibility classification: Acyclovir (ACV): <3 uM, 3-40 uM, >40 uM; Pencyclovir (PCV): <10 uM, 10-40 uM, >40 uM; Foscarnet (FOS): <250 uM, 250-400 uM, >400 uM; Cidofovir (CDV): <24 uM, 24-30 uM, >30 uM for HSV-1 and ACV: <6.5 μM, 6.5-40 μM, >40 μM; PCV: <38 μM, 38-40 μM, >40 μM; FOS: <250 μM, 250-400 μM, >400 μM for HSV-2, for sensitive, intermediate and resistant samples, respectively.
[0275] Table 4 presents ‘natural’ polymorphisms and resistance-associated substitutions and indels in HSV-1 TK gene and their effect on antiviral susceptibility. R=resistance (ACV and PCV IC.sub.50>40 μM; FOS IC.sub.50>400 μM; CDV IC.sub.50>30 μM); IR=intermediate resistance (ACV IC.sub.50>3 μM<40 μM; PCV IC.sub.50>10 μM<40 μM; FOS IC.sub.50>250<400 μM; CDV IC.sub.50>24 μM<30 μM); S=susceptible (ACV IC.sub.50<3 μM; PCV IC.sub.50<10 μM; FOS IC.sub.50<250 μM; CDV IC.sub.50<24 μM); ND=not done; .sup.amutation in non-conserved region in a sample which also contains Y239H in TK gene at a position associated with resistance; .sup.bsample also contains S914L and A1169AT mutations in non-conserved region of DNA pol gene; % ample also contains A1169AT mutations in non-conserved region of DNA pol gene; .sup.dsample also contains known resistance-associated mutation T287M in TK gene.
TABLE-US-00005 TABLE 4 Amino Drug susceptibility (IC.sub.50 value) Study acid Nucleotide Aciclovir Penciclovir Foscarnet Cidofovir ID no. change change (ACV) (PCV) (FOS) (CDV) HPG052 Q34* 100C>T IR (8.45 μM) IR (25 μM) S (76.9 μM) S (4.77 μM) HPG050 Q34* 100C>T R (>40 μM) R (>160 μM) S (169 μM) ND HPG005 A37V 110C>T S (1.46 μM) S (5.19 μM) S (36.9 μM) S (2.21 μM) HPG045 L49R 146T>G IR (31.5 μM) R (>160 μM) S (75 μM) S (<1.56 μM) HPG037 L69M.sup.a 205>A IR (24.8 μM) R (>160 μM) S (80 μM) S (4.39 μM) HPG013 M121I 363G>A IR (18.77 μM) R (105.29 μM) S (77.98 μM) S (3.11 μM) HPG010 Q125* 373C>T IR (9.21 μM) R (87.14 μM) S (46.66 μM) S (<1.56 μM) HPG011 Q125* 373C>T IR (13.85 μM) R (108.24 μM) S (55.95 μM) S (<1.56 μM) HPG046 I166N 497T>A IR (11.4 μM) R (98.5 μM) S (<50 μM) S (2.1 μM) HPG029 Y172S 515A>C R (>40 μM) R (>160 μM) S (110.81 μM) S (2.99 μM) HPG003 Y172S 515A>C IR (28 μM) R (106 μM) S (79 μM) S (<1.56 μM) HPG005 A192T 574G>A S (1.46 μM) S (5.19 μM) S (<50 μM) S (2.21 μM) HPG008 V204G 611T>G IR (22.5 μM) R (>160 μM) S (58.33 μM) S (3.56 μM) HPG027 V204G.sup.b 611T>G R (>40 μM) R (>160 μM) S (216 μM) S (3.93 μM) HPG055 V204G.sup.c 611T>G IR (5.1 μM) S (9.34 μM) S (<50 μM) S (<1.56 μM) HPG037 Y239H 715T>C IR (24.8 μM) R (>160 μM) S (80 μM) S (4.39 μM) HPG037 R256W 766C>T IR (24.8 μM) R (>160 μM) S (80 μM) S (4.39 μM) HPG033 I358L.sup.d 1072A>C IR (38.9 μM) R (>160 μM) S (88.3 μM) S (15 μM) HPG039 85* or 169delC, 170delC, IR (25.6 μM) R (158 μM) S (59.8 μM) S (2.97 μM) H58fs 171delC, or 172delC HPG043 183* 437_438insA (or IR (28.5 μM) R (>160 μM) S (69 μM) S (1.65 μM) E146fs or D228*) HPG032 183* 437_438insA (or IR (27.4 μM) R (>160 μM) S (100 μM) S (3.61 μM) E146fs or D228*) HPG018 frameshift 881delC, 882delC, IR (24.7 μM) R (117 μM) S (66.7 μM) S (3.03 μM) 883delC, 884delC, or 885delC HPG042 frameshift 881delC, 882delC, IR (>40 μM) R (>160 μM) S (60.7 μM) S (<1.56 μM) 883delC, 884delC, or 885delC
[0276] Table 5 presents ‘natural’ polymorphisms and resistance-associated substitutions and indels in HSV-2 TK gene and their effect on antiviral susceptibility. R=resistance (ACV and PCV IC.sub.50>40 μM; FOS IC.sub.50>400 μM); IR=intermediate resistance (ACV IC.sub.50>6.5 μM<40 μM; PCV IC.sub.50>38 μM<40 μM; FOS IC.sub.50>250<400 μM); S=susceptible (ACV IC.sub.50<6.5 μM; PCV IC.sub.50<38 μM; FOS IC.sub.50<250 μM); ND=not done; .sup.bmutation in non-conserved region of TK gene but virus was not isolated and therefore phenotypic drug susceptibility testing could not be performed
