Syncytial oncolytic herpes simplex mutants as effective cancer therapies

文档序号：1894228 发布日期：2021-11-26 浏览：27次中文

阅读说明：本技术 作为有效的癌症疗法的合胞溶瘤性单纯疱疹突变体 (Syncytial oncolytic herpes simplex mutants as effective cancer therapies ) 是由蒂莫西·P·克里普凯文·A·卡西迪王品怡朱莉娅·K·哈雷于 2020-03-13 设计创作，主要内容包括：本发明提供了一种非天然的单纯疱疹病毒(“HSV”),包含所述HSV、或基本上由所述HSV组成、或由所述HSV组成的组合物,以及制备所述HSV、或用所述HSV感染细胞的方法。本文还提供了在有需要的对象中治疗癌症或抑制癌细胞的生长或转移的方法。(The present invention provides a non-natural herpes simplex virus ("HSV"), compositions comprising, consisting essentially of, or consisting of the HSV, and methods of making the HSV, or infecting a cell with the HSV. Also provided herein are methods of treating cancer or inhibiting the growth or metastasis of cancer cells in a subject in need thereof.)

1. A non-native herpes simplex virus ("HSV"), wherein the virus comprises one or more mutations in virulence genes from the group consisting of:

(a) a glycoprotein E ("gE") coding gene,

(b) infected cell protein 0 ("ICP 0") encoding gene,

(d) ICP8 encoding a gene, or

(e) ICP34.5 encodes a gene.

2. The non-natural HSV of claim 1, wherein the HSV further comprises a gene encoding a dysfunctional ICP34.5 protein and/or a gene encoding a dysfunctional ICP6 protein.

3. The non-natural HSV of claim 2, wherein the gene encoding a dysfunctional ICP34.5 protein comprises a polynucleotide having a sequence selected from: SEQ ID number 5, SEQ ID number 7, and mutated sequences that are at least 95% identical to SEQ ID number 5 or SEQ ID number 7 and remain at the nucleotides in either SEQ ID number 5 or SEQ ID number 7.

4. The non-natural HSV of claim 2, wherein the gene encoding a dysfunctional ICP6 protein comprises a polynucleotide having a sequence selected from: SEQ ID number 45, SEQ ID number 47, and mutated sequences that are at least 95% identical to SEQ ID number 45 or SEQ ID number 47 and remain at nucleotides in either of SEQ ID number 45 or SEQ ID number 47.

5. The non-natural HSV of any one of claims 1-4, wherein a mutation in the virulence gene comprises an insertion, deletion, truncation, frameshift, substitution, or point mutation.

6. The non-natural HSV of any one of claims 1-5, wherein the HSV lacks a gene encoding a functional ICP34.5 protein and/or a functional ICP6 protein.

7. The non-natural HSV of any one of claims 1-6, wherein the mutation is a non-synonymous mutation in a virulence gene.

8. The non-natural HSV of any one of claims 1-7, or an equivalent thereof, wherein said mutation or equivalent encodes:

(a) alanine to threonine mutation at position 151 of the gE protein,

(b) an arginine to histidine mutation at position 258 of the ICP0 protein,

(d) a threonine to methionine mutation at position 1155 of the ICP8 protein, or

(e) Proline to histidine mutation at position 119 of ICP34.5 protein.

9. The non-natural HSV of any one of claims 1-8, comprising one or more of:

(a) a polynucleotide encoding an amino acid sequence selected from SEQ ID numbers 2, 6, 8, 10 and 52, and/or a polynucleotide having a sequence selected from SEQ ID numbers 1, 5, 7, 9 and 51;

(b) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID numbers 2, 6, 8, 10, and 52;

(c) a polynucleotide encoding an amino acid sequence selected from SEQ ID numbers 13, 15, 17 and 19, and/or a polynucleotide having a sequence selected from SEQ ID numbers 12, 14, 16 and 18;

(d) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID numbers 13, 15, 17, and 19;

(e) a polynucleotide encoding an amino acid sequence selected from SEQ ID numbers 21, 23 and 26, and/or a polynucleotide having a sequence selected from SEQ ID numbers 20, 22, 24, 25 and 53, or a sequence thereof which does not contain one or two or more introns;

(f) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID numbers 21, 23 and 26;

(g) a polynucleotide encoding an amino acid sequence selected from SEQ ID numbers 28, 30, 32 and 34, and/or a polynucleotide having a sequence selected from SEQ ID numbers 27, 29, 31 and 33;

(h) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID numbers 28, 30, 32, and 34;

(i) a polynucleotide encoding an amino acid sequence selected from SEQ ID numbers 36, 38, 40 and 42, and/or a polynucleotide having a sequence selected from SEQ ID numbers 35, 37, 39 and 41;

(j) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID numbers 36, 38, 40, and 42;

(k) a polynucleotide encoding an amino acid sequence selected from SEQ ID numbers 44, 46, 48 and 50, and/or a polynucleotide having a sequence selected from SEQ ID numbers 43, 45, 47 and 49;

(l) A polypeptide having an amino acid sequence selected from the group consisting of SEQ ID numbers 44, 46, 48 and 50.

10. The non-natural HSV of any of claims 1-9, further comprising a polynucleotide having a sequence identical to at least one fragment of a virulence gene from 17TermA HSV.

11. The non-natural HSV of any of claims 1-10, further comprising a polynucleotide having a sequence identical to at least one fragment of a virulence gene from rRp450 HSV.

12. The non-natural HSV of any of claims 1-11, wherein the HSV is derived from an HSV type 1 ("HSV-1") or an HSV type 2 ("HSV-2") strain.

13. The non-natural HSV of any of claims 1-11, wherein the HSV is derived from the HSV-1 KOS strain.

14. The non-natural HSV of any of claims 1-13, further comprising a transgene.

15. A composition comprising the non-natural HSV of any of claims 1-14 and a carrier.

16. The composition of claim 14, wherein the carrier is a pharmaceutically acceptable carrier.

17. The composition of claim 15 or 16, further comprising a cryopreservative agent that facilitates freezing and thawing of non-natural HSV without significant loss of virulence.

18. A non-human mammal infected with the non-natural HSV of any one of claims 1-14.

19. A cell infected with the non-natural HSV of any one of claims 1-14, optionally a lymphocyte.

20. A method of infecting a cell comprising contacting the cell with the non-natural HSV of any of claims 1-14 or the composition of any of claims 15-17.

21. The method of claim 20, wherein the cell is a lymphocyte.

22. The method of claim 20 or 21, wherein the cell has been infected with epstein-barr virus ("EBV").

23. A method of making a viral vector comprising introducing a transgene into the non-natural HSV of any one of claims 1-13.

24. A method for inhibiting growth or metastasis of a cancer cell, comprising contacting the cell with an effective amount of the non-natural HSV vector of any of claims 1-14 or the composition of any of claims 15-17.

25. The method of claim 24, wherein the contacting is in vitro or in vivo.

26. The method of claim 24, wherein said contacting is in vivo by administering the non-natural HSV to a subject.

27. A method for treating cancer in a subject, comprising administering to the subject an effective amount of the non-natural HSV vector of any of claims 1-14 or the composition of any of claims 15-17.

28. The method of any one of claims 24-27, wherein the cancer is of the following type, or the cancer cell is selected from the following types of cells: pancreatic cancer, renal cancer, small cell lung cancer, brain cancer, neural cancer, bone cancer, lymphoma, myeloma, colon cancer, uterine cancer, breast cancer, leukemia, liver cancer, prostate cancer, skin cancer, or melanoma.

29. The method of claim 27 or 28, wherein HSV vector or pharmaceutical composition is administered by injection, infusion, instillation, and/or inhalation.

30. The method of any one of claims 27-28, wherein the administering comprises a therapy comprising: first line therapy, second line therapy, third line therapy, fourth line therapy or fifth line therapy.

31. The method of claim 27, further comprising administering to the subject an effective amount of an anti-cancer therapy.

32. The method of any one of claims 27-31, wherein the subject is a mammal.

33. The method of claim 32, wherein the mammal is selected from the group consisting of: a human, mouse, rat, guinea pig, non-human primate, dog, cat, horse, cow, pig, goat, or sheep.

34. The method of claim 32, wherein the subject is a human.

35. A method of producing the HSV vector of any of claims 1-13, comprising

(a) Introducing a 17Terma HSV vector and an rRp450 HSV vector into a host cell;

(b) growing the host cell for at least 3 passages; and

36. The method of claim 35, wherein the HSV is introduced into a host cell by transfection, infection, transformation, electroporation, injection, microinjection, or a combination thereof.

37. The method of claim 35 or 36, wherein the host cell is grown for at least 3, 10, 20, 30, 40, or 50 passages.

38. The method of any one of claims 35-37, wherein the host cell comprises a complementary gene product to support replication of an introduced HSV vector.

39. The method of claim 38, wherein the complementary genes encode an ICP6 protein and/or an ICP34.5 protein.

40. The method of any one of claims 35-39, further comprising introducing a transgene into the HSV vector.

41. HSV particles made by the method of any one of claims 35-40.

42. A method of inducing cell lysis comprising contacting a cell with the non-natural HSV of any of claims 1-14 or the composition of any of claims 15-17.

43. The method of claim 42, wherein the cell is a cancer cell.

44. A kit comprising the HSV vector of any of claims 1-14 or the composition of any of claims 15-17, and instructions for use.

45. A method of making the HSV vector of any of claims 1-13, comprising

(a) Introducing a polynucleotide encoding the viral genome of an HSV vector into a host cell;

(b) growing the host cell; and

46. The method of claim 45, wherein the polynucleotide is introduced into the host cell by transfection, infection, transformation, electroporation, injection, microinjection, or a combination thereof.

47. The method of claim 45 or 46, wherein the nucleic acid sequence encoding the viral genome is introduced into the host cell in a vector.

48. The method of claim 47, wherein the vector is HSV or a plasmid.

49. The method of any one of claims 45-48, wherein the host cell comprises a complementary gene product to support replication of an introduced HSV vector.

50. The method of any one of claims 45-49, wherein the isolated HSV is substantially free of host cells, cell debris, culture medium, or any other agent used in culturing the host cells.

51. The method of any one of claims 45-50, wherein the separating is by centrifugation, filtration, chromatography, or any combination thereof.

Background

Oncolytic herpes simplex virus (oHSV) (e.g., Imlygic recently approved by FDA for the treatment of melanoma)^TM) Has promising anti-tumor effect. These vectors have two main mechanisms of action: (1) lysis phase, determined by direct infection and cell lysis; and (2) an immune phase, driven by stimulation of anti-tumor immunity. However, not all cancers respond similarly, as viral transmission in some cancers is inherently slow. During the culture process, the cells are tolerant to the virus to varying degrees. In animals, tumor stroma and immunityChanges in cellular composition result in changes in the ability of the virus to spread and immunoreact. Therefore, strategies to increase the efficacy of the lysis phase to achieve optimal therapeutic effects are still needed. The present invention fulfills these needs and provides related advantages.

Disclosure of Invention

The present invention provides a non-natural herpes simplex virus ("HSV"), wherein the virus comprises, consists essentially of, or consists of a mutation in one or more of the following: (a) a glycoprotein E ("gE") encoding gene, (b) an infected cellular protein 0 ("ICP 0") encoding gene, (c) a DNA packaging terminal enzyme subunit 1 encoding gene, (d) an ICP8 encoding gene, or (E) an ICP34.5 encoding gene.

In another aspect, the present invention provides a composition or pharmaceutical composition comprising, consisting essentially of, or consisting of the non-natural HSV of the present invention. In another aspect, the invention provides a method of infecting a cell, the method comprising, consisting essentially of, or consisting of: contacting a cell with a non-natural HSV or a composition or pharmaceutical composition comprising a non-natural HSV.

In one aspect, the present invention provides a method of making the non-natural HSV of the present invention, the method comprising, consisting essentially of, or consisting of: mutating a gene in a non-native HSV viral particle or introducing a transgene into a non-native HSV. In another aspect, a method of producing a non-natural HSV vector comprises, consists essentially of, or consists of: (a) introducing a 17Terma HSV vector and an rRp450 HSV vector into a host cell; (b) growing the host cell for at least 3 passages; and (c) isolating HSV particles produced by the host cell.

Also provided is a method of inhibiting growth or metastasis of a cancer cell or metastatic cancer cell, comprising, consisting essentially of, or consisting of the steps of: as described herein, a cell is contacted with an effective amount of a non-natural HSV vector or a composition or pharmaceutical composition containing a non-natural HSV vector. The contacting is in vitro or in vivo. In one aspect, the contacting is performed in vivo by administering a non-natural HSV or a composition or a pharmaceutical composition to a subject. The method is carried out in vitro by contacting the non-natural HSV with the cell. The in vitro methods can be used to test new therapies, or as a personalized analysis to determine whether the therapy is appropriate for the cancer to be treated. Additional cancer therapies may be combined with treatments that may be performed simultaneously or sequentially with the disclosed methods.

The cancer cell to be treated may be a solid tumor or a blood cancer, such as a carcinoma or sarcoma, and non-limiting examples thereof include pancreatic cancer, kidney cancer, small cell lung cancer, brain cancer, neuroblastoma, nerve cancer, bone cancer, lymphoma, myeloma, colon cancer, uterine cancer, breast cancer, leukemia, liver cancer, prostate cancer, skin cancer, or melanoma. The cell belongs to any species, such as mammals and humans, and when performed in vitro, it may be from a cultured cell line or primary cell, e.g. from a tissue biopsy. The cells may be adult or juvenile cells or cancer stem cells (i.e., cancer cells having characteristics associated with normal stem cells, particularly the ability to produce all cell types found in a particular cancer sample) or cancer cells that do not have characteristics associated with normal stem cells. In one embodiment, the cell expresses an N-myc proto-oncogene protein (MYCN), and/or expresses MYCN at a higher level than a non-cancerous cell.

In another aspect, the invention also provides a method for treating cancer or a metastatic cancer, or inhibiting the growth or metastasis of cancer cells, in a subject in need thereof, the method comprising, consisting essentially of, or consisting of: administering to a subject an effective amount of a non-natural HSV, composition, or pharmaceutical composition of the present invention. The subject to be treated may be of any species, such as mammals and humans, for example, canine, equine, bovine, feline, simian, rat or mouse. Administration can be as first line therapy, second line therapy, third line therapy, fourth line therapy or fifth line therapy. Additional cancer therapies may be combined with treatments that may be performed simultaneously or sequentially with the disclosed methods. The cancer cell to be treated may be a solid tumor or a blood cancer, such as a carcinoma or sarcoma, and non-limiting examples thereof include pancreatic cancer, kidney cancer, small cell lung cancer, brain cancer, neuroblastoma, nerve cancer, bone cancer, lymphoma, myeloma, colon cancer, uterine cancer, breast cancer, leukemia, liver cancer, prostate cancer, skin cancer, or melanoma.

The methods of the invention may be combined with appropriate diagnostics to monitor the remission or progression of a disease. Several methods for such monitoring are known in the art.

In one aspect, the invention provides a method of inducing cell lysis, the method comprising, consisting essentially of, or consisting of: contacting the cell with an effective amount of a non-natural HSV, composition, or pharmaceutical composition of the present invention. The contacting is in vitro or in vivo. In one aspect, the contacting is performed in vivo by administering a non-natural HSV or a composition or a pharmaceutical composition to a subject. The method is carried out in vitro by contacting the non-natural HSV with the cell. The in vitro methods can be used to test for new therapies, or as a personalized analysis to determine whether the therapy is appropriate for the subject to be treated. Additional cytolytic therapy may be combined with treatments that may be performed simultaneously or sequentially with the disclosed methods.

The cancer cell to be treated may be a solid tumor or a blood cancer, such as a carcinoma or sarcoma, and non-limiting examples thereof include pancreatic cancer, kidney cancer, small cell lung cancer, brain cancer, neuroblastoma, nerve cancer, bone cancer, lymphoma, myeloma, colon cancer, uterine cancer, breast cancer, leukemia, liver cancer, prostate cancer, skin cancer, or melanoma. The cell belongs to any species, such as mammals and humans, and when performed in vitro, it may be from a cultured cell line or primary cell, e.g. from a tissue biopsy. The cell may be an adult or juvenile cell, or a cancer stem cell, or a cancer cell that does not have the characteristics associated with a normal stem cell. The therapy may be combined with appropriate assays to test the effectiveness of the therapy, e.g., cancer remission or progression.

In another aspect, the invention also provides a kit comprising, consisting essentially of, or consisting of a non-natural HSV, a composition, and/or a pharmaceutical composition of the invention.

Description of the drawings.

FIGS. 1A-1B show schematic diagrams for the generation of Mut-3 and Mut-3. delta.34.5 (FIG. 1A) and Mut-3 SNPs (FIG. 1B). HSV Mut-3 was isolated from serial passages mixed with 17Terma and rRp450 in the non-permissive (non-permissive) line ("directed evolution"); and an attenuated mutant Mut-3 Δ 34.5 was constructed by gene editing (labeled as "CRISPR/Cas 9" step) (fig. 1A). FIG. 1B shows the sequence alignment of Mut-3 with its parent virus. Non-synonymous mutations that differ from any of the parents of Mut-3 are masked with backslash, including UL15, UL29, US8, RL1, and RL 2. The same genomic sequence as 17TermA is shown as blank boxes in the bottom panel; those identical to rRp450 are labeled positive slashes.

FIGS. 2A-2C show the plaque size and receptor usage for Mut-3, Mut-3 Δ 34.5, rRp450, and 17 TerMA. The applicants performed plaque analysis on four viruses simultaneously and scanned and analyzed plaque images after 3 days by a Keyence HS multifunctional fluorescence microscope BZ-II analyzer. (fig. 2A) raw (top) and mask (bottom, numbered plaque) images of four viruses. (FIG. 2B) the average plaque size (left) and plaque number (right) for the four viruses were calculated. The plaque size of Mut-3 and Mut-3. delta.34.5 is significantly larger than that of the parental viruses rRp450 and 17 Terma. (FIG. 2C) in vitro cytotoxicity/MTS assay of CHO cell sets. Applicants used four viruses to infect CHO-K1 (as labeled), CHO-Nectin-1 (as labeled), CHO-Nectin-2 (as labeled), and CHO-HVEM (as labeled) at different multiplicity of infection (MOI). Applicants measured cell viability by the colorimetric cell proliferation/MTS assay 3 days after viral infection (pvi) relative to untreated controls. Only CHO-Nectin-1 and CHO-HVEM, but not CHO-K1 or CHO-Nectin-2 (used primarily for HSV-2 entry), were sensitive to treatment with the four viruses, indicating that Mut-3 and Mut-3. delta. 34.5 did not bypass the receptor barrier and could still rely on the classical HSV entry receptor to infect the cells.

FIGS. 3A-3C: mut-3 Δ 34.5 in killing human and mouse neuroblastoma cellsRatio 17Terma (gamma)₁34.5-null) is more effective, not due to increased production of infectious viral particles in vitro. (FIG. 3A) in vitro cytotoxicity/MTS assay of neuroblastoma cell lines. Applicants used Mut-3 (AS a marker), Mut-3 Δ 34.5 (AS a marker), or 17Terma (AS a marker) to infect human (SK-N-AS) and mouse (975A 2) neuroblastoma cells at different MOIs. Applicants measured cell viability by MTS assay 4 days after viral infection (pvi) relative to untreated controls. N =6, error bars indicate SEM. P<0.0001, two-factor analysis of variance. (FIG. 3B) in vitro viral replication assay. Applicants infected neuroblastoma cell lines with 17Terma (as labeled) or Mut-3 Δ 34.5 (as labeled) at MOI =0.1 (upper panel, human line SK-N-as) or MOI =0.5 (lower panel, murine line 975A 2) and washed cells with PBS 2 hours after viral infection (pvi). Applicants obtained cell lysates 2, 24, 48, and 72 hours after viral infection (pvi) and determined viral yields by plaque assay. N = 3. Error bars indicate SD. P<0.05，**p<0.001，****p<0.0001, two-factor analysis of variance. (FIG. 3C) comparison of Mut-3. DELTA.34.5 versus the relatively free released virus particles in 17 TerMA-infected neuroblastoma cultures. Applicants performed an analysis similar to that described in (fig. 3B), by collecting supernatant and particulates, plaque analysis was performed on two separate fractions over a period of time, rather than as a whole as in fig. 3B. Applicants calculated the relative free virions as the ratio of whole culture (supernatant + particles) to infectious particles in the supernatant. Mut-3 Δ 34.5 showed significantly more relative release of virions at 48 hours than 17 TerMA. N = 3. Error bars indicate SD. P<0.05。

FIGS. 4A-4H TEM analysis of Mut-3. DELTA.34.5 and 17TerMA uptake in neuroblastoma cells. Neuroblastoma cells SK-N-AS and 975A were infected with Mut-3. DELTA.34.5 and 17Terma at 37 ℃ for 220 minutes at an MOI of 50. (FIGS. 4A, 4B, 4E and 4F) the inset shows that Mut-3. DELTA.34.5 virions are predominantly present in endocytic vesicles (arrows other than the right arrow in FIG. 4B), and fusion to the plasma membrane is rarely found (right arrow in FIG. 4B). (FIGS. 4C, 4D, 4G and 4H) the insets show that 17TerMA virions are predominantly present in endocytic vesicles (arrows other than the bottom arrow in FIG. 4G), with little fusion to the plasma membrane (bottom arrow in FIG. 4G). TEM analysis was done by Hitachi H-7650 TEM. N: and (4) cell nucleus. Scale bar: 500 nm.

FIGS. 5A-5C: attenuated 17 Δ 34.5 virions showed efficacy comparable to 17TermA in neuroblastoma cells. Applicants generated 17 Δ 34.5 by CRISPR-Cas9 gene editing technology to replace wild type strain 17 with the EGFP expression cassette⁺G134.5 gene in (1). (FIG. 5A) plaque image of 17 Δ 34.5 clone on Vero cells. After 3 rounds of plaque purification, both B4 and G1 clones produced 100% GFP-positive (left panel) non-syncytia (right panel, phase contrast) plaques. Two days after viral infection (pvi), by EVOS^®The FL imaging system takes an image. Scale bar: 400 μm. (FIG. 5B) in vitro cytotoxicity/MTS assay of neuroblastoma cell lines. Applicant used wild type strain 17⁺(AS labeled), 17TerMA (AS labeled), 17D34.5 clone B4 (AS labeled), or 17D34.5 clone G1 (AS labeled) infected neuroblastoma cell lines SK-N-AS and 975A2 at different MOIs. Applicants measured cell viability by MTS assay 3 days after viral infection (pvi) relative to untreated controls. The potency of the two 17D34.5 clones was significantly lower than its wild-type counterpart strain 17+, but comparable to 17 TermA. The DNA sequences of the EGFP expression cassette and EGFP are shown in FIG. 5C (SEQ ID NO: 11).

FIGS. 6A-6C Mut-3. DELTA.34.5 shows faster viral gene transfer and cell killing compared to 17. DELTA.34.5. Applicants treated (FIG. 6A) Vero (FIG. 6B) SK-N-AS (FIG. 6C) 975A2 cells with mock CTL, Mut-3 Δ 34.5 or 17 Δ 34.5 at different MOIs and monitored GFP positive regions (top panel) and cell fusion (bottom panel) over time using IncuCyte ZOOM live cell imaging. N =6 wells per condition, and N = 2 measurements per well per time point. After viral infection (pvi). The Mann-Whitney U test was used to compare the time to reach the maximum GFP region between viruses (FIG. 6A). Error bar representationSD。

FIG. 7 Mut-3. delta. 34.5 controls the growth of human neuroblastoma in vivo more effectively than 17 Terma. Nude athymic mice bearing sub-q SK-N-AS tumors were intratumorally injected with Phosphate Buffered Saline (PBS) control (AS labeled, N = 8), three doses of 1e7 pfu of 17TermA (AS labeled, N = 8), or Mut-3 Δ 34.5 (AS labeled, N = 9). Kaplan-Meier survival curves were plotted. Statistical significance between 17Terma and Mut-3 Δ 34.5 was scored using the log rank test. P < 0.05.

FIGS. 8A-8C Mut-3 Δ ICP6 (an attenuated version of Mut-3) induces higher cytotoxicity in the human neuroblastoma cell line CHP-134 compared to oncolytic herpes virus rRp 450. (FIG. 8A) a Mut-3 Δ ICP6 map constructed by the CRISPR-Cas9 gene editing strategy in which the UL39 gene encoding ICP-6 was replaced with a CMV-driven GFP reporter cassette. MTS cell activity assay compares the cytotoxicity information of Mut-3, rRp450 and Mut3- Δ ICP6/D7-1 in human neuroblastoma cell lines. Cells were seeded at 4000 cells/well in 96-well dishes, cultured overnight at 37 ℃, and then infected with each of the listed viruses at a multiplicity of infection (MOI) of 0.001, 0.01, 0.1, and 1 infectious viral particle per cell. Assays were performed using Cell Titer96 AQuesous nonradioactive Cell proliferation assay/MTS (Promega, Madison, Wis.) on day 3 or 4 post-infection, according to the manufacturer's instructions. Each sample was run in quadruplicate and results are shown as percent cell survival relative to uninfected controls. Error bars indicate standard deviation. Statistical significance was assessed using the t-test. P is less than or equal to 0.05, p is less than or equal to 0.01, and p is less than or equal to 0.001. Fig. 8B SK-N-AS and fig. 8C CHP-134 show the results in a picture-wise manner.

FIGS. 9A-9B Mut-3 Δ ICP6 induced higher cytotoxicity in the mouse neuroblastoma cell lines Neuro-2a and 975A2 compared to rRp 450. FIGS. 9A-9B show the results of MTS cell activity assays comparing cytotoxicity information of Mut-3, rRp450 and Mut3- Δ ICP6/D7-1 in mouse neuroblastoma cell lines. Cells were seeded at 4000 cells/well in 96-well dishes, cultured overnight at 37 ℃, and then infected with each of the listed viruses at a multiplicity of infection (MOI) of 0.001, 0.01, 0.1, and 1 infectious viral particle per cell. Assays were performed using Cell Titer96 AQuesous nonradioactive Cell proliferation assay/MTS (Promega, Madison, Wis.) on day 3 or 4 post-infection, according to the manufacturer's instructions. Each sample was run in quadruplicate and results are shown as percent cell survival relative to an uninfected control group. Statistical significance was assessed using the t-test. P is less than or equal to 0.01, and p is less than or equal to 0.001. FIG. 9A: neuro-2a and FIG. 9B: 975A 2.

FIGS. 10A-10B Mut-3 Δ ICP6 produced significantly higher virus yields in mouse neuroblastoma cell line 975A2 than rRp450 over the 48 and 72 hour infection period. Mouse 975A2 (FIG. 10A) and human SK-N-AS (FIG. 10B) neuroblastoma cells were plated at 2 x 10 per well⁵The individual cells were seeded in 12-well culture dishes, cultured overnight at 37 ℃, and SK-N-AS cells were infected at MOI 0.01 (fig. 10B) or 975a2 cells at MOI 0.5 (fig. 10A) for 2 hours in 200 μ L serum-free medium, with gentle shaking every 20 minutes. Cells were washed once with PBS and covered with 1 ml of complete medium. Cells and supernatants were then collected at 2, 24, 48 and 72 hours post infection, freeze-thawed three times, and serially diluted and titrated on Vero cells to determine infectious virus yield by standard plaque assay. Each sample was run in triplicate. Error bars indicate standard deviation. Statistical significance was assessed using the t-test. P is less than or equal to 0.05, and p is less than or equal to 0.01.

FIGS. 11A-11B the effects of Mut-3. DELTA.34.5 and Mut-3. DELTA.ICP 6 on differentiated human keratinocytes were significantly lower than for the Mut-3 virus. Human keratinocytes (HFK) were grown in EpiLife medium (Cascade Biologics) supplemented with human keratinocyte growth supplements according to the manufacturer's instructions. Undifferentiated HFK (fig. 11A) was seeded at a density of 2000 cells per well in 96-well culture plates and cultured overnight. The cultures were then infected with Mut-3, Mut-3. delta. 34.5/C8G5 or Mut-3. delta. ICP 6D 7-1 at MOIs of 0.0004, 0.004, 0.04, 0.4 and 4. Cell viability was determined by MTS assay 3 days after virus infection. 24 hours after inoculation, the culture medium was inoculated by adding 10% FBS and 1 mmol/l CaCl₂Differentiated HFKs were generated and allowed to incubate for an additional 48 hours prior to infection. Differentiated cell viability was determined by MTS assay 4 days after virus infection (fig. 11B). Each sample was done in quadruplicateAnd (5) operating. Error bars indicate standard deviation. Statistical significance was assessed using the t-test. P is less than or equal to 0.05, and p is less than or equal to 0.01.

FIG. 12: intravenous injection of the 1e6 pfu wild-type KOS virus strain was lethal to naive (nadive) Blab/c mice. Description of the safety study of Mut-3 Δ 34.5 and Mut-3 Δ ICP 6. Applicants initiated a virus biodistribution study by injecting 1e8 plaque forming units (pfu) of each virus into naive, non-tumor-bearing Balb/c mice (20 males, 20 females) via the tail vein. Then, applicants sacrificed these animals on days 1, 14, 28, 56, and 85 (n =4 per time point) and their peripheral blood, testis (male), ovary (female), spleen, lung, kidney, heart, lung, and brain were collected for analysis. During the first two weeks following virus administration, applicants observed each mouse daily and then twice weekly until the scheduled sacrifice date. The body weight of each mouse before infection with the virus was recorded, once a week thereafter. Meanwhile, wild-type KOS virus was administered as a positive (non-safety) control [ dose range: each mouse was 1e5 to 1e7 pfu (n = 3) ] individual groups of mice. The applicant also performed pathological analyses of the organs obtained, comparing samples obtained from mice inoculated with the mutant virus and mice inoculated with the wild-type KOS virus. Applicants also utilized plaque assays to detect the replication potential of these viruses. The results show the survival curves of mice receiving a single dose of 1e5, 1e6, or 1e7 pfu wild-type KOS virus.

FIGS. 13A-13B: the naive Balb/C mice can tolerate the Mut-3. Δ 34.5 (C8G 5) or Mut-3. Δ ICP6 (D7-1) of up to 1e8 pfu by intravenous injection. Applicants completed the safety and biodistribution studies and found by physical examination that mice receiving Mut-3 Δ 34.5 (C8G 5) or Mut-3 Δ ICP6 (D7-1) up to 1e8 pfu remained healthy until the designated day of sacrifice (up to 85 days). In female mice, plaque analysis showed the presence of infectious virus in heart, kidney, liver, ovary and spleen only 24 hours after infection. Fig. 13A and 13B show the results of female and male mice, respectively, in a graphical manner.

FIG. 14 replicates of HSV could be detected in the brain, kidney and ovary of mice receiving 1e6 pfu or more wild type KOS virus. FIG. 14 is a table summarizing the results of plaque analysis in tissues of mice injected with 1e6 or 1e7 pfu wild-type KOS virus. The mice were sacrificed 5-6 days after virus injection (pvi) after onset of symptoms (kyphosis, sleep-inducing limb paralysis, etc.). "+" indicates detectable plaque and "-" indicates its absence. The grey shaded boxes indicate that these data are not available.

FIG. 15 shows that replicating HSV can be detected in almost all tissues obtained (except lungs) from mice receiving C8G5 or D7-1 at 1e8 pfu 24 hours post viral infection (pvi). The table summarizes plaque analysis of tissues of mice injected with Mut Δ 34.5 or Mut3 Δ ICP6 24 hours after virus infection. "+" indicates detectable plaque and "-" indicates its absence. The grey shaded boxes indicate that these data are not available.

FIG. 16 shows that no replicating HSV was detected in any of the tissues obtained from mice that received C8G5 or D7-1 at 14 days post viral infection (pvi). The table summarizes plaque analysis of tissues of mice injected with Mut Δ 34.5 or Mut3 Δ ICP6 at 14 days after virus infection. "-" indicates no detectable plaque.

FIG. 17 shows that no replicating HSV was detected in any of the tissues obtained from mice receiving C8G5 or D7-1 at 28 days post viral infection (pvi). The table summarizes plaque analysis of tissues of mice injected with Mut Δ 34.5 or Mut3 Δ ICP6 at 28 days after virus infection. "-" indicates no detectable plaque. The grey shaded boxes indicate that these data are not available.

FIG. 18 is a table summarizing no detected replicating HSV in any of the tissues obtained from mice receiving 1e8 pfu of C8G5 or D7-1 56 days post viral infection (pvi). The table summarizes plaque analysis of tissues of mice injected with Mut Δ 34.5 or Mut3 Δ ICP6 56 days after virus infection. "-" indicates no detectable plaque.

FIG. 19 shows that no replicating HSV was detected in any of the tissues obtained from mice that received C8G5 or D7-1 at 1e8 pfu 85 days post viral infection (pvi). The table summarizes plaque analysis of tissues of mice injected with Mut Δ 34.5 or Mut3 Δ ICP6 85 days after virus infection. "-" indicates no detectable plaque.

FIGS. 20A-20D show that the plaque size of Mut-3. delta. gE is much smaller than that of Mut-3 or Mut-3D34.5 (C8G 5). FIG. 20A: mut-3 Δ gE was constructed by CRISPR-Cas9 gene editing strategy, in which the Us8 gene encoding glycoprotein gE was replaced by a CMV driven GFP reporter cassette. FIG. 20B: non-syncytia GFP-positive plaque phenotype of 28D5-B4 and 28D5-H1 of the Mut-3 Δ gE clone-Vero cells were infected with serial dilutions of 28D5-B4 or 28D5-H1 for 2 hours and overlaid in medium. Photographs of plaques were taken 3 days after viral infection (pvi). FIG. 20C: qPCR analysis of the Mut-3 Δ gE clones 28D5-B4 and 28D5-H1 showed a Us8/gE deletion. Young hamster kidney (BHK) cells were infected with the Mut-3. DELTA.gE clone 28D5-B4 or 28D5-H1, and lysates were collected when > 50% cytopathic effect was observed. 50-100ng of genomic DNA isolated from virus-infected BHK cell lysates was used for the qPCR reaction. Results are shown as gE/Us8 or GFP fold relative to Us8a control HSV gene. FIG. 20D: comparison of the plaque size between the Mut-3 Δ gE clone 28D5-B4 and 28D5-H1 and the Mut-3 or Mut-3 Δ 34.5/C8G5 was taken from the standard plaque analysis in Vero cells. The plaque photos of Mut-3, Mut-3. Δ 34.5, Mut-3. Δ gE clone 28D5-B4 and clone 28D5-H1 were taken 3 days after virus infection (pvi).

Fig. 21A-21D provide study results showing that plaque size for 17 Δ gE is much smaller than 17syn + or 17 Δ 34.5. FIG. 21A: 17 Δ gE was constructed by the CRISPR-Cas9 gene editing strategy, in which the Us8 gene encoding glycoprotein gE was replaced by a CMV-driven GFP reporter cassette. FIG. 21B: non-syncytia and small GFP positive plaque phenotype of 17 Δ gE clone 12G5 Vero cells were infected with serial dilutions of 17 Δ gE 12G5 for 2 hours and plated in overlay medium. Photographs of plaques were taken 3 days after viral infection (pvi). FIG. 21C: qPCR analysis of 17. delta. gE clone 12G5 showed a deletion of Us 8/gE. Young hamster kidney (BHK) cells were infected with the 17. delta. gE clone 12G5 and lysates thereof were collected when > 50% cytopathic effect was observed. 50-100ng of genomic DNA isolated from virus-infected BHK cell lysates was used for the qPCR reaction. Results are shown as gE/Us8 or GFP fold relative to the ICP6 control HSV gene. FIG. 21D: comparison of plaque size between 17 Δ gE clone 12G 517 syn + or 17 Δ 34.5 clone B4 was taken from a standard plaque assay performed in Vero cells. Photographs of plaque were taken 3 days after viral infection (pvi).

Detailed Description

Embodiments in accordance with the present invention are described more fully below. Aspects of the present invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

Unless defined otherwise, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of this application and the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein. Although not explicitly defined below, such terms should be construed according to their common meaning.

The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

The practice of the present technology will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the skill of the art.

The various features of the invention described herein may be used in any combination, unless the context indicates otherwise. Furthermore, the present invention also contemplates that in some embodiments, any feature or combination of features described herein may be excluded or omitted. For purposes of illustration, if the specification states that the compound includes components A, B and C, it is specifically intended that any one or combination of A, B or C can be omitted and disclaimed individually or in any combination.

Unless expressly stated otherwise, all specified embodiments, features and terms are intended to include the described embodiments, features or terms and their biological equivalents.

All numerical designations, such as pH, temperature, time, concentration, and molecular weight, including ranges, are approximate and vary (+) or (-) by 1.0 or 0.1, or +/-15%, or 10%, or 5%, or 2%, as the case may be. It is to be understood that, although not always explicitly stated, all numerical designations are preceded by the term "about". It is also to be understood that, although not always explicitly stated, the reagents described herein are merely exemplary reagents and that equivalents of such reagents are known in the art.

As used herein, the term "comparable to" refers to having the same level as a reference level, or within a range of +/-50%, or 45%, or 40%, or 35%, or 20%, or 25%, or 20%, or 15%, or 10%, or 5%, or 2% variation from the reference level.

Throughout the present invention, various publications, patents and published patent specifications are cited by identifying citations or arabic numerals. Full citations for publications identified by arabic numerals may be found before the claims. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference in their entirety to more fully describe the state of the art to which this invention pertains.

Definition of

The practice of the present technology will employ, unless otherwise indicated, conventional techniques of organic chemistry, pharmacology, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the skill of the art. See, for example, Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd edition (1989); current Protocols In Molecular Biology (F.M. Ausubel, et al. eds., (1987)); the series Methods in Enzymology (Academic Press, Inc.: PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, a Laboratory Manual, and Animal Cell Culture (R.I. Freeshine, ed. (1987)).

As used in the description of the invention and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements, but do not exclude other elements. As used herein, transitional phrases consisting essentially of … … (and grammatical variants thereof) are to be construed as including materials or steps that include those that do not materially affect the basic and novel characteristics of the described embodiments. These features are described in method embodiments. Thus, the term "consisting essentially of … …" as used herein should not be construed as equivalent to "comprising". "consisting of … …" shall mean excluding trace elements in excess of other ingredients and the basic method steps for administering the compositions disclosed herein. Aspects defined by each of these transition terms are within the scope of the invention.

The term "about," as used herein, when referring to a measurable value (e.g., an amount or concentration, etc.), is meant to encompass a change of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the stated amount.

The term or "acceptable", "effective" or "sufficient" when used to describe the selection of any ingredient, range, dosage form, etc., disclosed herein means that the ingredient, range, dosage form, etc., is suitable for the purposes disclosed.

Also as used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

The term "cell" as used herein may refer to a prokaryotic or eukaryotic cell, optionally obtained from a subject or a commercially available source.

"eukaryotic cell" refers to all kingdoms of life except prokaryotes. They can be easily distinguished by the membrane-attached nuclei. Animals, plants, fungi and protists are eukaryotes or organisms whose cells make up a complex structure through the inner membrane and cytoskeleton. The most typical membranous structures are nuclei. Unless otherwise specified, the term "host" includes eukaryotic hosts, including, for example, yeast, higher plant, insect, and mammalian cells. Non-limiting examples of eukaryotic cells or hosts include simians, bovines, porcines, mice, rats, avians, reptiles, and humans, such as HEK293 cells and 293T cells.

"prokaryotic cells" generally have no nucleus or any other membrane-attached organelle and are divided into two domains, bacteria and archaea. In addition to chromosomal DNA, these cells may also contain genetic information in a cycle called episome. The bacterial cells are very small, roughly corresponding to the size of the animal's mitochondria (about 1-2 μm in diameter, 10 μm long). Prokaryotic cells have three main shapes: rod-like, spherical and spiral. Bacterial cells do not undergo a complex replication process as eukaryotes do, but divide by binary divisions. Examples include, but are not limited to, bacillus, escherichia coli, and salmonella.

The term "encoding" as applied to a nucleic acid sequence refers to a polynucleotide that is described as "encoding" a polypeptide, which in its native state or when manipulated by methods well known to those skilled in the art, can be transcribed and/or translated to produce an mRNA for the polypeptide and/or fragments thereof. The antisense strand is the complement of such a nucleic acid, from which the coding sequence can be deduced.

The terms "equivalent" or "bioequivalent" are used interchangeably in reference to a particular molecular, biological or cellular material and mean a material that has minimal homology while retaining a desired structure or function. Non-limiting examples of equivalent polypeptides include polypeptides having at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95% identity to the polypeptide or polypeptide sequence, or polypeptides encoded by polynucleotides that hybridize under highly stringent conditions to a polynucleotide encoding the polypeptide sequence, or the complement thereof. Highly stringent conditions are described herein and incorporated herein by reference. Alternatively, an equivalent thereof is a polypeptide encoded by a polynucleotide having at least 70%, or at least 75%, or 80%, or at least 85%, or at least 90%, or at least 95% identity, or at least 97% sequence identity, or at least 99% identity to a reference polynucleotide (e.g., a wild-type polynucleotide), or a complement thereof. In one aspect, the equivalent polypeptide or polynucleotide has the same or substantially similar biological function as the reference polypeptide or polynucleotide, respectively, e.g., cytolytic function, anti-tumor, anti-metastatic, or anti-cancer function, as determined by an appropriate cellular assay or animal model as described herein.

