Oncolytic viruses and their uses

Abstract

This application relates to the technical field of biomedicine, and specifically discloses oncolytic viruses and their applications. [Solution] The oncolytic virus comprises an M protein, and the M protein comprises amino acid substitutions at one or more of the 32nd, 33rd, 49th, 54th, 133rd, and 225th positions compared to the amino acid sequence shown in SEQ ID NO: 1. The present application also relates to an expression vector for the oncolytic virus, a virus-producing cell that produces the oncolytic virus, a pharmaceutical composition comprising the oncolytic virus, and a preparation method and use of the oncolytic virus, the oncolytic virus expression vector, the virus-producing cell, and / or the pharmaceutical composition. The present application can effectively improve the safety and cure rate of the oncolytic virus.

Description

[Technical Field]

[0001] The present application relates to the field of biomedicine, and more particularly to oncolytic viruses and their uses. [Background technology]

[0002] Oncolytic viruses are replication-competent tumor-killing viruses that have now become widely accepted as an important field in tumor immunotherapy. Oncolytic viruses specifically target infected tumor cells, for example, by inactivating or deleting tumor suppressor genes in tumor cells, thereby selectively infecting tumor cells. After infecting tumor cells, oncolytic viruses replicate extensively within the tumor cells, ultimately destroying and thus killing the tumor cells. At the same time, oncolytic viruses can provide the immune-stimulating signals necessary to strengthen the host's own anti-cancer response, attracting more immune cells to continue killing the remaining tumor cells.

[0003] Oncolytic viruses have great potential for application in the field of tumor immunotherapy, but wild-type oncolytic viruses can always cause problems such as inflammation of the nervous system, and there is still a significant pathogenic risk in the process of using wild-type viruses to infect tumor cells. Therefore, in order to further promote the clinical application of oncolytic viruses, it is necessary to modify wild-type oncolytic viruses to obtain attenuated oncolytic viruses. The use of attenuated oncolytic viruses in clinical applications can thereby reduce the pathogenic risk of oncolytic viruses and improve the safety of oncolytic viruses.

[0004] However, in the process of modifying oncolytic viruses, although wild-type oncolytic viruses can be randomly modified to reduce their toxicity, the modified oncolytic viruses may have a low cure rate, and the modified oncolytic viruses cannot be packaged, which is disadvantageous for promoting the clinical application of oncolytic viruses. Therefore, it is necessary to provide a modified oncolytic virus with good safety performance and a high cure rate, which has important scientific research value and application significance in the field of tumor immunotherapy. Summary of the Invention

[0005] In order to improve the safety and cure rate of oncolytic viruses, the present application provides oncolytic viruses and their uses.

[0006] In a first aspect, the present application provides an oncolytic virus, which employs the following technical solutions:

[0007] An oncolytic virus comprising an M protein, wherein the M protein comprises an amino acid substitution at one or more of positions 32, 33, 49, 54, 133, and 225 compared to the amino acid sequence shown in SEQ ID NO: 1.

[0008] In some specific embodiments, the amino acid substitution in the M protein comprises an asparagine to serine mutation at position 32 (N32S), and / or a methionine to alanine mutation at position 33 (M33A), and / or an asparagine to aspartic acid mutation at position 49 (N49D), and / or a histidine to tyrosine mutation at position 54 (H54Y), and / or an alanine to threonine mutation at position 133 (A133T), and / or a valine to isoleucine mutation at position 225 (V225I).

[0009] Furthermore, the M protein further comprises amino acid substitutions at one or more of positions 21, 51, 111, 221, and 226.

[0010] In some specific embodiments, the amino acid substitution in the M protein further comprises a glycine to glutamic acid mutation at position 21 (G21E), and / or the amino acid substitution in the M protein comprises a methionine to arginine mutation at position 51 (M51R), and / or a methionine to alanine mutation at position 51 (M51A), and / or a leucine to alanine mutation at position 111 (L111A), and / or a valine to phenylalanine mutation at position 221 (V221F), and / or a serine to arginine mutation at position 226 (S226R).