TABLE-US-00006 TABLE 5 Amino Drug susceptibility (IC.sub.50 value) Study acid Nucleotide Aciclovir Penciclovir Foscarnet Cidofovir ID no. change change (ACV) (PCV) (FOS) (CDV) HPG038 R34C 100C>T S (2.04 μM) S (9.64 μM) S (73.7 μM) ND HPG036 E84K 250G>A R (>40 μM) R (>160 μM) S (78.75 μM) ND HPG044 M86L 256A>C R (>40 μM) R (>160 μM) S (73.8 μM) ND HPG048 Q90stop 268C>T R (>40 μM) R (>160 μM) S (56.9 μM) ND HPG030 Q90stop 268C>T R (>40 μM) R (>160 μM) S (92.3 μM) ND HPG001 A125T 373G>A S (1.53 μM) S (8.2 μM) S (236 μM) ND HPG023 Q186H 558G>T IR (12.08 μM) S (31.07 μM) S (77.98 μM) ND HPG017 Q186H 558G>T IR (10.23 μM) R (64.67 μM) S (117.82 μM) ND HPG022 E213D 639A>C S (4.46 μM) S (26.86 μM) S (82.05 μM) ND HPG024 E213D 639A>C S (2.5 μM) S (9.08 μM) S (88.72 μM) ND HPG034 L313S 938A>C IR (35.8 μM) R (>160 μM) S (73.5 μM) ND HPG049 L365P.sup.b 1094T>C ND ND ND ND HPG057 98stop, or 276delG, 278delG R (>40 μM) R (>160 μM) S (98.5 μM) ND A94fs 279delG, or 280delG HPG028 98stop, or 276delG, 278delG R (>40 μM) R (>160 μM) S (98.8 μM) ND A94fs 279delG, or 280delG HPG058 183stop, or 458delC, 459delC, IR (36.2 μM) R (>160 μM) S (59 μM) ND P154fs 460delC, or 461delC
[0277] Table 6 presents ‘natural’ resistance-associated substitutions and indels in HSV-1 pol gene and their effect on antiviral susceptibility. R=resistance (ACV and PCV IC.sub.50>40 μM; FOS IC.sub.50>400 μM; CDV IC.sub.50>30 μM); IR=intermediate resistance (ACV IC.sub.50>3 μM<40 μM; PCV IC.sub.50>10 μM<40 μM; FOS IC.sub.50>250<400 μM; CDV IC.sub.50>24 μM<30 μM); S=susceptible (ACV IC.sub.50<3 μM; PCV IC.sub.50<10 μM; FOS IC.sub.50<250 μM; CDV IC.sub.50<24 μM). Also contain A1169T mutation in non-conserved region; .sup.amutation in non-conserved region in a sample which also contains the deletion (C) nt 881-885 in TK gene associated with resistance; .sup.bmutation in non-conserved region in a sample which also contains the mutation M1211 in TK gene; .sup.cmutation in non-conserved region in a sample which also contains the mutation R176stop in TK gene associated with resistance; .sup.amutation in non-conserved region in a sample which also contains the mutation Q261stop in TK gene associated with resistance; .sup.amutation in non-conserved region in a sample which also contains the deletion (G) nt 430-436 in TK gene associated with resistance; .sup.fmutation in non-conserved region in a sample which also contains the mutation S724N in DNA pol gene associated with resistance; .sup.gmutation in non-conserved region in a sample which also contains the mutation L49R in TK gene; .sup.imutation in non-conserved region in a sample which also contains the deletion (C) nt 548-553 in TK gene associated with resistance; .sup.jmutation in non-conserved region in a sample which also contains deletion (G) nt 430-436 in TK gene associated with resistance; .sup.kmutation in non-conserved region in a sample which also contains deletion (G) nt 430-436 in TK gene associated with resistance; .sup.lmutations in conserved region (and for L802R on site associated with resistance) in a sample which also contains mutation R176W in TK gene associated with resistance; .sup.mmutation in conserved region in a sample which also contains deletion (G) nt 430-436 in TK gene associated with resistance; .sup.nmutation in non-conserved region in a sample which also contains mutation R216C in TK gene associated with resistance; .sup.omutation in non-conserved region in a sample which also contains mutation deletion (C) nt 896-900 in TK gene associated with resistance; .sup.p mutation in non-conserved region in a sample which also contains mutation V204G in TK gene associated with resistance; .sup.qmutation in non-conserved region in a sample which also contains mutation A93V in TK gene associated with resistance; .sup.rmutation in non-conserved region in a sample which also contains mutation insert (G) nt 430-436 in TK gene associated with resistance; .sup.smutation in non-conserved region in a sample which also contains mutation T287M in TK gene associated with resistance; .sup.tmutation in non-conserved region in a sample which also contains mutation deletion (C) nt 881-885 in TK gene, also Q substitution at this site is a known polymorphism