Non-limiting examples of equivalent polypeptides include polynucleotides that are at least 60%, or at least 65%, or at least 70%, or at least 75%, or 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97% identical to a reference polynucleotide. Equivalents also include polynucleotides that hybridize to a reference polynucleotide under highly stringent conditions, or the complement thereof.

A polynucleotide or polynucleotide region (or polypeptide region) has a percentage (e.g., 80%, 85%, 90% or 95%) of "sequence identity" with another sequence, meaning that when aligned, the percentage of bases (or amino acids) are the same when comparing the two sequences. The alignment and percent homology or sequence identity can be determined using software programs known in the art, for example, the software programs described in Current Protocols in Molecular Biology (Ausubel et al, eds. 1987) Supplement 30, section 7.7.18, Table 7.7.1. In some embodiments, default parameters are used for alignment. Using default parameters, a non-limiting exemplary alignment program can be performed using BLAST. Specifically, exemplary programs include BLASTN and BLASTP using the following default parameters: genetic code = standard; filter = none; strand = two strands; cutoff = 60; desired value = 10; matrix = BLOSUM 62; =50 sequences are described; the sorting mode = high score; database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS transitions + SwissProtein + SPupdate + PIR. For detailed information about these programs, the following web sites can be accessed: ncbi.nlm.nih.gov/cgi-bin/BLAST. Sequence identity and percent identity can be determined by incorporating them into clustalW (available on the website: genome. jp/tools/clustalW/last visit date, 1/13/2017).

"homology" or "identity" or "similarity" refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence, which may be aligned for comparison. When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An "unrelated" or "non-homologous" sequence is one that has less than 40% identity, or less than 25% identity, to one of the sequences of the present invention.

"homology" or "identity" or "similarity" may also refer to two nucleic acid molecules that hybridize under stringent conditions.

"hybridization" refers to the reaction of one or more polynucleotides to form a complex that is stabilized by hydrogen bonding between nucleotide residue bases. Hydrogen bonding can occur by Watson-Crick base pairing, Hoogstein binding, or any other sequence specific means. The compound comprises, consists essentially of, or consists of: two strands forming a double-stranded structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these. The hybridization reaction may constitute a step in a broader process, such as the initiation of a PCR reaction, or enzymatic cleavage of a polynucleotide by a ribozyme.

Examples of stringent hybridization conditions include: an incubation temperature of about 25 ℃ to about 37 ℃; the hybridization buffer concentration is about 6 XSSC to about 10 XSSC; formamide concentrations of about 0% to 25%; and a wash solution of from about 4 XSSC to about 8 XSSC. Examples of moderately stringent hybridization conditions include: a culture temperature of about 40 ℃ to about 50 ℃; the buffer concentration is about 9 XSSC to 2 XSSC; formamide concentration of about 30% to 50%; and a wash solution of about 5 XSSC to about 2 XSSC. Highly stringent hybridization means that the oligonucleotide hybridizes to the target sequence without mismatches (or complete complementarity). Examples of high stringency conditions include: an incubation temperature of about 55 ℃ to about 68 ℃; the buffer concentration is about 1 XSSC to 0.1 XSSC; formamide concentrations of about 55% to 75%; and about 1 XSSC, 0.1 XSSC, or deionized water. Typically, the hybridization incubation time is 5 minutes to 24 hours with 1,2 or more wash steps, and the wash incubation time is about 1,2 or 15 minutes. SSC is 0.15M NaCl and 15 mM sodium citrate buffer. It should be understood that SSC equivalents using other buffering systems may be used.

As used herein, "expression" refers to the process by which a polynucleotide is transcribed into mRNA and/or the process by which transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression may include splicing of mRNA in eukaryotic cells.

"Gene" refers to a polynucleotide comprising at least one Open Reading Frame (ORF) and capable of encoding a particular polypeptide or protein after transcription and translation. "Gene product" or optionally "gene expression product" refers to the amino acids (e.g., peptides or polypeptides) produced when a gene is transcribed and translated.

"under transcriptional control" is a term well known in the art and indicates that transcription of a polynucleotide sequence (typically a DNA sequence) is dependent on its operable linkage to elements that help initiate or promote transcription. By "operably linked" is meant that the polynucleotides are arranged in a manner that allows them to function in a cell. In one aspect, the invention provides a promoter operably linked to a downstream sequence (e.g., an HSV virulence gene or mutant thereof).

The term "isolated" as used herein refers to a molecule, biological product, or cellular material that is substantially free of other materials.

As used herein, the term "functional" may be used to modify any molecule, biological, or cellular material to achieve a particular specific effect.

As used herein, the terms "nucleic acid sequence" and "polynucleotide" are used interchangeably to refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derivatized nucleotide bases.

The term "wild-type" refers to a gene or gene product that has the characteristics of the gene or gene product when isolated from a natural source. In some embodiments, the wild-type gene or gene product, even for one strain of virus, comprises a slightly different sequence.

The term "mutant" refers to a gene or gene product that exhibits an alteration in sequence and/or functional properties (i.e., altered characteristics) as compared to the wild-type gene or gene product or a gene or gene product from other mutant strains. In one embodiment, the other mutant strain comprises a 17Terma or rR450 strain.

The term "mutation" refers to a variation in the DNA sequence from a wild-type or other mutant strain. Mutations may or may not produce functional properties in an organism. There are many types of mutations, including but not limited to insertions, deletions, truncations, frameshifts, substitutions, or point mutations.

The term "point mutation" refers to a mutation having a single nucleotide base change, insertion or deletion of genetic material, DNA or RNA.

"deletion" refers to a mutation in which a portion of a chromosome or a DNA sequence is deleted.

"frameshift" refers to a mutation caused by an insertion or deletion of multiple nucleotides in a DNA sequence that are not divisible by three.

"substitution" refers to a mutation in which one or several nucleotides of a gene are substituted.

"truncation" refers to the elimination of mutations at the N-or C-terminal portion of a protein by proteolysis or manipulation of the structural gene, or premature termination of protein extension due to the presence of a stop codon in the structural gene as a result of a nonsense mutation.

In some embodiments, the mutation is a non-synonymous mutation. The term "non-synonymous mutation" refers to a mutation that alters the amino acid sequence of a protein, as opposed to a synonymous mutation that does not alter the amino acid sequence.

As used herein, the term "promoter" refers to any sequence that regulates the expression of a coding sequence (e.g., a gene). For example, a promoter may be constitutive, inducible, repressible, or tissue specific. A "promoter" is a control sequence, which is a region of a polynucleotide sequence that controls the initiation and rate of transcription. It may contain genetic elements to which regulatory proteins and molecules can bind, such as RNA polymerase and other transcription factors. Non-limiting exemplary promoters include the ROS Sarcoma Virus (RSV) LTR promoter (optionally with the RSV enhancer), the Cytomegalovirus (CMV) promoter, the SV40 promoter, the dihydrofolate reductase promoter, the β -actin promoter, the phosphoglycerate kinase (PGK) promoter, the U6 promoter, or the EF1 promoter. In some embodiments, the promoter is a chicken β -actin ("CBA") promoter.

Additional non-limiting exemplary promoters with specific target specificity are provided below, including but not limited to CMV, EF1a, SV40, PGK1 (human or mouse), P5, Ubc, human β -actin, CAG, TRE, UAS, Ac5, polyhedrin, CaMKIIa, Gal1, TEF1, GDS, ADH1, CaMV35S, Ubi, H1, U6, α -1-antitrypsin. Synthetically derived promoters may be used for ubiquitous or tissue-specific expression. In addition, virus-derived promoters, some of which are described above, can be used in the methods disclosed herein, such as CMV, HIV, adenovirus, and AAV promoters. In some embodiments, the promoter is coupled to an enhancer to increase transcription efficiency.

An enhancer is a regulatory element that increases the expression of a target sequence. A "promoter/enhancer" is a polynucleotide comprising sequences that provide both promoter and enhancer functions. For example, the long terminal repeat of a retrovirus contains both promoter and enhancer functions. Enhancers/promoters may be "endogenous" or "exogenous" or "heterologous". An "endogenous" enhancer/promoter is one that is naturally linked to a given gene in the genome. An "exogenous" or "heterologous" enhancer/promoter is an enhancer/promoter that is juxtaposed to a gene by genetic manipulation (i.e., molecular biology techniques) such that transcription of the gene is controlled by the linked enhancer/promoter.

As used herein, the term "tumor-specific promoter or tissue-specific promoter" refers to a promoter that allows for expression of a gene under the control of the promoter, particularly in a desired tumor cell or tissue. Non-limiting examples of tissue-specific promoters that may be used in the present invention include the prostate-specific antigen (PSA) promoter, the prostate-specific membrane antigen (PSMA) promoter, the casein promoter, the IgG promoter, the chorionic embryonic antigen promoter, the elastase promoter, the porphobilinogen deaminase promoter, the insulin promoter, the growth hormone factor promoter, the acetylcholine receptor promoter, the alcohol dehydrogenase promoter, and the alpha or beta globin promoter.

Non-limiting examples of tumor specific promoters for use in the present invention include telomerase reverse transcriptase promoter, glial fibrillary acidic protein promoter, E2F promoter, survivin promoter, COX-2 promoter, EGD-2 promoter, ELF-1 promoter, hypoxia specific promoter, carcinoembryonic antigen promoter, and matrilysin 3 promoter.

The term "cryoprotectant" refers to a compound or material capable of protecting one or more tissues, viruses, or other biological agents from damage or injury. Examples of cryoprotectants include, but are not limited to, chondroitin sulfate, glycosaminoglycan dimethyl sulfoxide, cell-penetrating organic solutes, polysaccharides, glycerol, Dulbecco's Minimal Essential Medium (DMEM), glutamine, D-glucose, sodium pyruvate, fetal bovine serum, papaverine, DMSO, glycerol, trehalose, KH2PO4, K2HPO4, KCl, mannitol, NaHCO3, sodium ascorbate, 1, 2-propanediol, formamide, 2, 3-butanediol, probucol, curcumin, and mixtures thereof.

The terms "protein", "peptide" and "polypeptide" are used interchangeably and in their broadest sense refer to a compound consisting of two or more subunits of an amino acid, amino acid analog or peptidomimetic. The subunits may be linked by peptide bonds. In another aspect, the subunits may be linked by other linkages, such as esters, ethers, and the like. A protein or peptide must comprise at least two amino acids, and there is no limitation as to the maximum number of amino acids a protein or peptide sequence may comprise, consist essentially of, or consist of. As used herein, the term "amino acid" refers to natural and/or unnatural or synthetic amino acids, including glycine and the D and L optical isomers, amino acid analogs, and peptidomimetics.

As used herein, the term "vector" refers to a non-chromosomal nucleic acid comprising, consisting essentially of, or consisting of an intact replicon, such that the vector can be replicated when placed in a cell, e.g., by a transformation process. The vector may be a viral vector or a non-viral vector. Viral vectors include retroviruses, adenoviruses, herpes simplex virus ("HSV"), baculoviruses, modified baculoviruses, papovaviruses, or other modified native viruses. Exemplary non-viral vectors for delivering nucleic acids include: naked DNA; DNA complexed with cationic liposomes, alone or in combination with cationic polymers; anionic and cationic liposomes; DNA-protein complexes and particles, including DNA condensed with cationic polymers (e.g., isopolylysine, limited length oligopeptides, and polyethyleneimine), in some cases contained in, or consisting essentially of, or consisting of, liposomes; and to the use of a ternary complex comprising, consisting essentially of, or consisting of a virus and polylysine DNA.

A "viral vector" is defined as a recombinantly produced virus or viral particle comprising, consisting essentially of, or consisting of a polynucleotide to be delivered to a host cell in vivo, ex vivo, or in vitro. Examples of viral vectors include retroviral vectors, AAV vectors, lentiviral vectors, adenoviral vectors, alphaviral vectors, and the like. Alphavirus vectors, such as those based on the Semliki Forest virus and those based on the Sindbis virus, have also been developed for gene therapy and immunotherapy. See Schlesinger and Dubensky (1999) curr. Opin. Biotechnol. 5: 434-.

In another embodiment, expression of an HSV virulence protein (e.g., wild-type or mutant) is regulated by a promoter that is an inducible promoter. In a particularly related embodiment, the promoter is an inducible tetracycline promoter. The Tet-Off and Tet-On gene expression systems allow researchers to have ready access to regulated high level gene expression systems described as Tet-Off and Tet-On. In the Tet-Off system, gene expression is turned on when tetracycline (Tc) or doxycycline (Dox; a Tc derivative) is removed from the medium. In contrast, in the Tet-on system, expression is turned on by the addition of Dox. Both systems allow gene expression to be tightly regulated by varying concentrations of Tc or Dox. The maximum expression level in the Tet system is very high and performs well compared to that obtained with a strong constitutive mammalian promoter (e.g., CMV). Unlike other inducible mammalian expression systems, gene regulation in the Tet system is highly specific, so interpretation of results is not complicated by pleiotropic effects or non-specific induction. In E.coli, the Tet repressor protein (TetR) negatively regulates the gene of the tetracycline resistance operator on the Tn10 transposable element. In the absence of Tc, TetR blocks transcription of these genes by binding to the tet operator (tetO). TetR and tetO provide the basis for regulation and induction in mammalian experimental systems. In the Tet-On system, the regulatory protein is based On the "reverse" Tet repressor (rTetR) which is generated by four amino acid changes in TetR (Hillen & Berens, Mechanisms undersizing expression of Tn10 encoded tetracycline restriction. Annu Rev Microbiol. 1994;48:345-69; Gossen et al, transcription activity by tetracyclines in mammalian cells. science. 1995 Jun 23;268(5218): 1766-9). The resulting protein rtTA (reverse tTA is also known as tetracycline activator) is encoded by the pTet-On regulatory plasmid.

In a related embodiment, the vector further comprises, consists essentially of, or consists of: a nucleic acid encoding a tetracycline activator protein; and a promoter that regulates the expression of tetracycline activator protein.

Other inducible systems that can be used in the vectors, isolated cells, viral packaging systems, and methods described herein include modulation of ecdysone, estrogen, progesterone, dimerization chemistry inducers, and isopropyl-beta-D1-thiogalactopyranoside (IPTG).

As used herein, the term "recombinant expression system" or "recombinant vector" refers to a system for expressing one or more genetic constructs formed by recombination for the expression of certain genetic material.

A "gene delivery vector" is defined as any molecule capable of carrying a polynucleotide for insertion into a host cell. Examples of gene delivery vectors are: liposomes, micelles, biocompatible polymers, including natural polymers and synthetic polymers; a lipoprotein; a polypeptide; a polysaccharide; a lipopolysaccharide; enveloping the artificial virus; metal particles; and bacterial or viral, such as baculoviruses, adenoviruses and retroviruses, bacteriophages, cosmids, plasmids, fungal vectors and other recombinant vectors commonly used in the art, which have been described for expression in a variety of eukaryotic and prokaryotic hosts, and which are useful for gene therapy as well as simple protein expression.

The polynucleotides disclosed herein can be delivered to a cell or tissue using a gene delivery vector. As used herein, "gene delivery," "gene transfer," "transduction," and the like refer to the introduction of an exogenous polynucleotide (sometimes referred to as a "transgene") into a host cell, regardless of the method used for introduction. Such methods include a variety of well-known techniques, such as vector-mediated gene transfer (e.g., by viral infection/transfection or various other protein or lipid-based gene delivery complexes) and techniques that facilitate the delivery of "naked" polynucleotides (e.g., electroporation, "gene gun" delivery and various other techniques for introducing polynucleotides). The introduced polynucleotide may be stably or transiently maintained in the host cell. Stable maintenance generally requires that the introduced polynucleotide comprise an origin of replication compatible with the host cell, or that the replicon integrate into the host cell, e.g., an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome. Many vectors are known that are capable of mediating gene transfer into mammalian cells, as is known in the art and described herein.

A "plasmid" is an extrachromosomal DNA molecule that is separated from chromosomal DNA and is capable of replication independently of the chromosomal DNA. In many cases, it is circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within a microbial population, and generally offer a selective advantage under given environmental conditions. The plasmid may carry a gene that confers resistance to the natural antibiotic in a competitive environmental niche, or the protein produced may also act as a toxin under similar circumstances.

The "plasmid" used in genetic engineering is referred to as a "plasmid vector". Many commercial plasmids are available for such use. The gene to be replicated is inserted into a copy of the plasmid, which contains the gene conferring resistance to a particular antibiotic to the cell and a multiple cloning site (MCS or polylinker), a short region containing several commonly used restriction sites, allowing easy insertion of a DNA fragment at that location. Another major use of plasmids is the production of large quantities of protein. In this case, the researchers developed bacteria containing plasmids carrying the genes of interest. Just as bacteria produce proteins to confer antibiotic resistance, they can also be induced to produce large amounts of protein from an inserted gene.

The term "herpes simplex virus" or "HSV" as used herein refers to a herpes simplex virus, including wild-type or mutant herpes simplex viruses, which produces the effects of the present invention. In one embodiment, the mutant non-native HSV is obtained by mutating or modifying any gene of a wild-type HSV or by inserting any foreign gene. The serotype HSV includes, consists essentially of, or consists of HSV type 1 (or HSV-1) or HSV type 2 (or HSV-2). HSV-1 is an enveloped double-stranded DNA virus. In one embodiment, HSV-1 infects human cells. In another embodiment, a sequence, gene, or multiple genes may be incorporated into HSV-1. The size of the pooled sequence may be about 1 base, 5 bases, 10 bases, 100 bases, 1kb, 10kb, 100kb or 150 kb. HSV-1 induces cell lysis at a relatively low multiplicity of infection (MOI) and its proliferation can be inhibited by antiviral drugs. In one embodiment, HSV viral DNA resides outside of the chromosome without being incorporated into the genome of the host cell. HSV-1 can comprise a variety of strains (e.g., KOS and McKrae). See Wang et al, (2013) Virus Res. 173(2): 436- & 440. In one embodiment, HSV-1 is the HSV-1 KOS strain. In another embodiment, HSV-1 is the HSV-1 McKrae strain.

There are several HSV mutants, for example 17Terma HSV and rRp450 HSV. The term "17 Terma HSV" refers to a mutant HSV-1 virus which contains the entire ICP34.5 gene, but has a stop codon inserted 100 bp before the coding region, resulting in premature termination of protein expression and expression of a 30 amino acid truncated protein. The 17Terma HSV mutant showed growth defects due to the truncated ICP34.5 protein. See, Orvedahl et al, (2007) Cell Host & Microbe, 1:1, 23-25. The term "rRp 450" refers to an attenuated herpes simplex 1 vector lacking in virally encoded ribonucleotide reductase or ICP 6. See, Aghi M et al, (1999) Cancer Res, 59(16): 3861-5.

The HSV genome encodes a variety of virulence proteins, including, but not limited to, glycoprotein E ("gE"), infected cellular protein 0 ("ICP 0"), infected cellular protein 6 ("ICP 6"), DNA-packaging terminal enzyme subunit 1, infected cellular protein 8 ("ICP 8"), and infected cellular protein 34.5 ("ICP 34.5"). An exemplary HSV1 genome can be found in the NCBI reference sequence: NC _001806.2, last visit time 3/13/2020.

The term "gE-encoding gene" refers to a gene encoding a gE protein or a DNA fragment thereof. Exemplary gE encoding genes can be found in NCBI reference sequence: NC-001806.2 at positions 33-2555. The term "ICP 6 protein" refers to the infected cellular protein 6 encoded by the HSV genome. ICP6 is a subunit of ribonucleotide reductase ("RR") that is a key enzyme in nucleotide metabolism and viral DNA synthesis in non-dividing cells.

A "dysfunctional" protein refers to a protein in which the function of the original protein is impaired or non-functional. In one embodiment, the dysfunctional protein is caused by a deletion or substitution in the coding sequence. For example, if the ICP6 gene is dysfunctional, deleted or inactivated, HSV cannot replicate in normal, non-dividing cells. However, in actively dividing cells with increased RR activity, the viral deficient enzymatic activity is compensated for, enabling the virus to replicate. The DNA and amino acid sequences of ICP34.5 are provided in SEQ ID Nos. 1,2 and 5-10. The DNA and amino acid sequence of gE is provided in SEQ ID Nos. 12-19. The DNA and amino acid sequences of ICP0 are provided in SEQ ID Nos. 20-26. The DNA and amino acid sequences of the DNA packaging terminal enzyme subunit 1 are provided in SEQ ID Nos. 35-42. The DNA and amino acid sequences of ICP8 are provided in SEQ ID Nos. 27-34. The DNA and amino acid sequences of ICP6 are provided in SEQ ID Nos 43-50.

The term "ICP 0-encoding gene" refers to a gene encoding ICP0 protein or a DNA fragment thereof. Exemplary DNA and amino acid sequences for ICP0 are provided in SEQ ID Nos. 20-26. The term "gene encoding DNA-packaging terminal enzyme subunit 1" refers to a gene encoding a DNA-packaging terminal enzyme subunit 1 protein or peptide, or a DNA fragment thereof. Exemplary DNA and amino acid sequences of DNA packaging terminal enzyme subunit 1 are provided in SEQ ID Nos. 35-42. The term "ICP 8-encoding gene" refers to a gene encoding ICP8 protein or a DNA fragment thereof. Exemplary DNA and amino acid sequences for ICP8 are provided in SEQ ID Nos. 27-34. The term "ICP 34.5-encoding gene" refers to a gene encoding ICP34.5 protein or a DNA fragment thereof. Exemplary DNA and amino acid sequences for ICP34.5 are provided in SEQ ID Nos. 1,2, and 5-10. The term "glycoprotein E (" gE ") encoding gene" refers to a gene or DNA fragment thereof that encodes a gE protein. Exemplary DNA and amino acid sequences of gE are provided in SEQ ID Nos. 12-19.

The term "deletion or inactivation of a gene" means deletion of all or part of the gene or inhibition of expression of the gene by substitution of some bases, modification, insertion of an unnecessary sequence, or the like. Deletion or inactivation of HSV genes (e.g., gE, ICP0, and ICP 8) can be performed by one of skill in the art in a known method or a method based thereon. For example, a method using homologous recombination can be adopted. For example, HSV genes can be isolated and inactivated by cloning a DNA fragment containing a portion of the HSV gene and sequences unrelated to the HSV gene in a suitable plasmid vector, and then introducing it into HSV, thereby causing homologous recombination in certain regions of the HSV gene. Alternatively, mutations or deletions in HSV genes can be caused by spontaneous mutations in the virus passage.

In terms of gene transfer mediated by a DNA viral vector (e.g., herpes simplex virus), vector construction refers to polynucleotides and transgenes comprising, consisting essentially of, or consisting of a viral genome or portion thereof. Thus, in one aspect, the non-natural HSV further comprises a transgene encoding a therapeutic polynucleotide or protein.

Vectors comprising a promoter and a cloning site operably linked to a polynucleotide are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo, and are commercially available from sources such as Agilent Technologies (Santa Clara, Calif.) and Promega Biotech (Madison, Wis.). To optimize expression and/or in vitro transcription, it may be desirable to remove, add, or alter the 5 'and/or 3' untranslated portions of the clones to eliminate additional, potentially inappropriate alternative translation initiation codons or other sequences that may interfere with or reduce expression, whether at the transcriptional or translational level. Alternatively, the consensus ribosome binding site can be inserted immediately 5' of the start codon to enhance expression.

Gene delivery vectors also include DNA/liposome complexes, micelles, and targeted viral protein DNA complexes. Liposomes further comprising, consisting essentially of, or consisting of a targeting antibody or fragment thereof can be used in the methods disclosed herein. In addition to delivering the polynucleotide to a cell or population of cells, the protein described herein can be introduced directly into the cell or population of cells by non-limiting protein transfection techniques, or culture conditions that can enhance the expression and/or promote the activity of the protein disclosed herein are other non-limiting techniques.

As used herein, the term "signal peptide" or "signal polypeptide" means an amino acid sequence that is typically present at the N-terminus of a newly synthesized secreted or membrane polypeptide or protein. Its function is to direct the polypeptide to a specific cellular location, e.g., across the cell membrane, into the cell membrane, or into the nucleus. In some embodiments, the signal peptide is removed after localization. Examples of signal peptides are well known in the art. Non-limiting examples are those described in U.S. patent nos. 8,853,381, 5,958,736, and 8,795,965.

In one aspect, the HSV is detectably labeled. As used herein, the term "label" refers to a directly or indirectly detectable compound or composition, e.g., a polynucleotide or protein, e.g., an antibody, coupled directly or indirectly to a composition to be detected to produce a "labeled" composition. The term also includes sequences coupled to the polynucleotide that provide a signal upon expression of the inserted sequence, such as Green Fluorescent Protein (GFP), and the like. The label may be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable. The label may be suitable for small scale detection or more suitable for high throughput screening. Thus, suitable labels include, but are not limited to, radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes, and proteins, including enzymes. The label can simply be detected or quantified. A response that is simply detected typically includes, consists essentially of, or consists of a response that merely confirms its presence, while a quantified response typically includes a response having a quantifiable (e.g., digitally reportable) value, such as an intensity, polarization, and/or other characteristic. In luminescence or fluorescence assays, a luminophore or fluorophore associated with an assay component actually involved in binding that directly produces a detectable response may be used, or a luminophore or fluorophore associated with another (e.g., reporter or indicator) component may be used indirectly.

Examples of luminescent labels that produce a signal include, but are not limited to, bioluminescence and chemiluminescence. The detectable luminescent response typically comprises, consists essentially of, or consists of a change or occurrence in the luminescent signal. Suitable methods and luminophores for luminescent labelling of analytical components are known in the art, for example as described in Haughland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th ed.). Examples of luminescent probes include, but are not limited to, aequorin and luciferase.

Examples of suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, haematochrome, coumarin, methylcoumarin, pyrene, apple green, stilbene, Lucy Falset, Cascade blue TM., and Texas Red. Other suitable optical dyes are described, for example, in Haughland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th ed.).

In another aspect, the fluorescent label is functionalized to facilitate covalent attachment to cellular components present in or on the surface of the cell or tissue, such as cell surface markers. Suitable functional groups include, but are not limited to, isothiocyanate, amino, haloacetyl, maleimide, succinimidyl ester, and sulfonyl halide, all of which can be used to attach a fluorescent label to a second molecule. The choice of the fluorescent-labeled functional group depends on the attachment site of the linker, reagent, label, or second label.

Attachment of the fluorescent label may be directly to the cellular component or compound, or may be through a linker. Suitable binding pairs for indirectly linking a fluorescent label to an intermediate include, but are not limited to, antigens/antibodies such as rhodamine/anti-rhodamine, biotin/avidin, and biotin/streptavidin.

The phrase "solid support" refers to a non-aqueous surface, such as a "culture plate", "gene chip" or "microarray". Such gene chips or microarrays can be used for diagnostic and therapeutic purposes by a number of techniques known to those skilled in the art. In one technique, oligonucleotides are ligated and arrayed on a gene chip for determination of DNA sequences by hybridization methods, such as the methods outlined in U.S. Pat. Nos. 6,025,136 and 6,018,041. The polynucleotides of the invention may be modified into probes and may be used to detect genetic sequences. Such techniques are described, for example, in U.S. Pat. nos. 5,968,740 and 5,858,659. Probes can also be attached or immobilized to the surface of an electrode for electrochemical detection of Nucleic acid sequences, as described in Kayem et al, U.S. Pat. No. 952,172 and Kelley et al (1999) Nucleic Acids Res.27: 4830-4837.

"composition" refers to the active polypeptide, polynucleotide or antibody and another inert (e.g., detectable label) or active (e.g., gene delivery vector) compounds or compositions of combination.

"pharmaceutical composition" is meant to include the combination of an active polypeptide, polynucleotide or antibody with a carrier (inert or active carrier, e.g., a solid carrier) that renders the composition suitable for diagnostic or therapeutic use ex vivo, in vivo or in vitro.

As used herein, the term "pharmaceutically acceptable carrier" includes any standard pharmaceutical carrier, such as phosphate buffered saline solutions, water, and emulsions, such as oil/water or water/oil emulsions, as well as various types of wetting agents. The composition may also include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see Martin (1975) Remington's pharm. sci., 15th ed. (Mack pub. co., Easton).

A "subject" for diagnosis or treatment is a cell or an animal, such as a mammal or a human. Subjects are not limited to a particular species, and include non-human animals that are diagnosed or treated, as well as animals that are affected by infection or animal models, e.g., simians, murines (e.g., rats, mice, chinchillas), canines (e.g., dogs), lagomorphs (e.g., rabbits), livestock, sport animals, and pets. Human patients are also included in the term.

As used herein, the term "tissue" refers to the tissue of a living or dead organism, or any tissue derived from or designed to mimic a living or dead organism. The tissue may be healthy, diseased, and/or have a genetic mutation. Biological tissue may include any single tissue (e.g., a collection of cells that may be connected to one another) or tissues that make up an organ or body part or region of an organism. The tissue may comprise, consist essentially of, or consist of homogeneous cellular material, or may be a composite structure, such as found in body regions including the breast, which may include, for example, lung tissue, skeletal tissue, and/or muscle tissue. Exemplary tissues include, but are not limited to, tissues derived from the liver, lung, thyroid, skin, pancreas, blood vessels, bladder, kidney, brain, biliary tract, duodenum, abdominal aorta, iliac vein, heart, and intestinal tract, including any combination thereof.

As used herein, "treating" a disease in a subject refers to (1) preventing the appearance of symptoms or disease in a subject susceptible to or not yet exhibiting symptoms of the disease; (2) inhibiting or arresting the development of the disease; or (3) ameliorating or causing regression of the disease or disease symptoms. As understood in the art, "treatment" is a method for obtaining beneficial or desired results, including clinical results. For purposes of the present technology, beneficial or desired results may include one or more of, but are not limited to: alleviating or ameliorating one or more symptoms; reduction in the extent of the disorder (including disease); a stable (i.e., not worsening) condition (including disease); delay or alleviation of disorders (including diseases); progression, amelioration, or remission of a disorder (including disease); status and remission (whether partial or total), whether detectable or not.

As used herein, the term "effective amount" refers to an amount sufficient to achieve the desired effect. In the case of therapeutic or prophylactic use, the effective amount will depend on the type and severity of the disease in question and on the characteristics of the individual subject, such as general health, age, sex, body weight and tolerance to pharmaceutical compositions. In the context of gene therapy, in some embodiments, an effective amount is an amount sufficient to result in the recovery of some or all of the function of a gene that is lacking in a subject. In one aspect, an effective amount is an amount that provides a multiplicity of infection (MOI) of 0.001 to 1 infectious viral particle per cell, ranging between the two. Non-limiting examples include a multiplicity of infection (MOI) of at least 0.001, or at least 0.01, or at least 0.1, or at least 1, or 0.01 to 1, or 0.1 to 1, or about 0.01 to 0.1, or less than 1, or less than 0.1, or less than 0.01 infectious viral particles per cell. In other embodiments, an effective amount of HSV viral particles is an amount sufficient to cause cell lysis in a subject. In some embodiments, the effective amount is an amount required to increase galactose metabolism in a subject in need thereof. One skilled in the art will be able to determine appropriate amounts based on these and other factors.

In some embodiments, the effective amount will depend on the size and nature of the application. But also on the nature and sensitivity of the target object and the method used. One skilled in the art will be able to determine an effective amount based on these and other considerations. According to an embodiment, an effective amount may comprise, consist essentially of, or consist of one or more administrations of the composition.

As used herein, the term "administering" or "administration" refers to the delivery of a substance to a subject (such as an animal or human). Administration may be once, continuously or intermittently throughout the course of treatment, e.g. intratumorally or intravenously. Methods of determining the most effective mode of administration and dosage are known to those skilled in the art and will vary with the composition used for treatment, the purpose of the treatment, and the age, health, or sex of the subject being treated. Single or multiple administrations may be carried out with the dose level and pattern being selected by the treating physician, and in the case of pets and animals, by the treating veterinarian. Appropriate dosage formulations and methods of administration are known in the art. The route of administration can be determined, and the method of determining the most effective route of administration is known to those skilled in the art and will vary with the composition used for treatment, the purpose of the treatment, the health or disease stage of the subject being treated, and the target cell or tissue. Non-limiting examples of routes of administration include direct and systemic, e.g., intravenous, intra-arterial, intramuscular, intracardiac, intrathecal, sub-ventricular, epidural, intracerebral, intratumoral, intracranial, intracerebroventricular, subretinal, intravitreal, intraarticular, intraocular, intraperitoneal, intrauterine, intradermal, subcutaneous, transdermal, transmucosal, and inhalation.

Oncolytic herpes simplex virus (oHSV)

The therapeutic effect of oHSV on tumors derives from direct cell killing (lytic phase) and enhancement of anti-cancer immunity (immune phase). These viruses have been constructed in various ways to selectively target cancer cells. To achieve cancer selectivity, the most common mutation is the neurovirulence gene γ₁34.5/RL1 deletion. By gamma₁34.5/RL1 expression of ICP34.5 is critical for HSV-1 to antagonize the host cell antiviral protein kinase RNA activation (PKR) pathway, which normally phosphorylates e-IF2 α in response to viral infection and stops protein translation. ICP34.5 redirects cellular protein phosphatase-1 (PP 1) to dephosphorylated e-IF2 α, allowing efficient viral replication. Many cancer cells are deficient in PKR responses and therefore support replication of HSV vectors, including γ₁34.5-null mutant. Some vectors are constructed by mutating key metabolic viral genes such as Ribonucleotide Reductase (RR), the large subunit of which is encoded by the ICP6/UL39 gene. Because many malignant cells increase the expression and activity of the RR, the ICP6-null mutant selectively replicates in highly proliferating cancer cells due to the presence of a large pool of ribonucleotides.

HSV entry, intercellular spread and syncytia formation

The entry of HSV-1 involves a series of steps in which viral glycoproteins interact with cell surface molecules. First, glycoproteins B and C (gB and gC) attach to cellular heparan sulfate proteoglycans, and then gD binds to viral entry receptors (i.e., nectin-1, Herpes Virus Entry Mediator (HVEM), or 3-O-heparan sulfate (3-OS-HS)). This process further allows gH/gL to interact with gB and trigger fusion of the viral envelope with the target cell membrane, resulting in release of the virion capsid and interlayer proteins into the cell. Several reports have shown that membrane fusion is also critical for subsequent lateral spread of the virus between adjacent cells. When the virus spreads from an infected cell to an adjacent uninfected cell through the cell contact area, intercellular spreading of the virus occurs, even in the presence of virus neutralizing antibodies. The presence of gE/gI enhances intercellular dissemination, while the gE subtype mutants reduce intercellular fusion and plaque size. Syncytia are the result of fusion of multiple adjacent cells into multinucleated giant cells. Mutations that trigger syncytia are found in at least four HSV viral genes (i.e., gB, gK, UL20, and UL 24).

The invention provides a strategy for developing clinically relevant viral vectors by combining 'directed evolution' and CRISPR/Cas9 technologies. This effective combination represents a substantial departure from prior art approaches. Without being bound by theory, it is expected that unexpected mechanisms would result in increased viral efficacy in killing tumors. This is expected to reveal previously unknown mutations leading to a highly fused phenotype and enhanced efficacy, as well as stepping stones for the development of the next generation of oHSV. The oHSV thus produced has enhanced lytic phase, longer sustainability and maximized therapeutic effect, which meets the objectives of the national cancer institute clinical and transformation exploration/development studies.

Although the focus is on the "lytic phase" of viral therapy, a longer duration of viral response promotes an inflammatory response in vivo, thereby favoring the "immune phase" of subsequent viral therapy for optimal anti-tumor effect. Efficacy studies in childhood cancer models can solve the problem of using non-natural HSV to treat childhood cancer. The resulting modified viruses are also expected to play a role in a variety of adult cancers.

Modes for carrying out the invention

Although HSV can target or infect a wide range of cells to induce lysis, viral infection itself has led to cytotoxicity and other diseases such as encephalitis, oesophagitis and pneumonia. A large number of HSV genes affect pathogenicity. For example, γ 34.5 (RL 1) can cause neuropathy. ICP6 (UL 39), ribonucleotide reductase, thymidine kinase (UL 23), uracil DNA glycosidase (UL 2), dUTPase (UL 50) and DNA polymerase (UL 30) are involved in HSV nucleotide metabolism and virulence. Therefore, there is a need to prepare attenuated but replication-competent HSV particles to achieve their tumor-inhibiting function while minimizing their side effects.

Provided herein is a novel virus, designated Mut-3, which displays a vast syncytial plaquePhenotype. Applicants isolated Mut-3 from serial passages mixing 17Terma and rRp450 in a non-permissive line ("directed evolution") and constructed an attenuated mutant Mut-3. delta.34.5 by gene editing ("CRISPR/Cas 9" procedure) (FIG. 1A). The whole genome sequence analysis shows that Mut-3 obtains gamma₁34.5/RL1 and UL39 (which encodes ICP 6) in their entirety, making their genotypes similar to wild-type (WT) virus. Without being bound by theory, applicants found that Mut-3 has even higher lytic activity than many WT viruses, suggesting that its enhanced potency may be associated with other genomic alterations (in addition to its complete viral genome). Five non-synonymous mutations were found in Mut-3 that differ from either parental virus, including an alanine to threonine mutation at position 151 in the gene encoding gE (a 151T). Attenuated versions of Mut-3 viruses are provided, replacing gamma with Green Fluorescent Protein (GFP) by CRISPR/Cas9 gene editing₁34.5/RL1, named Mut-3 Δ 34.5 (FIG. 1A, bottom, labeled "CRISPR/Cas 9" step). Referring to FIG. 1B, a summary of the results of the sequence comparison of Mut-3 with its parent virus is shown. Non-synonymous mutations in Mut-3 that differ from either parent are masked with backslash, including UL15, UL29, US8, RL1, and RL 2. The same genomic sequence as 17TermA is shown as a blank box; the same parts as rRp450 are covered with forward slashes.

Accordingly, the present invention provides a non-native herpes simplex virus ("HSV"), wherein the virus comprises, consists essentially of, or consists of a mutation in a virulence gene from one or more of the following groups: (a) a glycoprotein E ("gE") encoding gene, (b) an infected cellular protein 0 ("ICP 0") encoding gene, (c) a DNA packaging terminal enzyme subunit 1 encoding gene, (d) an ICP8 encoding gene, or (E) an ICP34.5 encoding gene. In one embodiment, the HSV further comprises, consists essentially of, or consists of: a gene encoding a dysfunctional ICP34.5 protein and/or a gene encoding a dysfunctional ICP6 protein. In another embodiment, the gene encoding a dysfunctional ICP34.5 protein comprises, consists essentially of, or consists of the sequence of seq id no: polynucleotides having a sequence at least 95% identical to SEQ ID nos. 1, 5, 7, 9 or 51, and equivalents thereof, provided that the equivalents retain mutated or altered amino acids or nucleotides. In another embodiment, the gene encoding a dysfunctional ICP6 protein comprises, consists essentially of, or consists of: polynucleotides having a sequence at least 95% identical to SEQ ID nos. 43, 45, 47 or 49 and equivalents thereof, provided that the equivalents retain mutated or altered amino acids or nucleotides compared to the wt sequence.

In certain embodiments, the gE-encoding gene of the non-native herpes simplex virus comprises, consists essentially of, or consists of: polynucleotides having a sequence at least 80%, 85%, 90%, 95%, 97% or 99% identical to SEQ ID NOs 12, 14, 16, 18, and equivalents thereof, provided that the equivalents retain mutated or altered amino acids or nucleotides compared to the wt sequence. In another embodiment, HSV comprising such equivalents enter cells and/or transmit and/or replicate DNA intercellularly at levels comparable to non-native HSV comprising a mutant gE having the amino acid sequence of SEQ ID NO 13. Non-limiting examples of assessing HSV entry into cells, spreading between cells, and replicating DNA can be found in the examples. In another embodiment, the polynucleotide of the gE-encoding gene encodes a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs 13, 15, 17, and 19.

In certain embodiments, the gene encoding ICP0 of a non-native herpes simplex virus comprises, consists essentially of, or consists of the sequence of seq id no: a sequence selected from the group consisting of SEQ ID NOs 20, 22, 24, 25 and 53, a sequence of any of SEQ ID NOs 20, 22, 24, 25 and 53 without one or two introns, a polynucleotide having a sequence at least 80%, 85%, 90%, 95%, 97% or 99% identical to SEQ ID NOs 20, 22, 24, 25 and 53 and equivalents thereof, provided that the equivalents retain mutated or altered amino acids or nucleotides compared to the wt sequence. In one embodiment, introns are noted in the sequence table and below: nucleotide (nt) 58 to nt 861 of SEQ ID NO: 20, nt 1529 to nt 1663 of SEQ ID NO: 20, nt 58 to nt 822 of SEQ ID NO: 22, nt 1490 to nt 1625 of SEQ ID NO: 22, 58 to nt 862 of SEQ ID NO: 24, nt 1530 to nt 1668 of SEQ ID NO: 24, nt 58 to nt 861 of SEQ ID NO: 25, nt 1529 to nt 1663 of SEQ ID NO: 25, nt 58 to nt 822 of SEQ ID NO: 53, nt 1490 to nt 1625 of SEQ ID NO: 53. In another embodiment, the equivalent encodes an ICP0 polypeptide having functions (e.g., promoting transcription of viral genes, disrupting the nuclear structure of a nucleus known as a nuclear locus or promyelocytic leukemia (PML) nucleus, and binding to neuron-specific proteins alters expression of host and viral genes) at levels comparable to wild-type ICP0 or mutant ICP0 having the amino acid sequence of SEQ ID NO: 21. Examples of evaluating such functions can be found in Lee HR, Kim DJ, Lee JM, et al (June 2004), "Absiliity of the human cytomegavirus IE1 protein to modulation of PML crystals with both important functional activities in transcriptional regulation and activity in focused fibrous cells". J. Virol. 78 (12): 6527-42; gu H, Liang Y, Mandel G, Roizman B (May 2005), "Components of the REST/CoREST/hip deacetylase repressor complex dispersed, modified, and translocated in HSV-1-infested cells". Proc. Natl. Acad. Sci. U.S. A.102 (21): 7571-6; and Pinnoji RC, Bedadala GR, George B, Holland TC, Hill JM, Hsia SC (2007), "Ressor element-1 sharpening transformation factor/neural responsive silicon factor (REST/NRSF) can regulate HSV-1 outcome transformation vitamin a bone modification". Virol. J.4: 56. Additionally or alternatively, HSV comprising such equivalents enter cells and/or spread and/or replicate DNA intercellularly at levels comparable to non-native HSV comprising mutant ICP0 having the amino acid sequence of SEQ ID NO: 21. Non-limiting examples of assessing HSV entry into cells, spreading between cells, and replicating DNA can be found in the examples. In another embodiment, the polynucleotide of the ICP0 encoding gene encodes a polypeptide having an amino acid sequence selected from SEQ ID NOs 21, 23 and 26.