[0011] In one specific embodiment, the M protein amino acid substitution comprises a glycine to glutamic acid mutation at position 21 (G21E).

[0012] In one specific embodiment, the M protein amino acid substitution comprises an asparagine to serine mutation at position 32 (N32S).

[0013] In one specific embodiment, the M protein amino acid substitution comprises a methionine to alanine mutation at position 33 (M33A).

[0014] In one specific embodiment, the M protein amino acid substitution comprises an asparagine to aspartic acid mutation at position 49 (N49D).

[0015] In one specific embodiment, the amino acid substitution in the M protein comprises a histidine to tyrosine mutation at position 54 (H54Y).

[0016] In one specific embodiment, the amino acid substitution in the M protein comprises a leucine to alanine mutation at position 111 (L111A).

[0017] In one specific embodiment, the M protein amino acid substitution comprises an alanine to threonine mutation at position 133 (A133T).

[0018] In one specific embodiment, the M protein amino acid substitution comprises a valine to isoleucine mutation at position 225 (V225I).

[0019] In one specific embodiment, the M protein amino acid substitution comprises a methionine to arginine mutation at position 51 (M51R).

[0020] In one specific embodiment, the M protein amino acid substitution comprises a methionine to alanine mutation at position 51 (M51A).

[0021] In one specific embodiment, the amino acid substitution in the M protein comprises a valine to phenylalanine mutation at position 221 (V221F).

[0022] In one specific embodiment, the amino acid substitution in the M protein comprises a serine to arginine mutation at position 226 (S226R).

[0023] In one specific embodiment, the amino acid substitution in the M protein comprises a serine to glycine mutation at position 226 (S226G).

[0024] In one specific embodiment, the M protein has a G21E amino acid substitution.

[0025] In one specific embodiment, the M protein has the following amino acid substitutions: G21E, N32S.

[0026] In one specific embodiment, the M protein has the following amino acid substitutions: G21E, N32S, M33A.

[0027] In one specific embodiment, the M protein has the following amino acid substitutions: G21E, N32S, M33A, N49D.

[0028] In one specific embodiment, the M protein has the following amino acid substitutions: G21E, N32S, M33A, N49D, H54Y.

[0029] In one specific embodiment, the M protein has the following amino acid substitutions: G21E, N32S, M33A, N49D, H54Y, L111A.

[0030] In one specific embodiment, the M protein has the following amino acid substitutions: G21E, N32S, M33A, N49D, H54Y, L111A, A133T.

[0031] In one specific embodiment, the M protein has the following amino acid substitutions: G21E, N32S, M33A, N49D, H54Y, L111A, A133T, V225I.

[0032] In one specific embodiment, the M protein has the following amino acid substitutions: G21E, N32S, M33A, N49D, M51R, H54Y, L111A, A133T, V225I.

[0033] In a specific embodiment, the M protein has the following amino acid substitutions: G21E, N32S, M33A, N49D, M51R, H54Y, L111A, A133T, V221F, V225I.

[0034] In a specific embodiment, the M protein has the following amino acid substitutions: G21E, N32S, M33A, N49D, M51R, H54Y, L111A, A133T, V221F, V225I, S226R.

[0035] In one specific embodiment, the M protein has the following amino acid substitutions: N32S, M33A, N49D, M51R, H54Y, L111A, A133T, V221F, V225I, S226R.

[0036] In one specific embodiment, the M protein has the following amino acid substitutions: M33A, N49D, M51R, H54Y, L111A, A133T, V221F, V225I, S226R.

[0037] In one specific embodiment, the M protein has the following amino acid substitutions: N49D, M51R, H54Y, L111A, A133T, V221F, V225I, S226R.

[0038] In one specific embodiment, the M protein has the following amino acid substitutions: M51R, H54Y, L111A, A133T, V221F, V225I, S226R.