TABLE-US-00007 TABLE 6 Drug susceptibility (IC.sub.50 value) Amino Study acid Nucleotide Aciclovir Penciclovir Foscarnet Cidofovir ID no. change change (ACV) (PCV) (FOS) (CDV) HPG042.sup.‡ G22R.sup.a 64G>A R (>40 μM) R (>160 μM) S (60.7 μM) S (<1.56 μM) HPG014 T54A 160A>G S (1.41 μM) S (5.66 μM) S (140.54 μM) S (17.24 μM) HPG013.sup.‡ D83A.sup.b 248A>C IR (18.77 μM) R (105.29 μM) S (77.98 μM) S (3.11 μM) HPG051 D83A.sup.c 248A>C IR (29.2 μM) R (>160 μM) S (82.6 μM) S (4.94 μM) HPG006 G121S.sup.d 361G>A R (>40 μM) R (>160 μM) S (115 μM) S (6.14 μM) HPG031 G139R.sup.e 415G>C or IR (27.65 μM) R (155.68 μM) S (108.47 μM) S (4.8 μM) 415G>A HPG035 S239L.sup.f 716C>T IR (5.43 μM) IR (14.6 μM) R (701 μM) R (39.58 μM) HPG045.sup.‡ L419I.sup.g 1255C>A IR (31.5 μM) R (>160 μM) S (75 μM) S (<1.56 μM) HPG047.sup.‡ E680A.sup.i 2039A>C IR (22.43 μM) R (>160 μM) S (92.36 μM) S (2.88 μM) HPG021 R681Q.sup.j 2042G>A R (>40 μM) R (>160 μM) S (156 μM) S (<1.56 μM) HPG019.sup.‡ K750T.sup.k 2249A>C R (>40 μM) R (>160 μM) S (190 μM) S (20.35 μM) HPG053 L802R + 2405T>G R (>40 μM) R (>160 μM) R (625.97 μM) S (7.72 μM) A834S.sup.l 2500G>T HPG021 T839A.sup.m 2515A>G R (>40 μM) R (>160 μM) S (156 μM) S (<1.56 μM) HPG025 E850Q.sup.n 2548G>C IR (22.2 μM) R (118 μM) S (99.1 μM) S (<1.56 μM) HPG026.sup.‡ E850Q.sup.n 2548G>C IR (18.13 μM) IR (35 μM) S (68.3 μM) S (<1.56 μM) HPG020.sup.‡ S911N 2732G>A S (0.56 μM) S (2.48 μM) S (99.3 μM) S (4.38 μM) HPG041.sup.‡ S911N 2732G>A S (0.5 μM) S (3 μM) S (89.51 μM) S (17.97 μM) HPG040.sup.‡ S911N.sup.o 2732G>A R (>40 μM) R (>160 μM) S (137 μM) S (11.3 μM) HPG053.sup.‡ S911N.sup.l 2732G>A R (>40 μM) R (>160 μM) R (625.97 μM) S (7.72 μM) HPG027.sup.‡ S914L.sup.p 2741C>T R (>40 μM) R (>160 μM) S (216 μM) S (3.93 μM) HPG042.sup.‡ A972V.sup.a 2915C>T R (>40 μM) R (>160 μM) S (60.7 μM) S (<1.56 μM) HPG012 G985E.sup.q 2954G>A R (>40 μM) IR (9.67 μM) S (92.13 μM) S (2.84 μM) HPG002.sup.‡ E992K.sup.r 2974G>A IR (21.6 μM) R (>160 μM) S (<50 μM) S (6.09 μM) HPG009.sup.‡ E992K 2974G>A S (1.3 μM) S (4.05 μM) S(77.47 μM) S (9.44 μM) HPG054.sup.‡ E992K.sup.r 2974G>A IR (8.25 μM) R (>160 μM) S (52.9 μM) S (3.85 μM) HPG014 R993C 2977C>T S (1.41 μM) S (5.66 μM) S (140.54 μM) S (17.24 μM) HPG015 966stop, or 2892_2893insT, S (2.4 μM) S (8.48 μM) S (162 μM) R (45.08 μM) F965_I9 2893_2894insT, 66insF 2894_2895insT, or 2895_2896insT HPG033.sup.‡ N1046T.sup.s 3137A>C IR (38.9 μM) R (>160 μM) S (88.3 μM) S (15 μM) HPG014 A1115T 3343G>A S (1.41 μM) S (5.66 μM) S (140.54 μM) S (17.24 μM) HPG031 E1120A.sup.e 3359A>C IR (27.65 μM) R (155.68 μM) S (108.47 μM) S (4.8 μM) HPG016 A1169T 3505G>A S (3.55 μM) S (7.19 μM) S (154.4 μM) S (16.52 μM) HPG018.sup.‡ P1199T.sup.t 3595C>A IR (24.7 μM) R (117 μM) S (66.7 μM) S (3.03 μM)
[0278] Table 7 presents resistance-associated substitutions in HSV-2 pol gene and their effect on antiviral susceptibility. R=resistance (ACV and PCV IC.sub.50>40 μM; FOS IC.sub.50>400 μM); IR=intermediate resistance (ACV IC.sub.50>6.5 μM<40 μM; PCV IC.sub.50>38 μM<40 μM; FOS IC.sub.50>250<400 μM); S=susceptible (ACV IC.sub.50<6.5 μM; PCV IC.sub.50<38 μM; FOS IC.sub.50<250 μM); .sup.amutation in non-conserved region in a sample which also contains mutation S729N in DNA pol associated with resistance; .sup.bmutation in non-conserved region in a sample which also contains mutation Q90stop in TK gene; .sup.cmutation in non-conserved region in a sample which also contains mutation deletion (G) nt 276-280 in TK gene.
TABLE-US-00008 TABLE 7 Amino Drug susceptibility (IC.sub.50 value) Study acid Nucleotide Aciclovir Penciclovir Foscarnet Cidofovir ID no. change change (ACV) (PCV) (FOS) (CDV) HPG022 D174Y 520G>T S (4.46 μM) S (26.86 μM) S (82.05 μM) ND HPG056 M447L 1339A>C S (1.56 μM) S (8.61 μM) S (88.05 μM) ND HPG004 M494T.sup.a 1481T>C R (>40 μM) R (>160 μM) IR (348.71 μM) ND HPG007 R628G 1882C>G IR (28.6 μM) R (>160 μM) R (783 μM) ND HPG001 H714R 2141A>G S (1.53 μM) S (8.2 μM) S (236 μM) ND HPG048 E761K.sup.b 2281G>A R (>40 μM) R (>160 μM) S (56.9 μM) ND HPG030 E761K.sup.b 2281G>A R (>40 μM) R (>160 μM) S (92.3 μM) ND HPG028 R775C.sup.c; 2323C>T; R (>40 μM) R (>160 μM) S (98.8 μM) ND R775W.sup.c 2325C>G HPG028 E776Q.sup.c 2326G>C R (>40 μM) R (>160 μM) S (98.8 μM) ND
[0279] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.