In certain embodiments, the gene encoding ICP8 of a non-native herpes simplex virus comprises, consists essentially of, or consists of the sequence of seq id no: polynucleotides having a sequence selected from SEQ ID NOs 27, 29, 31, 33, having a sequence at least 80%, 85%, 90%, 95%, 97% or 99% identical to SEQ ID NOs 27, 29, 31 and 33, and equivalents thereof, provided that the equivalents retain mutated or altered amino acids or nucleotides compared to the wt sequence. In another embodiment, the equivalent encodes an ICP8 polypeptide having a level of function (e.g., annealing to single stranded DNA (ssdna), unwinding a small fragment of double stranded DNA, or disrupting the stability of double stranded DNA at the onset of replication) comparable to wild-type ICP8 or mutant ICP8 having the amino acid sequence of SEQ ID NO: 28. Such function can be assessed by methods available in the art, e.g., Boehmer, PE; Lehman, IR (1993), "Herpes simplex virus type 1 ICP8: Helix-destabilizing properties". Journal of virology. 67 (2): 711-5. Additionally or alternatively, HSV comprising such equivalents enter cells and/or spread and/or replicate DNA intercellularly at levels comparable to non-native HSV comprising mutant ICP8 having the amino acid sequence of SEQ ID NO 28. Non-limiting examples of assessing HSV entry into cells, spreading between cells, and replicating DNA can be found in the examples. In another embodiment, the polynucleotide of the ICP8 encoding gene encodes a polypeptide having an amino acid sequence selected from SEQ ID NOs 28, 30, 32 and 34.

In certain embodiments, the gene encoding subunit 1 of the DNA-packaging terminal enzyme of a non-native herpes simplex virus comprises, consists essentially of, or consists of the sequence set forth in seq id no: polynucleotides having a sequence selected from SEQ ID NOs 35, 37, 39, 41, having a sequence at least 80%, 85%, 90%, 95%, 97% or 99% identical to SEQ ID NOs 35, 37, 39 and 41 and equivalents thereof, provided that the equivalents retain mutated or altered amino acids or nucleotides compared to the wt sequence. In another embodiment, HSV comprising such equivalents enter cells and/or spread and/or replicate DNA intercellularly at levels comparable to non-native HSV comprising the mutant DNA packaging terminal enzyme subunit 1 having the amino acid sequence of SEQ ID NO: 36. Non-limiting examples of assessing HSV entry into cells, spreading between cells, and replicating DNA can be found in the examples. In another embodiment, the polynucleotide of the DNA packaging terminal enzyme subunit 1 encoding gene encodes a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs 36, 38, 40, and 42.

In certain embodiments, the HSV comprises, consists essentially of, or consists of a polynucleotide having a sequence selected from the group consisting of SEQ ID NOs 12, 20, 27, and 35. In another embodiment, HSV has no functional ICP34.5 (i.e., encodes dysfunctional ICP34.5 or non-encoding ICP 34.5). Additionally or alternatively, HSV does not have functional ICP6 (i.e. encodes dysfunctional ICP6 or non-encoding ICP 6). In another embodiment, the mutation in the virulence gene comprises, consists essentially of, or consists of: insertions, deletions, truncations, frameshifts, substitutions or point mutations, for example of the ICP34.5 gene and/or of the ICP6 gene. In another embodiment, HSV lacks genes encoding a functional ICP34.5 protein and/or a functional ICP6 protein. In another embodiment, the mutation is a non-synonymous mutation in a virulence gene.

In one embodiment, the mutation on a non-natural HSV of the present invention encodes one or more of: (a) an alanine to threonine mutation of the gE protein at position 151, (b) an arginine to histidine mutation of the ICP0 protein at position 258, (c) an alanine to threonine mutation of the DNA packaging terminal enzyme subunit 1 protein at position 376, (d) a threonine to methionine mutation of the ICP8 protein at position 1155, or (e) a proline to histidine mutation of the ICP34.5 protein at position 119. In another embodiment, the non-natural HSV comprises, consists essentially of, or consists of the sequence of seq id no: one or more of SEQ ID number 2, SEQ ID number 13, SEQ ID number 21, SEQ ID number 28 or SEQ ID number 36 and equivalents thereof, provided that the equivalents retain mutated or altered amino acids or nucleotides compared to the wt sequence.

In certain embodiments, there is provided a non-natural herpes simplex virus ("HSV"), wherein the virus comprises, consists essentially of, or consists of a mutation in one or more of the following: (a) gE, (b) ICP0, (c) DNA packaging terminal enzyme subunit 1, (d) ICP8, or (e) ICP 34.5. In certain embodiments, the HSV does not comprise a functional ICP34.5 protein (e.g., ICP34.5 of the 17TermA strain or the rRp450 strain). In another embodiment, the HSV does not comprise any ICP34.5 protein. Additionally or alternatively, the HSV does not comprise a functional ICP6 protein (e.g., ICP6 of the 17TermA strain or rRp450 strain). In another embodiment, the HSV does not comprise any ICP6 protein.

In one embodiment, the mutation on a non-natural HSV of the present invention is one or more of: (a) an alanine to threonine mutation of the gE protein at position 151, (b) an arginine to histidine mutation of the ICP0 protein at position 258, (c) an alanine to threonine mutation of the DNA packaging terminal enzyme subunit 1 protein at position 376, (d) a threonine to methionine mutation of the ICP8 protein at position 1155, or (e) a proline to histidine mutation of the ICP34.5 protein at position 119.

In certain embodiments, the gE of the non-natural HSV comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs 13, 15, 17, and 19. In another embodiment, the non-natural HSV further comprises a polynucleotide encoding a gE amino acid sequence, e.g., a polynucleotide having a sequence selected from the group consisting of SEQ ID NOs 12, 14, 16, 18, and equivalents thereof. In certain embodiments, the ICP0 of a non-natural HSV comprises, consists essentially of, or consists of the sequence: an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 23 and 26. In another embodiment, the non-natural HSV further comprises a polynucleotide encoding an amino acid sequence ICP0, e.g., a polynucleotide having a sequence selected from the group consisting of SEQ ID NOs 20, 22, 24, 25, 53, and equivalents thereof. In certain embodiments, ICP8 of a non-natural HSV comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs 28, 30, 32, and 34. In another embodiment, the non-natural HSV further comprises a polynucleotide encoding an amino acid sequence ICP8, e.g., a polynucleotide having a sequence selected from the group consisting of SEQ ID NOs 27, 29, 31, 33, and equivalents thereof. In certain embodiments, DNA-packaging terminal enzyme subunit 1 of a non-natural HSV comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs 36, 38, 40, and 42. In another embodiment, the non-natural HSV further comprises a polynucleotide encoding an amino acid sequence of DNA-packaging terminal enzyme subunit 1, e.g., a polynucleotide having a sequence selected from the group consisting of SEQ ID NOs 35, 37, 39, 41, or equivalents thereof.

In certain embodiments, the non-natural HSV comprises, consists essentially of, or consists of one or more polypeptides having an amino acid sequence selected from the group consisting of SEQ ID NOs 13, 21, 28, and 36. In another embodiment, the non-natural HSV comprises, consists essentially of, or consists of one or more polynucleotides encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs 13, 21, 28, and 36.

In certain embodiments, the non-natural HSV comprises, consists essentially of, or consists of the sequence of seq id no: (a) a polynucleotide encoding an amino acid sequence selected from SEQ ID numbers 2, 6, 8, 10 and 52, and/or a polynucleotide having a sequence selected from SEQ ID numbers 1, 5, 7, 9 and 51; (b) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID numbers 2, 6, 8, 10, and 52; (c) a polynucleotide encoding an amino acid sequence selected from SEQ ID numbers 13, 15, 17 and 19, and/or a polynucleotide having a sequence selected from SEQ ID numbers 12, 14, 16 and 18; (d) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID numbers 13, 15, 17, and 19; (e) polynucleotides encoding amino acid sequences selected from SEQ ID numbers 21, 23 and 26, and/or polynucleotides having sequences selected from SEQ ID numbers 20, 22, 24, 25 and 53 or sequences thereof that do not contain one or two or more introns; (f) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID numbers 21, 23 and 26; (g) a polynucleotide encoding an amino acid sequence selected from SEQ ID numbers 28, 30, 32 and 34, and/or a polynucleotide having a sequence selected from SEQ ID numbers 27, 29, 31 and 33; (h) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID numbers 28, 30, 32, and 34; (i) a polynucleotide encoding an amino acid sequence selected from SEQ ID numbers 36, 38, 40 and 42, and/or a polynucleotide having a sequence selected from SEQ ID numbers 35, 37, 39 and 41; (j) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID numbers 36, 38, 40, and 42; (k) a polynucleotide encoding an amino acid sequence selected from SEQ ID numbers 44, 46, 48 and 50, and/or a polynucleotide having a sequence selected from SEQ ID numbers 43, 45, 47 and 49; (l) A polypeptide having an amino acid sequence selected from the group consisting of SEQ ID numbers 44, 46, 48 and 50.

In another embodiment, the non-natural HSV comprises, consists essentially of, or consists of a polynucleotide having a sequence identical to at least one fragment of a virulence gene from 17TermA HSV and equivalents thereof. In another embodiment, the non-natural HSV comprises, consists essentially of, or consists of a polynucleotide having a sequence identical to at least one fragment of a virulence gene from the rRp450 HSV and equivalents thereof. In certain embodiments, the non-natural HSV is derived from an HSV type 1 ("HSV-1") or HSV type 2 ("HSV-2") strain. In one embodiment, the non-natural HSV is derived from the HSV-1 KOS strain. In another embodiment, the non-natural HSV further comprises, or consists essentially of, or consists of a transgene.

As the HSV disclosed herein retains its lytic function, in another aspect, the invention also provides a method for treating cancer or inhibiting the growth or metastasis of cancer cells in a subject in need thereof, the method comprising, consisting essentially of, or consisting of: administering to the subject an effective amount of a non-natural HSV of the present invention, or a composition comprising or consisting essentially of a non-natural HSV. In one aspect, the cancer comprises pancreatic cancer, renal cancer, small cell lung cancer, brain cancer, neural cancer, neuroblastoma, bone cancer, lymphoma, myeloma, colon cancer, uterine cancer, breast cancer, leukemia, liver cancer, prostate cancer, skin cancer, or melanoma. The subject to be treated may be an adult or pediatric patient, such as a mammalian or human patient. In another embodiment, the non-natural HSV vector or composition or pharmaceutical composition is administered locally or systemically by injection, infusion, instillation, and/or inhalation. In another embodiment, the subject is a mammal. In certain embodiments, the mammal is a mouse, rat, guinea pig, non-human primate, dog, cat, horse, cow, pig, goat, or sheep. In another embodiment, the subject is a human.

In another aspect, the invention provides a method of inducing cell lysis, the method comprising, consisting essentially of, or consisting of: contacting a cell with a non-natural HSV or a composition comprising or consisting essentially of a non-natural HSV. In one embodiment, the cell is a cancer cell. In another aspect, the cell is a cultured cell (for use as a preclinical model or preclinical analysis) or a cell isolated from a subject. The cells may be cultured, or within an isolated tissue. Non-limiting examples of such cells include cells from: pancreatic cancer, renal cancer, small cell lung cancer, brain cancer, neuroblastoma, neural cancer, bone cancer, lymphoma, myeloma, colon cancer, uterine cancer, breast cancer, leukemia, liver cancer, prostate cancer, skin cancer, or melanoma. These cells can be isolated from a mammal, for example, a human can be an adult or juvenile (child).

In another aspect, the invention provides a method of infecting a cell, the method comprising, consisting essentially of, or consisting of: contacting the cell with the non-natural HSV. In one embodiment, the cell is a eukaryotic cell. In another embodiment, the cell is a lymphocyte. In one embodiment, the cell is a cancer cell, such as a blood cancer or a solid tumor cell, such as a carcinoma or sarcoma. In another aspect, the cell is a cultured cell (for use as a preclinical model or preclinical analysis) or a cell isolated from a subject. The cells may be cultured, or within an isolated tissue. Non-limiting examples of such cells include cells from: pancreatic cancer, renal cancer, small cell lung cancer, brain cancer, neuroblastoma, neural cancer, bone cancer, lymphoma, myeloma, colon cancer, uterine cancer, breast cancer, leukemia, liver cancer, prostate cancer, skin cancer, or melanoma. These cells can be isolated from a mammal, for example, a human can be an adult or juvenile (child).

Applicants have found that lymphocytes infected with the conventional Epstein-Barr virus ("EBV") are resistant to conventional oncolytic HSV ("oHSV"). Without being bound by theory, this resistance is due, at least in part, to low level expression of HSV entry receptors on EBV-infected lymphocytes. Surprisingly, the HSV claimed herein is capable of inducing lysis in EBV-infected lymphocytes that are resistant to the wild-type strain 17, KOS and McKrae HSV viruses. Thus, in one embodiment, the cells comprise, consist essentially of, or consist of cells infected with EBV or alternatively resistant to EBV. In another embodiment, the cell comprises, consists essentially of, or consists of a virulence element of an epstein-barr virus ("EBV"). In another embodiment, the cell comprises, consists essentially of, or consists of a lymphocyte infected with EBV. In another embodiment, the claimed HSV has a higher infection rate on EBV-infected cells than the control group. In one embodiment, the control group comprises, consists essentially of, or consists of conventional oHSV. In another embodiment, the conventional oHSV comprises the wild type strain 17 HSV, KOS HSV, or McKrae HSV.

Preparation of HSV mutants or derivatives

The preparation of HSV mutants or derivatives involves alteration or mutation of a gene or combination of genes encoded by a parent HSV strain. For example, a derivative may have an HSV-l or HSV-2 genomic sequence modified by nucleotide substitutions (e.g., 1,2 or 3 to 10, 25, 50, or 100 substitutions). The HSV-l or HSV-2 genome may be selectively or alternatively modified by one or more insertions and/or deletions and/or extensions at one or both ends. Methods of genetic modification are known in the art, such as CRISPR, recombinant construction or point mutation. One of ordinary skill in the art would know how to make HSV mutants as desired.

In addition to the targeted gene modification method, HSV mutants can also be produced spontaneously. For example, the culture of viruses such as HSV involves a technique known as serial passage. To culture and maintain the virus, appropriate cells are infected with the virus, the virus replicates within the cells, and the virus is harvested; fresh cells are then reinfected, and this process constitutes a cycle of serial passages. For example, for HSV, each cycle may take several days. As mentioned above, such serial passage may result in changes in viral strain characteristics or gene sequence, in which case characteristics are selected that are advantageous for clinical use of HSV. For example, the enhanced properties may include the ability to replicate rapidly, or to propagate along axons to infect human cells. In addition, spontaneous mutations can be made by infecting cells with one or more HSV.

Accordingly, the present invention provides a method for preparing HSV, or a mutant or derivative thereof, by mutating a gene therein. In another embodiment, the method comprises, consists essentially of, or consists of: transgenes were induced into non-native HSV.

In another aspect, provided herein is a method of making an HSV viral particle, the method comprising, consisting essentially of, or consisting of: (a) introducing a 17Terma HSV vector and an rRp450 HSV vector into a host cell; (b) growing the host cell for at least 3 passages; and (c) isolating HSV particles produced by the host cell. In one embodiment, the HSV is introduced into the host cell by transfection, infection, transformation, electroporation, injection, microinjection, or a combination thereof. In another embodiment, the host cell is grown for at least 3 passages, 10 passages, 20 passages, 30 passages, 40 passages, or 50 passages. In some embodiments, the host cell comprises, consists essentially of, or consists of a complementing gene product to support replication of the introduced HSV vector. In another embodiment, the complementary genes encode ICP6 protein and/or ICP34.5 protein. In another embodiment, the HSV particle so produced comprises, consists essentially of, or consists of an HSV vector of the present invention.

In certain embodiments, provided herein is a method of making a non-natural HSV viral particle of the present invention. The method comprises, consists essentially of, or consists of: (a) introducing a non-native HSV vector into a host cell; (b) growing the host cell; and (c) isolating HSV particles produced by the host cell.

In certain embodiments, provided herein is a method of making a non-natural HSV viral particle of the present invention, comprising, consisting essentially of, or consisting of: (a) introducing a polynucleotide encoding the viral genome of a non-native HSV vector into a host cell; (b) growing the host cell; and (c) collecting and isolating HSV particles produced by the host cell. In one embodiment, the nucleic acid sequence encoding the viral genome is introduced into the host cell by transfection, infection, transformation, electroporation, injection, microinjection, or a combination thereof. In one embodiment, the nucleic acid sequence encoding the viral genome is introduced into a host cell in a vector. In another embodiment, the vector is a viral vector (e.g., HSV) or a non-viral vector (e.g., a plasmid or nanoparticle). In another embodiment, the vector is HSV. In certain embodiments, the host cell comprises, consists essentially of, or consists of a complementing gene product to support replication of the introduced HSV vector. In one embodiment, such complementary gene products are provided in a host cell by a helper virus. In another embodiment, the complementary genes encode ICP6 protein and/or ICP34.5 protein. In another embodiment, the HSV particle so produced comprises, consists essentially of, or consists of an HSV vector of the present invention.

In one embodiment, the separation step refers to a process of substantially separating HSV from other materials, such as host cells, cell debris, culture medium, or any other agent used in culturing host cells, by, for example, centrifugation, filtration, chromatography, or any combination thereof. Non-limiting examples can be found in Sia et al, Optimal publication methods for pharmaceutical-based viral vectors, minor chemistry for system routes of vector administration, J Virol methods, 2007 Feb, 139(2), 166-74.

Composition comprising a metal oxide and a metal oxide

In another aspect, the present invention provides a composition comprising, consisting essentially of, or consisting of a non-natural HSV as described herein. Compositions (including pharmaceutical compositions) comprising, consisting essentially of, or consisting of the agents or viral particles described herein can be manufactured by conventional mixing, dissolving, granulating, milling, emulsifying, encapsulating, entrapping, or lyophilizing processes. The compositions may be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, adjuvants or adjuvants that facilitate processing of the viral particles provided herein into pharmaceutically acceptable formulations.

The reagents and viral particles of this technology can be administered by parenteral (e.g., intramuscular, intraperitoneal, intravenous, intracerebroventricular ("ICV"), intracerebral injection or infusion, subcutaneous injection or implantation), oral, nasal inhalation spray, vaginal, rectal, sublingual, urethral (e.g., urethral suppository) or topical routes of administration (e.g., gel, ointment, cream, aerosol, etc.), and can be formulated, alone or together, into suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and solvents appropriate for each route of administration.

In one embodiment, the present invention relates to a composition comprising, consisting essentially of, or consisting of a non-natural HSV viral particle and a vector as described herein.

In another embodiment, the invention relates to a pharmaceutical composition comprising, consisting essentially of, or consisting of a non-native viral particle as described herein and a pharmaceutically acceptable carrier. In another embodiment, the composition comprises, consists essentially of, or consists of a cryoprotectant that facilitates freezing and thawing of non-natural HSV without significant loss of toxicity.

In another embodiment, the present invention relates to a pharmaceutical composition comprising, consisting essentially of, or consisting of a therapeutically effective amount of a non-natural HSV viral particle as described herein and a pharmaceutically acceptable carrier.

Pharmaceutical compositions for administration of HSV viral particles may conveniently be presented in dosage unit form and may be prepared by any of the methods well-known in the pharmaceutical arts. For example, a pharmaceutical composition can be prepared by uniformly and intimately bringing the HSV viral particles provided herein into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired dosage form. In pharmaceutical compositions, the compounds provided herein are included in an amount sufficient to produce the desired therapeutic effect. For example, the pharmaceutical compositions of the present invention may take a form suitable for virtually any mode of administration, including, for example, topical, ocular, oral, mucosal, systemic, nasal, injection, infusion, transdermal, rectal, and vaginal administration, or a form suitable for administration by inhalation or insufflation.

For topical administration, the non-natural HSV viral particles may be formulated as solutions, gels, ointments, creams, suspensions, and the like, as are well known in the art.

Systemic formulations include those designed for administration by injection (e.g., subcutaneous, intravenous, infusion, intramuscular, intradural or intraperitoneal injection), as well as those designed for transdermal, transmucosal, oral or pulmonary administration.

Effective injectable formulations include sterile suspensions, solutions or emulsions of the HSV viral particles provided herein in aqueous or oleaginous solvents. The composition may also comprise formulating agents, such as suspending, stabilizing and/or dispersing agents. Formulations for injection may be presented in unit dosage form, for example, in ampoules or in multi-dose containers, and may contain an added preservative.

Alternatively, the injectable formulations can be provided in powder form for reconstitution with a suitable solvent prior to use, including, but not limited to, sterile, pyrogen-free water, buffers, and dextrose solutions. To this end, the HSV viral particles provided herein can be dried by any technique known in the art (e.g., lyophilization) and reconstituted prior to use.

For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art.

For oral administration, the pharmaceutical compositions may take the form of, for example, lozenges, tablets or capsules prepared by conventional methods using pharmaceutically acceptable adjuvants, such as binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (such as lactose, microcrystalline cellulose or dibasic calcium phosphate); lubricants (e.g., magnesium stearate, talc, or silicon dioxide); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Tablets may be coated by methods known in the art, for example with sugar, a film or an enteric coating.

Compositions for oral administration may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions, and such compositions may contain agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the HSV viral particles provided herein, mixed with non-toxic pharmaceutically acceptable adjuvants suitable for the manufacture of tablets. These adjuvants may be: for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents (e.g., corn starch or alginic acid); binding agents (e.g., starch, gelatin, or acacia); and lubricating agents (e.g., magnesium stearate, stearic acid, or talc). The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated by techniques well known to those skilled in the art. The pharmaceutical compositions of this technology may also be in the form of oil-in-water emulsions.

Liquid preparations for oral administration may take the form of, for example, elixirs, solutions, syrups or suspensions, or may be presented as a dry product for use with water or other suitable solvent. Such liquid preparations may be prepared by conventional means using pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous solvents (such as almond oil, oily esters, ethyl alcohol, cremophore (TM) or fractionated vegetable oils); and preservatives (e.g., methyl or propyl paraben or sorbic acid). The formulations may also optionally contain buffer salts, preservatives, flavouring agents, colouring agents and sweetening agents.

In one embodiment, there is provided a method of making the non-natural HSV of the present invention, the method comprising, consisting essentially of, or consisting of: mutating a gene in a non-native HSV viral particle, or introducing a transgene into a non-native HSV. In another aspect, a method of making a non-natural HSV vector comprises, consists essentially of, or consists of: (a) introducing a 17Terma HSV vector and an rRp450 HSV vector into a host cell; (b) growing the host cell for at least 3 passages; and (c) isolating HSV particles produced by the host cell.

Also provided is a method of inhibiting growth or metastasis of a cancer cell or metastatic cancer cell, the method comprising, consisting essentially of, or consisting of: as described herein, a cell is contacted with an effective amount of a non-natural HSV vector or a composition or pharmaceutical composition containing a non-natural HSV vector. The contacting is in vitro or in vivo. In one aspect, the contacting is performed in vivo by administering a non-natural HSV or composition or pharmaceutical composition to a subject. The method is carried out in vitro by contacting the non-natural HSV with the cell. The in vitro methods can be used to test new therapies, or as a personalized analysis to determine whether the therapy is appropriate for the cancer to be treated. Additional cancer therapies may be combined with treatments that may be performed simultaneously or sequentially with the disclosed methods.

Furthermore, the present invention also provides a method for treating cancer or inhibiting the growth or metastasis of cancer cells in a subject in need thereof, the method comprising, consisting essentially of, or consisting of: administering to the subject an effective amount of a non-natural HSV, composition, or pharmaceutical composition of the present invention. The subject to be treated may be of any species, such as mammals and humans, for example, canine, equine, bovine, feline, simian, rat or mouse. Administration can be as first line therapy, second line therapy, third line therapy, fourth line therapy or fifth line therapy. Additional cancer therapies may be combined with treatments that may be performed simultaneously or sequentially with the disclosed methods. The cancer cell to be treated may be a solid tumor or a blood cancer, such as a carcinoma or sarcoma, and non-limiting examples thereof include pancreatic cancer, kidney cancer, small cell lung cancer, brain cancer, neuroblastoma, nerve cancer, bone cancer, lymphoma, myeloma, colon cancer, uterine cancer, breast cancer, leukemia, liver cancer, prostate cancer, skin cancer, or melanoma.

The methods of the invention may be combined with appropriate diagnostics to monitor the remission or progression of a disease. Several methods for such monitoring are known in the art.

Further provided is a method of inducing cell lysis, the method comprising, consisting essentially of, or consisting of: contacting the cell with an effective amount of a non-natural HSV, composition, or pharmaceutical composition of the present invention. The contacting is in vitro or in vivo. In one aspect, the contacting is performed in vivo by administering a non-natural HSV or composition or pharmaceutical composition to a subject. The method is carried out in vitro by contacting the non-natural HSV with the cell. The in vitro methods can be used to test for new therapies, or as a personalized analysis to determine whether the therapy is appropriate for the subject to be treated. Additional cytolytic therapy may be combined with treatments that may be performed simultaneously or sequentially with the disclosed methods.

The cells to be treated may be solid tumors or blood cancers, such as carcinoma or sarcoma, and non-limiting examples thereof include pancreatic cancer, kidney cancer, small cell lung cancer, brain cancer, neuroblastoma, neural cancer, bone cancer, lymphoma, myeloma, colon cancer, uterine cancer, breast cancer, leukemia, liver cancer, prostate cancer, skin cancer, or melanoma. The cell belongs to any species, such as mammals and humans, and when performed in vitro, it may be from a cultured cell line or primary cell, e.g. from a tissue biopsy. The cell may be an adult or juvenile cell, or a cancer stem cell, or a cancer cell that does not have the characteristics associated with a normal stem cell. The therapy may be combined with appropriate assays to test the effectiveness of the therapy, e.g., cancer remission or progression.

Application of HSV (herpes Simplex Virus) particles in preparation of medicines

The HSV and compositions of the present invention may also be used in the preparation of medicaments for the treatment of various pathologies described herein. Methods and techniques for preparing pharmaceutical compositions are known in the art. For illustrative purposes only, pharmaceutical formulations and routes of delivery are detailed herein.

Thus, one of skill in the art will readily appreciate that any one or more of the compositions described above, including many specific examples, can be used to prepare medicaments for the treatment of various diseases described herein by applying standard pharmaceutical manufacturing procedures. Such drugs may be delivered to a subject by using delivery methods known in the pharmaceutical art.

Administration of additional therapeutic agents

The methods disclosed herein may comprise, consist essentially of, or consist of administering an effective amount of an additional therapeutic agent to enhance or enhance the therapeutic effect of the disclosed methods. In one embodiment, the additional therapeutic agent comprises, consists essentially of, or consists of surgical resection of a tumor, an antineoplastic agent (e.g., a small molecule or immunotherapy or cytolytic therapy).

Administration of the therapies or agents of the invention to a patient will follow a general dosing regimen with a particular primary or secondary treatment, taking into account the toxicity of the treatment, if any. It is expected that the treatment cycle will be repeated as necessary. It is also contemplated that various standard therapies as well as surgical intervention may be applied in combination with the therapies.

As will be apparent to those skilled in the art, combination therapy may be employed as a combination therapy for simultaneous or sequential administration.

Reagent kit

In some embodiments, the agents or non-natural HSV described herein can be assembled into pharmaceutical or diagnostic or research kits to facilitate their use in therapeutic, diagnostic or research applications. Kits may include one or more of the reagents described herein, along with instructions describing the intended use and proper use of the reagents. In particular, the kit may include instructions for mixing one or more components of the kit and/or separating and mixing the sample and applying it to the subject. In certain embodiments, the agents in the kit are pharmaceutical formulations and dosages suitable for the particular application and method of administration. Kits for research purposes may contain the appropriate concentrations or amounts of the components for conducting the various experiments.

The design of the kit may facilitate the use of the methods described herein and may take a variety of forms. Where applicable, each composition of the kit may be provided in liquid form (e.g., a solution) or solid form (e.g., a dry powder). In some cases, some compositions may be combinable or otherwise processable (e.g., into an active form), such as by addition of a suitable solvent or other species (e.g., water or cell culture medium), which may or may not be provided with a kit. In some embodiments, the composition can be provided in a preservation solution (e.g., a cryopreservation solution). Non-limiting examples of preservation solutions include DMSO, paraformaldehyde, and CryoStor @ (Stem Cell Technologies, Vancouver, Canada). In some embodiments, the preservation solution comprises an amount of a metalloprotease inhibitor.

As used herein, "instructions" may define the components of an instruction and/or promotion and generally relate to the packaging of, or the written instructions associated with, the method, recombinant vector or composition. The instructions may also include any verbal or electronic instructions provided in any manner such that the user will clearly recognize that the instructions will be associated with the kit, e.g., audiovisual (e.g., videotape, DVD, etc.), internet and/or network-based communications, etc. The written instructions may be in a format prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which instructions may also reflect manufacture, use or sale approved by the animal's regulatory agency.

In some embodiments, a kit comprises any one or more of the components described herein in one or more containers. For example, in one embodiment, a kit can include instructions for mixing one or more components of the kit and/or separating and mixing samples and applying to a subject. The kit may comprise a container holding the reagents described herein. HSV can be in the form of a liquid, gel or solid (powder). HSV can be prepared aseptically, packaged in syringes and shipped refrigerated. Alternatively, it may be stored in a vial or other container. The second container may contain other reagents prepared by sterilization. Alternatively, the kit may include the active agents premixed and shipped in syringes, vials, tubes, or other containers. The kit may have one or more or all of the components required to administer the medicament to the subject, such as a syringe, a topical administration device, or an IV needle and bag.

Screening assays

The invention also provides screening assays to identify potential therapeutic agents for known and novel compounds and combinations. For example, one skilled in the art can also determine whether HSV provides a therapeutic benefit in vitro by contacting HSV with a sample cell or tissue to be treated. The cells or tissues may be from any species, e.g., simian, canine, bovine, ovine, rat, mouse, or human.

The contacting can also be performed in an appropriate animal model or in a human patient. When performed in vitro, HSV can be added directly to the cell culture medium. When performed in vitro, the method may be used to screen for new combination therapies, formulations or treatment regimens prior to administration to an animal or human patient.

On the other hand, the test entails contacting a first sample ("control sample") containing the appropriate cells or tissues with an effective amount of HSV as disclosed herein, and contacting a second sample ("test sample") of the appropriate cells or tissues with the HSV, agent, or combination to be analyzed. In one aspect, in the case of cancer, growth inhibition of the first and second cell samples is determined. The agent is a potential therapeutic if the growth inhibition of the second sample is substantially the same as or greater than the first sample. In one aspect, substantially the same or greater inhibition of cell growth is a difference of less than about 1%, or less than about 5%, or less than about 10%, or greater than about 20%, or greater than about 50%, or greater than about 90%. The contacting can be performed in vitro or in vivo. Methods for determining inhibition of cell growth are well known in the art.

In another aspect, the test agent is contacted with a third sample comprising cells or tissue corresponding to the normal of the control and test samples, and the agent is selected to treat the second cell or tissue sample without adversely affecting the third sample. For purposes of the assays described herein, suitable cells or tissues are described herein, such as cancer or other diseases described herein. Examples of such include, but are not limited to, cancer cells or tissues obtained by biopsy or blood.

The efficacy of the test composition is determined using methods known in the art, including but not limited to cell viability assays.

In another aspect, the assay requires at least two cell types, the first being a suitable control cell.

These assays may also be used to predict whether a subject will be appropriately treated by the present invention, by delivering HSV to a sample containing the cells to be treated and performing a therapeutic analysis, which will vary with the pathology, or for screening for new drugs and combinations. In one aspect, the cells or tissue are obtained from a subject or patient by biopsy. The invention also provides kits for determining whether a pathological cell or patient will be appropriately treated by the therapy by providing at least one composition of the invention and instructions for use.

Test cells can be grown in small multi-well plates and used to detect the biological activity of test compounds. For purposes of the present invention, a successful HSV or other agent will prevent the growth of, or kill, cancer cells, but the control cell type is not injured.

The following examples are intended to illustrate, but not limit, the invention.

Examples

Generation of HSV mutants

Non-permissive cell lines were infected with 17TerMA and rRp450 ("directed evolution") and cultured after serial passages to isolate HSV Mut-3 mutants containing WT-like genotypes. The HSV Mut-3 mutant was then used to construct an attenuated mutant, Mut-3. DELTA.34.5, by gene editing (labeled "CRISPR/Cas 9") (FIG. 1A). FIG. 1B shows a sequence comparison of Mut-3 with its parent virus. Non-synonymous mutations in Mut-3 that differ from either parent are indicated by backslash, including UL15, UL29, US8, RL1, and RL 2. The same genomic sequence as 17TermA is shown as a blank box; the same parts as rRp450 are indicated with forward slashes.

An efficient oHSV mutant Mut-3 containing the WT-like genotype was isolated. Both the Mut-3 and the attenuated version Mut-3. Δ 34.5 are dependent on typical HSV entry proteins, nectin-1 or Herpes Virus Entry Mediators (HVEM) to achieve a successful infection (not shown). The Mut-3 Δ 34.5 virus gene transfer kinetics measured by the beginning of the detectable GFP positive region was much earlier than 17 Δ 34.5 (Δ 34.5-null virus derived from wild type strain 17 by the same CRISPR/Cas9 gene editing strategy). In addition, a Mut-3 Δ 34.5 infection can lead to more cell death as measured by its smaller cell fusion area. However, the replication of Mut-3 Δ 34.5 seems to be lower than that of the parental virus 17TerMA of 17 Δ 34.5 or Mut-3 Δ 34.5-null. These results indicate that even in the attenuated Mut-3. Δ 34.5 version, unique genomic changes lead to a syncytium phenotype (not shown) and efficacy is still enhanced. In a study of Epidermal Growth Factor Receptor (EGFR) retargeting HSV, it has been reported that the introduction of syncytial mutations does not impair the specificity of entry and transmission. Without being bound by theory, applicants believe that the root cause of the syncytial phenotype in Mut-3. 34.5 may lead to altered kinetics when the virus fuses to the cell membrane, thereby affecting entry and/or virus-mediated intercellular fusion, resulting in faster cell killing and reduced production of viral particles.

No mutations were found in the typical glycoproteins gB, gD, gH and gL involved in HSV-1 entry. However, unlike either parent, there are five genes that contain non-synonymous (NS) mutations in Mut-3: RL1, RL2, UL15, UL29, and US 8/gE. Both Mut-3 and its attenuated version, Mut-3 Δ 34.5, show a fused phenotype, indicating that this phenomenon is independent of RL 1. Whole genome analysis also showed that this phenotype was not associated with the previously reported mutations, since the amino acid sequence of Mut-3 was either identical to rRp450 and the reference strain 17 genome (non-syncytial virus) in gK/UL53 and UL20, or identical to 17Terma (non-syncytial virus) in gB/UL27 and UL 24. The A151T mutation of gE/Us8 is the only glycoprotein (virion surface protein) in Mut-3 that has NS changes compared to its parent virus. Although gE is not associated with either viral attachment or entry, gE/gI dimerization mediates intercellular propagation of the virus because mutations in gE are associated with smaller plaque sizes. The applicant hypothesized that A151T of gE in Mut-3. 34.5 may be the root cause of its syncytia phenotype and synergistic phenotype. According to the applicant's research priorities (from top to bottom), the applicant lists all five NS mutations found in the five genes of Mut-3, as well as the corresponding functions of each gene, the possible effects associated with the synergistic phenotype of Mut-3 and the studies proposed by the applicant. Three other mutations (except RL 1) were also identified as was done for gE A151T.

Table 1 HSV protein mutations and their function, nt: a nucleotide; a, a: an amino acid.

Comparison of HSV mutants

Plaque analysis was performed simultaneously for the four viruses shown in FIG. 1A, and plaque images were scanned and analyzed by a Keyence HS multifunctional fluorescence microscope BZ-II analyzer after 3 days. As shown in FIGS. 1A and 1B, the plaque size of Mut-3 and Mut-3. delta.34.5 is significantly larger than that of the parental viruses rRp450 and 17 TerMA. In the in vitro cytotoxicity/MTS assay of the CHO cell set, CHO-K1, CHO-Nectin-1, CHO-Nectin-2 and CHO-HVEM were infected with four viruses at different multiplicity of infection (MOI). Cell viability was measured by colorimetric cell proliferation and MTS assays 3 days after viral infection (pvi) relative to untreated controls. Only CHO-Nectin-1 and CHO-HVEM, but not CHO-K1 or CHO-Nectin-2 (mainly for HSV-2 entry), were sensitive to treatment with four viruses (FIG. 2C). Without being bound by theory, the results indicate that Mut-3 and Mut-3 Δ 34.5 do not bypass the receptor barrier, and still rely on classical HSV to enter the receptor to infect the cell.

The increased efficacy of Mut-3 Δ 34.5 in killing human and mouse neuroblastoma cells compared to 17TerMA was not due to increased production of infectious virus (FIG. 3). Transmission electron microscopy ("TEM") analysis also showed that after infection of neuroblastoma cells, Mut-3 Δ 34.5 virions were predominantly present in endocytic vesicles, while 17TermA virions were predominantly present in endocytic vesicles (fig. 4).

An attenuated 17 Δ 34.5 mutant was generated by CRISPR-Cas9 gene editing technology to replace the g134.5 gene in wild type strain 17+ with the EGFP expression cassette. The 17 Δ 34.5 mutant was less potent than its wild-type strain 17+, but comparable to 17Terma (FIG. 5). The attenuation of 17 Δ 34.5 was further confirmed when Mut-3 Δ 34.5 showed faster viral gene transfer and cell killing compared to 17 Δ 34.5 (fig. 6). Furthermore, Mut-3 Δ 34.5 controls human neuroblastoma growth in vivo more effectively than 17Terma (FIG. 7).

Us8/gE revertants in Mut-3. delta.34.5 for loss of function studies

Introducing single nucleotide changes in the HSV genome by CRISPR/Cas9 technology alone is difficult because there are multiple copies of the genome during viral replication and there is no selectable marker associated with this gene editing. One construct may be the result of a two-step process incorporating CRISPR/Cas9 technology: replacement of the entire gE coding region with a reporter gene (e.g., mCherry or Red Fluorescent Protein (RFP)) to construct gE-nullMut-3 Δ 34.5; then 2) replace the reporter gene with the WT-gE coding region, yielding a gE-WT Mut-3. 34.5 revertant. As described above, the extent to which the gE-null intermediate and the gE revertant, Mut-3.5, lose phenotype can be observed. The invention also provides such constructs.

Us8/gE A151T mutations in the Mut-3 parent 17TermA for functional gain

As described above, gE mutant 17TermA can be constructed in two steps: 1) the gE coding region was completely knocked out and replaced with a reporter gene (e.g. GFP); then 2) the reporter gene was replaced by knocking-in the gE coding region containing the a151T mutation. The phenotype at 34.5 to Mut-3 will be determined. The invention also provides such constructs.

If gE A151T is responsible for only a partial or unobserved phenotype, or even so, the present invention also provides additional mutations of Mut-3. Mutants were constructed by systematically employing similar methods of gain-of-function and loss-of-function, either alone or in combination, as described herein and shown in table 1. The safety and efficacy of attenuated Mut-3 Δ 34.5 can be tested in a mouse model. Accordingly, the present invention provides animal models for testing mutants and methods of testing.

The Mut-3 strain is a potent WT-like HSV mutant, recombinantly constructed from 17TerMA and rRp 450. By deletion of the viral virulence protein gamma₁34.5/RL1, an attenuated version Mut-3 Δ 34.5 was created to ensure safety in clinical use. In applicants' pilot toxicology studies, up to 1e8 plaque forming units (pfu) were injected intravenouslyAfter more than 85 days, no adverse clinical symptoms or significant changes in body weight were observed in Balb/c mice (not shown). Furthermore, Mut-3 Δ 34.5 also showed anti-tumor efficacy compared to 17TerMA in highly invasive neuroblastoma models in vitro (FIG. 3A) and in vivo (FIG. 7).