TABLE-US-00009 SEQUENCES SEQ ID NO: 1 (HSV-1 TK nucleic acid sequence) ATGGCTTCGTACCCCTGCCATCAACACGCGTCTGCGTTCGACCAGGCTGCGCGTTCTC GCGGCCATAACAACCGACGTACGGCGTTGCGCCCTCGCCGGCAGCAAAAAGCCACGG AAGTCCGCCTGGAGCAGAAAATGCCCACGCTACTGCGGGTTTATATAGACGGTCCCCA CGGGATGGGGAAAACCACCACCACGCAACTGCTGGTGGCCCTGGGTTCGCGCGACGA TATCGTCTACGTACCCGAGCCGATGACTTACTGGCGGGTGTTGGGGGCTTCCGAGACA ATCGCGAACATCTACACCACACAACACCGCCTCGACCAGGGTGAGATATCGGCCGGGG ACGCGGCGGTGGTAATGACAAGCGCCCAGATAACAATGGGCATGCCTTATGCCGTGAC CGACGCCGTTCTGGCTCCTCATATCGGGGGGGAGGCTGGGAGCTCACATGCCCCGCC CCCGGCCCTCACCCTCATCTTCGACCGCCATCCCATCGCCGCCCTCCTGTGCTACCCG GCCGCGCGATACCTTATGGGCAGCATGACCCCCCAGGCCGTGCTGGCGTTCGTGGCC CTCATCCCGCCGACCTTGCCCGGCACAAACATCGTGTTGGGGGCCCTTCCGGAGGACA GACACATCGACCGCCTGGCCAAACGCCAGCGCCCCGGCGAGCGGCTTGACCTGGCTA TGCTGGCCGCGATTCGCCGCGTTTATGGGCTGCTTGCCAATACGGTGCGGTATCTGCA GGGCGGCGGGTCGTGGCGGGAGGATTGGGGACAGCTTTCGGGGGCGGCCGTGCCGC CCCAGGGTGCCGAGCCCCAGAGCAACGCGGGCCCACGACCCCATATCGGGGACACGT TATTTACCCTGTTTCGGGCCCCCGAGTTGCTGGCCCCCAACGGCGACCTGTATAACGT GTTTGCCTGGGCTTTGGACGTCTTGGCCAAACGCCTCCGTCCCATGCACGTCTTTATCC TGGATTACGACCAATCGCCCGCCGGCTGCCGGGACGCCCTGCTGCAACTTACCTCCGG GATGGTCCAGACCCACGTCACCACCCCAGGCTCCATACCGACGATCTGCGACCTGGCG CGCACGTTTGCCCGGGAGATGGGGGAGGCTAACTGA SEQ ID NO: 2 (HSV-1 TK polypeptide sequence) MASYPCHQHASAFDQAARSRGHNNRRTALRPRRQQKATEVRLEQKMPTLLRVYIDGPHG MGKTTTTQLLVALGSRDDIVYVPEPMTYWRVLGASETIANIYTTQHRLDQGEISAGDAAVVM TSAQITMGMPYAVTDAVLAPHIGGEAGSSHAPPPALTLIFDRHPIAALLCYPAARYLMGSMT PQAVLAFVALIPPTLPGTNIVLGALPEDRHIDRLAKRQRPGERLDLAMLAAIRRVYGLLANTV RYLQGGGSWREDWGQLSGAAVPPQGAEPQSNAGPRPHIGDTLFTLFRAPELLAPNGDLYN VFAWALDVLAKRLRPMHVFILDYDQSPAGCRDALLQLTSGMVQTHVTTPGSIPTICDLARTF AREMGEAN SEQ ID NO: 3 (HSV-1 DNA pol nucleic acid sequence) ATGTTTTCCGGTGGCGGCGGCCCGCTGTCCCCCGGAGGAAAGTCGGCGGCCAGGGCG GCGTCCGGGTTTTTTGCGCCCGCCGGCCCTCGCGGAGCCAGCCGGGGACCCCCGCCT TGTTTGAGGCAAAACTTTTACAACCCCTACCTCGCCCCAGTCGGGACGCAACAGAAGCC GACCGGGCCAACCCAGCGCCATACGTACTATAGCGAATGCGATGAATTTCGATTCATCG CCCCGCGGGTGCTGGACGAGGATGCCCCCCCGGAGAAGCGCGCCGGGGTGCACGAC GGTCACCTCAAGCGCGCCCCCAAGGTGTACTGCGGGGGGGACGAGCGCGACGTCCTC CGCGTCGGGTCGGGCGGCTTCTGGCCGCGGCGCTCGCGCCTGTGGGGCGGCGTGGA CCACGCCCCGGCGGGGTTCAACCCCACCGTCACCGTCTTTCACGTGTACGACATCCTG GAGAACGTGGAGCACGCGTACGGCATGCGCGCGGCCCAGTTCCACGCGCGGTTTATG GACGCCATCACACCGACGGGGACCGTCATCACGCTCCTGGGCCTGACTCCGGAAGGC CACCGGGTGGCCGTTCACGTTTACGGCACGCGGCAGTACTTTTACATGAACAAGGAGG AGGTCGACAGGCACCTACAATGCCGCGCCCCACGAGATCTCTGCGAGCGCATGGCCG CGGCCCTGCGCGAGTCCCCGGGCGCGTCGTTCCGCGGCATCTCCGCGGACCACTTCG AGGCGGAGGTGGTGGAGCGCACCGACGTGTACTACTACGAGACGCGCCCCGCTCTGT TTTACCGCGTCTACGTCCGAAGCGGGCGCGTGCTGTCGTACCTGTGCGACAACTTCTG CCCGGCCATCAAGAAGTACGAGGGTGGGGTCGACGCCACCACCCGGTTCATCCTGGA CAACCCCGGGTTCGTCACCTTCGGCTGGTACCGTCTCAAACCGGGCCGGAACAACACG CTAGCCCAGCCGCGGGCCCCGATGGCCTTCGGGACATCCAGCGACGTCGAGTTTAACT