Biodistribution of Mut-3. DELTA.34.5 in naive, non-tumor bearing mice

Applicants' own studies show that naive Balb/c mice can tolerate up to 1e8 pfu of intravenous injection (iv) delivered Mut-3 Δ 34.5 virus with no signs of disease for more than 85 days. The biodistribution study began with intravenous injection of the highest dose previously tested (Mut-3. 34.5 virus per mouse at 1e8 pfu) in naive non-tumor-bearing Balb/c mice of both sexes (30 per sex). Peripheral blood was collected and the mice were sacrificed. Testis, ovary, spleen, lung, kidney, heart, lung and brain were collected at pvi 24 hours, 14 days, 28 days, 56 days and 85 days (n =6 per time point). Half of the organs were preserved embedded in formalin for pathological analysis, and the other half was homogenized for qPCR analysis and plaque analysis of HSV genomes to obtain the viral load in each organ. Mice were observed daily for two weeks after dosing and then twice weekly until the scheduled day of sacrifice. Body weight of each mouse was measured before and weekly after viral infection. Mice were sacrificed to show the presence or absence of adverse clinical symptoms or weight loss>20% and the organs were analyzed for viral activity as described above. Meanwhile, wild-type KOS virus [ dose range: 1X 10 per mouse⁵To 1X 10⁷pfu（n=3）]Administered to each group of mice as a positive (non-safety) control. Applicants have previously discovered that 1X 10⁶pfu KOS virus dose was uniformly lethal in FVBN mice within 2 to 3 days. qPCR was used to analyze HSV genomic copies, plaque analysis was used to assess viral activity, and pathology was performed on organs of positive control mice showing signs of disease. These results can be used as positive indicators/thresholds for evaluating the data collected from mice treated with Mut-3 Δ 34.5. Further evaluation of pathological changes in tissue/organ in the Mut-3. 34.5 treatment group to showViral load comparable to the positive control group. Each sex 6 mice per group were used to assess biodistribution and safety and tolerability at different periods. The biodistribution of Mut-3 Δ 34.5 was measured by HSV genome copy number per nanogram of genomic DNA in different organs at different times, using descriptive statistics, and compared with univariate analysis (if applicable).

Mut-3 Δ 34.5 and other γ ₁ 34.5/RL1 Virus (17 Terma and T-VEC) on various childhood cancer cell lines In vitro cytotoxicity of

Better killing of Mut-3 Δ 34.5 was observed compared to 17TerMA in human and mouse neuroblastoma cells (FIG. 3A). Using the same MTS in the in vitro assay shown in fig. 3A, the same assay was used for other childhood cancer cells, such as sarcomas, Malignant Peripheral Nerve Sheath Tumors (MPNST) and brain tumors (various models are available to applicants) to determine if the increased potency phenotype is applicable to different tumor types. The most potent cell line of each cell type was used for in vivo efficacy studies.

Examination of Mut-3. 34.5 in human child tumor models in comparison with other oHSV therapies 17Terma and T-VEC The therapeutic effects of

One of the highly responsive models for each tumor type (three in total) was selected for efficacy studies in xenografts using 5 to 6 week old female athymic nude mice. When the tumor reaches 150-300 mm³At times, mice were pooled and randomized into 3 groups (n =11 per group): i) phosphate Buffered Saline (PBS) control group, ii) Mut-3. Δ 34.5 virus, or iii) 17TerMA or T-VEC virus. For efficacy between viruses, at least 11 mice were used per group and the large difference in survival and tumor growth was examined with a minimum of 80% efficacy (20% vs 80%, at day 20). According to our previous study, 1X 10 in 100. mu.l PBS was performed every other day for each mouse⁷pfu virus or PBS only (control) intratumoral treatment, three total injections. Mice were monitored for tumors within 80 days after virus injectionVolume (twice weekly) and body weight (once weekly). Endpoint indicators included tumor volumes in excess of 2500 mm³Tumor diameter of 2 cm, or weight loss>20 percent. Animal survival can be shown by Kaplan-Meier curves and compared between groups by log rank test.

Mut-3. DELTA.34.5 in mouse child tumor models compared to other oHSV therapies (17 Terma and T-VEC) Therapeutic effect

One of the highly responsive models for each tumor type (two in total) was selected for the applicant's efficacy study in (5 to 6) age-matched C57BL6 mice. Similarly, mice were pooled and randomized into 3 groups, each group, n = 11: i) PBS, ii) Mut-3. Δ 34.5, iii) 17TerMA or T-VEC. Every other day 1X 10 of each mouse⁸pfu virus or PBS only intratumoral treatment, three total injections, were used as a previous study by the applicant. Endpoint indicators included tumor volumes in excess of 2500 mm³Tumor diameter of 2 cm, or weight loss>20 percent. Animal survival can be shown by Kaplan-Meier curves and compared between groups by log rank test.

Summary of pathological analysis

Tissues from 4 wild-type KOS-injected mice (1 e6 and 1e7 pfu, 2 mice per dose) were submitted for pathological analysis by OSU comparison of Pathology and Mouse phenotype Shared resources (comprehensive Pathology & Mouse Phenotyping Resource). Tissues from Mut-3D34.5/C8G5 and Mut-3DICP6/D7-1 injected mice (pvi time points 24 hours and 14 days, 2 mice per time point) were then submitted for pathological analysis.

Pathology report (female): KOS injection mice

The brain dry lymphoplasmacytic encephalitis is consistent with published central nervous system pathology reports of HSV-1 injected Balb/c mice. All the examined mice submitted this time had this lesion, although one submitted brain included only a small portion of the brainstem, so the lesion appeared to be lighter than the other three mice. Adrenal sections were preserved along with kidney sections from both mice. The adrenal cortex and medulla of these mice had marked necrosis.

Pathology report (female): 24-hour and 14-day Mut-3. Δ 34.5/C8G5 and Mut-3. Δ ICP6/D7-1 small injection Mouse

Samples of mice sacrificed 24 hours post-HSV infection all had the following moderate to significant severity lesions: necrosis around portal vein to hepatic zone, including single hepatocyte and localized diffuse hepatic cord necrosis; marked splenic red and moderate white marrow necrosis, with follicular less than expected. Apoptosis/necrosis of luteal cells of follicular and ovarian tissues increased in several 24-hour mice. In the control group of mice evaluated several months ago, the expected amount thereof was not noticed, but was exceeded in the mice mentioned here.

In 14-day mice, oval cell proliferation is a common chronic response to liver damage, possibly in response to infection. A few lung specimens had necrotic and inflammatory lesions not found in 24 hour animals. Overall, the 14-day mice had fewer and milder lesions than the 24-hour mice.

Mutant viruses developed in the laboratory may cause necrosis of a variety of cell types, particularly within 24 hours, including liver cells, spleen cells in red and white marrow, and cells in the ovary.

Equivalents of the formula

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

The embodiments illustratively described herein suitably may be practiced in the absence of any element, limitation, or restriction which is not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "containing," and the like are to be construed broadly and not restrictively. Furthermore, the terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention, in the use of such terms and expressions, of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the disclosure.

Thus, it should be understood that although the present disclosure has been specifically disclosed by particular embodiments and optional features, those skilled in the art may resort to modifications, improvements, and variations of the examples disclosed herein, and that such modifications, improvements, and variations are considered to be within the scope of the present disclosure. The materials, methods, and examples provided herein are representative of particular embodiments, are exemplary, and are not intended to limit the scope of the present disclosure.

The scope of the present disclosure has been described broadly and generally herein. Each of the narrower species and subgeneric groups that fall within the generic disclosure also form part of the disclosure. This includes the generic description with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

All publications, patent applications, patents, and other references mentioned herein, including all formulas and figures, are incorporated by reference in their entirety to the same extent as if each was incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.

Other embodiments are set forth in the following claims.

SEQ ID NO. 1: DNA sequence of ICP34.5 in strains Mut3 and Mut-3. ICP 6. The c356a mutation is shown in bold and italics in the sequence below.

ATGGCCCGCCGCCGCCGCCATCGCGGCCCCCGCCGCCCCCGGCCGCCCGG

GCCCACGGGCGCGGTCCCAACCGCACAGTCCCAGGTAACCTCCACGCCCA

ACTCGGAACCCGTGGTCAGGAGCGCGCCCGCGGCCGCCCCGCCGCCGCCC

CCCGCCAGTGGGCCCCCGCCTTCTTGTTCGCTGCTGCTGCGCCAGTGGCT

CCACGTTCCCGAGTCCGCGTCCGACGACGACGACGACGACTGGCCGGACA

GCCCCCCGCCCGAGCCGGCGCCAGAGGCCCGGCCCACCGCCGCCGCCCCC

CGCCCCCGGTCCCCACCGCCCGGCGCGGGCCCGGGGGGCGGGGCTAACCC

CTCCCACCCCCCCTCACGCCCCTTCCGCCTTCCGCCGCGCCTCGCCCTCC

GCCTGCGCGTCACCGCAGAGCACCTGGCGCGCCTGCGCCTGCGACGCGCG

GGCGGGGAGGGGGCGCCGAAGCCCCCCGCGACCCCCGCGACCCCCGCGAC

CCCCACGCGGGTGCGCTTCTCGCCCCACGTCCGGGTGCGCCACCTGGTGG

TCTGGGCCTCGGCCGCCCGCCTGGCGCGCCGCGGCTCGTGGGCCCGCGAG

CGGGCCGACCGGGCTCGGTTCCGGCGCCGGGTGGCGGAGGCCGAGGCGGT

CATCGGGCCGTGCCTGGGGCCCGAGGCCCGTGCCCGGGCCCTGGCCCGCG

GAGCCGGCCCGGCGAACTCGGTCTAA

SEQ ID number 2: amino acid sequence of ICP34.5 in Mut3 and Mut-3 ICP 6. The P119H mutation is shown in bold and italics in the sequence below.

MARRRRHRGPRRPRPPGPTGAVPTAQSQVTSTPNSEPVVRSAPAAAPPPP

PASGPPPSCSLLLRQWLHVPESASDDDDDDWPDSPPPEPAPEARPTAAAP

RPRSPPPGAGPGGGANPSHPPSRPFRLPPRLALRLRVTAEHLARLRLRRA

GGEGAPKPPATPATPATPTRVRFSPHVRVRHLVVWASAARLARRGSWARE

RADRARFRRRVAEAEAVIGPCLGPEARARALARGAGPANSV

SEQ ID number 3: the DNA coding sequence of EGFP. The coding region of ICP34.5 was replaced by the EGFP expression cassette in Mut3 IC34.5 (FIG. 5C, SEQ ID number 11).

ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGT

CGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGG

GCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACC

ACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTA

CGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACT

TCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTC

TTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGG

CGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGG

ACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAAC

GTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAA

GATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACC

AGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCAC

TACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGA

TCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCA

TGGACGAGCTGTACAAGAAGCTTAGCCATGGCTTCCCGCCGGAGGTGGAG

GAGCAGGATGATGGCACGCTGCCCATGTCTTGTGCCCAGGAGAGCGGGAT

GGACCGTCACCCTGCAGCCTGTGCTTCTGCTAGGATCAATGTGTAG

SEQ ID number 4: the amino acid coding sequence of EGFP.

MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICT

TGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIF

FKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN

VYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH

YLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKKLSHGFPPEVE

EQDDGTLPMSCAQESGMDRHPAACASARINV

SEQ ID number 5: 17 DNA sequence of ICP34.5 in Terma strain.

ATGGCCCGCCGCCGCCGCCATCGCGGCCCCCGCCGCCCCCGGCCGCCCGG

GCCCACGGGCGCCGTCCCAACCGCACAGTCCCAGGTAACCTAGACTAGTC

TAGCGTAACCTCCACGCCCAACTCGGAACCCGCGGTCAGGAGCGCGCCCG

CGGCCGCCCCGCCGCCGCCCCCCGCCGGTGGGCCCCCGCCTTCTTGTTCG

CTGCTGCTGCGCCAGTGGCTCCACGTTCCCGAGTCCGCGTCCGACAACGA

CGATGACGACGACTGGCCGGACAGCCCCCCGCCCGAGCCGGCGCCAGAGG

CCCGGCCCACCGCCGCCGCCCCCCGGCCCCGGCCCCCACCGCCCGGCGTG

GGCCCGGGGGGCGGGGCTGACCCCTCCCACCCCCCCTCGCGCCCCTTCCG

CCTTCCGCCGCGCCTCGCCCTCCGCCTGCGCGTCACCGCGGAGCACCTGG

CGCGCCTGCGCCTGCGACGCGCGGGCGGGGAGGGGGCGCCGGAGCCCCCC

GCGACCCCCGCGACCCCCGCGACCCCCGCGACCCCCGCGACCCCCGCGCG

GGTGCGCTTCTCGCCCCACGTCCGGGTGCGCCACCTGGTGGTCTGGGCCT

CGGCCGCCCGCCTGGCGCGCCGCGGCTCGTGGGCCCGCGAGCGGGCCGAC

CGGGCTCGGTTCCGGCGCCGGGTGGCGGAGGCCGAGGCGGTCATCGGGCC

GTGCCTGGGGCCCGAGGCCCGTGCCCGGGCCCTGGCCCGCGGAGCCGGCC

CGGCGAACTCGGTCTAA

SEQ ID number 6: 17 amino acid sequence of ICP34.5 in TermA strain.

MARRRRHRGPRRPRPPGPTGAVPTAQSQVT*

(stop codon-predicted sequence not expressed)

SEQ ID number 7: rRp450 DNA sequence of ICP34.5 in strain 450.

ATGGCCCGCCGCCGCCATCGCGGCCCCCGCCGCCCCCGGCCGCCCGGGCC

CACGGGCGCGGTCCCAACCGCACAGTCCCAGGTAACCTCCACGCCCAACT

CGGAACCCGTGGTCAGGAGCGCGCCCGCGGCCGCCCCGCCGCCGCCCCCC

GCCAGTGGGCCCCCGCCTTCTTGTTCGCTGCTGCTGCGCCAGTGGCTCCA

CGTTCCCGAGTCCGCGTCCGACGACGACGACGACGACTGGCCGGACAGCC

CCCCGCCCGAGCCGGCGCCAGAGGCCCGGCCCACCGCCGCCGCCCCCCGC

CCCCGGTCCCCACCGCCCGGCGCGGGCCCGGGGGGCGGGGCTAACCCCTC

CCCCCCCCCCTCACGCCCCTTCCGCCTTCCGCCGCGCCTCGCCCTCCGCC

TGCGCGTCACCGCAGAGCACCTGGCGCGCCTGCGCCTGCGACGCGCGGGC

GGGGAGGGGGCGCCGAAGCCCCCCGCGACCCCCGCGACCCCCGCGACCCC

CACGCGGGTGCGCTTCTCGCCCCACGTCCGGGTGCGCCACCTGGTGGTCT

GGGCCTCGGCCGCCCGCCTGGCGCGCCGCGGCTCGTGGGCCCGCGAGCGG

GCCGACCGGGCTCGGTTCCGGCGCCGGGTGGCGGAGGCCGAGGCGGTCAT

CGGGCCGTGCCTGGGGCCCGAGGCCCGTGCCCGGGCCCTGGCCCGCGGAG

CCGGCCCGGCGAACTCGGTCTAA

SEQ ID number 8: rRp450 strain ICP 34.5.

MARRRHRGPRRPRPPGPTGAVPTAQSQVTSTPNSEPVVRSAPAAAPPPPP

ASGPPPSCSLLLRQWLHVPESASDDDDDDWPDSPPPEPAPEARPTAAAPR

PRSPPPGAGPGGGANPSPPPSRPFRLPPRLALRLRVTAEHLARLRLRRAG

GEGAPKPPATPATPATPTRVRFSPHVRVRHLVVWASAARLARRGSWARER

ADRARFRRRVAEAEAVIGPCLGPEARARALARGAGPANSV

SEQ ID number 9: DNA sequence of ICP34.5 in wild type 17 strain.

ATGGCCCGCCGCCGCCGCCATCGCGGCCCCCGCCGCCCCCGGCCGCCCGG

GCCCACGGGCGCGGTCCCAACCGCACAGTCCCAGGTAACCTCCACGCCCA

ACTCGGAACCCGTGGTCAGGAGCGCGCCCGCGGCCGCCCCGCCGCCGCCC

CCCGCCAGTGGGCCCCCGCCTTCTTGTTCGCTGCTGCTGCGCCAGTGGCT

CCACGTTCCCGAGTCCGCGTCCGACGACGACGACGACGACTGGCCGGACA

GCCCCCCGCCCGAGCCGGCGCCAGAGGCCCGGCCCACCGCCGCCGCCCCC

CGCCCCCGGTCCCCACCGCCCGGCGCGGGCCCGGGGGGCGGGGCTAACCC

CTCCCACCCCCCCTCACGCCCCTTCCGCCTTCCGCCGCGCCTCGCCCTCC

GCCTGCGCGTCACCGCAGAGCACCTGGCGCGCCTGCGCCTGCGACGCGCG

GGCGGGGAGGGGGCGCCGAAGCCCCCCGCGACCCCCGCGACCCCCGCGAC

CCCCACGCGGGTGCGCTTCTCGCCCCACGTCCGGGTGCGCCACCTGGTGG

TCTGGGCCTCGGCCGCCCGCCTGGCGCGCCGCGGCTCGTGGGCCCGCGAG

CGGGCCGACCGGGCTCGGTTCCGGCGCCGGGTGGCGGAGGCCGAGGCGGT

CATCGGGCCGTGCCTGGGGCCCGAGGCCCGTGCCCGGGCCCTGGCCCGCG

GAGCCGGCCCGGCGAACTCGGTCTAA

SEQ ID number 51: DNA sequence of ICP34.5 in wild type 17 strain.

ATGGCCCGCCGCCGCCGCCATCGCGGCCCCCGCCGCCCCCGGCCGCCCGGGCCCACGGGCGCCGTCCCAACCGCACAGTCCCAGGTAACCTCCACGCCCAACTCGGAACCCGCGGTCAGGAGCGCGCCCGCGGCCGCCCCGCCGCCGCCCCCCGCCGGTGGGCCCCCGCCTTCTTGTTCGCTGCTGCTGCGCCAGTGGCTCCACGTTCCCGAGTCCGCGTCCGACGACGACGATGACGACGACTGGCCGGACAGCCCCCCGCCCGAGCCGGCGCCAGAGGCCCGGCCCACCGCCGCCGCCCCCCGGCCCCGGCCCCCACCGCCCGGCGTGGGCCCGGGGGGCGGGGCTGACCCCTCCCACCCCCCCTCGCGCCCCTTCCGCCTTCCGCCGCGCCTCGCCCTCCGCCTGCGCGTCACCGCGGAGCACCTGGCGCGCCTGCGCCTGCGACGCGCGGGCGGGGAGGGGGCGCCGGAGCCCCCCGCGACCCCCGCGACCCCCGCGACCCCCGCGACCCCCGCGACCCCCGCGCGGGTGCGCTTCTCGCCCCACGTCCGGGTGCGCCACCTGGTGGTCTGGGCCTCGGCCGCCCGCCTGGCGCGCCGCGGCTCGTGGGCCCGCGAGCGGGCCGACCGGGCTCGGTTCCGGCGCCGGGTGGCGGAGGCCGAGGCGGTCATCGGGCCGTGCCTGGGGCCCGAGGCCCGTGCCCGGGCCCTGGCCCGCGGAGCCGGCCCGGCGAACTCGGTCTAA

SEQ ID number 10: amino acid sequence of ICP34.5 in wild type 17 strain.

MARRRRHRGPRRPRPPGPTGAVPTAQSQVTSTPNSEPVVRSAPAAAPPPP

PASGPPPSCSLLLRQWLHVPESASDDDDDDWPDSPPPEPAPEARPTAAAP

RPRSPPPGAGPGGGANPSHPPSRPFRLPPRLALRLRVTAEHLARLRLRRA

GGEGAPKPPATPATPATPTRVRFSPHVRVRHLVVWASAARLARRGSWARE

RADRARFRRRVAEAEAVIGPCLGPEARARALARGAGPANSV

SEQ ID number 52: amino acid sequence of ICP34.5 in wild type 17 strain.

MARRRRHRGPRRPRPPGPTGAVPTAQSQVTSTPNSEPAVRSAPA

AAPPPPPAGGPPPSCSLLLRQWLHVPESASDDDDDDDWPDSPPPEPAPEARPTAAAPR

PRPPPPGVGPGGGADPSHPPSRPFRLPPRLALRLRVTAEHLARLRLRRAGGEGAPEPP

ATPATPATPATPATPARVRFSPHVRVRHLVVWASAARLARRGSWARERADRARFRRRV

AEAEAVIGPCLGPEARARALARGAGPANSV

11, SEQ ID NO: DNA sequence of EGFP expression cassette.

AACACATTAATTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATA

AAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGT

TACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTC

ACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATG

TCTGCTCGAAGCGGCCGGCCGCCCCGACTCTAGACTACACATTGATCCTA

GCAGAAGCACAGGCTGCAGGGTGACGGTCCATCCCGCTCTCCTGGGCACA

AGACATGGGCAGCGTGCCATCATCCTGCTCCTCCACCTCCGGCGGGAAGC

CATGGCTAAGCTTCTTGTACAGCTCGTCCATGCCGAGAGTGATCCCGGCG

GCGGTCACGAACTCCAGCAGGACCATGTGATCGCGCTTCTCGTTGGGGTC

TTTGCTCAGGGCGGACTGGGTGCTCAGGTAGTGGTTGTCGGGCAGCAGCA

CGGGGCCGTCGCCGATGGGGGTGTTCTGCTGGTAGTGGTCGGCGAGCTGC

ACGCTGCCGTCCTCGATGTTGTGGCGGATCTTGAAGTTCACCTTGATGCC

GTTCTTCTGCTTGTCGGCCATGATATAGACGTTGTGGCTGTTGTAGTTGT

ACTCCAGCTTGTGCCCCAGGATGTTGCCGTCCTCCTTGAAGTCGATGCCC

TTCAGCTCGATGCGGTTCACCAGGGTGTCGCCCTCGAACTTCACCTCGGC

GCGGGTCTTGTAGTTGCCGTCGTCCTTGAAGAAGATGGTGCGCTCCTGGA

CGTAGCCTTCGGGCATGGCGGACTTGAAGAAGTCGTGCTGCTTCATGTGG

TCGGGGTAGCGGCTGAAGCACTGCACGCCGTAGGTCAGGGTGGTCACGAG

GGTGGGCCAGGGCACGGGCAGCTTGCCGGTGGTGCAGATGAACTTCAGGG

TCAGCTTGCCGTAGGTGGCATCGCCCTCGCCCTCGCCGGACACGCTGAAC

TTGTGGCCGTTTACGTCGCCGTCCAGCTCGACCAGGATGGGCACCACCCC

GGTGAACAGCTCCTCGCCCTTGCTCACCATCCGGGAATTGCGGCCGCGGG

TACAATTCCGCAGCTTTTAGAGCAGAAGTAACACTTCCGTACAGGCCTAG

AAGTAAAGGCAACATCCACTGAGGAGCAGTTCTTTGATTTGCACCACCAC

CGGATCCGGGACCTGAAATAAAAGACAAAAAGACTAAACTTACCAGTTAA

CTTTCTGGTTTTTCAGTTCCTCGAGTACCGGATCCTCTAGAGTCCGGAGG

CTGGATCGGTCCCGGTGTCTTCTATGGAGGTCAAAACAGCGTGGATGGCG

TCTCCAGGCGATCTGACGGTTCACTAAACGAGCTCTGCTTATATAGACCT

CCCACCGTACACGCCTACCGCCCATTTGCGTCAATGGGGCGGAGTTGTTA

CGACATTTTGGAAAGTCCCGTTGATTTTGGTGCCAAAACAAACTCCCATT

GACGTCAATGGGGTGGAGACTTGGAAATCCCCGTGAGTCAAACCGCTATC

CACGCCCATTGATGTACTGCCAAAACCGCATCACCATGGTAATAGCGATG

ACTAATACGTAGATGTACTGCCAAGTAGGAAAGTCCCATAAGGTCATGTA

CTGGGCATAATGCCAGGCGGGCCATTTACCGTCATTGACGTCAATAGGGG

GCGTACTTGGCATATGATACACTTGATGTACTGCCAAGTGGGCAGTTTAC

CGTAAATACTCCACCCATTGACGTCAATGGAAAGTCCCTATTGGCGTTAC

TATGGGAACATACGTCATTATTGACGTCAATGGGCGGGGGTCGTTGGGCG

GTCAGCCAGGCGGGCCATTTACCGTAAGTTATGTAACGACCTGCAGGCAT

GCAAGCTCGAATTCGAACACGCAGATGCAGTCGGGGCGGCAGATCTTAAT

TAATGGCTGGTTGTTTGTTGT

12, SEQ ID NO: DNA sequence of gE (glycoprotein E) in the strain Mut-3, Mut-3. Δ 34.5 or Mut-3. Δ ICP 6. The g451a mutation is shown in bold and italics in the sequence below.

ATGGATCGCGGGGCGGTGGTGGGGTTTCTTCTCGGTGTTTGTGTTGTATC

GTGCTTGGCGGGAACGCCCAAAACGTCCTGGAGACGGGTGAGTGTCGGCG

AGGACGTTTCGTTGCTTCCAGCTCCGGGGCCTACGGGGCGCGGCCCGACC

CAGAAACTACTATGGGCCGTGGAACCCCTGGATGGGTGCGGCCCCTTACA

CCCGTCGTGGGTCTCGCTGATGCCCCCCAAGCAGGTGCCCGAGACGGTCG

TGGATGCGGCGTGCATGCGCGCTCCGGTCCCGCTGGCGATGGCGTACGCC

CCCCCGGCCCCATCTGCGACCGGGGGTCTACGAACGGACTTCGTGTGGCA

GGAGCGCGCGGCCGTGGTTAACCGGAGTCTGGTTATTCACGGGGTCCGAG

AGACGGACAGCGGCCTGTATACCCTGTCCGTGGGCGACATAAAGGACCCG

ACTCGCCAAGTGGCCTCGGTGGTCCTGGTGGTGCAACCGGCCCCAGTTCC

GACCCCACCCCCGACCCCAGCCGATTACGACGAGGATGACAATGACGAGG

GCGAGGACGAAAGTCTCGCCGGCACTCCCGCCAGCGGGACCCCCCGGCTC

CCGCCTCCCCCCGCCCCCCCGAGGTCTTGGCCCAGCGCCCCCGAAGTCTC

ACATGTGCGTGGGGTGACCGTGCGTATGGAGACTCCGGAAGCTATCCTGT

TTTCCCCCGGGGAGACGTTCAGCACGAACGTCTCCATCCATGCCATCGCC

CACGACGACCAGACCTACTCCATGGACGTCGTCTGGTTGAGGTTCGACGT

GCCGACCTCGTGTGCCGAGATGCGAATATACGAATCGTGTCTGTATCACC

CGCAGCTCCCAGAATGTCTGTCCCCGGCCGACGCGCCGTGCGCCGCGAGT

ACGTGGACGTCTCGCCTGGCCGTCCGCAGCTACGCGGGGTGTTCCAGAAC

AAACCCCCCACCGCGCTGTTCGGCCGAGGCTCACATGGAGCCCGTCCCGG

GGCTGGCGTGGCAGGCGGCCTCCGTCAATCTGGAGTTCCGGGACGCGTCC

CCACAACACTCCGGCCTGTATCTGTGTGTGGTGTACGTCAACGACCATAT

TCACGCCTGGGGCCACATTACCATCAGCACCGCGGCGCAGTACCGGAACG

CGGTGGTGGAACAGCCCCTCCCACAGCGCGGCGCGGATTTGGCCGAGCCC

ACCCACCCGCACGTCGGGGCCCCTCCCCACGCGCCCCCAACCCACGGCGC

CCTGCGGTTAGGGGCGGTGATGGGGGCCGCCCTGCTGCTGTCTGCGCTGG

GGTTGTCGGTGTGGGCGTGTATGACCTGTTGGCGCAGGCGTGCCTGGCGG

GCGGTTAAAAGCAGGGCCTCGGGTAAGGGGCCCACGTACATTCGCGTGGC

CGACAGCGAGCTGTACGCGGACTGGAGCTCGGACAGCGAGGGAGAACGCG

ACCAGGTCCCGTGGCTGGCCCCCCCGGAGAGACCCGACTCTCCCTCCACC

AATGGATCCGGCTTTGAGATCTTATCACCAACGGCTCCGTCTGTATACCC

CCGTAGCGACGGGCATCAATCTCGCCGCCAGCTCACAACCTTTGGATCCG

GAAGGCCCGATCGCCGTTACTCCCAGGCCTCCGATTCGTCCGTCTTCTGG

TAA

13 in SEQ ID NO: the amino acid sequence of gE (glycoprotein E) in the strain Mut-3, Mut-3. Δ 34.5 or Mut-3. Δ ICP 6. The a151T mutation is shown in bold and italics in the sequence below.

MDRGAVVGFLLGVCVVSCLAGTPKTSWRRVSVGEDVSLLPAPGPTGRGPT

QKLLWAVEPLDGCGPLHPSWVSLMPPKQVPETVVDAACMRAPVPLAMAYA

PPAPSATGGLRTDFVWQERAAVVNRSLVIHGVRETDSGLYTLSVGDIKDP

TRQVASVVLVVQPAPVPTPPPTPADYDEDDNDEGEDESLAGTPASGTPRL

PPPPAPPRSWPSAPEVSHVRGVTVRMETPEAILFSPGETFSTNVSIHAIA

HDDQTYSMDVVWLRFDVPTSCAEMRIYESCLYHPQLPECLSPADAPCAAS

TWTSRLAVRSYAGCSRTNPPPRCSAEAHMEPVPGLAWQAASVNLEFRDAS

PQHSGLYLCVVYVNDHIHAWGHITISTAAQYRNAVVEQPLPQRGADLAEP

THPHVGAPPHAPPTHGALRLGAVMGAALLLSALGLSVWACMTCWRRRAWR

AVKSRASGKGPTYIRVADSELYADWSSDSEGERDQVPWLAPPERPDSPST

NGSGFEILSPTAPSVYPRSDGHQSRRQLTTFGSGRPDRRYSQASDSSVFW

14, SEQ ID NO: 17 DNA sequence of gE in Terma strain.

ATGGATCGCGGGGCGGTGGTGGGGTTTCTTCTCGGTGTTTGTGTTGTATC

GTGCTTGGCGGGAACGCCCAAAACGTCCTGGAGACGGGTGAGTGTCGGCG

AGGACGTTTCGTTGCTTCCAGCTCCGGGGCCTACGGGGCGCGGCCCGACC

CAGAAACTACTATGGGCCGTGGAACCCCTGGATGGGTGCGGCCCCTTACA

CCCGTCGTGGGTCTCGCTGATGCCCCCCAAGCAGGTGCCCGAGACGGTCG

TGGATGCGGCGTGCATGCGCGCTCCGGTCCCGCTGGCGATGGCGTACGCC

CCCCCGGCCCCATCTGCGACCGGGGGTCTACGAACGGACTTCGTGTGGCA

GGAGCGCGCGGCCGTGGTTAACCGGAGTCTGGTTATTCACGGGGTCCGAG

AGACGGACAGCGGCCTGTATACCCTGTCCGTGGGCGACATAAAGGACCCG

GCTCGCCAAGTGGCCTCGGTGGTCCTGGTGGTGCAACCGGCCCCAGTTCC

GACCCCACCCCCGACCCCAGCCGATTACGACGAGGATGACAATGACGAGG

GCGAGGACGAAAGTCTCGCCGGCACTCCCGCCAGCGGGACCCCCCGGCTC

CCGCCTCCCCCCGCCCCCCCGAGGTCTTGGCCCAGCGCCCCCGAAGTCTC

ACATGTGCGTGGGGTGACCGTGCGTATGGAGACTCCGGAAGCTATCCTGT

TTTCCCCCGGGGAGACGTTCAGCACGAACGTCTCCATCCATGCCATCGCC

CACGACGACCAGACCTACTCCATGGACGTCGTCTGGTTGAGGTTCGACGT

GCCGACCTCGTGTGCCGAGATGCGAATATACGAATCGTGTCTGTATCACC

CGCAGCTCCCAGAATGTCTGTCCCCGGCCGACGCGCCGTGCGCCGCGAGT

ACGTGGACGTCTCGCCTGGCCGTCCGCAGCTACGCGGGGTGTTCCAGAAC

AAACCCCCCACCGCGCTGTTCGGCCGAGGCTCACATGGAGCCCGTCCCGG

GGCTGGCGTGGCAGGCGGCCTCCGTCAATCTGGAGTTCCGGGACGCGTCC

CCACAACACTCCGGCCTGTATCTGTGTGTGGTGTACGTCAACGACCATAT

TCACGCCTGGGGCCACATTACCATCAGCACCGCGGCGCAGTACCGGAACG

CGGTGGTGGAACAGCCCCTCCCACAGCGCGGCGCGGATTTGGCCGAGCCC

ACCCACCCGCACGTCGGGGCCCCTCCCCACGCGCCCCCAACCCACGGCGC

CCTGCGGTTAGGGGCGGTGATGGGGGCCGCCCTGCTGCTGTCTGCACTGG

GGTTGTCGGTGTGGGCGTGTATGACCTGTTGGCGCAGGCGTGCCTGGCGG

GCGGTTAAAAGCAGGGCCTCGGGTAAGGGGCCCACGTACATTCGCGTGGC

CGACAGCGAGCTGTACGCGGACTGGAGCTCGGACAGCGAGGGAGAACGCG

ACCAGGTCCCGTGGCTGGCCCCCCCGGAGAGACCCGACTCTCCCTCCACC

AATGGATCCGGCTTTGAGATCTTATCACCAACGGCTCCGTCTGTATACCC

CCGTAGCGATGGGCATCAATCTCGCCGCCAGCTCACAACCTTTGGATCCG

GAAGGCCCGATCGCCGTTACTCCCAGGCCTCCGATTCGTCCGTCTTCTGG

TAA

15, SEQ ID NO: 17 amino acid sequence of gE in the TermA strain.

MDRGAVVGFLLGVCVVSCLAGTPKTSWRRVSVGEDVSLLPAPGPTGRGPT

QKLLWAVEPLDGCGPLHPSWVSLMPPKQVPETVVDAACMRAPVPLAMAYA

PPAPSATGGLRTDFVWQERAAVVNRSLVIHGVRETDSGLYTLSVGDIKDP

ARQVASVVLVVQPAPVPTPPPTPADYDEDDNDEGEDESLAGTPASGTPRL

PPPPAPPRSWPSAPEVSHVRGVTVRMETPEAILFSPGETFSTNVSIHAIA

HDDQTYSMDVVWLRFDVPTSCAEMRIYESCLYHPQLPECLSPADAPCAAS

TWTSRLAVRSYAGCSRTNPPPRCSAEAHMEPVPGLAWQAASVNLEFRDAS

PQHSGLYLCVVYVNDHIHAWGHITISTAAQYRNAVVEQPLPQRGADLAEP

THPHVGAPPHAPPTHGALRLGAVMGAALLLSALGLSVWACMTCWRRRAWR

AVKSRASGKGPTYIRVADSELYADWSSDSEGERDQVPWLAPPERPDSPST

NGSGFEILSPTAPSVYPRSDGHQSRRQLTTFGSGRPDRRYSQASDSSVFW

SEQ ID number 16: rRp450 DNA sequence of gE in strain 450.

ATGGATCGCGGGGCGGTGGTGGGGTTTCTTCTCGGTGTTTGTGTTGTATC

GTGCTTGGCGGGAACGCCCAAAACGTCCTGGAGACGGGTGAGTGTCGGCG

AGGACGTTTCGTTGCTTCCAGCTCCGGGGCCTACGGGGCGCGGCCCGACC

CAGAAACTACTATGGGCCGTGGAACCCCTGGATGGGTGCGGCCCCTTACA

CCCGTCGTGGGTCTCGCTGATGCCCCCCAAGCAGGTGCCCGAGACGGTCG

TGGATGCGGCGTGCATGCGCGCTCCGGTCCCGCTGGCGATGGCGTACGCC

CCCCCGGCCCCATCTGCGACCGGGGGTCTACGAACGGACTTCGTGTGGCA

GGAGCGCGCGGCCGTGGTTAACCGGAGTCTGGTTATTCACGGGGTCCGAG

AGACGGACAGCGGCCTGTATACCCTGTCCGTGGGCGACATAAAGGACCCG

GCTCGCCAAGTGGCCTCGGTGGTCCTGGTGGTGCAACCGGCCCCAGTTCC

GACCCCACCCCCGACCCCAGCCGATTACGACGAGGATGACAATGACGAGG

GCGAGGACGAAAGTCTCGCCGGCACTCCCGCCAGCGGGACCCCCCGGCTC

CCGCCTCCCCCCGCCCCCCCGAGGTCTTGGCCCAGCGCCCCCGAAGTCTC

ACATGTGCGTGGGGTGACCGTGCGTATGGAGACTCCGGAAGCTATCCTGT

TTTCCCCCGGGGAGACGTTCAGCACGAACGTCTCCATCCATGCCATCGCC

CACGACGACCAGACCTACTCCATGGACGTCGTCTGGTTGAGGTTCGACGT

GCCGACCTCGTGTGCCGAGATGCGAATATACGAATCGTGTCTGTATCACC

CGCAGCTCCCAGAATGTCTGTCCCCGGCCGACGCGCCGTGCGCCGCGAGT

ACGTGGACGTCTCGCCTGGCCGTCCGCAGCTACGCGGGGTGTTCCAGAAC

AAACCCCCCACCGCGCTGTTCGGCCGAGGCTCACATGGAGCCCGTCCCGG

GGCTGGCGTGGCAGGCGGCCTCCGTCAATCTGGAGTTCCGGGACGCGTCC

CCACAACACTCCGGCCTGTATCTGTGTGTGGTGTACGTCAACGACCATAT

TCACGCCTGGGGCCACATTACCATCAGCACCGCGGCGCAGTACCGGAACG

CGGTGGTGGAACAGCCCCTCCCACAGCGCGGCGCGGATTTGGCCGAGCCC

ACCCACCCGCACGTCGGGGCCCCTCCCCACGCGCCCCCAACCCACGGCGC

CCTGCGGTTAGGGGCGGTGATGGGGGCCGCCCTGCTGCTGTCTGCGCTGG

GGTTGTCGGTGTGGGCGTGTATGACCTGTTGGCGCAGGCGTGCCTGGCGG

GCGGTTAAAAGCAGGGCCTCGGGTAAGGGGCCCACGTACATTCGCGTGGC

CGACAGCGAGCTGTACGCGGACTGGAGCTCGGACAGCGAGGGAGAACGCG

ACCAGGTCCCGTGGCTGGCCCCCCCGGAGAGACCCGACTCTCCCTCCACC

AATGGATCCGGCTTTGAGATCTTATCACCAACGGCTCCGTCTGTATACCC

CCGTAGCGACGGGCATCAATCTCGCCGCCAGCTCACAACCTTTGGATCCG

GAAGGCCCGATCGCCGTTACTCCCAGGCCTCCGATTCGTCCGTCTTCTGG

TAA

SEQ ID number 17: rRp450 strain, and the amino acid sequence of gE in strain (la) 89450.

MDRGAVVGFLLGVCVVSCLAGTPKTSWRRVSVGEDVSLLPAPGPTGRGPT

QKLLWAVEPLDGCGPLHPSWVSLMPPKQVPETVVDAACMRAPVPLAMAYA

PPAPSATGGLRTDFVWQERAAVVNRSLVIHGVRETDSGLYTLSVGDIKDP

ARQVASVVLVVQPAPVPTPPPTPADYDEDDNDEGEDESLAGTPASGTPRL

PPPPAPPRSWPSAPEVSHVRGVTVRMETPEAILFSPGETFSTNVSIHAIA

HDDQTYSMDVVWLRFDVPTSCAEMRIYESCLYHPQLPECLSPADAPCAAS

TWTSRLAVRSYAGCSRTNPPPRCSAEAHMEPVPGLAWQAASVNLEFRDAS

PQHSGLYLCVVYVNDHIHAWGHITISTAAQYRNAVVEQPLPQRGADLAEP

THPHVGAPPHAPPTHGALRLGAVMGAALLLSALGLSVWACMTCWRRRAWR

AVKSRASGKGPTYIRVADSELYADWSSDSEGERDQVPWLAPPERPDSPST

NGSGFEILSPTAPSVYPRSDGHQSRRQLTTFGSGRPDRRYSQASDSSVFW

SEQ ID number 18: DNA sequence of gE in wild type 17 strain.

ATGGATCGCGGGGCGGTGGTGGGGTTTCTTCTCGGTGTTTGTGTTGTATC

GTGCTTGGCGGGAACGCCCAAAACGTCCTGGAGACGGGTGAGTGTCGGCG

AGGACGTTTCGTTGCTTCCAGCTCCGGGGCCTACGGGGCGCGGCCCGACC

CAGAAACTACTATGGGCCGTGGAACCCCTGGATGGGTGCGGCCCCTTACA

CCCGTCGTGGGTCTCGCTGATGCCCCCCAAGCAGGTGCCCGAGACGGTCG

TGGATGCGGCGTGCATGCGCGCTCCGGTCCCGCTGGCGATGGCGTACGCC

CCCCCGGCCCCATCTGCGACCGGGGGTCTACGAACGGACTTCGTGTGGCA

GGAGCGCGCGGCCGTGGTTAACCGGAGTCTGGTTATTCACGGGGTCCGAG

AGACGGACAGCGGCCTGTATACCCTGTCCGTGGGCGACATAAAGGACCCG

GCTCGCCAAGTGGCCTCGGTGGTCCTGGTGGTGCAACCGGCCCCAGTTCC

GACCCCACCCCCGACCCCAGCCGATTACGACGAGGATGACAATGACGAGG

GCGAGGACGAAAGTCTCGCCGGCACTCCCGCCAGCGGGACCCCCCGGCTC

CCGCCTCCCCCCGCCCCCCCGAGGTCTTGGCCCAGCGCCCCCGAAGTCTC

ACATGTGCGTGGGGTGACCGTGCGTATGGAGACTCCGGAAGCTATCCTGT

TTTCCCCCGGGGAGACGTTCAGCACGAACGTCTCCATCCATGCCATCGCC

CACGACGACCAGACCTACTCCATGGACGTCGTCTGGTTGAGGTTCGACGT

GCCGACCTCGTGTGCCGAGATGCGAATATACGAATCGTGTCTGTATCACC

CGCAGCTCCCAGAATGTCTGTCCCCGGCCGACGCGCCGTGCGCCGCGAGT

ACGTGGACGTCTCGCCTGGCCGTCCGCAGCTACGCGGGGTGTTCCAGAAC

AAACCCCCCACCGCGCTGTTCGGCCGAGGCTCACATGGAGCCCGTCCCGG

GGCTGGCGTGGCAGGCGGCCTCCGTCAATCTGGAGTTCCGGGACGCGTCC

CCACAACACTCCGGCCTGTATCTGTGTGTGGTGTACGTCAACGACCATAT

TCACGCCTGGGGCCACATTACCATCAGCACCGCGGCGCAGTACCGGAACG

CGGTGGTGGAACAGCCCCTCCCACAGCGCGGCGCGGATTTGGCCGAGCCC

ACCCACCCGCACGTCGGGGCCCCTCCCCACGCGCCCCCAACCCACGGCGC

CCTGCGGTTAGGGGCGGTGATGGGGGCCGCCCTGCTGCTGTCTGCGCTGG

GGTTGTCGGTGTGGGCGTGTATGACCTGTTGGCGCAGGCGTGCCTGGCGG

GCGGTTAAAAGCAGGGCCTCGGGTAAGGGGCCCACGTACATTCGCGTGGC

CGACAGCGAGCTGTACGCGGACTGGAGCTCGGACAGCGAGGGAGAACGCG

ACCAGGTCCCGTGGCTGGCCCCCCCGGAGAGACCCGACTCTCCCTCCACC

AATGGATCCGGCTTTGAGATCTTATCACCAACGGCTCCGTCTGTATACCC

CCGTAGCGACGGGCATCAATCTCGCCGCCAGCTCACAACCTTTGGATCCG

GAAGGCCCGATCGCCGTTACTCCCAGGCCTCCGATTCGTCCGTCTTCTGG

TAA

SEQ ID number 19: the amino acid sequence of gE in wild type 17 strain.

MDRGAVVGFLLGVCVVSCLAGTPKTSWRRVSVGEDVSLLPAPGPTGRGPT

QKLLWAVEPLDGCGPLHPSWVSLMPPKQVPETVVDAACMRAPVPLAMAYA

PPAPSATGGLRTDFVWQERAAVVNRSLVIHGVRETDSGLYTLSVGDIKDP

ARQVASVVLVVQPAPVPTPPPTPADYDEDDNDEGEDESLAGTPASGTPRL

PPPPAPPRSWPSAPEVSHVRGVTVRMETPEAILFSPGETFSTNVSIHAIA

HDDQTYSMDVVWLRFDVPTSCAEMRIYESCLYHPQLPECLSPADAPCAAS

TWTSRLAVRSYAGCSRTNPPPRCSAEAHMEPVPGLAWQAASVNLEFRDAS

PQHSGLYLCVVYVNDHIHAWGHITISTAAQYRNAVVEQPLPQRGADLAEP

THPHVGAPPHAPPTHGALRLGAVMGAALLLSALGLSVWACMTCWRRRAWR

AVKSRASGKGPTYIRVADSELYADWSSDSEGERDQVPWLAPPERPDSPST

NGSGFEILSPTAPSVYPRSDGHQSRRQLTTFGSGRPDRRYSQASDSSVFW

SEQ ID number 20: DNA sequences of ICP0 in the strains Mut3, Mut-3 Δ 34.5 and Mut-3 Δ ICP 6. The a848c (intronic) and g1712a (for R258H) mutations are shown in bold and italics in the following sequences. Two introns are included (i.e., nucleotides (nt) 58 to nt 861 and nt 1529 to nt 1663 of the following sequences, see also the sequences in parentheses).

ATGGAGCCCCGCCCCGGAGCGAGTACCCGCCGGCCTGAGGGCCGCCCCCA

GCGCGAG(GTGAGGGGCCGGGCGCCATGTCTGGGGCGCCATGTCTGGGGCG

CCATGTCTGGGGCGCCATGTCTGGGGCGCCATGTTGGGGGGCGCCATGTT

GGGGGGCGCCATGTTGGGGGACCCCCGACCCTTACACTGGAACCGGCCGC

CATGTTGGGGGACCCCCACTCATACACGGGAGCCGGGCGCCATGTTGGGG

CGCCATGTTAGGGGGCGTGGAACCCCGTGACACTATATATACAGGGACCG

GGGGCGCCATGTTAGGGGGCGCGGAACCCCCTGACCCTATATATACAGGG

ACCGGGGTCGCCCTGTTAGGGGTCGCCATGTGACCCCCTGACTTTATATA

TACAGACCCCCAACACCTACACATGGCCCCTTTGACTCAGACGCAGGGCC

CGGGGTCGCCGTGGGACCCCCCTGACTCATACACAGAGACACGCCCCCAC

AACAAACACACAGGGACCGGGGTCGCCGTGTTAGGGGGCGTGGTCCCCAC

TGACTCATACGCAGGGCCCCCTTACTCACACGCATCTAGGGGGGTGGGGA

GGAGCCGCCCGCCATATTTGGGGGACGCCGTGGGACCCCCGACTCCGGTG

CGTCTGGAGGGCGGGAGAAGAGGGAAGAAGAGGGGTCGGGATCCAAAGGA

CGGACCCAGACCACCTTTGGTTGCAGACCCCTTTCTCCCCCCTCTTCCGA

GGCCAGCAGGGGGGCAGGACTTTGTGAGGCGGGGGGGGAGGGGGAACTCG

TGGGCGCTGATTGACGCGGGAAATCCCCCCATTCTTACCCGCCCCCCCTT

TTTCCCCTCAG)CCCGCCCCGGATGTCTGGGTGTTTCCCTGCGACCGAGAC

CTGCCGGACAGCAGCGACTCGGAGGCGGAGACCGAAGTGGGGGGGCGGGG

GGACGCCGACCACCATGACGACGACTCCGCCTCCGAGGCGGACAGCACGG

ACACGGAACTGTTCGAGACGGGGCTGCTGGGGCCGCAGGGCGTGGATGGG

GGGGCGGTCTCGGGGGGGAGCCCCCCCCGCGAGGAAGACCCCGGCAGTTG

CGGGGGCGCCCCCCCTCGAGAGGACGGGGGGAGCGACGAGGGCGACGTGT

GCGCCGTGTGCACGGATGAGATCGCGCCCCACCTGCGCTGCGACACCTTC

CCGTGCATGCACCGCTTCTGCATCCCGTGCATGAAAACCTGGATGCAATT

GCGCAACACCTGCCCGCTGTGCAACGCCAAGCTGGTGTACCTGATAGTGG

GCGTGACGCCCAGCGGGTCGTTCAGCACCATCCCGATCGTGAACGACCCC

CAGACCCGCATGGAGGCCGAGGAGGCCGTCAGGGCGGGCACGGCCGTGGA

CTTTATCTGGACGGGCAATCAGCGGTTCGCCCCGCGGTACCTGACCCTGG

GGGGGCACACGGTGAGGGCCCTGTCGCCCACCCACCCGGAACCCACCACG

GACGAGGATGACGACGACCTGGACGACG(GTGAGGCGGGGGGCGGCAAGGA

CCCTGGGGGAGGAGGAGGAGGAGGGGGGGGAGGGAGGAATAGGCGGGCGG

GCGAGGAAAGGGCGGGCCGGGGAGGGGGCGTAACCTGATCGCGCCCCCCG

TTGTCTCTTGCAG)CAGACTACGTACCGCCCGCCCCCCGCCGGACGCCCCG

CGCCCCCCCACACAGAGGCGCCGCCGCGCCCCCCGTGACGGGCGGGGCGT

CTCACGCAGCCCCCCAGCCGGCCGCGGCTCGGACAGCGCCCCCCTCGGCG

CCCATCGGGCCACACGGCAGCAGTAACACCAACACCACCACCAACAGCAG

CGGCGGCGGCGGCTCCCGCCAGTCGCGAGCCGCGGCGCCGCGGGGGGCGT

CTGGCCCCTCCGGGGGGGTTGGGGTTGGGGTTGGGGTTGTTGAAGCGGAG

GCGGGGCGGCCGAGGGGCCGGACGGGCCCCCTTGTCAACAGACCCGCCCC

CCTTGCAAACAACAGAGACCCCATAGTGATCAGCGACTCCCCCCCGGCCT

CTCCCCACAGGCCCCCCGCGGCGCCCATGCCAGGCTCCGCCCCCCGCCCC

GGGCCCCCCGCGTCCGCGGCCGCGTCGGGACCCGCGCGCCCCCGCGCGGC

CGTGGCCCCGTGCGTGCGAGCGCCGCCTCCGGGGCCCGGCCCCCGCGCCC

CGGCCCCCGGGGCGGAGCCGGCCGCCCGCCCCGCGGACGCGCGCCGTGTG

CCCCAGTCGCACTCGTCCCTGGCTCAGGCCGCGAACCAAGAACAGAGTCT

GTGCCGGGCGCGTGCGACGGTGGCGCGCGGCTCGGGGGGGCCGGGCGTGG

AGGGTGGGCACGGGCCCTCCCGCGGCGCCGCCCCCTCCGGCGCCGCCCCG

CTCCCCTCCGCCGCCTCTGTCGAGCAGGAGGCGGCGGTGCGTCCGAGGAA

GAGGCGCGGGTCGGGCCAGGAAAACCCCTCCCCCCAGTCCACGCGTCCCC

CCCTCGCGCCGGCAGGGGCCAAGAGGGCGGCGACGCACCCCCCCTCCGAC

TCAGGGCCGGGGGGGCGCGGCCAGGGTGGGCCCGGGACCCCCCTGACGTC

CTCGGCGGCCTCCGCCTCTTCCTCCTCTGCCTCTTCCTCCTCGGCCCCGA

CCCCCGCGGGGGCCGCCTCTTCCGCCGCCGGGGCCGCGTCCTCCTCCGCT

TCCGCCTCCTCGGGCGGGGCCGTCGGTGCCCTGGGAGGGAGACAAGAGGA

AACCTCCCTCGGCCCCCGCGCTGCTTCTGGGCCGCGGGGGCCGAGGAAGT

GTGCCCGGAAGACGCGCCACGCGGAGACTTCCGGGGCCGTCCCCGCGGGC

GGCCTCACGCGCTACCTGCCCATCTCGGGGGTCTCTAGCGTGGTCGCCCT

GTCGCCTTACGTGAACAAGACGATCACGGGGGACTGCCTGCCCATCCTGG

ACATGGAGACGGGGAACATCGGGGCGTACGTGGTCCTGGTGGACCAGACG

GGAAACATGGCGACCCGGCTGCGGGCCGCGGTCCCCGGCTGGAGCCGCCG

CACCCTGCTCCCCGAGACCGCGGGTAACCACGTGATGCCCCCCGAGTACC

CGACGGCCCCCGCGTCGGAGTGGAACAGCCTCTGGATGACCCCCGTGGGG

AACATGCTGTTCGACCAGGGCACCCTAGTGGGCGCCCTGGACTTCCGCAG

CCTGCGGTCTCGGCACCCGTGGTCCGGGGAGCAGGGGGCGTCGACCCGGG

ACGAGGGAAAACAATAA

SEQ ID number 21: the amino acid sequences of ICP0 in Mut3, Mut-3 Δ 34.5 and Mut-3 Δ ICP 6. The R258H mutation is shown in italics and bold in the sequence below.

SEQ ID number 22: DNA sequence of ICP0 in 17TerMA strain. Two introns are included (i.e., nucleotides (nt) 58 to nt 822 and nt 1490 to nt 1625 of the following sequences, see also the sequences in parentheses).

ATGGAGCCCCGCCCCGGAGCGAGTACCCGCCGGCCTGAGGGCCGCCCCCA

GCGCGAG(GTGAGGGGCCGGGCGCCATGTCTGGGGCGCCATATTGGGGGGC

GCCATATTGGGGGGCGCCATGTTGGGGGACCCCCGACCCTTACACTGGAA

CCGGCCGCCATGTTGGGGGACCCCCACTCATACACGGGAGCCGGGCGCCA

TGTTGGGGCGCCATGTTAGGGGGCGTGGAACCCCGTGACACTATATATAC

AGGGACCGGGGGCGCCATGTTAGGGGGTGCGGAACCCCCTGACCCTATAT

ATACAGGGACCGGGGTCGCCCTGTTGGGGGTCGCCATGTGACCCCCTGAC

TTTATATATACAGACCCCCAACACATACACATGGCCCCTTTGACTCAGAC

GCAGGGCCCGGGGTCGCCGTGGGACCCCCTGACTCATACACAGAGACACG

CCCCCACAACAAACACACAGGGACCGGGGTCGCCGTGTTGGGGGCGTGGT

CCCCACTGACTCATACGCAGGCCCCCCTTACTCACACGCATCTAGGGGGG

TGGGGAGGAGCCGCCCGCCATATTTGGGGGACGCCGTGGGACCCCCGACT

CCGGTGCGTCTGGAGGGCGGGAGAAGAGGGAAGAAGAGGGGTCGGGATCC

AAAGGACGGACCCAGACCACCTTTGGTTGCAGACCCCTTTCTCCCCCCTC

TTCCGAGGCCAGCAGGGGGGCAGGACTTTGTGAGGCGGGGGGGGGAGAGG

GGGAACTCGTGGGTGCTGATTGACGCGGGAAATCCCCCCCCATTCTTACC

CGCCCCCCTTTTTTCCCCTTAG)CCCGCCCCGGATGTCTGGGTGTTTCCCT

GCGACCGAGACCTGCCGGACAGCAGCGACTCTGAGGCGGAGACCGAAGTG

GGGGGGCGGGGGGACGCCGACCACCATGACGACGACTCCGCCTCCGAGGC

GGACAGCACGGACACGGAACTGTTCGAGACGGGGCTGCTGGGGCCGCAGG

GCGTGGATGGGGGGGCGGTCTCGGGGGGGAGCCCCCCCCGCGAGGAAGAC

CCCGGCAGTTGCGGGGGCGCCCCCCCTCGAGAGGACGGGGGGAGCGACGA

GGGTGACGTGTGCGCCGTGTGCACGGATGAGATCGCGCCCCACCTGCGCT

GCGACACCTTCCCGTGCATGCACCGCTTCTGCATCCCGTGCATGAAAACC

TGGATGCAATTGCGCAACACCTGCCCGCTGTGCAACGCCAAGCTGGTGTA

CCTGATAGTGGGCGTGACGCCCAGCGGGTCGTTCAGCACCATCCCGATCG

TGAACGACCCCCAGACCCGCATGGAGGCCGAGGAGGCCGTCAGGGCGGGC

ACGGCCGTGGACTTTATCTGGACGGGCAATCAGCGGTTCGCCCCGCGGTA

CCTGACCCTGGGGGGGCACACGGTGAGGGCCCTGTCGCCCACCCACCCGG

AACCCACCACGGACGAGGATGACGACGACCTGGACGACG(GTGAGGCGGGG

GGCGGCAAGGACCCTGGGGGAGGAGGAGGAGGAGGGGGGGGGAGGGAGGA

ATAGGCGGGCGGGCGAGGAAAGGGCGGGCCGGGGAGGGGGCGTAACCTGA

TCGCGCCCCCCGTTGTCTCTTGCAG)CAGACTACGTACCGCCCGCCCCCCG

CCGGACGCCCCGCGCCCCCCCACGCAGAGGCGCCGCCGCGCCCCCCGTGA

CGGGCGGGGCGTCTCACGCAGCCCCCCAGCCGGCCGCGGCTCGGACAGCG

CCCCCCTCGGCGCCCATCGGGCCACACGGCAGCAGTAACACCAACACCAC

CACCAACAGCAGCGGCGGCGGCGGCTCCCGCCAGTCGCGAGCCGCGGCGC

CGCGGGGGGCGTCTGGCCCCTCCGGGGGGGTTGGGGTTGGGGTTGGGGTT

GTTGAAGCGGAGGCGGGGCGGCCGAGGGGCCGGACGGGCCCCCTTGTCAA

CAGACCCGCCCCCCTTGCAAACAACAGAGACCCCATAGTGATCAGCGACT

CCCCCCCGGCCTCTCCCCACAGGCCCCCCGCGGCGCCCATGCCAGGCTCC

GCCCCCCGCCCCGGGCCCCCCGCGTCCGCGGCCGCGTCGGGACCCGCGCG

CCCCCGCGCGGCCGTGGCCCCGTGCGTGCGAGCGCCGCCTCCGGGGCCCG

GCCCCCGCGCCCCGGCCCCCGGGGCGGAGCCGGCCGCCCGCCCCGCGGAC

GCGCGCCGTGTGCCCCAGTCGCACTCGTCCCTGGCTCAGGCCGCGAACCA

AGAACAGAGTCTGTGCCGGGCGCGTGCGACGGTGGCGCGCGGCTCGGGGG

GGCCGGGCGTGGAGGGTGGGCACGGGCCCTCCCGCGGCGCCGCCCCCTCC

GGCGCCGCCCCGCTCCCCTCCGCCGCCTCTGTCGAGCAGGAGGCGGCGGT

GCGTCCGAGGAAGAGGCGCGGGTCGGGCCAGGAAAACCCCTCCCCCCAGT

CCACGCGTCCCCCCCTCGCGCCGGCAGGGGCCAAGAGGGCGGCGACGCAC

CCCCCCTCCGACTCAGGGCCGGGGGGGCGCGGCCAGGGTGGGCCCGGGAC

CCCCCTGACGTCCTCGGCGGCCTCCGCCTCTTCCTCCTCTGCCTCTTCCT

CCTCGGCCCCGACCCCCGCGGGGGCCGCCTCTTCCGCCGCCGGGGCCGCG

TCCTCCTCCGCTTCCGCCTCCTCGGGCGGGGCCGTCGGTGCCCTGGGAGG

GAGACAAGAGGAAACCTCCCTCGGCCCCCGCGCTGCTTCTGGGCCGCGGG

GGCCGAGGAAGTGTGCCCGGAAGACGCGCCACGCGGAGACTTCCGGGGCC

GTCCCCGCGGGCGGCCTCACGCGCTACCTGCCCATCTCGGGGGTCTCTAG

CGTGGTCGCCCTGTCGCCTTACGTGAACAAGACTATCACGGGGGACTGCC

TGCCCATCCTGGACATGGAGACGGGGAACATCGGGGCGTACGTGGTCCTG

GTGGACCAGACGGGAAACATGGCGACCCGGCTGCGGGCCGCGGTCCCCGG

CTGGAGCCGCCGCACCCTGCTCCCCGAGACCGCGGGTAACCACGTGATGC

CCCCCGAGTACCCGACGGCCCCCGCGTCGGAGTGGAACAGCCTCTGGATG

ACCCCCGTGGGGAACATGCTGTTCGACCAGGGCACCCTAGTGGGCGCCCT

GGACTTCCGCAGCCTGCGGTCTCGGCACCCGTGGTCCGGGGAGCAGGGGG

CGTCGACCCGGGACGAGGGAAAACAATAA

SEQ ID number 23: 17Terma strain ICP 0.

SEQ ID number 24: rRp450 strain, and the DNA sequence of ICP 0. Two introns are included (i.e., nucleotides (nt) 58 to nt 862 and nt 1530 to nt 1668 of the following sequences, see also the sequences in parentheses).

ATGGAGCCCCGCCCCGGAGCGAGTACCCGCCGGCCTGAGGGCCGCCCCCA

GCGCGAG(GTGAGGGGCCGGGCGCCATGTCTGGGGCGCCATGTCTGGGGCG

CCATGTCTGGGGCGCCATGTCTGGGGCGCCATGTTGGGGGGCGCCATGTT

GGGGGGCGCCATGTTGGGGGACCCCCGACCCTTACACTGGAACCGGCCGC

CATGTTGGGGGACCCCCACTCATACACGGGAGCCGGGCGCCATGTTGGGG

CGCCATGTTAGGGGGCGTGGAACCCCGTGACACTATATATACAGGGACCG

GGGGCGCCATGTTAGGGGGCGCGGAACCCCCTGACCCTATATATACAGGG

ACCGGGGTCGCCCTGTTAGGGGTCGCCATGTGACCCCCTGACTTTATATA

TACAGACCCCCAACACCTACACATGGCCCCTTTGACTCAGACGCAGGGCC

CGGGGTCGCCGTGGGACCCCCCTGACTCATACACAGAGACACGCCCCCAC

AACAAACACACAGGGACCGGGGTCGCCGTGTTAGGGGGCGTGGTCCCCAC

TGACTCATACGCAGGGCCCCCTTACTCACACGCATCTAGGGGGGTGGGGA

GGAGCCGCCCGCCATATTTGGGGGACGCCGTGGGACCCCCGACTCCGGTG

CGTCTGGAGGGCGGGAGAAGAGGGAAGAAGAGGGGTCGGGATCCAAAGGA

CGGACCCAGACCACCTTTGGTTGCAGACCCCTTTCTCCCCCCTCTTCCGA

GGCCAGCAGGGGGGCAGGACTTTGTGAGGCGGGGGGGGAGGGGGAACTCG

TGGGCGCTGATTGACGCGGGAAATCCCCCCATTCTTACCCGCCCCCCCTT

TTTTCCCCTCAG)CCCGCCCCGGATGTCTGGGTGTTTCCCTGCGACCGAGA

CCTGCCGGACAGCAGCGACTCGGAGGCGGAGACCGAAGTGGGGGGGCGGG

GGGACGCCGACCACCATGACGACGACTCCGCCTCCGAGGCGGACAGCACG

GACACGGAACTGTTCGAGACGGGGCTGCTGGGGCCGCAGGGCGTGGATGG

GGGGGCGGTCTCGGGGGGGAGCCCCCCCCGCGAGGAAGACCCCGGCAGTT

GCGGGGGCGCCCCCCCTCGAGAGGACGGGGGGAGCGACGAGGGCGACGTG

TGCGCCGTGTGCACGGATGAGATCGCGCCCCACCTGCGCTGCGACACCTT

CCCGTGCATGCACCGCTTCTGCATCCCGTGCATGAAAACCTGGATGCAAT

TGCGCAACACCTGCCCGCTGTGCAACGCCAAGCTGGTGTACCTGATAGTG

GGCGTGACGCCCAGCGGGTCGTTCAGCACCATCCCGATCGTGAACGACCC

CCAGACCCGCATGGAGGCCGAGGAGGCCGTCAGGGCGGGCACGGCCGTGG

ACTTTATCTGGACGGGCAATCAGCGGTTCGCCCCGCGGTACCTGACCCTG

GGGGGGCACACGGTGAGGGCCCTGTCGCCCACCCACCCTGAGCCCACCAC

GGACGAGGATGACGACGACCTGGACGACG(GTGAGGCGGGGGGGCGGCGAG

GACCCTGGGGGAGGAGGAGGAGGGGGGGGGGGGGGAGGAATAGGCGGGCG

GGCGGGCGAGGAAAGGGCGGGCCGGGGAGGGGGCGTAACCTGATCGCGCC

CCCCGTTGTCTCTTGCAG)CAGACTACGTACCGCCCGCCCCCCGCCGGACG

CCCCGCGCCCCCCCACGCAGAGGCGCCGCCGCGCCCCCCGTGACGGGCGG

GGCGTCTCACGCAGCCCCCCAGCCGGCCGCGGCTCGGACAGCGCCCCCCT

CGGCGCCCATCGGGCCACACGGCAGCAGTAACACTAACACCACCACCAAC

AGCAGCGGCGGCGGCGGCTCCCGCCAGTCGCGAGCCGCGGTGCCGCGGGG

GGCGTCTGGCCCCTCCGGGGGGGTTGGGGTTGTTGAAGCGGAGGCGGGGC

GGCCGAGGGGCCGGACGGGCCCCCTTGTCAACAGACCCGCCCCCCTTGCA

AACAACAGAGACCCCATAGTGATCAGCGACTCCCCCCCGGCCTCTCCCCA

CAGGCCCCCCGCGGCGCCCATGCCAGGCTCCGCCCCCCGCCCCGGTCCCC

CCGCGTCCGCGGCCGCGTCGGGCCCCGCGCGCCCCCGCGCGGCCGTGGCC

CCGTGTGTGCGGGCGCCGCCTCCGGGGCCCGGCCCCCGCGCCCCGGCCCC

CGGGGCGGAGCCGGCCGCCCGCCCCGCGGACGCGCGCCGTGTGCCCCAGT

CGCACTCGTCCCTGGCTCAGGCCGCGAACCAAGAACAGAGTCTGTGCCGG

GCGCGTGCGACGGTGGCGCGCGGCTCGGGGGGGCCGGGCGTGGAGGGTGG

ACACGGGCCCTCCCGCGGCGCCGCCCCCTCCGGCGCCGCCCCCTCCGGCG

CCCCCCCGCTCCCCTCCGCCGCCTCTGTCGAGCAGGAGGCGGCGGTGCGT

CCGAGGAAGAGGCGCGGGTCGGGCCAGGAAAACCCCTCCCCCCAGTCCAC

GCGTCCCCCCCTCGCGCCGGCAGGGGCCAAGAGGGCGGCGACGCACCCCC

CCTCCGACTCAGGGCCGGGGGGGCGCGGCCAGGGAGGGCCCGGGACCCCC

CTGACGTCCTCGGCGGCCTCCGCCTCTTCCTCCTCCGCCTCTTCCTCCTC

GGCCCCGACTCCCGCGGGGGCCACCTCTTCCGCCACCGGGGCCGCGTCCT

CCTCCGCTTCCGCCTCCTCGGGCGGGGCCGTCGGTGCCCTGGGAGGGAGA

CAAGAGGAAACCTCCCTCGGCCCCCGCGCTGCTTCTGGGCCGCGGGGGCC

GAGGAAGTGTGCCCGGAAGACGCGCCACGCGGAGACTTCCGGGGCCGTCC

CCGCGGGCGGCCTCACGCGCTACCTGCCCATCTCGGGGGTCTCTAGCGTG

GTCGCCCTGTCGCCTTACGTGAACAAGACGATCACGGGGGACTGCCTGCC

CATCCTGGACATGGAGACGGGGAACATCGGGGCGTACGTGGTCCTGGTGG

ACCAGACGGGAAACATGGCGACCCGGCTGCGGGCCGCGGTCCCCGGCTGG

AGCCGCCGCACCCTGCTCCCCGAGACCGCGGGTAACCACGTGACGCCCCC

CGAGTACCCGACGGCCCCCGCGTCGGAGTGGAACAGCCTCTGGATGACCC

CCGTGGGGAACATGCTGTTCGACCAGGGCACCCTAGTGGGCGCCCTGGAC

TTCCGCAGCCTGCGGTCTCGGCACCCGTGGTCCGGGGAGCAGGGGGCGTC

GACCCGGGACGAGGGAAAACAATAA

SEQ ID number 25: DNA sequence of ICP0 in wild type 17 strain. Two introns are included (i.e., nucleotides (nt) 58 to nt 861 and nt 1529 to nt 1663 of the following sequences, see also the sequences in parentheses).

ATGGAGCCCCGCCCCGGAGCGAGTACCCGCCGGCCTGAGGGCCGCCCCCA

GCGCGAG(GTGAGGGGCCGGGCGCCATGTCTGGGGCGCCATGTCTGGGGCG

CCATGTCTGGGGCGCCATGTCTGGGGCGCCATGTTGGGGGGCGCCATGTT

GGGGGGCGCCATGTTGGGGGACCCCCGACCCTTACACTGGAACCGGCCGC

CATGTTGGGGGACCCCCACTCATACACGGGAGCCGGGCGCCATGTTGGGG

CGCCATGTTAGGGGGCGTGGAACCCCGTGACACTATATATACAGGGACCG

GGGGCGCCATGTTAGGGGGCGCGGAACCCCCTGACCCTATATATACAGGG

ACCGGGGTCGCCCTGTTAGGGGTCGCCATGTGACCCCCTGACTTTATATA

TACAGACCCCCAACACCTACACATGGCCCCTTTGACTCAGACGCAGGGCC

CGGGGTCGCCGTGGGACCCCCCTGACTCATACACAGAGACACGCCCCCAC

AACAAACACACAGGGACCGGGGTCGCCGTGTTAGGGGGCGTGGTCCCCAC

TGACTCATACGCAGGGCCCCCTTACTCACACGCATCTAGGGGGGTGGGGA

GGAGCCGCCCGCCATATTTGGGGGACGCCGTGGGACCCCCGACTCCGGTG

CGTCTGGAGGGCGGGAGAAGAGGGAAGAAGAGGGGTCGGGATCCAAAGGA

CGGACCCAGACCACCTTTGGTTGCAGACCCCTTTCTCCCCCCTCTTCCGA

GGCCAGCAGGGGGGCAGGACTTTGTGAGGCGGGGGGGGAGGGGGAACTCG

TGGGCGCTGATTGACGCGGGAAATCCCCCCATTCTTACCCGCCCCCCTTT

TTTCCCCTCAG)CCCGCCCCGGATGTCTGGGTGTTTCCCTGCGACCGAGAC

CTGCCGGACAGCAGCGACTCGGAGGCGGAGACCGAAGTGGGGGGGCGGGG

GGACGCCGACCACCATGACGACGACTCCGCCTCCGAGGCGGACAGCACGG

ACACGGAACTGTTCGAGACGGGGCTGCTGGGGCCGCAGGGCGTGGATGGG

GGGGCGGTCTCGGGGGGGAGCCCCCCCCGCGAGGAAGACCCCGGCAGTTG

CGGGGGCGCCCCCCCTCGAGAGGACGGGGGGAGCGACGAGGGCGACGTGT

GCGCCGTGTGCACGGATGAGATCGCGCCCCACCTGCGCTGCGACACCTTC

CCGTGCATGCACCGCTTCTGCATCCCGTGCATGAAAACCTGGATGCAATT

GCGCAACACCTGCCCGCTGTGCAACGCCAAGCTGGTGTACCTGATAGTGG

GCGTGACGCCCAGCGGGTCGTTCAGCACCATCCCGATCGTGAACGACCCC

CAGACCCGCATGGAGGCCGAGGAGGCCGTCAGGGCGGGCACGGCCGTGGA

CTTTATCTGGACGGGCAATCAGCGGTTCGCCCCGCGGTACCTGACCCTGG

GGGGGCACACGGTGAGGGCCCTGTCGCCCACCCACCCGGAACCCACCACG

GACGAGGATGACGACGACCTGGACGACG(GTGAGGCGGGGGGCGGCAAGGA

CCCTGGGGGAGGAGGAGGAGGAGGGGGGGGAGGGAGGAATAGGCGGGCGG

GCGAGGAAAGGGCGGGCCGGGGAGGGGGCGTAACCTGATCGCGCCCCCCG

TTGTCTCTTGCAG)CAGACTACGTACCGCCCGCCCCCCGCCGGACGCCCCG

CGCCCCCCCACGCAGAGGCGCCGCCGCGCCCCCCGTGACGGGCGGGGCGT

CTCACGCAGCCCCCCAGCCGGCCGCGGCTCGGACAGCGCCCCCCTCGGCG

CCCATCGGGCCACACGGCAGCAGTAACACCAACACCACCACCAACAGCAG

CGGCGGCGGCGGCTCCCGCCAGTCGCGAGCCGCGGCGCCGCGGGGGGCGT

CTGGCCCCTCCGGGGGGGTTGGGGTTGGGGTTGGGGTTGTTGAAGCGGAG

GCGGGGCGGCCGAGGGGCCGGACGGGCCCCCTTGTCAACAGACCCGCCCC

CCTTGCAAACAACAGAGACCCCATAGTGATCAGCGACTCCCCCCCGGCCT

CTCCCCACAGGCCCCCCGCGGCGCCCATGCCAGGCTCCGCCCCCCGCCCC

GGGCCCCCCGCGTCCGCGGCCGCGTCGGGACCCGCGCGCCCCCGCGCGGC

CGTGGCCCCGTGCGTGCGAGCGCCGCCTCCGGGGCCCGGCCCCCGCGCCC

CGGCCCCCGGGGCGGAGCCGGCCGCCCGCCCCGCGGACGCGCGCCGTGTG

CCCCAGTCGCACTCGTCCCTGGCTCAGGCCGCGAACCAAGAACAGAGTCT

GTGCCGGGCGCGTGCGACGGTGGCGCGCGGCTCGGGGGGGCCGGGCGTGG

AGGGTGGGCACGGGCCCTCCCGCGGCGCCGCCCCCTCCGGCGCCGCCCCG

CTCCCCTCCGCCGCCTCTGTCGAGCAGGAGGCGGCGGTGCGTCCGAGGAA

GAGGCGCGGGTCGGGCCAGGAAAACCCCTCCCCCCAGTCCACGCGTCCCC

CCCTCGCGCCGGCAGGGGCCAAGAGGGCGGCGACGCACCCCCCCTCCGAC

TCAGGGCCGGGGGGGCGCGGCCAGGGTGGGCCCGGGACCCCCCTGACGTC

CTCGGCGGCCTCCGCCTCTTCCTCCTCTGCCTCTTCCTCCTCGGCCCCGA

CCCCCGCGGGGGCCGCCTCTTCCGCCGCCGGGGCCGCGTCCTCCTCCGCT

TCCGCCTCCTCGGGCGGGGCCGTCGGTGCCCTGGGAGGGAGACAAGAGGA

AACCTCCCTCGGCCCCCGCGCTGCTTCTGGGCCGCGGGGGCCGAGGAAGT

GTGCCCGGAAGACGCGCCACGCGGAGACTTCCGGGGCCGTCCCCGCGGGC

GGCCTCACGCGCTACCTGCCCATCTCGGGGGTCTCTAGCGTGGTCGCCCT

GTCGCCTTACGTGAACAAGACGATCACGGGGGACTGCCTGCCCATCCTGG

ACATGGAGACGGGGAACATCGGGGCGTACGTGGTCCTGGTGGACCAGACG

GGAAACATGGCGACCCGGCTGCGGGCCGCGGTCCCCGGCTGGAGCCGCCG

CACCCTGCTCCCCGAGACCGCGGGTAACCACGTGATGCCCCCCGAGTACC

CGACGGCCCCCGCGTCGGAGTGGAACAGCCTCTGGATGACCCCCGTGGGG

AACATGCTGTTCGACCAGGGCACCCTAGTGGGCGCCCTGGACTTCCGCAG

CCTGCGGTCTCGGCACCCGTGGTCCGGGGAGCAGGGGGCGTCGACCCGGG

ACGAGGGAAAACAATAA

SEQ ID number 53: DNA sequence of ICP0 in wild type 17 strain. Two introns are included (i.e., nucleotides (nt) 58 to nt 822 and nt 1490 to nt 1625 of the following sequences, see also the sequences in parentheses).

ATGGAGCCCCGCCCCGGAGCGAGTACCCGCCGGCCTGAGGGCCGCCCCCA

GCGCGAG(GTGAGGGGCCGGGCGCCATGTCTGGGGCGCCATATTGGGGGGC

GCCATATTGGGGGGCGCCATGTTGGGGGACCCCCGACCCTTACACTGGAA

CCGGCCGCCATGTTGGGGGACCCCCACTCATACACGGGAGCCGGGCGCCA

TGTTGGGGCGCCATGTTAGGGGGCGTGGAACCCCGTGACACTATATATAC

AGGGACCGGGGGCGCCATGTTAGGGGGTGCGGAACCCCCTGACCCTATAT

ATACAGGGACCGGGGTCGCCCTGTTGGGGGTCGCCATGTGACCCCCTGAC

TTTATATATACAGACCCCCAACACATACACATGGCCCCTTTGACTCAGAC

GCAGGGCCCGGGGTCGCCGTGGGACCCCCTGACTCATACACAGAGACACG

CCCCCACAACAAACACACAGGGACCGGGGTCGCCGTGTTGGGGGCGTGGT

CCCCACTGACTCATACGCAGGCCCCCCTTACTCACACGCATCTAGGGGGG

TGGGGAGGAGCCGCCCGCCATATTTGGGGGACGCCGTGGGACCCCCGACT

CCGGTGCGTCTGGAGGGCGGGAGAAGAGGGAAGAAGAGGGGTCGGGATCC

AAAGGACGGACCCAGACCACCTTTGGTTGCAGACCCCTTTCTCCCCCCTC

TTCCGAGGCCAGCAGGGGGGCAGGACTTTGTGAGGCGGGGGGGGGAGAGG

GGGAACTCGTGGGTGCTGATTGACGCGGGAAATCCCCCCCCATTCTTACC

CGCCCCCCTTTTTTCCCCTTAG)CCCGCCCCGGATGTCTGGGTGTTTCCCT

GCGACCGAGACCTGCCGGACAGCAGCGACTCTGAGGCGGAGACCGAAGTG

GGGGGGCGGGGGGACGCCGACCACCATGACGACGACTCCGCCTCCGAGGC

GGACAGCACGGACACGGAACTGTTCGAGACGGGGCTGCTGGGGCCGCAGG

GCGTGGATGGGGGGGCGGTCTCGGGGGGGAGCCCCCCCCGCGAGGAAGAC

CCCGGCAGTTGCGGGGGCGCCCCCCCTCGAGAGGACGGGGGGAGCGACGA

GGGCGACGTGTGCGCCGTGTGCACGGATGAGATCGCGCCCCACCTGCGCT

GCGACACCTTCCCGTGCATGCACCGCTTCTGCATCCCGTGCATGAAAACC

TGGATGCAATTGCGCAACACCTGCCCGCTGTGCAACGCCAAGCTGGTGTA

CCTGATAGTGGGCGTGACGCCCAGCGGGTCGTTCAGCACCATCCCGATCG

TGAACGACCCCCAGACCCGCATGGAGGCCGAGGAGGCCGTCAGGGCGGGC

ACGGCCGTGGACTTTATCTGGACGGGCAATCAGCGGTTCGCCCCGCGGTA

CCTGACCCTGGGGGGGCACACGGTGAGGGCCCTGTCGCCCACCCACCCGG

AGCCCACCACGGACGAGGATGACGACGACCTGGACGACG(GTGAGGCGGGG

GGCGGCAAGGACCCTGGGGGAGGAGGAGGAGGAGGGGGGGGGAGGGAGGA

ATAGGCGGGCGGGCGAGGAAAGGGCGGGCCGGGGAGGGGGCGTAACCTGA

TCGCGCCCCCCGTTGTCTCTTGCAG)CAGACTACGTACCGCCCGCCCCCCG

CCGGACGCCCCGCGCCCCCCCACGCAGAGGCGCCGCCGCGCCCCCCGTGA

CGGGCGGGGCGTCTCACGCAGCCCCCCAGCCGGCCGCGGCTCGGACAGCG

CCCCCCTCGGCGCCCATCGGGCCACACGGCAGCAGTAACACCAACACCAC

CACCAACAGCAGCGGCGGCGGCGGCTCCCGCCAGTCGCGAGCCGCGGCGC

CGCGGGGGGCGTCTGGCCCCTCCGGGGGGGTTGGGGTTGGGGTTGGGGTT

GTTGAAGCGGAGGCGGGGCGGCCGAGGGGCCGGACGGGCCCCCTTGTCAA

CAGACCCGCCCCCCTTGCAAACAACAGAGACCCCATAGTGATCAGCGACT

CCCCCCCGGCCTCTCCCCACAGGCCCCCCGCGGCGCCCATGCCAGGCTCC

GCCCCCCGCCCCGGGCCCCCCGCGTCCGCGGCCGCGTCGGGACCCGCGCG

CCCCCGCGCGGCCGTGGCCCCGTGCGTGCGAGCGCCGCCTCCGGGGCCCG

GCCCCCGCGCCCCGGCCCCCGGGGCGGAGCCGGCCGCCCGCCCCGCGGAC

GCGCGCCGTGTGCCCCAGTCGCACTCGTCCCTGGCTCAGGCCGCGAACCA

AGAACAGAGTCTGTGCCGGGCGCGTGCGACGGTGGCGCGCGGCTCGGGGG

GGCCGGGCGTGGAGGGTGGGCACGGGCCCTCCCGCGGCGCCGCCCCCTCC

GGCGCCGCCCCGCTCCCCTCCGCCGCCTCTGTCGAGCAGGAGGCGGCGGT

GCGTCCGAGGAAGAGGCGCGGGTCGGGCCAGGAAAACCCCTCCCCCCAGT

CCACGCGTCCCCCCCTCGCGCCGGCAGGGGCCAAGAGGGCGGCGACGCAC

CCCCCCTCCGACTCAGGGCCGGGGGGGCGCGGCCAGGGTGGGCCCGGGAC

CCCCCTGACGTCCTCGGCGGCCTCCGCCTCTTCCTCCTCTGCCTCTTCCT

CCTCGGCCCCGACCCCCGCGGGGGCCGCCTCTTCCGCCGCCGGGGCCGCG

TCCTCCTCCGCTTCCGCCTCCTCGGGCGGGGCCGTCGGTGCCCTGGGAGG

GAGACAAGAGGAAACCTCCCTCGGCCCCCGCGCTGCTTCTGGGCCGCGGG

GGCCGAGGAAGTGTGCCCGGAAGACGCGCCACGCGGAGACTTCCGGGGCC

GTCCCCGCGGGCGGCCTCACGCGCTACCTGCCCATCTCGGGGGTCTCTAG

CGTGGTCGCCCTGTCGCCTTACGTGAACAAGACTATCACGGGGGACTGCC

TGCCCATCCTGGACATGGAGACGGGGAACATCGGGGCGTACGTGGTCCTG

GTGGACCAGACGGGAAACATGGCGACCCGGCTGCGGGCCGCGGTCCCCGG

CTGGAGCCGCCGCACCCTGCTCCCCGAGACCGCGGGTAACCACGTGATGC

CCCCCGAGTACCCGACGGCCCCCGCGTCGGAGTGGAACAGCCTCTGGATG

ACCCCCGTGGGGAACATGCTGTTCGACCAGGGCACCCTAGTGGGCGCCCT

GGACTTCCGCAGCCTGCGGTCTCGGCACCCGTGGTCCGGGGAGCAGGGGG

CGTCGACCCGGGACGAGGGAAAACAATAA

SEQ ID number 26: amino acid sequence of ICP0 in wild type 17 strain.