GTACGGCGGACAACCTGGCCATCGAGGGGGGCATGAGCGACCTACCGGCATACAAGC TCATGTGCTTCGATATCGAATGCAAGGCGGGGGGGGAGGACGAGCTGGCCTTTCCGGT GGCCGGGCACCCGGAGGACCTGGTCATCCAGATATCCTGTCTGCTCTACGACCTGTCC ACCACCGCCCTGGAGCACGTCCTCCTGTTTTCGCTCGGTTCCTGCGACCTCCCCGAAT CCCACCTGAACGAGCTGGCGGCCAGGGGCCTGCCCACGCCCGTGGTTCTGGAATTCG ACAGCGAATTCGAGATGCTGTTGGCCTTCATGACCCTTGTGAAACAGTACGGCCCCGA GTTCGTGACCGGGTACAACATCATCAACTTCGACTGGCCCTTCTTGCTGGCCAAGCTGA CGGACATTTACAAGGTCCCCCTGGACGGGTACGGCCGCATGAACGGCCGGGGCGTGT TTCGCGTGTGGGACATAGGCCAGAGCCACTTCCAGAAGCGCAGCAAGATAAAGGTGAA CGGCATGGTGAACATCGACATGTACGGGATTATAACCGACAAGATCAAGCTCTCGAGCT ACAAGCTCAACGCCGTGGCCGAAGCCGTCCTGAAGGACAAGAAGAAGGACCTGAGCTA TCGCGACATCCCCGCCTACTACGCCGCCGGGCCCGCGCAACGCGGGGTGATCGGCGA GTACTGCATACAGGATTCCCTGCTGGTGGGCCAGCTGTTTTTTAAGTTTTTGCCCCATCT GGAGCTCTCGGCCGTCGCGCGCTTGGCGGGTATTAACATCACCCGCACCATCTACGAC GGCCAGCAGATCCGCGTCTTTACGTGCCTGCTGCGCCTGGCCGACCAGAAGGGCTTTA TTCTGCCGGACACCCAGGGGCGATTTAGGGGCGCCGGGGGGGAGGCGCCCAAGCGT CCGGCCGCAGCCCGGGAGGACGAGGAGCGGCCAGAGGAGGAGGGGGAGGACGAGG ACGAACGCGAGGAGGGCGGGGGCGAGCGGGAGCCGGAGGGCGCGCGGGAGACCGC CGGCAGGCACGTGGGGTACCAGGGGGCCAGGGTCCTTGACCCCACTTCCGGGTTTCA CGTGAACCCCGTGGTGGTGTTCGACTTTGCCAGCCTGTACCCCAGCATCATCCAGGCC CACAACCTGTGCTTCAGCACGCTCTCCCTGAGGGCCGACGCAGTGGCGCACCTGGAG GCGGGCAAGGACTACCTGGAGATCGAGGTGGGGGGGCGACGGCTGTTCTTCGTCAAG GCTCACGTGCGAGAGAGCCTCCTCAGCATCCTCCTGCGGGACTGGCTCGCCATGCGAA AGCAGATCCGCTCGCGGATTCCCCAGAGCAGCCCCGAGGAGGCCGTGCTCCTGGACA AGCAGCAGGCCGCCATCAAGGTCGTGTGTAACTCGGTGTACGGGTTCACGGGAGTGCA GCACGGACTCCTGCCGTGCCTGCACGTTGCCGCGACGGTGACGACCATCGGCCGCGA GATGCTGCTCGCGACCCGCGAGTACGTCCACGCGCGCTGGGCGGCCTTCGAACAGCT CCTGGCCGATTTCCCGGAGGCGGCCGACATGCGCGCCCCCGGGCCCTATTCCATGCG CATCATCTACGGGGACACGGACTCCATCTTTGTGCTGTGCCGCGGCCTCACGGCCGCC GGGCTGACGGCCGTGGGCGACAAGATGGCGAGCCACATCTCGCGCGCGCTGTTTCTG CCCCCCATCAAACTCGAGTGCGAAAAGACGTTCACCAAGCTGCTGCTGATCGCCAAGA AAAAGTACATCGGCGTCATCTACGGGGGTAAGATGCTCATCAAGGGCGTGGATCTGGT GCGCAAAAACAACTGCGCGTTTATCAACCGCACCTCCAGGGCCCTGGTCGACCTGCTG TTTTACGACGATACCGTCTCCGGAGCGGCCGCCGCGTTAGCCGAGCGCCCCGCGGAG GAGTGGCTGGCGCGACCCCTGCCCGAGGGACTGCAGGCGTTCGGGGCCGTCCTCGTA GACGCCCATCGGCGCATCACCGACCCGGAGAGGGACATCCAGGACTTTGTCCTCACCG CCGAACTGAGCAGACACCCGCGCGCGTACACCAACAAGCGCCTGGCCCACCTGACGG TGTATTACAAGCTCATGGCCCGCCGCGCGCAGGTCCCGTCCATCAAGGACCGGATCCC GTACGTGATCGTGGCCCAGACCCGCGAGGTAGAGGAGACGGTCGCGCGGCTGGCCGC CCTCCGCGAGCTAGACGCCGCCGCCCCAGGGGACGAGCCCGCCCCCCCCGCGGCCC TGCCCTCCCCGGCCAAGCGCCCCCGGGAGACGCCGTCGCCTGCCGACCCCCCGGGA GGCGCGTCCAAGCCCCGCAAGCTGCTGGTGTCCGAGCTGGCCGAGGATCCCGCATAC GCCATTGCCCACGGCGTCGCCCTGAACACGGACTATTACTTCTCCCACCTGTTGGGGG CGGCGTGCGTGACATTCAAGGCCCTGTTTGGGAATAACGCCAAGATCACCGAGAGTCT GTTAAAAAGGTTTATTCCCGAAGTGTGGCACCCCCCGGACGACGTGGCCGCGCGGCTC CGGACCGCAGGGTTCGGGGCGGTGGGTGCCGGCGCTACGGCGGAGGAAACTCGTCG