SEQ ID number 27: DNA sequences of ICP8 of strains Mut-3, Mut-3. Δ 34.5 and Mut-3. Δ ICP 6. The c3464t mutation is shown in bold and italics in the sequence below.

ATGGAGACAAAGCCCAAGACGGCAACCACCATCAAGGTCCCCCCCGGGCC

CCTGGGATACGTGTACGCTCGCGCGTGTCCGTCCGAAGGCATCGAGCTTC

TGGCGTTACTGTCGGCACGCAGCGGCGATTCCGACGTCGCCGTGGCGCCC

CTGGTCGTGGGCCTGACCGTGGAGAGCGGCTTTGAGGCCAACGTGGCCGT

GGTCGTGGGTTCTCGCACGACGGGGCTCGGGGGTACCGCGGTGTCCCTGA

AACTGACGCCCTCGCACTACAGCTCGTCCGTGTACGTCTTTCACGGCGGC

CGGCACCTGGACCCCAGCACCCAGGCCCCGAACCTGACGCGACTTTGCGA

GCGGGCACGCCGCCATTTTGGCTTTTCGGACTACACCCCCCGGCCCGGCG

ACCTCAAACACGAGACGACGGGGGAGGCGCTGTGTGAGCGCCTCGGCCTG

GACCCGGACCGCGCCCTCCTGTATCTGGTCGTTACCGAGGGCTTCAAGGA

GGCCGTGTGCATCAACAACACCTTTCTGCACCTGGGAGGCTCGGACAAGG

TAACCATAGGCGGGGCGGAGGTGCACCGCATACCCGTGTACCCGTTGCAG

CTGTTCATGCCGGATTTTAGCCGTGTCATCGCAGAGCCGTTCAACGCCAA

CCACCGATCGATCGGGGAGAATTTTACCTACCCGCTTCCGTTTTTTAACC

GCCCCCTCAACCGCCTCCTGTTCGAGGCGGTCGTGGGACCCGCCGCCGTG

GCACTGCGATGCCGAAACGTGGACGCCGTGGCCCGCGCGGCCGCCCACCT

GGCGTTTGACGAAAACCACGAGGGCGCCGCCCTCCCCGCCGACATTACGT

TCACGGCCTTCGAAGCCAGCCAGGGTAAGACCCCGCGGGGCGGGCGCGAC

GGCGGCGGCAAGGGCCCGGCGGGCGGGTTCGAACAGCGCCTGGCCTCCGT

CATGGCCGGAGACGCCGCCCTGGCCCTCGATTCTATCGTGTCGATGGCCG

TCTTTGACGAGCCGCCCACCGACATCTCCGCGTGGCCGCTGTTCGAGGGC

CAGGACACGGCCGCGGCCCGCGCCAACGCCGTCGGGGCGTACCTGGCGCG

CGCCGCGGGACTCGTGGGGGCCATGGTATTTAGCACCAACTCGGCCCTCC

ATCTCACCGAGGTGGACGACGCCGGCCCGGCGGACCCAAAGGACCACAGC

AAACCCTCCTTTTACCGCTTCTTCCTCGTGCCCGGGACCCACGTGGCGGC

CAACCCACAGGTGGACCGCGAGGGACACGTGGTGCCCGGGTTCGAGGGTC

GGCCCACCGCGCCCCTCGTCGGCGGAACCCAGGAATTTGCCGGCGAGCAC

CTGGCCATGCTGTGTGGGTTTTCCCCGGCGCTGCTGGCCAAGATGCTGTT

TTACCTGGAGCGCTGCGACGGCGGCGTGATCGTCGGGCGCCAGGAGATGG

ACGTGTTTCGATACGTCGCGGACTCCAACCAGACCGACGTGCCCTGTAAC

CTATGCACCTTCGACACGCGCCACGCCTGCGTACACACGACGCTCATGCG

CCTCCGGGCGCGCCATCCAAAGTTCGCCAGCGCCGCCCGCGGAGCCATCG

GCGTCTTCGGGACCATGAACAGCATGTACAGCGACTGCGACGTGCTGGGA

AACTACGCCGCCTTCTCGGCCCTGAAGCGCGCGGACGGATCCGAGACCGC

CCGGACCATCATGCAGGAGACGTACCGCGCGGCGACCGAGCGCGTCATGG

CCGAACTCGAGACCCTGCAGTACGTGGACCAGGCGGTCCCCACGGCCATG

GGGCGGCTGGAGACCATCATCACCAACCGCGAGGCCCTGCATACGGTGGT

GAACAACGTCAGGCAGGTCGTGGACCGCGAGGTGGAGCAGCTGATGCGCA

ACCTGGTGGAGGGGAGGAACTTCAAGTTTCGCGACGGTCTGGGCGAGGCC

AACCACGCCATGTCCCTGACGCTGGACCCGTACGCGTGCGGGCCGTGCCC

CCTGCTTCAGCTTCTCGGGCGGCGATCCAACCTCGCCGTGTACCAGGACC

TGGCCCTGAGTCAGTGCCACGGGGTGTTCGCCGGGCAGTCGGTCGAGGGG

CGCAACTTTCGCAATCAATTCCAACCGGTGCTGCGGCGGCGCGTGATGGA

CATGTTTAACAACGGGTTTCTGTCGGCCAAAACGCTGACGGTCGCGCTCT

CGGAGGGGGCGGCTATCTGCGCCCCCAGCCTAACGGCCGGCCAGACGGCC

CCCGCCGAGAGCAGCTTCGAGGGCGACGTTGCCCGCGTGACCCTGGGGTT

TCCCAAGGAGCTGCGCGTCAAGAGCCGCGTGTTGTTCGCGGGCGCGAGCG

CCAACGCGTCCGAGGCCGCCAAGGCGCGGGTCGCCAGCCTCCAGAGCGCC

TACCAGAAGCCCGACAAGCGCGTGGACATCCTCCTCGGACCGCTGGGCTT

TCTGCTGAAGCAGTTCCACGCGGCCATCTTCCCCAACGGCAAGCCCCCGG

GGTCCAACCAGCCGAACCCGCAGTGGTTCTGGACGGCCCTCCAACGCAAC

CAGCTTCCCGCCCGGCTCCTGTCGCGCGAGGACATCGAGACCATCGCGTT

CATTAAAAAGTTTTCCCTGGACTACGGCGCGATAAACTTTATTAACCTGG

CCCCCAACAACGTGAGCGAGCTGGCGATGTACTACATGGCAAACCAGATT

CTGCGGTACTGCGATCACTCGACATACTTCATCAACACCCTTACGGCCAT

CATCGCGGGGTCCCGCCGTCCCCCCAGCGTGCAGGCTGCGGCCGCGTGGT

CCGCGCAGGGCGGGGCGGGCCTGGAGGCCGGGGCCCGCGCGCTGATGGAC

GCCGTGGACGCGCATCCGGGCGCGTGGACGTCCATGTTCGCCAGCTGCAA

CCTGCTGCGGCCCGTCATGGCGGCGCGCCCCATGGTCGTGTTGGGGTTGA

GCATCAGCAAGTACTACGGCATGGCCGGCAACGACCGTGTGTTTCAGGCC

GGGAACTGGGCCAGCCTGATGGGCGGCAAAAACGCGTGCCCGCTCCTTAT

TTTTGACCGCACCCGCAAGTTCGTCCTGGCCTGTCCCCGGGCCGGGTTTG

TGTGCGCGGCCTCAAGCCTCGGCGGCGGAGCGCACGAAAGCTCGCTGTGC

GAGCAGCTCCGGGGCATTATCTCCGAGGGCGGGGCGGCCGTCGCCAGTAG

CGTGTTCGTGGCGACCGTGAAAAGCCTGGGGCCCCGCACCCAGCAGCTGC

AGATCGAGGACTGGCTGGCGCTCCTGGAGGACGAGTACCTAAGCGAGGAG

ATGATGGAGCTGACCGCGCGTGCCCTGGAGCGCGGCAACGGCGAGTGGTC

GACGGACGCGGCCCTGGAGGTGGCGCACGAGGCCGAGGCCCTAGTCAGCC

AACTCGGCAACGCCGGGGAGGTGTTTAACTTTGGGGATTTTGGCTGCGAG

GACGACAACGCGATGCCGTTCGGCGGCCCGGGGGCCCCGGGACCGGCATT

TGCCGGCCGCAAACGGGCGTTCCACGGGGATGACCCGTTTGGGGAGGGGC

CCCCCGACAAAAAGGGAGACCTGACGTTGGATATGCTGTGA

SEQ ID number 28: the amino acid sequences of the ICP8 strains of Mut-3, Δ 34.5 and Mut-3, ICP 6. The T1155M mutation is shown in bold and italics in the sequence below.

METKPKTATTIKVPPGPLGYVYARACPSEGIELLALLSARSGDSDVAVAP

LVVGLTVESGFEANVAVVVGSRTTGLGGTAVSLKLTPSHYSSSVYVFHGG

RHLDPSTQAPNLTRLCERARRHFGFSDYTPRPGDLKHETTGEALCERLGL

DPDRALLYLVVTEGFKEAVCINNTFLHLGGSDKVTIGGAEVHRIPVYPLQ

LFMPDFSRVIAEPFNANHRSIGENFTYPLPFFNRPLNRLLFEAVVGPAAV

ALRCRNVDAVARAAAHLAFDENHEGAALPADITFTAFEASQGKTPRGGRD

GGGKGPAGGFEQRLASVMAGDAALALDSIVSMAVFDEPPTDISAWPLFEG

QDTAAARANAVGAYLARAAGLVGAMVFSTNSALHLTEVDDAGPADPKDHS

KPSFYRFFLVPGTHVAANPQVDREGHVVPGFEGRPTAPLVGGTQEFAGEH

LAMLCGFSPALLAKMLFYLERCDGGVIVGRQEMDVFRYVADSNQTDVPCN

LCTFDTRHACVHTTLMRLRARHPKFASAARGAIGVFGTMNSMYSDCDVLG

NYAAFSALKRADGSETARTIMQETYRAATERVMAELETLQYVDQAVPTAM

GRLETIITNREALHTVVNNVRQVVDREVEQLMRNLVEGRNFKFRDGLGEA

NHAMSLTLDPYACGPCPLLQLLGRRSNLAVYQDLALSQCHGVFAGQSVEG

RNFRNQFQPVLRRRVMDMFNNGFLSAKTLTVALSEGAAICAPSLTAGQTA

PAESSFEGDVARVTLGFPKELRVKSRVLFAGASANASEAAKARVASLQSA

YQKPDKRVDILLGPLGFLLKQFHAAIFPNGKPPGSNQPNPQWFWTALQRN

QLPARLLSREDIETIAFIKKFSLDYGAINFINLAPNNVSELAMYYMANQI

LRYCDHSTYFINTLTAIIAGSRRPPSVQAAAAWSAQGGAGLEAGARALMD

AVDAHPGAWTSMFASCNLLRPVMAARPMVVLGLSISKYYGMAGNDRVFQA

GNWASLMGGKNACPLLIFDRTRKFVLACPRAGFVCAASSLGGGAHESSLC

EQLRGIISEGGAAVASSVFVATVKSLGPRTQQLQIEDWLALLEDEYLSEE

MMELTARALERGNGEWSTDAALEVAHEAEALVSQLGNAGEVFNFGDFGCE

DDNAMPFGGPGAPGPAFAGRKRAFHGDDPFGEGPPDKKGDLTLDML

SEQ ID number 29: DNA sequence of ICP8 in 17TerMA strain.

ATGGAGACAAAGCCCAAGACGGCAACCACCATCAAGGTCCCCCCCGGGCC

CCTGGGATACGTGTACGCTCGCGCGTGTCCGTCCGAAGGCATCGAGCTTC

TGGCGTTACTGTCGGCACGCAGCGGCGATTCCGACGTCGCCGTGGCGCCC

CTGGTCGTGGGCCTGACCGTGGAGAGCGGCTTTGAGGCCAACGTGGCCGT

GGTCGTGGGTTCTCGCACGACGGGGCTCGGGGGTACCGCGGTGTCCCTGA

AACTGACGCCCTCGCACTACAGCTCGTCCGTGTACGTCTTTCACGGCGGC

CGGCACCTGGACCCCAGCACCCAGGCCCCGAACCTGACGCGACTTTGCGA

GCGGGCACGCCGCCATTTTGGCTTTTCGGACTACACCCCCCGGCCCGGCG

ACCTCAAACACGAGACGACGGGGGAGGCGCTGTGTGAGCGCCTCGGCCTG

GACCCGGACCGCGCCCTCCTGTATCTGGTCGTTACCGAGGGCTTCAAGGA

GGCCGTGTGCATCAACAACACCTTTCTGCACCTGGGAGGCTCGGACAAGG

TAACCATAGGCGGGGCGGAGGTGCACCGCATACCCGTGTACCCGTTGCAG

CTGTTCATGCCGGATTTTAGCCGTGTCATCGCAGAGCCGTTCAACGCCAA

CCACCGATCGATCGGGGAGAATTTTACCTACCCGCTTCCGTTTTTTAACC

GCCCCCTCAACCGCCTCCTGTTCGAGGCGGTCGTGGGACCCGCCGCCGTG

GCACTGCGATGCCGAAACGTGGACGCCGTGGCCCGCGCGGCCGCCCACCT

GGCGTTTGACGAAAACCACGAGGGCGCCGCCCTCCCCGCCGACATTACGT

TCACGGCCTTCGAAGCCAGCCAGGGTAAGACCCCGCGGGGCGGGCGCGAC

GGCGGCGGCAAGGGCCCGGCGGGCGGGTTCGAACAGCGCCTGGCCTCCGT

CATGGCCGGAGACGCCGCCCTGGCCCTCGATTCTATCGTGTCGATGGCCG

TCTTTGACGAGCCGCCCACCGACATCTCCGCGTGGCCGCTGTTCGAGGGC

CAGGACACGGCCGCGGCCCGCGCCAACGCCGTCGGGGCGTACCTGGCGCG

CGCCGCGGGACTCGTGGGGGCCATGGTATTTAGCACCAACTCGGCCCTCC

ATCTCACCGAGGTGGACGACGCCGGCCCGGCGGACCCAAAGGACCACAGC

AAACCCTCCTTTTACCGCTTCTTCCTCGTGCCCGGGACCCACGTGGCGGC

CAACCCACAGGTGGACCGCGAGGGACACGTGGTGCCCGGGTTCGAGGGTC

GGCCCACCGCGCCCCTCGTCGGCGGAACCCAGGAATTTGCCGGCGAGCAC

CTGGCCATGCTGTGTGGGTTTTCCCCGGCGCTGCTGGCCAAGATGCTGTT

TTACCTGGAGCGCTGCGACGGCGGCGTGATCGTCGGGCGCCAGGAGATGG

ACGTGTTTCGATACGTCGCGGACTCCAACCAGACCGACGTGCCCTGTAAC

CTATGCACCTTCGACACGCGCCACGCCTGCGTACACACGACGCTCATGCG

CCTCCGGGCGCGCCATCCAAAGTTCGCCAGCGCCGCCCGCGGAGCCATCG

GCGTCTTCGGGACCATGAACAGCATGTACAGCGACTGCGACGTGCTGGGA

AACTACGCCGCCTTCTCGGCCCTGAAGCGCGCGGACGGATCCGAGACCGC

CCGGACCATCATGCAGGAGACGTACCGCGCGGCGACCGAGCGCGTCATGG

CCGAACTCGAGACCCTGCAGTACGTGGACCAGGCGGTCCCCACGGCCATG

GGGCGGCTGGAGACCATCATCACCAACCGCGAGGCCCTGCATACGGTGGT

GAACAACGTCAGGCAGGTCGTGGACCGCGAGGTGGAGCAGCTGATGCGCA

ACCTGGTGGAGGGGAGGAACTTCAAGTTTCGCGACGGTCTGGGCGAGGCC

AACCACGCCATGTCCCTGACGCTGGACCCGTACGCGTGCGGGCCGTGCCC

CCTGCTTCAGCTTCTCGGGCGGCGATCCAACCTCGCCGTGTACCAGGACC

TGGCCCTGAGTCAGTGCCACGGGGTGTTCGCCGGGCAGTCGGTCGAGGGG

CGCAACTTTCGCAATCAATTCCAACCGGTGCTGCGGCGGCGCGTGATGGA

CATGTTTAACAACGGGTTTCTGTCGGCCAAAACGCTGACGGTCGCGCTCT

CGGAGGGGGCGGCTATCTGCGCCCCCAGCCTAACGGCCGGCCAGACGGCC

CCCGCCGAGAGCAGCTTCGAGGGCGACGTTGCCCGCGTGACCCTGGGGTT

TCCCAAGGAGCTGCGCGTCAAGAGCCGCGTGTTGTTCGCGGGCGCGAGCG

CCAACGCGTCCGAGGCCGCCAAGGCGCGGGTCGCCAGCCTCCAGAGCGCC

TACCAGAAGCCCGACAAGCGCGTGGACATCCTCCTCGGACCGCTGGGCTT

TCTGCTGAAGCAGTTCCACGCGGCCATCTTCCCCAACGGCAAGCCCCCGG

GGTCCAACCAGCCGAACCCGCAGTGGTTCTGGACGGCCCTCCAACGCAAC

CAGCTTCCCGCCCGGCTCCTGTCGCGCGAGGACATCGAGACCATCGCGTT

CATTAAAAAGTTTTCCCTGGACTACGGCGCGATAAACTTTATTAACCTGG

CCCCCAACAACGTGAGCGAGCTGGCGATGTACTACATGGCAAACCAGATT

CTGCGGTACTGCGATCACTCGACATACTTCATCAACACCCTTACGGCCAT

CATCGCGGGGTCCCGCCGTCCCCCCAGCGTGCAGGCTGCGGCCGCGTGGT

CCGCGCAGGGCGGGGCGGGCCTGGAGGCCGGGGCCCGCGCGCTGATGGAC

GCCGTGGACGCGCATCCGGGCGCGTGGACGTCCATGTTCGCCAGCTGCAA

CCTGCTGCGGCCCGTCATGGCGGCGCGCCCCATGGTCGTGTTGGGGTTGA

GCATCAGCAAGTACTACGGCATGGCCGGCAACGACCGTGTGTTTCAGGCC

GGGAACTGGGCCAGCCTGATGGGCGGCAAAAACGCGTGCCCGCTCCTTAT

TTTTGACCGCACCCGCAAGTTCGTCCTGGCCTGTCCCCGGGCCGGGTTTG

TGTGCGCGGCCTCAAGCCTCGGCGGCGGAGCGCACGAAAGCTCGCTGTGC

GAGCAGCTCCGGGGCATTATCTCCGAGGGCGGGGCGGCCGTCGCCAGTAG

CGTGTTCGTGGCGACCGTGAAAAGCCTGGGGCCCCGCACCCAGCAGCTGC

AGATCGAGGACTGGCTGGCGCTCCTGGAGGACGAGTACCTAAGCGAGGAG

ATGATGGAGCTGACCGCGCGTGCCCTGGAGCGCGGCAACGGCGAGTGGTC

GACGGACGCGGCCCTGGAGGTGGCGCACGAGGCCGAGGCCCTAGTCAGCC

AACTCGGCAACGCCGGGGAGGTGTTTAACTTTGGGGATTTTGGCTGCGAG

GACGACAACGCGACGCCGTTCGGCGGCCCGGGGGCCCCGGGACCGGCATT

TGCCGGCCGCAAACGGGCGTTCCACGGGGATGACCCGTTTGGGGAGGGGC

CCCCCGACAAAAAGGGAGACCTGACGTTGGATATGCTGTGA

SEQ ID number 30: 17Terma strain ICP 8.

METKPKTATTIKVPPGPLGYVYARACPSEGIELLALLSARSGDSDVAVAP

LVVGLTVESGFEANVAVVVGSRTTGLGGTAVSLKLTPSHYSSSVYVFHGG

RHLDPSTQAPNLTRLCERARRHFGFSDYTPRPGDLKHETTGEALCERLGL

DPDRALLYLVVTEGFKEAVCINNTFLHLGGSDKVTIGGAEVHRIPVYPLQ

LFMPDFSRVIAEPFNANHRSIGENFTYPLPFFNRPLNRLLFEAVVGPAAV

ALRCRNVDAVARAAAHLAFDENHEGAALPADITFTAFEASQGKTPRGGRD

GGGKGPAGGFEQRLASVMAGDAALALDSIVSMAVFDEPPTDISAWPLFEG

QDTAAARANAVGAYLARAAGLVGAMVFSTNSALHLTEVDDAGPADPKDHS

KPSFYRFFLVPGTHVAANPQVDREGHVVPGFEGRPTAPLVGGTQEFAGEH

LAMLCGFSPALLAKMLFYLERCDGGVIVGRQEMDVFRYVADSNQTDVPCN

LCTFDTRHACVHTTLMRLRARHPKFASAARGAIGVFGTMNSMYSDCDVLG

NYAAFSALKRADGSETARTIMQETYRAATERVMAELETLQYVDQAVPTAM

GRLETIITNREALHTVVNNVRQVVDREVEQLMRNLVEGRNFKFRDGLGEA

NHAMSLTLDPYACGPCPLLQLLGRRSNLAVYQDLALSQCHGVFAGQSVEG

RNFRNQFQPVLRRRVMDMFNNGFLSAKTLTVALSEGAAICAPSLTAGQTA

PAESSFEGDVARVTLGFPKELRVKSRVLFAGASANASEAAKARVASLQSA

YQKPDKRVDILLGPLGFLLKQFHAAIFPNGKPPGSNQPNPQWFWTALQRN

QLPARLLSREDIETIAFIKKFSLDYGAINFINLAPNNVSELAMYYMANQI

LRYCDHSTYFINTLTAIIAGSRRPPSVQAAAAWSAQGGAGLEAGARALMD

AVDAHPGAWTSMFASCNLLRPVMAARPMVVLGLSISKYYGMAGNDRVFQA

GNWASLMGGKNACPLLIFDRTRKFVLACPRAGFVCAASSLGGGAHESSLC

EQLRGIISEGGAAVASSVFVATVKSLGPRTQQLQIEDWLALLEDEYLSEE

MMELTARALERGNGEWSTDAALEVAHEAEALVSQLGNAGEVFNFGDFGCE

DDNATPFGGPGAPGPAFAGRKRAFHGDDPFGEGPPDKKGDLTLDML

SEQ ID number 31: rRp450 strain, and the DNA sequence of ICP 8.

ATGGAGACAAAGCCCAAGACGGCAACCACCATCAAGGTCCCCCCCGGGCC

CCTGGGATACGTGTACGCTCGCGCGTGTCCGTCCGAAGGCATCGAGCTTC

TGGCGTTACTGTCGGCGCGCAGCGGCGATGCCGACGTCGCCGTGGCGCCC

CTGGTCGTGGGCCTGACCGTGGAGAGCGGCTTTGAGGCCAACGTAGCCGT

GGTCGTGGGTTCTCGCACGACGGGGCTCGGGGGTACCGCGGTGTCCCTGA

AACTGACGCCATCGCACTACAGCTCGTCCGTGTACGTCTTTCACGGCGGC

CGGCACCTGGACCCCAGCACCCAGGCCCCAAACCTGACGCGACTCTGCGA

GCGGGCACGCCGCCATTTTGGCTTTTCGGACTACACCCCCCGGCCCGGCG

ACCTCAAACACGAGACGACGGGGGAGGCGCTGTGTGAGCGCCTCGGCCTG

GACCCGGACCGCGCCCTCCTGTATCTGGTCGTTACCGAGGGCTTCAAGGA

GGCCGTGTGCATCAACAACACCTTTCTGCACCTGGGAGGCTCGGACAAGG

TAACCATAGGCGGGGCGGAGGTGCACCGCATACCCGTGTATCCGTTGCAG

CTGTTCATGCCGGATTTTAGCCGGGTCATCGCCGAGCCGTTCAACGCCAA

CCACCGATCGATCGGGGAGAATTTTACCTACCCGCTTCCGTTTTTTAACC

GCCCCCTCAACCGCCTCCTGTTCGAGGCGGTCGTGGGACCCGCCGCCGTG

GCACTGCGATGCCGAAACGTGGACGCCGTGGCCCGCGCGGCCGCCCACCT

GGCGTTTGACGAAAACCACGAGGGCGCCGCCCTCCCCGCCGACATTACGT

TCACGGCCTTCGAAGCCAGCCAGGGTAAGACCCCGCGGGGTGGGCGCGAC

GGCGGCGGCAAGGGCCCGGCGGGCGGGTTCGAACAGCGCCTGGCCTCCGT

CATGGCCGGAGACGCCGCCCTGGCCCTCGAGTCTATCGTGTCGATGGCCG

TCTTCGACGAGCCGCCCACCGACATCTCCGCGTGGCCGCTGTGCGAGGGC

CAGGACACGGCCGCGGCCCGCGACAACGCCGTCGGGGCGTACCTGGCGCG

CGCCGCGGGACTCGTGGGGGCCATGGTATTTAGCACCAACTCGGCCCTCC

ATCTCACCGAGGTGGACGACGCCGGTCCGGCGGACCCAAAGGACCACAGC

AAACCCTCCTTTTACCGCTTCTTCCTCGTGCCCGGGACCCACGTGGCGGC

CAACCCACAGGTGGACCGCGAGGGACACGTGGTGCCCGGGTTCGAGGGTC

GGCCCACCGCGCCCCTCGTCGGCGGAACCCAGGAATTTGCCGGCGAGCAC

CTGGCCATGCTGTGTGGGTTTTCCCCGGCGCTGCTGGCCAAGATGCTGTT

TTACCTGGAGCGCTGCGACGGCGGCGTGATCGTCGGGCGCCAGGAGATGG

ACGTGTTTCGATACGTCGCGGACTCCAACCAGACCGACGTGCCCTGCAAC

CTGTGCACCTTCGACACGCGCCACGCCTGCGTACACACGACGCTCATGCG

CCTCCGGGCGCGCCATCCCAAGTTCGCCAGCGCCGCCCGCGGAGCCATCG

GCGTCTTCGGGACCATGAACAGCATGTACAGCGACTGCGACGTGCTGGGA

AACTACGCCGCCTTCTCGGCCCTGAAGCGCGCGGACGGATCCGAGACCGC

CCGGACCATCATGCAGGAGACGTACCGCGCGGCGACCGAGCGCGTCATGG

CCGAACTCGAGACCCTGCAGTACGTGGACCAGGCGGTCCCCACGGCCATG

GGGCGGCTGGAGACCATCATCACCAACCGCGAGGCCCTGCATACGGTGGT

GAACAACGTCAGGCAGGTCGTGGACCGCGAGGTGGAGCAGCTGATGCGCA

ACCTGGTGGAGGGGAGGAACTTCAAGTTTCGCGACGGTCTGGGCGAGGCC

AACCACGCCATGTCCCTGACGCTGGACCCGTACGCGTGCGGGCCATGCCC

CCTGCTTCAGCTTCTCGGGCGGCGATCCAACCTCGCCGTGTATCAGGACC

TGGCCCTGAGCCAGTGCCACGGGGTGTTCGCCGGGCAGTCGGTCGAGGGG

CGCAACTTTCGCAATCAATTCCAACCGGTGCTGCGGCGGCGCGTGATGGA

CATGTTTAACAACGGGTTTCTGTCGGCCAAAACGCTGACGGTCGCGCTCT

CGGAGGGGGCGGCTATCTGCGCCCCCAGCCTAACGGCCGGCCAGACGGCC

CCCGCCGAGAGCAGCTTCGAGGGCGACGTTGCCCGCGTGACCCTGGGGTT

TCCCAAGGAGCTGCGCGTCAAGAGCCGCGTGTTGTTCGCGGGCGCGAGCG

CCAACGCGTCCGAGGCCGCCAAGGCGCGGGTCGCCAGCCTCCAGAGCGCC

TACCAGAAGCCCGACAAGCGCGTGGACATCCTCCTCGGACCGCTGGGCTT

TCTGCTGAAGCAGTTCCACGCGGCCATCTTCCCCAACGGCAAGCCCCCGG

GGTCCAACCAGCCGAACCCGCAGTGGTTCTGGACGGCCCTCCAACGCAAC

CAGCTTCCCGCCCGGCTCCTGTCGCGCGAGGACATCGAGACCATCGCGTT

CATTAAAAAGTTTTCCCTGGACTACGGCGCGATAAACTTTATTAACCTGG

CCCCCAACAACGTGAGCGAGCTGGCGATGTACTACATGGCAAACCAGATT

CTGCGGTACTGCGATCACTCGACATACTTCATCAACACCCTCACGGCCAT

CATCGCGGGGTCCCGCCGTCCCCCCAGCGTGCAGGCGGCGGCCGCGTGGT

CCGCGCAGGGCGGGGCGGGCCTGGAGGCCGGGGCCCGCGCGCTGATGGAC

GCCGTGGACGCGCATCCGGGCGCGTGGACGTCCATGTTCGCCAGCTGCAA

CCTGCTGCGGCCCGTCATGGCGGCGCGCCCCATGGTCGTGTTGGGGTTGA

GCATCAGCAAATACTACGGCATGGCCGGCAACGACCGTGTGTTTCAGGCC

GGGAACTGGGCCAGCCTGATGGGCGGCAAAAACGCGTGCCCGCTCCTTAT

TTTTGACCGCACCCGCAAGTTCGTCCTGGCCTGTCCCCGGGCCGGGTTTG

TGTGCGCGGCCTCGAACCTCGGCGGCGGAGCGCACGAAAGCTCGCTGTGC

GAGCAGCTCCGGGGCATTATCTCCGAGGGCGGGGCGGCCGTCGCCAGTAG

CGTGTTCGTGGCGACCGTGAAAAGCCTGGGGCCCCGCACCCAGCAGCTGC

AGATCGAGGACTGGCTGGCGCTCCTGGAGGACGAGTACCTAAGCGAGGAG

ATGATGGAGCTGACCGCGCGTGCCCTGGAGCGCGGCAACGGCGAGTGGTC

GACGGACGCGGCCCTGGAGGTGGCGCACGAGGCCGAGGCCCTAGTCAGCC

AACTCGGCAACGCCGGGGAGGTGTTTAACTTTGGGGATTTTGGCTGCGAG

GACGACAACGCGACGCCGTTCGGCGGCCCGGGGGCCCCGGGACCGGCATT

TGCCGGCCGCAAACGGGCGTTCCACGGGGATGACCCGTTTGGGGAGGGGC

CCCCCGACAAAAAGGGAGACCTGACGTTGGATATGCTGTGA

SEQ ID number 32: rRp450 strain, ICP 8.

METKPKTATTIKVPPGPLGYVYARACPSEGIELLALLSARSGDADVAVAP

LVVGLTVESGFEANVAVVVGSRTTGLGGTAVSLKLTPSHYSSSVYVFHGG

RHLDPSTQAPNLTRLCERARRHFGFSDYTPRPGDLKHETTGEALCERLGL

DPDRALLYLVVTEGFKEAVCINNTFLHLGGSDKVTIGGAEVHRIPVYPLQ

LFMPDFSRVIAEPFNANHRSIGENFTYPLPFFNRPLNRLLFEAVVGPAAV

ALRCRNVDAVARAAAHLAFDENHEGAALPADITFTAFEASQGKTPRGGRD

GGGKGPAGGFEQRLASVMAGDAALALESIVSMAVFDEPPTDISAWPLCEG

QDTAAARDNAVGAYLARAAGLVGAMVFSTNSALHLTEVDDAGPADPKDHS

KPSFYRFFLVPGTHVAANPQVDREGHVVPGFEGRPTAPLVGGTQEFAGEH

LAMLCGFSPALLAKMLFYLERCDGGVIVGRQEMDVFRYVADSNQTDVPCN

LCTFDTRHACVHTTLMRLRARHPKFASAARGAIGVFGTMNSMYSDCDVLG

NYAAFSALKRADGSETARTIMQETYRAATERVMAELETLQYVDQAVPTAM

GRLETIITNREALHTVVNNVRQVVDREVEQLMRNLVEGRNFKFRDGLGEA

NHAMSLTLDPYACGPCPLLQLLGRRSNLAVYQDLALSQCHGVFAGQSVEG

RNFRNQFQPVLRRRVMDMFNNGFLSAKTLTVALSEGAAICAPSLTAGQTA

PAESSFEGDVARVTLGFPKELRVKSRVLFAGASANASEAAKARVASLQSA

YQKPDKRVDILLGPLGFLLKQFHAAIFPNGKPPGSNQPNPQWFWTALQRN

QLPARLLSREDIETIAFIKKFSLDYGAINFINLAPNNVSELAMYYMANQI

LRYCDHSTYFINTLTAIIAGSRRPPSVQAAAAWSAQGGAGLEAGARALMD

AVDAHPGAWTSMFASCNLLRPVMAARPMVVLGLSISKYYGMAGNDRVFQA

GNWASLMGGKNACPLLIFDRTRKFVLACPRAGFVCAASNLGGGAHESSLC

EQLRGIISEGGAAVASSVFVATVKSLGPRTQQLQIEDWLALLEDEYLSEE

MMELTARALERGNGEWSTDAALEVAHEAEALVSQLGNAGEVFNFGDFGCE

DDNATPFGGPGAPGPAFAGRKRAFHGDDPFGEGPPDKKGDLTLDML

SEQ ID number 33: DNA sequence of ICP8 in wild type 17 strain.

ATGGAGACAAAGCCCAAGACGGCAACCACCATCAAGGTCCCCCCCGGGCC

CCTGGGATACGTGTACGCTCGCGCGTGTCCGTCCGAAGGCATCGAGCTTC

TGGCGTTACTGTCGGCACGCAGCGGCGATTCCGACGTCGCCGTGGCGCCC

CTGGTCGTGGGCCTGACCGTGGAGAGCGGCTTTGAGGCCAACGTGGCCGT

GGTCGTGGGTTCTCGCACGACGGGGCTCGGGGGTACCGCGGTGTCCCTGA

AACTGACGCCCTCGCACTACAGCTCGTCCGTGTACGTCTTTCACGGCGGC

CGGCACCTGGACCCCAGCACCCAGGCCCCGAACCTGACGCGACTTTGCGA

GCGGGCACGCCGCCATTTTGGCTTTTCGGACTACACCCCCCGGCCCGGCG

ACCTCAAACACGAGACGACGGGGGAGGCGCTGTGTGAGCGCCTCGGCCTG

GACCCGGACCGCGCCCTCCTGTATCTGGTCGTTACCGAGGGCTTCAAGGA

GGCCGTGTGCATCAACAACACCTTTCTGCACCTGGGAGGCTCGGACAAGG

TAACCATAGGCGGGGCGGAGGTGCACCGCATACCCGTGTACCCGTTGCAG

CTGTTCATGCCGGATTTTAGCCGTGTCATCGCAGAGCCGTTCAACGCCAA

CCACCGATCGATCGGGGAGAATTTTACCTACCCGCTTCCGTTTTTTAACC

GCCCCCTCAACCGCCTCCTGTTCGAGGCGGTCGTGGGACCCGCCGCCGTG

GCACTGCGATGCCGAAACGTGGACGCCGTGGCCCGCGCGGCCGCCCACCT

GGCGTTTGACGAAAACCACGAGGGCGCCGCCCTCCCCGCCGACATTACGT

TCACGGCCTTCGAAGCCAGCCAGGGTAAGACCCCGCGGGGCGGGCGCGAC

GGCGGCGGCAAGGGCCCGGCGGGCGGGTTCGAACAGCGCCTGGCCTCCGT

CATGGCCGGAGACGCCGCCCTGGCCCTCGATTCTATCGTGTCGATGGCCG

TCTTTGACGAGCCGCCCACCGACATCTCCGCGTGGCCGCTGTTCGAGGGC

CAGGACACGGCCGCGGCCCGCGCCAACGCCGTCGGGGCGTACCTGGCGCG

CGCCGCGGGACTCGTGGGGGCCATGGTATTTAGCACCAACTCGGCCCTCC

ATCTCACCGAGGTGGACGACGCCGGCCCGGCGGACCCAAAGGACCACAGC

AAACCCTCCTTTTACCGCTTCTTCCTCGTGCCCGGGACCCACGTGGCGGC

CAACCCACAGGTGGACCGCGAGGGACACGTGGTGCCCGGGTTCGAGGGTC

GGCCCACCGCGCCCCTCGTCGGCGGAACCCAGGAATTTGCCGGCGAGCAC

CTGGCCATGCTGTGTGGGTTTTCCCCGGCGCTGCTGGCCAAGATGCTGTT

TTACCTGGAGCGCTGCGACGGCGGCGTGATCGTCGGGCGCCAGGAGATGG

ACGTGTTTCGATACGTCGCGGACTCCAACCAGACCGACGTGCCCTGTAAC

CTATGCACCTTCGACACGCGCCACGCCTGCGTACACACGACGCTCATGCG

CCTCCGGGCGCGCCATCCAAAGTTCGCCAGCGCCGCCCGCGGAGCCATCG

GCGTCTTCGGGACCATGAACAGCATGTACAGCGACTGCGACGTGCTGGGA

AACTACGCCGCCTTCTCGGCCCTGAAGCGCGCGGACGGATCCGAGACCGC

CCGGACCATCATGCAGGAGACGTACCGCGCGGCGACCGAGCGCGTCATGG

CCGAACTCGAGACCCTGCAGTACGTGGACCAGGCGGTCCCCACGGCCATG

GGGCGGCTGGAGACCATCATCACCAACCGCGAGGCCCTGCATACGGTGGT

GAACAACGTCAGGCAGGTCGTGGACCGCGAGGTGGAGCAGCTGATGCGCA

ACCTGGTGGAGGGGAGGAACTTCAAGTTTCGCGACGGTCTGGGCGAGGCC

AACCACGCCATGTCCCTGACGCTGGACCCGTACGCGTGCGGGCCGTGCCC

CCTGCTTCAGCTTCTCGGGCGGCGATCCAACCTCGCCGTGTACCAGGACC

TGGCCCTGAGTCAGTGCCACGGGGTGTTCGCCGGGCAGTCGGTCGAGGGG

CGCAACTTTCGCAATCAATTCCAACCGGTGCTGCGGCGGCGCGTGATGGA

CATGTTTAACAACGGGTTTCTGTCGGCCAAAACGCTGACGGTCGCGCTCT

CGGAGGGGGCGGCTATCTGCGCCCCCAGCCTAACGGCCGGCCAGACGGCC

CCCGCCGAGAGCAGCTTCGAGGGCGACGTTGCCCGCGTGACCCTGGGGTT

TCCCAAGGAGCTGCGCGTCAAGAGCCGCGTGTTGTTCGCGGGCGCGAGCG

CCAACGCGTCCGAGGCCGCCAAGGCGCGGGTCGCCAGCCTCCAGAGCGCC

TACCAGAAGCCCGACAAGCGCGTGGACATCCTCCTCGGACCGCTGGGCTT

TCTGCTGAAGCAGTTCCACGCGGCCATCTTCCCCAACGGCAAGCCCCCGG

GGTCCAACCAGCCGAACCCGCAGTGGTTCTGGACGGCCCTCCAACGCAAC

CAGCTTCCCGCCCGGCTCCTGTCGCGCGAGGACATCGAGACCATCGCGTT

CATTAAAAAGTTTTCCCTGGACTACGGCGCGATAAACTTTATTAACCTGG

CCCCCAACAACGTGAGCGAGCTGGCGATGTACTACATGGCAAACCAGATT

CTGCGGTACTGCGATCACTCGACATACTTCATCAACACCCTTACGGCCAT

CATCGCGGGGTCCCGCCGTCCCCCCAGCGTGCAGGCTGCGGCCGCGTGGT

CCGCGCAGGGCGGGGCGGGCCTGGAGGCCGGGGCCCGCGCGCTGATGGAC

GCCGTGGACGCGCATCCGGGCGCGTGGACGTCCATGTTCGCCAGCTGCAA

CCTGCTGCGGCCCGTCATGGCGGCGCGCCCCATGGTCGTGTTGGGGTTGA

GCATCAGCAAGTACTACGGCATGGCCGGCAACGACCGTGTGTTTCAGGCC

GGGAACTGGGCCAGCCTGATGGGCGGCAAAAACGCGTGCCCGCTCCTTAT

TTTTGACCGCACCCGCAAGTTCGTCCTGGCCTGTCCCCGGGCCGGGTTTG

TGTGCGCGGCCTCAAGCCTCGGCGGCGGAGCGCACGAAAGCTCGCTGTGC

GAGCAGCTCCGGGGCATTATCTCCGAGGGCGGGGCGGCCGTCGCCAGTAG

CGTGTTCGTGGCGACCGTGAAAAGCCTGGGGCCCCGCACCCAGCAGCTGC

AGATCGAGGACTGGCTGGCGCTCCTGGAGGACGAGTACCTAAGCGAGGAG

ATGATGGAGCTGACCGCGCGTGCCCTGGAGCGCGGCAACGGCGAGTGGTC

GACGGACGCGGCCCTGGAGGTGGCGCACGAGGCCGAGGCCCTAGTCAGCC

AACTCGGCAACGCCGGGGAGGTGTTTAACTTTGGGGATTTTGGCTGCGAG

GACGACAACGCGACGCCGTTCGGCGGCCCGGGGGCCCCGGGACCGGCATT

TGCCGGCCGCAAACGGGCGTTCCACGGGGATGACCCGTTTGGGGAGGGGC

CCCCCGACAAAAAGGGAGACCTGACGTTGGATATGCTGTGA

SEQ ID number 34: amino acid sequence of ICP8 in wild type 17 strain.

METKPKTATTIKVPPGPLGYVYARACPSEGIELLALLSARSGDSDVAVAP

LVVGLTVESGFEANVAVVVGSRTTGLGGTAVSLKLTPSHYSSSVYVFHGG

RHLDPSTQAPNLTRLCERARRHFGFSDYTPRPGDLKHETTGEALCERLGL

DPDRALLYLVVTEGFKEAVCINNTFLHLGGSDKVTIGGAEVHRIPVYPLQ

LFMPDFSRVIAEPFNANHRSIGENFTYPLPFFNRPLNRLLFEAVVGPAAV

ALRCRNVDAVARAAAHLAFDENHEGAALPADITFTAFEASQGKTPRGGRD

GGGKGPAGGFEQRLASVMAGDAALALDSIVSMAVFDEPPTDISAWPLFEG

QDTAAARANAVGAYLARAAGLVGAMVFSTNSALHLTEVDDAGPADPKDHS

KPSFYRFFLVPGTHVAANPQVDREGHVVPGFEGRPTAPLVGGTQEFAGEH

LAMLCGFSPALLAKMLFYLERCDGGVIVGRQEMDVFRYVADSNQTDVPCN

LCTFDTRHACVHTTLMRLRARHPKFASAARGAIGVFGTMNSMYSDCDVLG

NYAAFSALKRADGSETARTIMQETYRAATERVMAELETLQYVDQAVPTAM

GRLETIITNREALHTVVNNVRQVVDREVEQLMRNLVEGRNFKFRDGLGEA

NHAMSLTLDPYACGPCPLLQLLGRRSNLAVYQDLALSQCHGVFAGQSVEG

RNFRNQFQPVLRRRVMDMFNNGFLSAKTLTVALSEGAAICAPSLTAGQTA

PAESSFEGDVARVTLGFPKELRVKSRVLFAGASANASEAAKARVASLQSA

YQKPDKRVDILLGPLGFLLKQFHAAIFPNGKPPGSNQPNPQWFWTALQRN

QLPARLLSREDIETIAFIKKFSLDYGAINFINLAPNNVSELAMYYMANQI

LRYCDHSTYFINTLTAIIAGSRRPPSVQAAAAWSAQGGAGLEAGARALMD

AVDAHPGAWTSMFASCNLLRPVMAARPMVVLGLSISKYYGMAGNDRVFQA

GNWASLMGGKNACPLLIFDRTRKFVLACPRAGFVCAASSLGGGAHESSLC

EQLRGIISEGGAAVASSVFVATVKSLGPRTQQLQIEDWLALLEDEYLSEE

MMELTARALERGNGEWSTDAALEVAHEAEALVSQLGNAGEVFNFGDFGCE

DDNATPFGGPGAPGPAFAGRKRAFHGDDPFGEGPPDKKGDLTLDML

SEQ ID number 35: DNA sequence of end enzyme subunit 1 is packaged in DNA of strains Mut-3, Mut-3. Δ 34.5 and Mut-3. Δ ICP 6. The g1126a mutation is shown in bold and italics in the sequence below.

ATGTTTGGTCAGCAGCTGGCGTCCGACGTCCAGCAGTACCTGGAGCGCCT

CGAGAAACAGAGGCAACTTAAGGTGGGCGCGGACGAGGCGTCGGCGGGCC

TCACCATGGGCGGCGATGCCCTACGAGTGCCCTTTTTAGATTTCGCGACC

GCGACCCCCAAGCGCCACCAGACCGTGGTCCCTGGCGTCGGGACGCTCCA

CGACTGCTGCGAGCACTCGCCGCTCTTCTCGGCCGTGGCGCGGCGGCTGC

TGTTTAATAGCCTGGTGCCGGCGCAACTAAAGGGGCGTGATTTCGGGGGC

GACCACACGGCCAAGCTGGAATTCCTGGCCCCCGAGTTGGTACGGGCGGT

GGCGCGACTGCGGTTTAAGGAGTGCGCGCCGGCGGACGTGGTGCCTCAGC

GTAACGCCTACTATAGCGTTCTGAATACGTTTCAGGCCCTCCACCGCTCC

GAAGCCTTTCGCCAGCTGGTGCACTTTGTGCGGGACTTTGCCCAGCTGCT

CAAAACCTCCTTCCGGGCCTCCAGCCTCACGGAGACCACGGGCCCCCCCA

AAAAACGGGCCAAGGTGGACGTGGCCACCCACGGCCGGACGTACGGCACG

CTGGAGCTGTTCCAAAAAATGATCCTTATGCACGCCACCTACTTTCTGGC

CGCCGTGCTCCTCGGGGACCACGCGGAGCAGGTCAACACGTTCCTGCGTC

TCGTGTTTGAGATCCCCCTGTTTAGCGACGCGGCCGTGCGCCACTTCCGC

CAGCGCGCCACCGTGTTTCTCGTCCCCCGGCGCCACGGCAAGACCTGGTT

TCTGGTGCCCCTCATCGCGCTGTCGCTGGCCTCCTTTCGGGGGATCAAGA

TCGGCTACACGGCGCACATCCGCAAGGCGACCGAGCCGGTGTTTGAGGAG

ATCGACGCCTGCCTGCGGGGCTGGTTCGGTTCGGCCCGAGTGGACCACGT

TAAAGGGGAAACCATCTCCTTCTCGTTTCCGGACGGGTCGCGCAGTACCA

TCGTGTTTGCCTCCAGCCACAACACAAACGGAATCCGAGGCCAGGACTTT

AACCTGCTCTTTGTCGACGAGGCCAACTTTATTCGCCCGGATGCGGTCCA

GACGATTATGGGCTTTCTCAACCAGACCAACTGCAAGATTATCTTCGTGT

CGTCCACCAACACCGGGAAGGCCAGTACGAGCTTTTTGTACAACCTCCGC

GGGGCCGCAGACGAGCTTCTCAACGTGGTGACCTATATATGCGATGATCA

CATGCCGAGGGTGGTGACGCACACAAACGCCACGGCCTGTTCTTGTTATA

TCCTCAACAAGCCCGTTTTCATCACGATGGACGGGGCGGTTCGCCGGACC

GCCGATTTGTTTCTGGCCGATTCCTTCATGCAGGAGATCATCGGGGGCCA

GGCCAGGGAGACCGGCGACGACCGGCCCGTTCTGACCAAGTCTGCGGGGG

AGCGGTTTCTGTTGTACCGCCCCTCGACCACCACCAACAGCGGCCTCATG

GCCCCCGATTTGTACGTGTACGTGGATCCCGCGTTCACGGCCAACACCCG

AGCCTCCGGGACCGGCGTCGCTGTCGTCGGGCGGTACCGCGACGATTATA

TCATCTTCGCCCTGGAGCACTTTTTTCTCCGCGCGCTCACGGGCTCGGCC

CCCGCCGACATCGCCCGCTGCGTCGTCCACAGTCTGACGCAGGTCCTGGC

CCTGCATCCCGGGGCGTTTCGCGGCGTCCGGGTGGCGGTCGAGGGAAATA

GCAGCCAGGACTCGGCCGTCGCCATCGCCACGCACGTGCACACAGAGATG

CACCGCCTACTGGCCTCGGAGGGGGCCGACGCGGGCTCGGGCCCCGAGCT

TCTCTTCTACCACTGCGAGCCTCCCGGGAGCGCGGTGCTGTACCCCTTTT

TCCTGCTCAACAAACAGAAGACGCCCGCCTTTGAACACTTTATTAAAAAG

TTTAACTCCGGGGGCGTCATGGCCTCCCAGGAGATCGTTTCCGCGACGGT

GCGCCTGCAGACCGACCCGGTCGAGTATCTGCTCGAGCAGCTAAATAACC

TCACCGAAACCGTCTCCCCCAACACTGACGTCCGTACGTATTCCGGAAAA

CGGAACGGCGCCTCGGATGACCTTATGGTCGCCGTCATTATGGCCATCTA

CCTCGCGGCCCAGGCCGGACCTCCGCACACATTCGCTCCTATCATACGCG

TCTCGTGA

SEQ ID number 36: the amino acid sequence of the end enzyme subunit 1 of the DNA packaging of the strains Mut-3, Mut-3. Δ 34.5 and Mut-3. Δ ICP 6. The a376T mutation is shown in bold and italics in the sequence below.

MFGQQLASDVQQYLERLEKQRQLKVGADEASAGLTMGGDALRVPFLDFAT

ATPKRHQTVVPGVGTLHDCCEHSPLFSAVARRLLFNSLVPAQLKGRDFGG

DHTAKLEFLAPELVRAVARLRFKECAPADVVPQRNAYYSVLNTFQALHRS

EAFRQLVHFVRDFAQLLKTSFRASSLTETTGPPKKRAKVDVATHGRTYGT

LELFQKMILMHATYFLAAVLLGDHAEQVNTFLRLVFEIPLFSDAAVRHFR

QRATVFLVPRRHGKTWFLVPLIALSLASFRGIKIGYTAHIRKATEPVFEE

IDACLRGWFGSARVDHVKGETISFSFPDGSRSTIVFASSHNTNGIRGQDF

NLLFVDEANFIRPDAVQTIMGFLNQTNCKIIFVSSTNTGKASTSFLYNLR

GAADELLNVVTYICDDHMPRVVTHTNATACSCYILNKPVFITMDGAVRRT

ADLFLADSFMQEIIGGQARETGDDRPVLTKSAGERFLLYRPSTTTNSGLM

APDLYVYVDPAFTANTRASGTGVAVVGRYRDDYIIFALEHFFLRALTGSA

PADIARCVVHSLTQVLALHPGAFRGVRVAVEGNSSQDSAVAIATHVHTEM

HRLLASEGADAGSGPELLFYHCEPPGSAVLYPFFLLNKQKTPAFEHFIKK

FNSGGVMASQEIVSATVRLQTDPVEYLLEQLNNLTETVSPNTDVRTYSGK

RNGASDDLMVAVIMAIYLAAQAGPPHTFAPIIRVS

SEQ ID number 37: 17Terma strain DNA packaging terminal enzyme subunit 1 DNA sequence.

ATGTTTGGTCAGCAGCTGGCGTCCGACGTCCAGCAGTACCTGGAGCGCCT

CGAGAAACAGAGGCAACTTAAGGTGGGCGCGGACGAGGCGTCGGCGGGCC

TCACCATGGGCGGCGATGCCCTACGAGTGCCCTTTTTAGATTTCGCGACC

GCGACCCCCAAGCGCCACCAGACCGTGGTCCCTGGCGTCGGGACGCTCCA

CGACTGCTGCGAGCACTCGCCGCTCTTCTCGGCCGTGGCGCGGCGGCTGC

TGTTTAATAGCCTGGTGCCGGCGCAACTAAAGGGGCGTGATTTCGGGGGC

GACCACACGGCCAAGCTGGAATTCCTGGCCCCCGAGTTGGTACGGGCGGT

GGCGCGACTGCGGTTTAAGGAGTGCGCGCCGGCGGACGTGGTGCCTCAGC

GTAACGCCTACTATAGCGTTCTGAATACGTTTCAGGCCCTCCACCGCTCC

GAAGCCTTTCGCCAGCTGGTGCACTTTGTGCGGGACTTTGCCCAGCTGCT

CAAAACCTCCTTCCGGGCCTCCAGCCTCACGGAGACCACGGGCCCCCCCA

AAAAACGGGCCAAGGTGGACGTGGCCACCCACGGCCGGACGTACGGCACG

CTGGAGCTGTTCCAAAAAATGATCCTTATGCACGCCACCTACTTTCTGGC

CGCCGTGCTCCTCGGGGACCACGCGGAGCAGGTCAACACGTTCCTGCGTC

TCGTGTTTGAGATCCCCCTGTTTAGCGACGCGGCCGTGCGCCACTTCCGC

CAGCGCGCCACCGTGTTTCTCGTCCCCCGGCGCCACGGCAAGACCTGGTT

TCTGGTGCCCCTCATCGCGCTGTCGCTGGCCTCCTTTCGGGGGATCAAGA

TCGGCTACACGGCGCACATCCGCAAGGCGACCGAGCCGGTGTTTGAGGAG

ATCGACGCCTGCCTGCGGGGCTGGTTCGGTTCGGCCCGAGTGGACCACGT

TAAAGGGGAAACCATCTCCTTCTCGTTTCCGGACGGGTCGCGCAGTACCA

TCGTGTTTGCCTCCAGCCACAACACAAACGGAATCCGAGGCCAGGACTTT

AACCTGCTCTTTGTCGACGAGGCCAACTTTATTCGCCCGGATGCGGTCCA

GACGATTATGGGCTTTCTCAACCAGGCCAACTGCAAGATTATCTTCGTGT

CGTCCACCAACACCGGGAAGGCCAGTACGAGCTTTTTGTACAACCTCCGC

GGGGCCGCAGACGAGCTTCTCAACGTGGTGACCTATATATGCGATGATCA

CATGCCGAGGGTGGTGACGCACACAAACGCCACGGCCTGTTCTTGTTATA

TCCTCAACAAGCCCGTTTTCATCACGATGGACGGGGCGGTTCGCCGGACC

GCCGATTTGTTTCTGGCCGATTCCTTCATGCAGGAGATCATCGGGGGCCA

GGCCAGGGAGACCGGCGACGACCGGCCCGTTCTGACCAAGTCTGCGGGGG

AGCGGTTTCTGTTGTACCGCCCCTCGACCACCACCAACAGCGGCCTCATG

GCCCCCGATTTGTACGTGTACGTGGATCCCGCGTTCACGGCCAACACCCG

AGCCTCCGGGACCGGCGTCGCTGTCGTCGGGCGGTACCGCGACGATTATA

TCATCTTCGCCCTGGAGCACTTTTTTCTCCGCGCGCTCACGGGCTCGGCC

CCCGCCGACATCGCCCGCTGCGTCGTCCACAGTCTGACGCAGGTCCTGGC

CCTGCATCCCGGGGCGTTTCGCGGCGTCCGGGTGGCGGTCGAGGGAAATA

GCAGCCAGGACTCGGCCGTCGCCATCGCCACGCACGTGCACACAGAGATG

CACCGCCTACTGGCCTCGGAGGGGGCCGACGCGGGCTCGGGCCCCGAGCT

TCTCTTCTACCACTGCGAGCCTCCCGGGAGCGCGGTGCTGTACCCCTTTT

TCCTGCTCAACAAACAGAAGACGCCCGCCTTTGAACACTTTATTAAAAAG

TTTAACTCCGGGGGCGTCATGGCCTCCCAGGAGATCGTTTCCGCGACGGT

GCGCCTGCAGACCGACCCGGTCGAGTATCTGCTCGAGCAGCTAAATAACC

TCACCGAAACCGTCTCCCCCAACACTGACGTCCGTACGTATTCCGGAAAA

CGGAACGGCGCCTCGGATGACCTTATGGTCGCCGTCATTATGGCCATCTA

CCTCGCGGCCCAGGCCGGACCTCCGCACACATTCGCTCCTATCATACGCG

TCTCGTGA

SEQ ID number 38: 17Terma strain DNA packaging terminal enzyme subunit 1 amino acid sequence.

MFGQQLASDVQQYLERLEKQRQLKVGADEASAGLTMGGDALRVPFLDFAT

ATPKRHQTVVPGVGTLHDCCEHSPLFSAVARRLLFNSLVPAQLKGRDFGG

DHTAKLEFLAPELVRAVARLRFKECAPADVVPQRNAYYSVLNTFQALHRS

EAFRQLVHFVRDFAQLLKTSFRASSLTETTGPPKKRAKVDVATHGRTYGT

LELFQKMILMHATYFLAAVLLGDHAEQVNTFLRLVFEIPLFSDAAVRHFR

QRATVFLVPRRHGKTWFLVPLIALSLASFRGIKIGYTAHIRKATEPVFEE

IDACLRGWFGSARVDHVKGETISFSFPDGSRSTIVFASSHNTNGIRGQDF

NLLFVDEANFIRPDAVQTIMGFLNQANCKIIFVSSTNTGKASTSFLYNLR

GAADELLNVVTYICDDHMPRVVTHTNATACSCYILNKPVFITMDGAVRRT

ADLFLADSFMQEIIGGQARETGDDRPVLTKSAGERFLLYRPSTTTNSGLM

APDLYVYVDPAFTANTRASGTGVAVVGRYRDDYIIFALEHFFLRALTGSA

PADIARCVVHSLTQVLALHPGAFRGVRVAVEGNSSQDSAVAIATHVHTEM

HRLLASEGADAGSGPELLFYHCEPPGSAVLYPFFLLNKQKTPAFEHFIKK

FNSGGVMASQEIVSATVRLQTDPVEYLLEQLNNLTETVSPNTDVRTYSGK

RNGASDDLMVAVIMAIYLAAQAGPPHTFAPIIRVS

SEQ ID number 39: rRp450 DNA sequence of the DNA packaging terminal enzyme subunit 1 of the virus strain.

ATGTTTGGTCAGCAGCTGGCGTCCGACGTCCAGCAGTACCTGGAGCGCCT

CGAGAAACAGAGGCAACTTAAGGTGGGCGCGGACGAGGCGTCGGCGGGCC

TCACAATGGGCGGCGATGCCCTACGAGTGCCCTTTTTAGATTTCGCGACC

GCGACCCCCAAGCGCCACCAGACCGTGGTCCCGGGCGTCGGGACGCTCCA

CGACTGCTGCGAGCACTCGCCGCTCTTCTCGGCCGTGGCGCGGCGGCTGC

TGTTTAATAGCCTGGTGCCGGCGCAACTAAAGGGGCGTGATTTCGGGGGC

GACCACACGGCCAAGCTGGAATTCCTGGCCCCCGAGTTGGTACGGGCGGT

GGCGCGACTGCGGTTTAAGGAGTGCGCGCCGGCGGACGTGGTGCCTCAGC

GTAACGCCTACTATAGCGTTCTGAACACGTTTCAGGCCCTCCACCGCTCC

GAAGCCTTTCGCCAGCTGGTGCACTTTGTGCGGGACTTTGCCCAGCTGCT

TAAAACCTCCTTCCGGGCCTCCAGCCTCACGGAGACCACGGGCCCCCCAA

AAAAACGGGCCAAGGTGGACGTGGCCACCCACGGCCGGACGTACGGCACG

CTGGAGCTGTTCCAAAAAATGATCCTTATGCACGCCACCTACTTTCTGGC

CGCCGTGCTCCTCGGGGACCACGCGGAGCAGGTCAACACGTTCCTGCGTC

TCGTGTTTGAGATCCCCCTGTTTAGCGACGCGGCCGTGCGCCACTTCCGC

CAGCGCGCCACCGTGTTTCTCGTCCCCCGGCGCCACGGCAAGACCTGGTT

TCTAGTGCCCCTCATCGCGCTGTCGCTGGCCTCCTTTCGGGGGATCAAGA

TCGGCTACACGGCGCACATCCGCAAGGCGACCGAGCCGGTGTTTGAGGAG

ATCGACGCCTGCCTGCGGGGCTGGTTCGGTTCGGCCCGAGTGGACCACGT

TAAAGGGGAAACCATCTCCTTCTCGTTTCCGGACGGGTCGCGCAGTACCA

TCGTGTTTGCCTCCAGCCACAACACAAACGGAATCCGAGGCCAGGACTTT

AACCTGCTCTTTGTCGACGAGGCCAACTTTATTCGCCCGGATGCGGTCCA

GACGATTATGGGCTTTCTCAACCAGGCCAACTGCAAGATTATCTTCGTGT

CGTCCACCAACACCGGGAAGGCCAGTACGAGCTTTTTGTACAACCTCCGC

GGGGCCGCCGACGAGCTTCTCAACGTGGTGACCTATATATGCGATGATCA

CATGCCGCGGGTGGTGACGCACACAAACGCCACGGCCTGTTCTTGTTATA

TCCTCAACAAGCCCGTTTTCATCACGATGGACGGGGCGGTTCGCCGGACC

GCCGATTTGTTTCTGGCCGATTCCTTCATGCAGGAGATCATCGGGGGCCA

GGCCAGGGAGACCGGCGACGACCGGCCCGTTCTGACCAAGTCTGCGGGGG

AGCGGTTTCTGTTGTACCGCCCCTCGACCACCACCAACAGCGGCCTCATG

GCCCCCGATTTGTACGTGTACGTGGATCCCGCGTTCACGGCCAACACCCG

AGCCTCCGGGACCGGCGTCGCTGTCGTCGGGCGGTACCGCGACGATTATA

TCATCTTCGCCCTGGAGCACTTTTTTCTCCGCGCGCTCACGGGCTCGGCC

CCCGCCGACATCGCCCGCTGCGTCGTCCACAGTCTGACGCAGGTCCTGGC

CCTGCATCCCGGGGCGTTTCGCGGCGTCCGGGTGGCGGTCGAGGGAAATA

GCAGCCAGGACTCGGCCGTCGCCATCGCCACGCACGTGCACACAGAGATG

CACCGCCTACTGGCCTCGGAGGGGGCCGACGCGGGCTCGGGCCCCGAGCT

TCTCTTCTACCACTGCGAGCCTCCCGGGAGCGCGGTGCTGTACCCCTTTT

TCCTGCTCAACAAACAGAAGACGCCCGCCTTTGAACACTTTATTAAAAAG

TTTAACTCCGGGGGCGTCATGGCCTCCCAGGAGATCGTTTCCGCGACGGT

GCGCCTGCAGACCGACCCGGTCGAGTATCTGCTCGAGCAGCTGAATAACC

TCACCGAAACCGTCTCCCCCAACACGGACGTCCGTACGTATTCCGGAAAA

CGGAACGGCGCCTCGGATGACCTTATGGTCGCCGTCATTATGGCCATCTA

CCTTGCGGCCCAGGCCGGACCTCCGCACACATTCGCTCCCATCACACGCG

TTTCGTGA

SEQ ID number 40: rRp450 strain of the virus.

MFGQQLASDVQQYLERLEKQRQLKVGADEASAGLTMGGDALRVPFLDFAT

ATPKRHQTVVPGVGTLHDCCEHSPLFSAVARRLLFNSLVPAQLKGRDFGG

DHTAKLEFLAPELVRAVARLRFKECAPADVVPQRNAYYSVLNTFQALHRS

EAFRQLVHFVRDFAQLLKTSFRASSLTETTGPPKKRAKVDVATHGRTYGT

LELFQKMILMHATYFLAAVLLGDHAEQVNTFLRLVFEIPLFSDAAVRHFR

QRATVFLVPRRHGKTWFLVPLIALSLASFRGIKIGYTAHIRKATEPVFEE

IDACLRGWFGSARVDHVKGETISFSFPDGSRSTIVFASSHNTNGIRGQDF

NLLFVDEANFIRPDAVQTIMGFLNQANCKIIFVSSTNTGKASTSFLYNLR

GAADELLNVVTYICDDHMPRVVTHTNATACSCYILNKPVFITMDGAVRRT

ADLFLADSFMQEIIGGQARETGDDRPVLTKSAGERFLLYRPSTTTNSGLM

APDLYVYVDPAFTANTRASGTGVAVVGRYRDDYIIFALEHFFLRALTGSA

PADIARCVVHSLTQVLALHPGAFRGVRVAVEGNSSQDSAVAIATHVHTEM

HRLLASEGADAGSGPELLFYHCEPPGSAVLYPFFLLNKQKTPAFEHFIKK

FNSGGVMASQEIVSATVRLQTDPVEYLLEQLNNLTETVSPNTDVRTYSGK

RNGASDDLMVAVIMAIYLAAQAGPPHTFAPITRVS

SEQ ID number 41: the DNA of wild type 17 strain packages the DNA sequence of terminal enzyme subunit 1.

ATGTTTGGTCAGCAGCTGGCGTCCGACGTCCAGCAGTACCTGGAGCGCCT

CGAGAAACAGAGGCAACTTAAGGTGGGCGCGGACGAGGCGTCGGCGGGCC

TCACCATGGGCGGCGATGCCCTACGAGTGCCCTTTTTAGATTTCGCGACC

GCGACCCCCAAGCGCCACCAGACCGTGGTCCCTGGCGTCGGGACGCTCCA

CGACTGCTGCGAGCACTCGCCGCTCTTCTCGGCCGTGGCGCGGCGGCTGC

TGTTTAATAGCCTGGTGCCGGCGCAACTAAAGGGGCGTGATTTCGGGGGC

GACCACACGGCCAAGCTGGAATTCCTGGCCCCCGAGTTGGTACGGGCGGT

GGCGCGACTGCGGTTTAAGGAGTGCGCGCCGGCGGACGTGGTGCCTCAGC

GTAACGCCTACTATAGCGTTCTGAATACGTTTCAGGCCCTCCACCGCTCC

GAAGCCTTTCGCCAGCTGGTGCACTTTGTGCGGGACTTTGCCCAGCTGCT

CAAAACCTCCTTCCGGGCCTCCAGCCTCACGGAGACCACGGGCCCCCCCA

AAAAACGGGCCAAGGTGGACGTGGCCACCCACGGCCGGACGTACGGCACG

CTGGAGCTGTTCCAAAAAATGATCCTTATGCACGCCACCTACTTTCTGGC

CGCCGTGCTCCTCGGGGACCACGCGGAGCAGGTCAACACGTTCCTGCGTC

TCGTGTTTGAGATCCCCCTGTTTAGCGACGCGGCCGTGCGCCACTTCCGC

CAGCGCGCCACCGTGTTTCTCGTCCCCCGGCGCCACGGCAAGACCTGGTT

TCTGGTGCCCCTCATCGCGCTGTCGCTGGCCTCCTTTCGGGGGATCAAGA

TCGGCTACACGGCGCACATCCGCAAGGCGACCGAGCCGGTGTTTGAGGAG

ATCGACGCCTGCCTGCGGGGCTGGTTCGGTTCGGCCCGAGTGGACCACGT

TAAAGGGGAAACCATCTCCTTCTCGTTTCCGGACGGGTCGCGCAGTACCA

TCGTGTTTGCCTCCAGCCACAACACAAACGGAATCCGAGGCCAGGACTTT

AACCTGCTCTTTGTCGACGAGGCCAACTTTATTCGCCCGGATGCGGTCCA

GACGATTATGGGCTTTCTCAACCAGGCCAACTGCAAGATTATCTTCGTGT

CGTCCACCAACACCGGGAAGGCCAGTACGAGCTTTTTGTACAACCTCCGC

GGGGCCGCAGACGAGCTTCTCAACGTGGTGACCTATATATGCGATGATCA

CATGCCGAGGGTGGTGACGCACACAAACGCCACGGCCTGTTCTTGTTATA

TCCTCAACAAGCCCGTTTTCATCACGATGGACGGGGCGGTTCGCCGGACC

GCCGATTTGTTTCTGGCCGATTCCTTCATGCAGGAGATCATCGGGGGCCA

GGCCAGGGAGACCGGCGACGACCGGCCCGTTCTGACCAAGTCTGCGGGGG

AGCGGTTTCTGTTGTACCGCCCCTCGACCACCACCAACAGCGGCCTCATG

GCCCCCGATTTGTACGTGTACGTGGATCCCGCGTTCACGGCCAACACCCG

AGCCTCCGGGACCGGCGTCGCTGTCGTCGGGCGGTACCGCGACGATTATA

TCATCTTCGCCCTGGAGCACTTTTTTCTCCGCGCGCTCACGGGCTCGGCC

CCCGCCGACATCGCCCGCTGCGTCGTCCACAGTCTGACGCAGGTCCTGGC

CCTGCATCCCGGGGCGTTTCGCGGCGTCCGGGTGGCGGTCGAGGGAAATA

GCAGCCAGGACTCGGCCGTCGCCATCGCCACGCACGTGCACACAGAGATG

CACCGCCTACTGGCCTCGGAGGGGGCCGACGCGGGCTCGGGCCCCGAGCT

TCTCTTCTACCACTGCGAGCCTCCCGGGAGCGCGGTGCTGTACCCCTTTT

TCCTGCTCAACAAACAGAAGACGCCCGCCTTTGAACACTTTATTAAAAAG

TTTAACTCCGGGGGCGTCATGGCCTCCCAGGAGATCGTTTCCGCGACGGT

GCGCCTGCAGACCGACCCGGTCGAGTATCTGCTCGAGCAGCTAAATAACC

TCACCGAAACCGTCTCCCCCAACACTGACGTCCGTACGTATTCCGGAAAA

CGGAACGGCGCCTCGGATGACCTTATGGTCGCCGTCATTATGGCCATCTA

CCTCGCGGCCCAGGCCGGACCTCCGCACACATTCGCTCCTATCATACGCG

TCTCGTGA

SEQ ID number 42: wild type 17 strain DNA packages the amino acid sequence of terminal enzyme subunit 1.

MFGQQLASDVQQYLERLEKQRQLKVGADEASAGLTMGGDALRVPFLDFAT

ATPKRHQTVVPGVGTLHDCCEHSPLFSAVARRLLFNSLVPAQLKGRDFGG

DHTAKLEFLAPELVRAVARLRFKECAPADVVPQRNAYYSVLNTFQALHRS

EAFRQLVHFVRDFAQLLKTSFRASSLTETTGPPKKRAKVDVATHGRTYGT

LELFQKMILMHATYFLAAVLLGDHAEQVNTFLRLVFEIPLFSDAAVRHFR

QRATVFLVPRRHGKTWFLVPLIALSLASFRGIKIGYTAHIRKATEPVFEE

IDACLRGWFGSARVDHVKGETISFSFPDGSRSTIVFASSHNTNGIRGQDF

NLLFVDEANFIRPDAVQTIMGFLNQANCKIIFVSSTNTGKASTSFLYNLR

GAADELLNVVTYICDDHMPRVVTHTNATACSCYILNKPVFITMDGAVRRT

ADLFLADSFMQEIIGGQARETGDDRPVLTKSAGERFLLYRPSTTTNSGLM

APDLYVYVDPAFTANTRASGTGVAVVGRYRDDYIIFALEHFFLRALTGSA

PADIARCVVHSLTQVLALHPGAFRGVRVAVEGNSSQDSAVAIATHVHTEM

HRLLASEGADAGSGPELLFYHCEPPGSAVLYPFFLLNKQKTPAFEHFIKK

FNSGGVMASQEIVSATVRLQTDPVEYLLEQLNNLTETVSPNTDVRTYSGK

RNGASDDLMVAVIMAIYLAAQAGPPHTFAPIIRVS

SEQ ID number 43: DNA sequences of ICP6 in the strains Mut-3 and Mut-3. 34.5. This sequence is identical to that in wild type 17 strain.

ATGGCCAGCCGCCCAGCCGCATCCTCTCCCGTCGAAGCGCGGGCCCCGGT

TGGGGGACAGGAGGCCGGCGGCCCCAGCGCAGCCACCCAGGGGGAGGCCG

CCGGGGCCCCTCTCGCCCACGGCCACCACGTGTACTGCCAGCGAGTCAAT

GGCGTGATGGTGCTTTCCGACAAGACGCCCGGGTCCGCGTCCTACCGCAT

CAGCGATAGCAACTTTGTCCAATGTGGTTCCAACTGCACCATGATCATCG

ACGGAGACGTGGTGCGCGGGCGCCCCCAGGACCCGGGGGCCGCGGCATCC

CCCGCTCCCTTCGTTGCGGTGACAAACATCGGAGCCGGCAGCGACGGCGG

GACCGCCGTCGTGGCATTCGGGGGAACCCCACGTCGCTCGGCGGGGACGT

CTACCGGTACCCAGACGGCCGACGTCCCCACCGAGGCCCTTGGGGGCCCC

CCTCCTCCTCCCCGCTTCACCCTGGGTGGCGGCTGTTGTTCCTGTCGCGA

CACACGGCGCCGCTCTGCGGTATTCGGGGGGGAGGGGGATCCAGTCGGCC

CCGCGGAGTTCGTCTCGGACGACCGGTCGTCCGATTCCGACTCGGATGAC

TCGGAGGACACGGACTCGGAGACGCTGTCACACGCCTCCTCGGACGTGTC

CGGCGGGGCCACGTACGACGACGCCCTTGACTCCGATTCGTCATCGGATG

ACTCCCTGCAGATAGATGGCCCCGTGTGTCGCCCGTGGAGCAATGACACC

GCGCCCCTGGATGTTTGCCCCGGGACCCCCGGCCCGGGCGCCGACGCCGG

TGGTCCCTCAGCGGTAGACCCACACGCGCCGACGCCAGAGGCCGGCGCTG

GTCTTGCGGCCGATCCCGCCGTGGCCCGGGACGACGCGGAGGGGCTTTCG

GACCCCCGGCCACGTCTGGGAACGGGCACGGCCTACCCCGTCCCCCTGGA

ACTCACGCCCGAGAACGCGGAGGCCGTGGCGCGCTTTCTGGGAGATGCCG

TGAACCGCGAACCCGCGCTCATGCTGGAGTACTTTTGCCGGTGCGCCCGC

GAGGAAACCAAGCGTGTCCCCCCCAGGACATTCGGCAGCCCCCCTCGCCT

CACGGAGGACGACTTTGGGCTTCTCAACTACGCGCTCGTGGAGATGCAGC

GCCTGTGTCTGGACGTTCCTCCGGTCCCGCCGAACGCATACATGCCCTAT

TATCTCAGGGAGTATGTGACGCGGCTGGTCAACGGGTTCAAGCCGCTGGT

GAGCCGGTCCGCTCGCCTTTACCGCATCCTGGGGGTTCTGGTGCACCTGC

GGATCCGGACCCGGGAGGCCTCCTTTGAGGAGTGGCTGCGATCCAAGGAA

GTGGCCCTGGATTTTGGCCTGACGGAAAGGCTTCGCGAGCACGAAGCCCA

GCTGGTGATCCTGGCCCAGGCTCTGGACCATTACGACTGTCTGATCCACA

GCACACCGCACACGCTGGTCGAGCGGGGGCTGCAATCGGCCCTGAAGTAT

GAGGAGTTTTACCTAAAGCGTTTTGGCGGGCACTACATGGAGTCCGTCTT

CCAGATGTACACCCGCATCGCCGGCTTTTTGGCCTGCCGGGCCACGCGCG

GCATGCGCCACATCGCCCTGGGGCGAGAGGGGTCGTGGTGGGAAATGTTC

AAGTTCTTTTTCCACCGCCTCTACGACCACCAGATCGTACCGTCGACCCC

CGCCATGCTGAACCTGGGGACCCGCAACTACTACACCTCCAGCTGCTACC

TGGTAAACCCCCAGGCCACCACAAACAAGGCGACCCTGCGGGCCATCACC

AGCAACGTCAGTGCCATCCTCGCCCGCAACGGGGGCATCGGGCTATGCGT

GCAGGCGTTTAACGACTCCGGCCCCGGGACCGCCAGCGTCATGCCCGCCC

TCAAGGTCCTTGACTCGCTGGTGGCGGCGCACAACAAAGAGAGCGCGCGT

CCGACCGGCGCGTGCGTGTACCTGGAGCCGTGGCACACCGACGTGCGGGC

CGTGCTCCGGATGAAGGGGGTCCTCGCCGGCGAAGAGGCCCAGCGCTGCG

ACAATATCTTCAGCGCCCTCTGGATGCCAGACCTGTTTTTCAAGCGCCTG

ATTCGCCACCTGGACGGCGAGAAGAACGTCACATGGACCCTGTTCGACCG

GGACACCAGCATGTCGCTCGCCGACTTTCACGGGGAGGAGTTCGAGAAGC

TCTACCAGCACCTCGAGGTCATGGGGTTCGGCGAGCAGATACCCATCCAG

GAGCTGGCCTATGGCATTGTGCGCAGTGCGGCCACGACCGGGAGCCCCTT

CGTCATGTTCAAAGACGCGGTGAACCGCCACTACATCTACGACACCCAGG

GGGCGGCCATCGCCGGCTCCAACCTCTGCACCGAGATCGTCCATCCGGCC

TCCAAGCGATCCAGTGGGGTCTGCAACCTGGGAAGCGTGAATCTGGCCCG

ATGCGTCTCCAGGCAGACGTTTGACTTTGGGCGGCTCCGCGACGCCGTGC

AGGCGTGCGTGCTGATGGTGAACATCATGATCGACAGCACGCTACAACCC

ACGCCCCAGTGCACCCGCGGCAACGACAACCTGCGGTCCATGGGAATCGG

CATGCAGGGCCTGCACACGGCCTGCCTGAAGCTGGGGCTGGATCTGGAGT

CTGTCGAATTTCAGGACCTGAACAAACACATCGCCGAGGTGATGCTGCTG

TCGGCGATGAAGACCAGCAACGCGCTGTGCGTTCGCGGGGCCCGTCCCTT

CAACCACTTTAAGCGCAGCATGTATCGCGCCGGCCGCTTTCACTGGGAGC

GCTTTCCGGACGCCCGGCCGCGGTACGAGGGCGAGTGGGAGATGCTACGC

CAGAGCATGATGAAACACGGCCTGCGCAACAGCCAGTTTGTCGCGCTGAT

GCCCACCGCCGCCTCGGCGCAGATCTCGGACGTCAGCGAGGGCTTTGCCC

CCCTGTTCACCAACCTGTTCAGCAAGGTGACCCGGGACGGCGAGACGCTG

CGCCCCAACACGCTCCTGCTAAAGGAACTGGAACGCACGTTTAGCGGGAA

GCGCCTCCTGGAGGTGATGGACAGTCTCGACGCCAAGCAGTGGTCCGTGG

CGCAGGCGCTCCCGTGCCTGGAGCCCACCCACCCCCTCCGGCGATTCAAG

ACCGCGTTTGACTACGACCAGAAGTTGCTGATCGACCTGTGTGCGGACCG

CGCCCCCTACGTCGACCATAGCCAATCCATGACCCTGTATGTCACGGAGA

AGGCGGACGGGACCCTCCCAGCCTCCACCCTGGTCCGCCTTCTGGTCCAC

GCATATAAGCGCGGACTAAAAACAGGGATGTACTACTGCAAGGTTCGCAA

GGCGACCAACAGCGGGGTCTTTGGCGGCGACGACAACATTGTCTGCACGA

GCTGCGCGCTGTGA

SEQ ID number 44: amino acid sequence of ICP6 in the strains Mut3 and Mut-3. 34.5.

MASRPAASSPVEARAPVGGQEAGGPSAATQGEAAGAPLAHGHHVYCQRVN

GVMVLSDKTPGSASYRISDSNFVQCGSNCTMIIDGDVVRGRPQDPGAAAS

PAPFVAVTNIGAGSDGGTAVVAFGGTPRRSAGTSTGTQTADVPTEALGGP

PPPPRFTLGGGCCSCRDTRRRSAVFGGEGDPVGPAEFVSDDRSSDSDSDD

SEDTDSETLSHASSDVSGGATYDDALDSDSSSDDSLQIDGPVCRPWSNDT

APLDVCPGTPGPGADAGGPSAVDPHAPTPEAGAGLAADPAVARDDAEGLS

DPRPRLGTGTAYPVPLELTPENAEAVARFLGDAVNREPALMLEYFCRCAR

EETKRVPPRTFGSPPRLTEDDFGLLNYALVEMQRLCLDVPPVPPNAYMPY

YLREYVTRLVNGFKPLVSRSARLYRILGVLVHLRIRTREASFEEWLRSKE

VALDFGLTERLREHEAQLVILAQALDHYDCLIHSTPHTLVERGLQSALKY

EEFYLKRFGGHYMESVFQMYTRIAGFLACRATRGMRHIALGREGSWWEMF

KFFFHRLYDHQIVPSTPAMLNLGTRNYYTSSCYLVNPQATTNKATLRAIT

SNVSAILARNGGIGLCVQAFNDSGPGTASVMPALKVLDSLVAAHNKESAR

PTGACVYLEPWHTDVRAVLRMKGVLAGEEAQRCDNIFSALWMPDLFFKRL

IRHLDGEKNVTWTLFDRDTSMSLADFHGEEFEKLYQHLEVMGFGEQIPIQ

ELAYGIVRSAATTGSPFVMFKDAVNRHYIYDTQGAAIAGSNLCTEIVHPA

SKRSSGVCNLGSVNLARCVSRQTFDFGRLRDAVQACVLMVNIMIDSTLQP

TPQCTRGNDNLRSMGIGMQGLHTACLKLGLDLESVEFQDLNKHIAEVMLL

SAMKTSNALCVRGARPFNHFKRSMYRAGRFHWERFPDARPRYEGEWEMLR

QSMMKHGLRNSQFVALMPTAASAQISDVSEGFAPLFTNLFSKVTRDGETL

RPNTLLLKELERTFSGKRLLEVMDSLDAKQWSVAQALPCLEPTHPLRRFK

TAFDYDQKLLIDLCADRAPYVDHSQSMTLYVTEKADGTLPASTLVRLLVH

AYKRGLKTGMYYCKVRKATNSGVFGGDDNIVCTSCAL

SEQ ID number 45: DNA sequence of ICP6 in 17TerMA strain.