AATGTTGCATAGAGCCTTTGATACTCTAGCATGAGCCCCCCGTCGAAGCTGATGTCCCT CATTTTACAATAAA SEQ ID NO: 4 (HSV-1 DNA pol polypeptide sequence) MFSGGGGPLSPGGKSAARAASGFFAPAGPRGASRGPPPCLRQNFYNPYLAPVGTQQKPT GPTQRHTYYSECDEFRFIAPRVLDEDAPPEKRAGVHDGHLKRAPKVYCGGDERDVLRVGS GGFWPRRSRLWGGVDHAPAGFNPTVTVFHVYDILENVEHAYGMRAAQFHARFMDAITPTG TVITLLGLTPEGHRVAVHVYGTRQYFYMNKEEVDRHLQCRAPRDLCERMAAALRESPGASF RGISADHFEAEVVERTDVYYYETRPALFYRVYVRSGRVLSYLCDNFCPAIKKYEGGVDATTR FlLDNPGFVTFGWYRLKPGRNNTLAQPRAPMAFGTSSDVEFNCTADNLAIEGGMSDLPAYK LMCFDIECKAGGEDELAFPVAGHPEDLVIQISCLLYDLSTTALEHVLLFSLGSCDLPESHLNE LAARGLPTPVVLEFDSEFEMLLAFMTLVKQYGPEFVTGYNIINFDWPFLLAKLTDIYKVPLDG YGRMNGRGVFRVWDIGQSHFQKRSKIKVNGMVNIDMYGIITDKIKLSSYKLNAVAEAVLKDK KKDLSYRDIPAYYAAGPAQRGVIGEYCIQDSLLVGQLFFKFLPHLELSAVARLAGINITRTIYD GQQIRVFTCLLRLADQKGFILPDTQGRFRGAGGEAPKRPAAAREDEERPEEEGEDEDEREE GGGEREPEGARETAGRHVGYQGARVLDPTSGFHVNPVVVFDFASLYPSIIQAHNLCFSTLS LRADAVAHLEAGKDYLEIEVGGRRLFFVKAHVRESLLSILLRDWLAMRKQIRSRIPQSSPEEA VLLDKQQAAIKVVCNSVYGFTGVQHGLLPCLHVAATVTTIGREMLLATREYVHARWAAFEQL LADFPEAADMRAPGPYSMRIIYGDTDSIFVLCRGLTAAGLTAVGDKMASHISRALFLPPIKLE CEKTFTKLLLIAKKKYIGVIYGGKMLIKGVDLVRKNNCAFINRTSRALVDLLFYDDTVSGAAAA LAERPAEEWLARPLPEGLQAFGAVLVDAHRRITDPERDIQDFVLTAELSRHPRAYTNKRLAH LTVYYKLMARRAQVPSIKDRIPYVIVAQTREVEETVARLAALRELDAAAPGDEPAPPAALPSP AKRPRETPSPADPPGGASKPRKLLVSELAEDPAYAIAHGVALNTDYYFSHLLGAACVTFKAL FGNNAKITESLLKRFIPEVWHPPDDVAARLRTAGFGAVGAGATAEETRRMLHRAFDTLA SEQ ID NO: 5 (HSV-2 TK nucleic acid sequence) ATGGCTTCTCACGCCGGCCAACAGCACGCGCCTGCGTTCGGTCAGGCTGCTCGTGCGA GCGGGCCTACCGACGGCCGCGCGGCGTCCCGTCCTAGCCATCGCCAGGGGGCCTCC GGAGCCCGCGGGGATCCGGAGCTGCCCACGCTGCTGCGGGTTTATATAGACGGACCC CACGGGGTGGGGAAGACCACCACCTCCGCGCAGCTGATGGAGGCCCTGGGGCCGCG CGACAATATCGTCTACGTCCCCGAGCCGATGACTTACTGGCAGGTGCTGGGGGCCTCC GAGACCCTGACGAACATCTACAACACGCAGCACCGTCTGGACCGCGGCGAGATATCGG CCGGGGAGGCGGCGGTGGTAATGACCAGCGCCCAGATAACAATGAGCACGCCTTATG CGGCGACGGACGCCGTTTTGGCTCCTCATATCGGGGGGGAGGCTGTGGGCCCGCAAG CCCCGCCCCCGGCCCTCACCCTTGTTTTCGACCGGCACCCTATCGCCTCCCTGCTGTG CTACCCGGCCGCGCGGTACCTCATGGGAAGCATGACCCCCCAGGCCGTGTTGGCGTT CGTGGCCCTCATGCCCCCGACCGCGCCCGGCACGAACCTGGTCCTGGGTGTCCTTCC GGAGGCCGAACACGCCGACCGCCTGGCCAGACGCCAACGCCCGGGCGAGCGGCTTG ACCTGGCCATGCTGTCCGCCATTCGCCGTGTCTACGATCTACTCGCCAACACGGTGCG GTACCTGCAGCGCGGCGGGAGGTGGCGGGAGGACTGGGGCCGGCTGACGGGGGTCG CCGCGGCGACCCCGCGCCCCGACCCCGAGGACGGCGCGGGGTCTCTGCCCCGCATC GAGGACACGCTGTTTGCCCTGTTCCGCGTTCCCGAGCTGCTGGCCCCCAACGGGGACT TGTACCACATTTTTGCCTGGGTCTTGGACGTCTTGGCCGACCGCCTCCTTCCGATGCAT CTATTTGTCCTGGATTACGATCAGTCGCCCGTCGGGTGTCGAGACGCCCTGTTGCGCC TCACCGCCGGGATGATCCCAACCCGCGTCACAACCGCCGGGTCCATCGCCGAGATAC GCGACCTGGCGCGCACGTTTGCCCGCGAGGTGGGGGGAGTTTAG SEQ ID NO: 6 (HSV-2 TK polypeptide sequence) MASHAGQQHAPAFGQAARASGPTDGRAASRPSHRQGASGARGDPELPTLLRVYIDGPHG VGKTTTSAQLMEALGPRDNIVYVPEPMTYWQVLGASETLTNIYNTQHRLDRGEISAGEAAVV MTSAQITMSTPYAATDAVLAPHIGGEAVGPQAPPPALTLVFDRHPIASLLCYPAARYLMGSM TPQAVLAFVALMPPTAPGTNLVLGVLPEAEHADRLARRQRPGERLDLAMLSAIRRVYDLLAN TVRYLQRGGRWREDWGRLTGVAAATPRPDPEDGAGSLPRIEDTLFALFRVPELLAPNGDL YHIFAWVLDVLADRLLPMHLFVLDYDQSPVGCRDALLRLTAGMIPTRVTTAGSIAEIRDLART FAREVGGV SEQ ID NO: 7 (HSV-2 DNA pol nucleic acid sequence) ATGTTTTGTGCCGCGGGCGGCCCGGCTTCCCCCGGGGGGAAGCCGGCGGCTCGGGC GGCGTCTGGGTTTTTTGCCCCCCACAACCCCCGGGGAGCCACCCAGACGGCACCGCC GCCTTGCCGCCGGCAGAACTTCTACAACCCCCACCTCGCTCAGACCGGAACGCAGCCA AAGGCCCTCGGGCCGGCTCAGCGCCATACGTACTACAGCGAGTGCGACGAATTTCGAT TTATCGCCCCGCGTTCGCTGGACGAGGACGCCCCCGCGGAGCAGCGCACCGGGGTCC ACGACGGCCGCCTCCGGCGCGCCCCTAAGGTGTACTGCGGGGGGGACGAGCGCGAC GTCCTCCGCGTGGGCCCGGAGGGCTTCTGGCCGCGTCGCTTGCGCCTGTGGGGCGGT GCGGACCATGCCCCCGAGGGGTTCGACCCCACCGTCACCGTCTTCCACGTGTACGACA TCCTGGAGCACGTGGAACACGCGTACAGCATGCGCGCCGCCCAGCTCCACGAGCGAT TTATGGACGCCATCACGCCCGCCGGGACCGTCATCACGCTTCTGGGTCTGACCCCCGA AGGCCATCGCGTCGCCGTTCACGTCTACGGCACGCGGCAGTACTTTTACATGAACAAG GCGGAGGTGGATCGGCACCTGCAGTGCCGTGCCCCGCGCGATCTCTGCGAGCGCCTG GCGGCGGCCCTGCGCGAGTCGCCGGGGGCGTCGTTCCGCGGCATCTCCGCGGACCA CTTCGAGGCGGAGGTGGTGGAGCGCGCCGACGTGTACTATTACGAAACGCGCCCGAC CCTGTACTACCGCGTCTTCGTGCGAAGCGGGCGCGCGCTGGCCTACCTGTGCGACAAC TTTTGCCCCGCGATCAGGAAGTACGAGGGGGGCGTCGACGCCACCACCCGGTTTATCC TGGACAACCCGGGGTTTGTCACCTTCGGCTGGTACCGCCTCAAGCCCGGCCGCGGGA ACGCGCCGGCCCAACCGCGCCCCCCGACGGCGTTCGGAACCTCGAGCGACGTCGAGT TTAACTGCACGGCGGACAACCTGGCCGTCGAGGGGGCCATGTGTGACCTGCCGGCCT ACAAGCTCATGTGCTTCGATATCGAATGCAAGGCCGGGGGGGAGGACGAGCTGGCCTT TCCGGTCGCGGAACGCCCGGAAGACCTCGTCATCCAGATCTCCTGTCTGCTCTACGAC CTGTCCACCACCGCCCTCGAGCACATCCTCCTGTTTTCGCTCGGATCCTGCGACCTCCC CGAGTCCCACCTCAGCGATCTCGCCTCCAGGGGCCTGCCGGCCCCCGTCGTCCTGGA GTTTGACAGCGAATTCGAGATGCTGCTGGCCTTCATGACCTTCGTCAAGCAGTACGGCC CCGAGTTCGTGACCGGGTACAACATCATCAACTTCGACTGGCCCTTCGTCCTGACCAAG CTGACGGAGATCTACAAGGTCCCGCTCGACGGGTACGGGCGCATGAACGGCCGGGGT GTGTTCCGCGTGTGGGACATCGGCCAGAGCCACTTTCAGAAGCGCAGCAAGATCAAGG TGAACGGGATGGTGAACATCGACATGTACGGCATCATCACCGACAAGGTCAAACTCTCC AGCTACAAGCTGAACGCCGTCGCCGAGGCCGTCTTGAAGGACAAGAAGAAGGATCTGA GCTACCGCGACATCCCCGCCTACTACGCCTCCGGGCCCGCGCAGCGCGGGGTGATCG GCGAGTATTGTGTGCAGGACTCGCTGCTGGTCGGGCAGCTGTTCTTCAAGTTTCTGCC GCACCTGGAGCTTTCCGCCGTCGCGCGCCTGGCGGGCATCAACATCACCCGCACCATC TACGACGGCCAGCAGATCCGCGTCTTCACGTGCCTCCTGCGCCTTGCGGGCCAGAAG GGCTTCATCCTGCCGGACACCCAGGGGCGGTTTCGGGGCCTCGACAAGGAGGCGCCC AAGCGCCCGGCCGTGCCTCGGGGGGAAGGGGAGCGGCCGGGGGACGGGAACGGGG ACGAGGATAAGGACGACGACGAGGACGGGGACGAGGACGGGGACGAGCGCGAGGAG GTCGCGCGCGAGACCGGGGGCCGGCACGTTGGGTACCAGGGGGCCCGGGTCCTCGA CCCCACCTCCGGGTTTCACGTCGACCCCGTGGTGGTGTTTGACTTTGCCAGCCTGTAC CCCAGCATCATCCAGGCCCACAACCTGTGCTTCAGTACGCTCTCCCTGCGGCCCGAGG CCGTCGCGCACCTGGAGGCGGACCGGGACTACCTGGAGATCGAGGTGGGGGGCCGA CGGCTGTTCTTCGTGAAGGCCCACGTACGCGAGAGCCTGCTGAGCATCCTGCTGCGCG