ATGGCCAGCCGCCCAGCCGCATCCTCTCCCGTCGAAGCGCGGGCCCCGGT

TGGGGGACAGGAGGCCGGCGGCCCCAGCGCAGCCACCCAGGGGGAGGCCG

CCGGGGCCCCTCTCGCCCACGGCCACCACGTGTACTGCCAGCGAGTCAAT

GGCGTGATGGTGCTTTCCGACAAGACGCCCGGGTCCGCGTCCTACCGCAT

CAGCGATAGCAACTTTGTCCAATGTGGTTCCAACTGCACCATGATCATCG

ACGGAGACGTGGTGCGCGGGCGCCCCCAGGACCCGGGGGCCGCGGCATCC

CCCGCTCCCTTCGTTGCGGTGACAAACATCGGAGCCGGCAGCGACGGCGG

GACCGCCGTCGTGGCATTCGGGGGAACCCCACGTCGCTCGGCGGGGACGT

CTACCGGTACCCAGACGGCCGACGTCCCCACCGAGGCCCTTGGGGGCCCC

CCTCCTCCTCCCCGCTTCACCCTGGGTGGCGGCTGTTGTTCCTGTCGCGA

CACACGGCGCCGCTCTGCGGTATTCGGGGGGGAGGGGGATCCAGTCGGCC

CCGCGGAGTTCGTCTCGGACGACCGGTCGTCCGATTCCGACTCGGATGAC

TCGGAGGACACGGACTCGGAGACGCTGTCACACGCCTCCTCGGACGTGTC

CGGCGGGGCCACGTACGACGACGCCCTTGACTCCGATTCGTCATCGGATG

ACTCCCTGCAGATAGATGGCCCCGTGTGTCGCCCGTGGAGCAATGACACC

GCGCCCCTGGATGTTTGCCCCGGGACCCCCGGCCCGGGCGCCGACGCCGG

TGGTCCCTCAGCGGTAGACCCACACGCGCCGACGCCAGAGGCCGGCGCTG

GTCTTGCGGCCGATCCCGCCGTGGCCCGGGACGACGCGGAGGGGCTTTCG

GACCCCCGGCCACGTCTGGGAACGGGCACGGCCTACCCCGTCCCCCTGGA

ACTCACGCCCGAGAACGCGGAGGCCGTGGCGCGCTTTCTGGGAGATGCCG

TGAACCGCGAACCCGCGCTCATGCTGGAGTACTTTTGCCGGTGCGCCCGC

GAGGAAACCAAGCGTGTCCCCCCCAGGACATTCGGCAGCCCCCCTCGCCT

CACGGAGGACGACTTTGGGCTTCTCAACTACGCGCTCGTGGAGATGCAGC

GCCTGTGTCTGGACGTTCCTCCGGTCCCGCCGAACGCATACATGCCCTAT

TATCTCAGGGAGTATGTGACGCGGCTGGTCAACGGGTTCAAGCCGCTGGT

GAGCCGGTCCGCTCGCCTTTACCGCATCCTGGGGGTTCTGGTGCACCTGC

GGATCCGGACCCGGGAGGCCTCCTTTGAGGAGTGGCTGCGATCCAAGGAA

GTGGCCCTGGATTTTGGCCTGACGGAAAGGCTTCGCGAGCACGAAGCCCA

GCTGGTGATCCTGGCCCAGGCTCTGGACCATTACGACTGTCTGATCCACA

GCACACCGCACACGCTGGTCGAGCGGGGGCTGCAATCGGCCCTGAAGTAT

GAGGAGTTTTACCTAAAGCGTTTTGGCGGGCACTACATGGAGTCCGTCTT

CCAGATGTACACCCGCATCGCCGGCTTTTTGGCCTGCCGGGCCACGCGCG

GCATGCGCCACATCGCCCTGGGGCGAGAGGGGTCGTGGTGGGAAATGTTC

AAGTTCTTTTTCCACCGCCTCTACGACCACCAGATCGTACCGTCGACCCC

CGCCATGCTGAACCTGGGGACCCGCAACTACTACACCTCCAGCTGCTACC

TGGTAAACCCCCAGGCCACCACAAACAAGGCGACCCTGCGGGCCATCACC

AGCAACGTCAGTGCCATCCTCGCCCGCAACGGGGGCATCGGGCTATGCGT

GCAGGCGTTTAACGACTCCGGCCCCGGGACCGCCAGCGTCATGCCCGCCC

TCAAGGTCCTTGACTCGCTGGTGGCGGCGCACAACAAAGAGAGCGCGCGT

CCGACCGGCGCGTGCGTGTACCTGGAGCCGTGGCACACCGACGTGCGGGC

CGTGCTCCGGATGAAGGGGGTCCTCGCCGGCGAAGAGGCCCAGCGCTGCG

ACAATATCTTCAGCGCCCTCTGGATGCCAGACCTGTTTTTCAAGCGCCTG

ATTCGCCACCTGGACGGCGAGAAGAACGTCACATGGACCCTGTTCGACCG

GGACACCAGCATGTCGCTCGCCGACTTTCACGGGGAGGAGTTCGAGAAGC

TCTACCAGCACCTCGAGGTCATGGGGTTCGGCGAGCAGATACCCATCCAG

GAGCTGGCCTATGGCATTGTGCGCAGTGCGGCCACGACCGGGAGCCCCTT

CGTCATGTTCAAAGACGCGGTGAACCGCCACTACATCTACGACACCCAGG

GGGCGGCCATCGCCGGCTCCAACCTCTGCACCGAGATCGTCCATCCGGCC

TCCAAGCGATCCAGTGGGGTCTGCAACCTGGGAAGCGTGAATCTGGCCCG

ATGCGTCTCCAGGCAGACGTTTGACTTTGGGCGGCTCCGCGACGCCGTGC

AGGCGTGCGTGCTGATGGTGAACATCATGATCGACAGCACGCTACAACCC

ACGCCCCAGTGCACCCGCGGCAACGACAACCTGCGGTCCATGGGAATCGG

CATGCAGGGCCTGCACACGGCCTGCCTGAAGCTGGGGCTGGATCTGGAGT

CTGCCGAATTTCAGGACCTGAACAAACACATCGCCGAGGTGATGCTGCTG

TCGGCGATGAAGACCAGCAACGCGCTGTGCGTTCGCGGGGCCCGTCCCTT

CAACCACTTTAAGCGCAGCATGTATCGCGCCGGCCGCTTTCACTGGGAGC

GCTTTCCGGACGCCCGGCCGCGGTACGAGGGCGAGTGGGAGATGCTACGC

CAGAGCATGATGAAACACGGCCTGCGCAACAGCCAGTTTGTCGCGCTGAT

GCCCACCGCCGCCTCGGCGCAGATCTCGGACGTCAGCGAGGGCTTTGCCC

CCCTGTTCACCAACCTGTTCAGCAAGGTGACCCGGGACGGCGAGACGCTG

CGCCCCAACACGCTCCTGCTAAAGGAACTGGAACGCACGTTTAGCGGGAA

GCGCCTCCTGGAGGTGATGGACAGTCTCGACGCCAAGCAGTGGTCCGTGG

CGCAGGCGCTCCCGTGCCTGGAGCCCACCCACCCCCTCCGGCGATTCAAG

ACCGCGTTTGACTACGACCAGAAGTTGCTGATCGACCTGTGTGCGGACCG

CGCCCCCTACGTCGACCATAGCCAATCCATGACCCTGTATGTCACGGAGA

AGGCGGACGGGACCCTCCCAGCCTCCACCCTGGTCCGCCTTCTGGTCCAC

GCATATAAGCGCGGACTAAAAACAGGGATGTACTACTGCAAGGTTCGCAA

GGCGACCAACAGCGGGGTCTTTGGCGGCGACGACAACATTGTCTGCATGA

GCTGCGCGCTGTGA

SEQ ID number 46: 17Terma strain ICP 6.

MASRPAASSPVEARAPVGGQEAGGPSAATQGEAAGAPLAHGHHVYCQRVN

GVMVLSDKTPGSASYRISDSNFVQCGSNCTMIIDGDVVRGRPQDPGAAAS

PAPFVAVTNIGAGSDGGTAVVAFGGTPRRSAGTSTGTQTADVPTEALGGP

PPPPRFTLGGGCCSCRDTRRRSAVFGGEGDPVGPAEFVSDDRSSDSDSDD

SEDTDSETLSHASSDVSGGATYDDALDSDSSSDDSLQIDGPVCRPWSNDT

APLDVCPGTPGPGADAGGPSAVDPHAPTPEAGAGLAADPAVARDDAEGLS

DPRPRLGTGTAYPVPLELTPENAEAVARFLGDAVNREPALMLEYFCRCAR

EETKRVPPRTFGSPPRLTEDDFGLLNYALVEMQRLCLDVPPVPPNAYMPY

YLREYVTRLVNGFKPLVSRSARLYRILGVLVHLRIRTREASFEEWLRSKE

VALDFGLTERLREHEAQLVILAQALDHYDCLIHSTPHTLVERGLQSALKY

EEFYLKRFGGHYMESVFQMYTRIAGFLACRATRGMRHIALGREGSWWEMF

KFFFHRLYDHQIVPSTPAMLNLGTRNYYTSSCYLVNPQATTNKATLRAIT

SNVSAILARNGGIGLCVQAFNDSGPGTASVMPALKVLDSLVAAHNKESAR

PTGACVYLEPWHTDVRAVLRMKGVLAGEEAQRCDNIFSALWMPDLFFKRL

IRHLDGEKNVTWTLFDRDTSMSLADFHGEEFEKLYQHLEVMGFGEQIPIQ

ELAYGIVRSAATTGSPFVMFKDAVNRHYIYDTQGAAIAGSNLCTEIVHPA

SKRSSGVCNLGSVNLARCVSRQTFDFGRLRDAVQACVLMVNIMIDSTLQP

TPQCTRGNDNLRSMGIGMQGLHTACLKLGLDLESAEFQDLNKHIAEVMLL

SAMKTSNALCVRGARPFNHFKRSMYRAGRFHWERFPDARPRYEGEWEMLR

QSMMKHGLRNSQFVALMPTAASAQISDVSEGFAPLFTNLFSKVTRDGETL

RPNTLLLKELERTFSGKRLLEVMDSLDAKQWSVAQALPCLEPTHPLRRFK

TAFDYDQKLLIDLCADRAPYVDHSQSMTLYVTEKADGTLPASTLVRLLVH

AYKRGLKTGMYYCKVRKATNSGVFGGDDNIVCMSCAL

SEQ ID number 47: rRp450 strain, rat cytochrome P4502B 1.

GAACCCCTTCGCCATGGAGCCCAGTATCTTGCTCCTCCTTGCTCTCCTTG

TGGGCTTCTTGTTACTCTTAGTCAGGGGACACCCAAAGTCCCGTGGCAAC

TTCCCACCAGGACCTCGTCCCCTTCCCCTCTTGGGGAACCTCCTGCAGTT

GGACAGAGGGGGCCTCCTCAATTCCTTCATGCAGCTTCGAGAAAAATATG

GAGATGTGTTCACAGTACACCTGGGACCAAGGCCTGTGGTCATGCTATGT

GGGACAGACACCATAAAGGAGGCTCTGGTGGGCCAAGCTGAGGATTTCTC

TGGTCGGGGAACAATCGCTGTGATTGAGCCAATCTTCAAGGAATATGGTG

TGATCTTTGCCAATGGGGAACGCTGGAAGGCCCTTCGGCGATTCTCTCTG

GCTACCATGAGAGACTTTGGGATGGGAAAGAGGAGTGTGGAAGAACGGAT

TCAGGAGGAAGCCCAATGTTTGGTGGAGGAACTGCGGAAATCCCAGGGAG

CCCCACTGGATCCCACCTTCCTCTTCCAGTGCATCACAGCCAACATCATC

TGCTCCATTGTGTTTGGAGAGCGCTTTGACTACACAGACCGCCAGTTCCT

GCGCCTGTTGGAGCTGTTCTACCGGACCTTTTCCCTCCTAAGTTCATTCT

CCAGCCAGGTGTTTGAGTTCTTCTCTGGGTTCCTGAAATACTTTCCTGGT

GCCCACAGACAAATCTCCAAAAACCTCCAGGAAATCCTCGATTACATTGG

CCATATTGTGGAGAAGCACAGGGCCACCTTAGACCCAAGCGCTCCACGAG

ACTTCATCGACACTTACCTTCTGCGCATGGAGAAGGAGAAGTCGAACCAC

CACACAGAGTTCCATCATGAGAACCTCATGATCTCCCTGCTCTCTCTCTT

CTTTGCTGGCACTGAGACCAGCAGCACCACACTCCGCTATGGTTTCCTGC

TGATGCTCAAGTACCCCCATGTCGCAGAGAAAGTCCAAAAGGAGATTGAT

CAGGTGATCGGCTCACACCGGCTACCAACCCTTGATGACCGCAGTAAAAT

GCCATACACTGATGCAGTTATCCATGAGATTCAGAGGTTTTCAGATCTTG

TCCCTATTGGAGTACCACACAGAGTCACCAAAGACACCATGTTCCGAGGG

TACCTGCTTCCCAAGAACACTGAAGTGTACCCCATCCTGAGTTCAGCTCT

CCATGACCCACAGTACTTTGACCACCCAGACAGCTTCAATCCTGAACACT

TCCTGGATGCCAATGGGGCACTGAAAAAGAGTGAAGCTTTCATGCCCTTC

TCCACAGGAAAGCGCATTTGTCTTGGCGAAGGCATTGCCCGAAATGAATT

GTTCCTCTTCTTCACCACCATCCTCCAGAACTTCTCTGTGTCAAGCCATT

TGGCTCCCAAGGACATTGACCTCACGCCCAAGGAGAGTGGCATTGGAAAA

ATACCTCCAACGTACCAGATCTGCTTCTCAGCTCGGTGATCCGGCTGAGG

CAGCCATGTGCCCCAGTTCTGTTGGGAATGGAACTTGTTTATTGCAGCTT

ATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCA

TTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATC

TTATCATGTCTGGATCCCCGGGCGAGCTCGAATTCCTCCTTTGAGGAGTG

GCTGCGATCCAAGGAAGTGGCCCTGGACTTTGGCCTGACGGAAAGGCTTC

GCGAGCACGAAGCCCAGCTGGTGATCCTGGCCCAGGCTCTGGACCATTAC

GACTGTCTGATCCACAGCACACCGCACACGCTGGTCGAGCGGGGGCTGCA

ATCGGCCCTGAAGTATGAGGAGTTTTACCTAAAGCGCTTTGGCGGGCACT

ACATGGAGTCCGTCTTCCAGATGTACACCCGCATCGCCGGCTTTTTGGCC

TGCCGGGCCACGCGCGGCATGCGCCACATCGCCCTGGGGCGAGAGGGGTC

GTGGTGGGAAATGTTCAAGTTCTTTTTCCACCGCCTCTACGACCACCAGA

TCGTACCGTCGACCCCCGCCATGCTGAACCTGGGGACCCGCAACTACTAC

ACCTCCAGCTGCTACCTGGTAAACCCCCAGGCCACCACAAACAAGGCGAC

CCTGCGGGCCATCACCAGCAACGTCAGCGCCATCCTCGCCCGCAACGGGG

GCATCGGGCTATGCGTGCAGGCGTTTAACGACTCCGGCCCCGGGACCGCT

AGCGTCATACCCGCCCTCAAGGTCCTCGACTCGCTGGTGGCGGCGCACAA

CAAAGAGAGCGCGCGTCCAACCGGCGCGTGCGTGTACCTGGAGCCGTGGC

ACACCGACGTGCGGGCCGTGCTCCGGATGAAGGGGGTCCTCGCCGGCGAA

GAGGCCCAGCGCTGCGACAATATCTTCAGCGCCCTCTGGATGCCAGACCT

GTTTTTCAAGCGCCTGATTCGCCACCTGGACGGCGAGAAGAACGTCACAT

GGACCCTGTTCGACCGGGACACCAGCATGTCGCTCGCCGACTTTCACGGG

GAGGAGTTCGAGAAGCTCTACCAGCACCTCGAGGTCATGGGGTTCGGCGA

GCAGATACCCATCCAGGAGCTGGCCTATGGCATTGTGCGCAGTGCGGCCA

CGACCGGGAGCCCCTTCGTCATGTTCAAAGACGCGGTGAACCGCCACTAC

ATCTACGACACCCAGGGGGCGGCCATCGCCGGCTCCAACCTCTGCACCGA

GATCGTCCATCCGGCCTCCAAGCGATCCAGTGGGGTCTGCAATCTGGGAA

GCGTGAATCTGGCCCGATGCGTCTCCAGGCAGACGTTTGACTTTGGGCGG

CTCCGCGACGCCGTGCAGGCGTGCGTGCTGATGGTGAACATCATGATCGA

CAGCACGCTACAACCCACGCCCCAGTGCACCCGCGGCAACGACAACCTGC

GGTCCATGGGAATCGGCATGCAGGGCCTGCACACGGCCTGCCTGAAGCTG

GGGCTGGATCTGGAGTCTGTCGAATTTCAGGACCTGAACAAACACATCGC

CGAGGTGATGCTGCTGTCGGCGATGAAGACCAGCAACGCGCTGTGCGTTC

GCGGGGCCCGTCCCTTCAACCACTTTAAGCGCAGCATGTATCGCGCCGGC

CGCTTTCACTGGGAGCGCTTTCCGGACGCCCGGCCGCGGTACGAGGGCGA

GTGGGAGATGCTACGCCAGAGCATGATGAAACACGGCCTGCGCAACAGCC

AGTTTGTCGCGCTGATGCCCACCGCCGCCTCGGCGCAGATCTCGGACGTC

AGCGAGGGCTTTGCCCCCCTGTTCACCAACCTGTTCAGCAAGGTGACCCG

GGACGGCGAGACGCTGCGCCCCAACACGCTCCTGCTAAAGGAACTGGAAC

GCACGTTTAGCGGGAAGCGCCTCCTGGAGGTGATGGACAGTCTCGACGCC

AAGCAGTGGTCCGTGGCGCAGGCGCTCCCGTGCCTGGAGCCCACCCACCC

CCTCCGGCGATTCAAGACCGCGTTTGACTACGACCAGAAGTTGCTGATCG

ACCTGTGTGCGGACCGCGCCCCCTACGTCGACCATAGCCAATCCATGACC

CTGTATGTCACGGAGAAGGCGGACGGGACCCTCCCAGCCTCCACCCTGGT

CCGCCTTCTGGTCCACGCATATAAGCGCGGACTAAAAACAGGGATGTACT

ACTGCAAGGTTCGCAAGGCGACCAACAGCGGGGTCTTTGGCGGCGACGAC

AACATTGTCTGCACGAGCTGCGCGCTGTGA

SEQ ID number 48: rRp450 strain, rat cytochrome P4502B 1.

MEPSILLLLALLVGFLLLLVRGHPKSRGNFPPGPRPLPLLGNLLQL

DRGGLLNSFMQLREKYGDVFTVHLGPRPVVMLCGTDTIKEALVGQAEDFS

GRGTIAVIEPIFKEYGVIFANGERWKALRRFSLATMRDFGMGKRSVEERI

QEEAQCLVEELRKSQGAPLDPTFLFQCITANIICSIVFGERFDYTDRQFL

RLLELFYRTFSLLSSFSSQVFEFFSGFLKYFPGAHRQISKNLQEILDYIG

HIVEKHRATLDPSAPRDFIDTYLLRMEKEKSNHHTEFHHENLMISLLSLF

FAGTETSSTTLRYGFLLMLKYPHVAEKVQKEIDQVIGSHRLPTLDDRSKM

PYTDAVIHEIQRFSDLVPIGVPHRVTKDTMFRGYLLPKNTEVYPILSSAL

HDPQYFDHPDSFNPEHFLDANGALKKSEAFMPFSTGKRICLGEGIARNEL

FLFFTTILQNFSVSSHLAPKDIDLTPKESGIGKIPPTYQICFSAR*

Stop codon-after prediction sequence is not expressed

SEQ ID number 49: DNA sequence of ICP6 in wild type 17 strain.

ATGGCCAGCCGCCCAGCCGCATCCTCTCCCGTCGAAGCGCGGGCCCCGGT

TGGGGGACAGGAGGCCGGCGGCCCCAGCGCAGCCACCCAGGGGGAGGCCG

CCGGGGCCCCTCTCGCCCACGGCCACCACGTGTACTGCCAGCGAGTCAAT

GGCGTGATGGTGCTTTCCGACAAGACGCCCGGGTCCGCGTCCTACCGCAT

CAGCGATAGCAACTTTGTCCAATGTGGTTCCAACTGCACCATGATCATCG

ACGGAGACGTGGTGCGCGGGCGCCCCCAGGACCCGGGGGCCGCGGCATCC

CCCGCTCCCTTCGTTGCGGTGACAAACATCGGAGCCGGCAGCGACGGCGG

GACCGCCGTCGTGGCATTCGGGGGAACCCCACGTCGCTCGGCGGGGACGT

CTACCGGTACCCAGACGGCCGACGTCCCCACCGAGGCCCTTGGGGGCCCC

CCTCCTCCTCCCCGCTTCACCCTGGGTGGCGGCTGTTGTTCCTGTCGCGA

CACACGGCGCCGCTCTGCGGTATTCGGGGGGGAGGGGGATCCAGTCGGCC

CCGCGGAGTTCGTCTCGGACGACCGGTCGTCCGATTCCGACTCGGATGAC

TCGGAGGACACGGACTCGGAGACGCTGTCACACGCCTCCTCGGACGTGTC

CGGCGGGGCCACGTACGACGACGCCCTTGACTCCGATTCGTCATCGGATG

ACTCCCTGCAGATAGATGGCCCCGTGTGTCGCCCGTGGAGCAATGACACC

GCGCCCCTGGATGTTTGCCCCGGGACCCCCGGCCCGGGCGCCGACGCCGG

TGGTCCCTCAGCGGTAGACCCACACGCGCCGACGCCAGAGGCCGGCGCTG

GTCTTGCGGCCGATCCCGCCGTGGCCCGGGACGACGCGGAGGGGCTTTCG

GACCCCCGGCCACGTCTGGGAACGGGCACGGCCTACCCCGTCCCCCTGGA

ACTCACGCCCGAGAACGCGGAGGCCGTGGCGCGCTTTCTGGGAGATGCCG

TGAACCGCGAACCCGCGCTCATGCTGGAGTACTTTTGCCGGTGCGCCCGC

GAGGAAACCAAGCGTGTCCCCCCCAGGACATTCGGCAGCCCCCCTCGCCT

CACGGAGGACGACTTTGGGCTTCTCAACTACGCGCTCGTGGAGATGCAGC

GCCTGTGTCTGGACGTTCCTCCGGTCCCGCCGAACGCATACATGCCCTAT

TATCTCAGGGAGTATGTGACGCGGCTGGTCAACGGGTTCAAGCCGCTGGT

GAGCCGGTCCGCTCGCCTTTACCGCATCCTGGGGGTTCTGGTGCACCTGC

GGATCCGGACCCGGGAGGCCTCCTTTGAGGAGTGGCTGCGATCCAAGGAA

GTGGCCCTGGATTTTGGCCTGACGGAAAGGCTTCGCGAGCACGAAGCCCA

GCTGGTGATCCTGGCCCAGGCTCTGGACCATTACGACTGTCTGATCCACA

GCACACCGCACACGCTGGTCGAGCGGGGGCTGCAATCGGCCCTGAAGTAT

GAGGAGTTTTACCTAAAGCGTTTTGGCGGGCACTACATGGAGTCCGTCTT

CCAGATGTACACCCGCATCGCCGGCTTTTTGGCCTGCCGGGCCACGCGCG

GCATGCGCCACATCGCCCTGGGGCGAGAGGGGTCGTGGTGGGAAATGTTC

AAGTTCTTTTTCCACCGCCTCTACGACCACCAGATCGTACCGTCGACCCC

CGCCATGCTGAACCTGGGGACCCGCAACTACTACACCTCCAGCTGCTACC

TGGTAAACCCCCAGGCCACCACAAACAAGGCGACCCTGCGGGCCATCACC

AGCAACGTCAGTGCCATCCTCGCCCGCAACGGGGGCATCGGGCTATGCGT

GCAGGCGTTTAACGACTCCGGCCCCGGGACCGCCAGCGTCATGCCCGCCC

TCAAGGTCCTTGACTCGCTGGTGGCGGCGCACAACAAAGAGAGCGCGCGT

CCGACCGGCGCGTGCGTGTACCTGGAGCCGTGGCACACCGACGTGCGGGC

CGTGCTCCGGATGAAGGGGGTCCTCGCCGGCGAAGAGGCCCAGCGCTGCG

ACAATATCTTCAGCGCCCTCTGGATGCCAGACCTGTTTTTCAAGCGCCTG

ATTCGCCACCTGGACGGCGAGAAGAACGTCACATGGACCCTGTTCGACCG

GGACACCAGCATGTCGCTCGCCGACTTTCACGGGGAGGAGTTCGAGAAGC

TCTACCAGCACCTCGAGGTCATGGGGTTCGGCGAGCAGATACCCATCCAG

GAGCTGGCCTATGGCATTGTGCGCAGTGCGGCCACGACCGGGAGCCCCTT

CGTCATGTTCAAAGACGCGGTGAACCGCCACTACATCTACGACACCCAGG

GGGCGGCCATCGCCGGCTCCAACCTCTGCACCGAGATCGTCCATCCGGCC

TCCAAGCGATCCAGTGGGGTCTGCAACCTGGGAAGCGTGAATCTGGCCCG

ATGCGTCTCCAGGCAGACGTTTGACTTTGGGCGGCTCCGCGACGCCGTGC

AGGCGTGCGTGCTGATGGTGAACATCATGATCGACAGCACGCTACAACCC

ACGCCCCAGTGCACCCGCGGCAACGACAACCTGCGGTCCATGGGAATCGG

CATGCAGGGCCTGCACACGGCCTGCCTGAAGCTGGGGCTGGATCTGGAGT

CTGTCGAATTTCAGGACCTGAACAAACACATCGCCGAGGTGATGCTGCTG

TCGGCGATGAAGACCAGCAACGCGCTGTGCGTTCGCGGGGCCCGTCCCTT

CAACCACTTTAAGCGCAGCATGTATCGCGCCGGCCGCTTTCACTGGGAGC

GCTTTCCGGACGCCCGGCCGCGGTACGAGGGCGAGTGGGAGATGCTACGC

CAGAGCATGATGAAACACGGCCTGCGCAACAGCCAGTTTGTCGCGCTGAT

GCCCACCGCCGCCTCGGCGCAGATCTCGGACGTCAGCGAGGGCTTTGCCC

CCCTGTTCACCAACCTGTTCAGCAAGGTGACCCGGGACGGCGAGACGCTG

CGCCCCAACACGCTCCTGCTAAAGGAACTGGAACGCACGTTTAGCGGGAA

GCGCCTCCTGGAGGTGATGGACAGTCTCGACGCCAAGCAGTGGTCCGTGG

CGCAGGCGCTCCCGTGCCTGGAGCCCACCCACCCCCTCCGGCGATTCAAG

ACCGCGTTTGACTACGACCAGAAGTTGCTGATCGACCTGTGTGCGGACCG

CGCCCCCTACGTCGACCATAGCCAATCCATGACCCTGTATGTCACGGAGA

AGGCGGACGGGACCCTCCCAGCCTCCACCCTGGTCCGCCTTCTGGTCCAC

GCATATAAGCGCGGACTAAAAACAGGGATGTACTACTGCAAGGTTCGCAA

GGCGACCAACAGCGGGGTCTTTGGCGGCGACGACAACATTGTCTGCACGA

GCTGCGCGCTGTGA

SEQ ID number 50: amino acid sequence of ICP6 in wild type 17 strain.

MASRPAASSPVEARAPVGGQEAGGPSAATQGEAAGAPLAHGHHVYCQRVN

GVMVLSDKTPGSASYRISDSNFVQCGSNCTMIIDGDVVRGRPQDPGAAAS

PAPFVAVTNIGAGSDGGTAVVAFGGTPRRSAGTSTGTQTADVPTEALGGP

PPPPRFTLGGGCCSCRDTRRRSAVFGGEGDPVGPAEFVSDDRSSDSDSDD

SEDTDSETLSHASSDVSGGATYDDALDSDSSSDDSLQIDGPVCRPWSNDT

APLDVCPGTPGPGADAGGPSAVDPHAPTPEAGAGLAADPAVARDDAEGLS

DPRPRLGTGTAYPVPLELTPENAEAVARFLGDAVNREPALMLEYFCRCAR

EETKRVPPRTFGSPPRLTEDDFGLLNYALVEMQRLCLDVPPVPPNAYMPY

YLREYVTRLVNGFKPLVSRSARLYRILGVLVHLRIRTREASFEEWLRSKE

VALDFGLTERLREHEAQLVILAQALDHYDCLIHSTPHTLVERGLQSALKY

EEFYLKRFGGHYMESVFQMYTRIAGFLACRATRGMRHIALGREGSWWEMF

KFFFHRLYDHQIVPSTPAMLNLGTRNYYTSSCYLVNPQATTNKATLRAIT

SNVSAILARNGGIGLCVQAFNDSGPGTASVMPALKVLDSLVAAHNKESAR

PTGACVYLEPWHTDVRAVLRMKGVLAGEEAQRCDNIFSALWMPDLFFKRL

IRHLDGEKNVTWTLFDRDTSMSLADFHGEEFEKLYQHLEVMGFGEQIPIQ

ELAYGIVRSAATTGSPFVMFKDAVNRHYIYDTQGAAIAGSNLCTEIVHPA

SKRSSGVCNLGSVNLARCVSRQTFDFGRLRDAVQACVLMVNIMIDSTLQP

TPQCTRGNDNLRSMGIGMQGLHTACLKLGLDLESVEFQDLNKHIAEVMLL

SAMKTSNALCVRGARPFNHFKRSMYRAGRFHWERFPDARPRYEGEWEMLR

QSMMKHGLRNSQFVALMPTAASAQISDVSEGFAPLFTNLFSKVTRDGETL

RPNTLLLKELERTFSGKRLLEVMDSLDAKQWSVAQALPCLEPTHPLRRFK

TAFDYDQKLLIDLCADRAPYVDHSQSMTLYVTEKADGTLPASTLVRLLVH

AYKRGLKTGMYYCKVRKATNSGVFGGDDNIVCTSCAL。

Sequence listing

<110> national institute of Children's Hospital

<120> syncytial oncolytic herpes simplex mutants as effective cancer therapies

<130> 21F-1902-CNP

<150> 62/932,725

<151> 2019-11-08

<150> 62/818,577

<151> 2019-03-14

<160> 53

<170> PatentIn version 3.5

<210> 1

<211> 726

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthesis of polynucleotides

<220>

<221> mutation

<222> (356)..(356)

<223> c356a

<400> 1

atggcccgcc gccgccgcca tcgcggcccc cgccgccccc ggccgcccgg gcccacgggc 60

gcggtcccaa ccgcacagtc ccaggtaacc tccacgccca actcggaacc cgtggtcagg 120

agcgcgcccg cggccgcccc gccgccgccc cccgccagtg ggcccccgcc ttcttgttcg 180

ctgctgctgc gccagtggct ccacgttccc gagtccgcgt ccgacgacga cgacgacgac 240

tggccggaca gccccccgcc cgagccggcg ccagaggccc ggcccaccgc cgccgccccc 300

cgcccccggt ccccaccgcc cggcgcgggc ccggggggcg gggctaaccc ctcccacccc 360

ccctcacgcc ccttccgcct tccgccgcgc ctcgccctcc gcctgcgcgt caccgcagag 420

cacctggcgc gcctgcgcct gcgacgcgcg ggcggggagg gggcgccgaa gccccccgcg 480

acccccgcga cccccgcgac ccccacgcgg gtgcgcttct cgccccacgt ccgggtgcgc 540

cacctggtgg tctgggcctc ggccgcccgc ctggcgcgcc gcggctcgtg ggcccgcgag 600

cgggccgacc gggctcggtt ccggcgccgg gtggcggagg ccgaggcggt catcgggccg 660

tgcctggggc ccgaggcccg tgcccgggcc ctggcccgcg gagccggccc ggcgaactcg 720

gtctaa 726

<210> 2

<211> 241

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic polypeptides

<220>

<221> MISC_FEATURE

<222> (119)..(119)

<223> P119H mutation

<400> 2

Met Ala Arg Arg Arg Arg His Arg Gly Pro Arg Arg Pro Arg Pro Pro

1 5 10 15

Gly Pro Thr Gly Ala Val Pro Thr Ala Gln Ser Gln Val Thr Ser Thr

20 25 30

Pro Asn Ser Glu Pro Val Val Arg Ser Ala Pro Ala Ala Ala Pro Pro

35 40 45

Pro Pro Pro Ala Ser Gly Pro Pro Pro Ser Cys Ser Leu Leu Leu Arg

50 55 60

Gln Trp Leu His Val Pro Glu Ser Ala Ser Asp Asp Asp Asp Asp Asp

65 70 75 80

Trp Pro Asp Ser Pro Pro Pro Glu Pro Ala Pro Glu Ala Arg Pro Thr

85 90 95

Ala Ala Ala Pro Arg Pro Arg Ser Pro Pro Pro Gly Ala Gly Pro Gly

100 105 110

Gly Gly Ala Asn Pro Ser His Pro Pro Ser Arg Pro Phe Arg Leu Pro

115 120 125

Pro Arg Leu Ala Leu Arg Leu Arg Val Thr Ala Glu His Leu Ala Arg

130 135 140

Leu Arg Leu Arg Arg Ala Gly Gly Glu Gly Ala Pro Lys Pro Pro Ala

145 150 155 160

Thr Pro Ala Thr Pro Ala Thr Pro Thr Arg Val Arg Phe Ser Pro His

165 170 175

Val Arg Val Arg His Leu Val Val Trp Ala Ser Ala Ala Arg Leu Ala

180 185 190

Arg Arg Gly Ser Trp Ala Arg Glu Arg Ala Asp Arg Ala Arg Phe Arg

195 200 205

Arg Arg Val Ala Glu Ala Glu Ala Val Ile Gly Pro Cys Leu Gly Pro

210 215 220

Glu Ala Arg Ala Arg Ala Leu Ala Arg Gly Ala Gly Pro Ala Asn Ser

225 230 235 240

Val

<210> 3

<211> 846

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthesis of polynucleotides

<400> 3

atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60

ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120

ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180

ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420

aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480

ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540

gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600

tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagaag 720

cttagccatg gcttcccgcc ggaggtggag gagcaggatg atggcacgct gcccatgtct 780

tgtgcccagg agagcgggat ggaccgtcac cctgcagcct gtgcttctgc taggatcaat 840

gtgtag 846

<210> 4

<211> 281

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic polypeptides

<400> 4

Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu

1 5 10 15

Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly

20 25 30

Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile

35 40 45

Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr

50 55 60

Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys

65 70 75 80

Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu

85 90 95

Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu

100 105 110

Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly

115 120 125

Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr

130 135 140

Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn

145 150 155 160

Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser

165 170 175

Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly

180 185 190

Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu

195 200 205

Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe

210 215 220

Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Lys

225 230 235 240

Leu Ser His Gly Phe Pro Pro Glu Val Glu Glu Gln Asp Asp Gly Thr

245 250 255

Leu Pro Met Ser Cys Ala Gln Glu Ser Gly Met Asp Arg His Pro Ala

260 265 270

Ala Cys Ala Ser Ala Arg Ile Asn Val

275 280

<210> 5

<211> 767

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthesis of polynucleotides

<400> 5

atggcccgcc gccgccgcca tcgcggcccc cgccgccccc ggccgcccgg gcccacgggc 60

gccgtcccaa ccgcacagtc ccaggtaacc tagactagtc tagcgtaacc tccacgccca 120

actcggaacc cgcggtcagg agcgcgcccg cggccgcccc gccgccgccc cccgccggtg 180

ggcccccgcc ttcttgttcg ctgctgctgc gccagtggct ccacgttccc gagtccgcgt 240

ccgacaacga cgatgacgac gactggccgg acagcccccc gcccgagccg gcgccagagg 300

cccggcccac cgccgccgcc ccccggcccc ggcccccacc gcccggcgtg ggcccggggg 360

gcggggctga cccctcccac cccccctcgc gccccttccg ccttccgccg cgcctcgccc 420

tccgcctgcg cgtcaccgcg gagcacctgg cgcgcctgcg cctgcgacgc gcgggcgggg 480

agggggcgcc ggagcccccc gcgacccccg cgacccccgc gacccccgcg acccccgcga 540

cccccgcgcg ggtgcgcttc tcgccccacg tccgggtgcg ccacctggtg gtctgggcct 600

cggccgcccg cctggcgcgc cgcggctcgt gggcccgcga gcgggccgac cgggctcggt 660

tccggcgccg ggtggcggag gccgaggcgg tcatcgggcc gtgcctgggg cccgaggccc 720

gtgcccgggc cctggcccgc ggagccggcc cggcgaactc ggtctaa 767

<210> 6

<211> 30

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic polypeptides

<400> 6

Met Ala Arg Arg Arg Arg His Arg Gly Pro Arg Arg Pro Arg Pro Pro

1 5 10 15

Gly Pro Thr Gly Ala Val Pro Thr Ala Gln Ser Gln Val Thr

20 25 30

<210> 7

<211> 723

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthesis of polynucleotides

<400> 7

atggcccgcc gccgccatcg cggcccccgc cgcccccggc cgcccgggcc cacgggcgcg 60

gtcccaaccg cacagtccca ggtaacctcc acgcccaact cggaacccgt ggtcaggagc 120

gcgcccgcgg ccgccccgcc gccgcccccc gccagtgggc ccccgccttc ttgttcgctg 180

ctgctgcgcc agtggctcca cgttcccgag tccgcgtccg acgacgacga cgacgactgg 240

ccggacagcc ccccgcccga gccggcgcca gaggcccggc ccaccgccgc cgccccccgc 300

ccccggtccc caccgcccgg cgcgggcccg gggggcgggg ctaacccctc cccccccccc 360

tcacgcccct tccgccttcc gccgcgcctc gccctccgcc tgcgcgtcac cgcagagcac 420

ctggcgcgcc tgcgcctgcg acgcgcgggc ggggaggggg cgccgaagcc ccccgcgacc 480

cccgcgaccc ccgcgacccc cacgcgggtg cgcttctcgc cccacgtccg ggtgcgccac 540

ctggtggtct gggcctcggc cgcccgcctg gcgcgccgcg gctcgtgggc ccgcgagcgg 600

gccgaccggg ctcggttccg gcgccgggtg gcggaggccg aggcggtcat cgggccgtgc 660

ctggggcccg aggcccgtgc ccgggccctg gcccgcggag ccggcccggc gaactcggtc 720

taa 723

<210> 8

<211> 240

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic polypeptides

<400> 8

Met Ala Arg Arg Arg His Arg Gly Pro Arg Arg Pro Arg Pro Pro Gly

1 5 10 15

Pro Thr Gly Ala Val Pro Thr Ala Gln Ser Gln Val Thr Ser Thr Pro

20 25 30

Asn Ser Glu Pro Val Val Arg Ser Ala Pro Ala Ala Ala Pro Pro Pro

35 40 45

Pro Pro Ala Ser Gly Pro Pro Pro Ser Cys Ser Leu Leu Leu Arg Gln

50 55 60

Trp Leu His Val Pro Glu Ser Ala Ser Asp Asp Asp Asp Asp Asp Trp

65 70 75 80

Pro Asp Ser Pro Pro Pro Glu Pro Ala Pro Glu Ala Arg Pro Thr Ala

85 90 95

Ala Ala Pro Arg Pro Arg Ser Pro Pro Pro Gly Ala Gly Pro Gly Gly

100 105 110

Gly Ala Asn Pro Ser Pro Pro Pro Ser Arg Pro Phe Arg Leu Pro Pro

115 120 125

Arg Leu Ala Leu Arg Leu Arg Val Thr Ala Glu His Leu Ala Arg Leu

130 135 140

Arg Leu Arg Arg Ala Gly Gly Glu Gly Ala Pro Lys Pro Pro Ala Thr

145 150 155 160

Pro Ala Thr Pro Ala Thr Pro Thr Arg Val Arg Phe Ser Pro His Val

165 170 175

Arg Val Arg His Leu Val Val Trp Ala Ser Ala Ala Arg Leu Ala Arg

180 185 190

Arg Gly Ser Trp Ala Arg Glu Arg Ala Asp Arg Ala Arg Phe Arg Arg

195 200 205

Arg Val Ala Glu Ala Glu Ala Val Ile Gly Pro Cys Leu Gly Pro Glu

210 215 220

Ala Arg Ala Arg Ala Leu Ala Arg Gly Ala Gly Pro Ala Asn Ser Val

225 230 235 240

<210> 9

<211> 726

<212> DNA

<213> herpes simplex virus type 1

<400> 9