ACTGGCTGGCCATGCGAAAGCAGATCCGCTCGCGGATCCCCCAGAGCACCCCCGAGG AGGCCGTCCTCCTCGACAAGCAACAGGCCGCCATCAAGGTGGTGTGCAACTCGGTGTA CGGGTTCACCGGGGTGCAGCACGGTCTTCTGCCCTGCCTGCACGTGGCCGCCACCGT GACGACCATCGGCCGCGAGATGCTCCTCGCGACGCGCGCGTACGTGCACGCGCGCTG GGCGGAGTTCGATCAGCTGCTGGCCGACTTTCCGGAGGCGGCCGGCATGCGCGCCCC CGGTCCGTACTCCATGCGCATCATCTACGGGGACACGGACTCCATTTTCGTTTTGTGCC GCGGCCTCACGGCCGCGGGCCTGGTGGCCATGGGCGACAAGATGGCGAGCCACATCT CGCGCGCGCTGTTCCTCCCCCCGATCAAGCTCGAGTGCGAAAAAACGTTCACCAAGCT GCTGCTCATCGCCAAGAAAAAGTACATCGGCGTCATCTGCGGGGGCAAGATGCTCATC AAGGGCGTGGATCTGGTGCGCAAAAACAACTGCGCGTTTATCAACCGCACCTCCAGGG CCCTGGTCGACCTGCTGTTTTACGACGATACCGTATCCGGAGCGGCCGCCGCGTTAGC CGAGCGCCCCGCAGAGGAGTGGCTGGCGCGACCCCTGCCCGAGGGACTGCAGGCGT TCGGGGCCGTCCTCGTAGACGCCCATCGGCGCATCACCGACCCGGAGAGGGACATCC AGGACTTTGTCCTCACCGCCGAACTGAGCAGACACCCGCGCGCGTACACCAACAAGCG CCTGGCCCACCTGACGGTGTATTACAAGCTCATGGCCCGCCGCGCGCAGGTCCCGTCC ATCAAGGACCGGATCCCGTACGTGATCGTGGCCCAGACCCGCGAGGTAGAGGAGACG GTCGCGCGGCTGGCCGCCCTCCGCGAGCTAGACGCCGCCGCCCCAGGGGACGAGCC CGCCCCCCCAGCGGCCCTGCCCTCCCCGGCCAAGCGCCCCCGGGAGACGCCGTCGC ATGCCGACCCCCCGGGAGGCGCGTCCAAGCCCCGCAAGCTGCTGGTGTCCGAGCTGG CGGAGGATCCCGGGTACGCCATCGCCCGGGGCGTTCCGCTCAACACGGACTATTACTT CTCGCACCTGCTGGGGGCGGCCTGCGTGACGTTCAAGGCCCTGTTTGGAAATAACGCC AAGATCACCGAGAGTCTGTTAAAGAGGTTTATTCCCGAGACGTGGCACCCCCCGGACG ACGTGGCCGCGCGGCTCAGGGCCGCGGGGTTCGGGCCGGCGGGGGCCGGCGCTAC GGCGGAGGAAACTCGTCGAATGTTGCATAGAGCCTTTGATACTCTAGCATGAGCCCCC CGTCGAAGCTGATGTCCCGCATCTTGCAATAAA SEQ ID NO: 8 (HSV-2 DNA pol polypeptide sequence) MFCAAGGPASPGGKPAARAASGFFAPHNPRGATQTAPPPCRRQNFYNPHLAQTGTQPKA LGPAQRHTYYSECDEFRFIAPRSLDEDAPAEQRTGVHDGRLRRAPKVYCGGDERDVLRVG PEGFWPRRLRLWGGADHAPEGFDPTVTVFHVYDILEHVEHAYSMRAAQLHERFMDAITPA GTVITLLGLTPEGHRVAVHVYGTRQYFYMNKAEVDRHLQCRAPRDLCERLAAALRESPGAS FRGISADHFEAEVVERADVYYYETRPTLYYRVFVRSGRALAYLCDNFCPAIRKYEGGVDATT RFILDNPGFVTFGWYRLKPGRGNAPAQPRPPTAFGTSSDVEFNCTADNLAVEGAMCDLPA YKLMCFDIECKAGGEDELAFPVAERPEDLVIQISCLLYDLSTTALEHILLFSLGSCDLPESHLS DLASRGLPAPVVLEFDSEFEMLLAFMTFVKQYGPEFVTGYNIINFDWPFVLTKLTEIYKVPLD GYGRMNGRGVFRVWDIGQSHFQKRSKIKVNGMVNIDMYGIITDKVKLSSYKLNAVAEAVLK DKKKDLSYRDIPAYYASGPAQRGVIGEYCVQDSLLVGQLFFKFLPHLELSAVARLAGINITRTI YDGQQIRVFTCLLRLAGQKGFILPDTQGRFRGLDKEAPKRPAVPRGEGERPGDGNGDEDK DDDEDGDEDGDEREEVARETGGRHVGYQGARVLDPTSGFHVDPVVVFDFASLYPSIIQAH NLCFSTLSLRPEAVAHLEADRDYLEIEVGGRRLFFVKAHVRESLLSILLRDWLAMRKQIRSRI PQSTPEEAVLLDKQQAAIKVVCNSVYGFTGVQHGLLPCLHVAATVTTIGREMLLATRAYVHA RWAEFDQLLADFPEAAGMRAPGPYSMRIIYGDTDSIFVLCRGLTAAGLVAMGDKMASHISR ALFLPPIKLECEKTFTKLLLIAKKKYIGVICGGKMLIKGVDLVRKNNCAFINRTSRALVDLLFYD DTVSGAAAALAERPAEEWLARPLPEGLQAFGAVLVDAHRRITDPERDIQDFVLTAELSRHPR AYTNKRLAHLTVYYKLMARRAQVPSIKDRIPYVIVAQTREVEETVARLAALRELDAAAPGDEP APPAALPSPAKRPRETPSHADPPGGASKPRKLLVSELAEDPGYAIARGVPLNTDYYFSHLLG AACVTFKALFGNNAKITESLLKRFIPETVVHPPDDVAARLRAAGFGPAGAGATAEETRRMLHR AFDTLA