[0039] In one specific embodiment, the M protein has the following amino acid substitutions: H54Y, L111A, A133T, V221F, V225I, S226R.

[0040] In one specific embodiment, the M protein has the following amino acid substitutions: L111A, A133T, V221F, V225I, S226R.

[0041] In one specific embodiment, the M protein has the following amino acid substitutions: A133T, V221F, V225I, S226R.

[0042] In one specific embodiment, the M protein has the following amino acid substitutions: V221F, V225I, S226R.

[0043] In one specific embodiment, the M protein has the following amino acid substitutions: V225I, S226R.

[0044] In one specific embodiment, the M protein has an amino acid substitution of S226R.

[0045] In one specific embodiment, the M protein has the following amino acid substitutions: N32S, N49D, H54Y, V225I.

[0046] In one specific embodiment, the M protein has the following amino acid substitutions: N32S, N49D, H54Y, V225I, S226G.

[0047] In one specific embodiment, the M protein has the following amino acid substitutions: N32S, N49D, M51R, H54Y, V221F, V225I, S226R.

[0048] In one specific embodiment, the M protein has the following amino acid substitutions: N32S, M33A, N49D, M51R, H54Y, V221F, V225I, S226R.

[0049] In one specific embodiment, the M protein has the following amino acid substitutions: N32S, N49D, M51R, H54Y, A133T, V221F, V225I, S226R.

[0050] In one specific embodiment, the M protein has the following amino acid substitutions: N32S, M33A, N49D, M51R, H54Y, A133T, V221F, V225I, S226R.

[0051] In one specific embodiment, the M protein has the following amino acid substitutions: G21E, N32S, N49D, M51A, H54Y, L111A, V225I, S226R.

[0052] In one specific embodiment, the M Protein comprises the amino acid sequence set forth in SEQ ID NO:2.

[0053] In one specific embodiment, the M Protein comprises the amino acid sequence set forth in SEQ ID NO:3.

[0054] In one specific embodiment, the M Protein comprises the amino acid sequence set forth in SEQ ID NO:4.

[0055] In one specific embodiment, the M Protein comprises the amino acid sequence set forth in SEQ ID NO:5.

[0056] In one specific embodiment, the M Protein comprises the amino acid sequence set forth in SEQ ID NO:6.

[0057] In one specific embodiment, the M Protein comprises the amino acid sequence set forth in SEQ ID NO:7.

[0058] In one specific embodiment, the M Protein comprises the amino acid sequence set forth in SEQ ID NO:8.

[0059] In one specific embodiment, the M Protein comprises the amino acid sequence set forth in SEQ ID NO:9.

[0060] In one specific embodiment, the M Protein comprises the amino acid sequence set forth in SEQ ID NO:10.

[0061] In one specific embodiment, the M Protein comprises the amino acid sequence set forth in SEQ ID NO:11.

[0062] In one specific embodiment, the M Protein comprises the amino acid sequence set forth in SEQ ID NO:12.

[0063] In one specific embodiment, the M Protein comprises the amino acid sequence set forth in SEQ ID NO:13.

[0064] In one specific embodiment, the M Protein comprises the amino acid sequence set forth in SEQ ID NO:14.

[0065] In one specific embodiment, the M Protein comprises the amino acid sequence set forth in SEQ ID NO:15.

[0066] In one specific embodiment, the M Protein comprises the amino acid sequence set forth in SEQ ID NO:16.

[0067] In one specific embodiment, the M Protein comprises the amino acid sequence set forth in SEQ ID NO:17.

[0068] In one specific embodiment, the M Protein comprises the amino acid sequence set forth in SEQ ID NO:18.

[0069] In one specific embodiment, the M Protein comprises the amino acid sequence set forth in SEQ ID NO:19.

[0070] In one specific embodiment, the M Protein comprises the amino acid sequence set forth in SEQ ID NO:20.

[0071] In one specific embodiment, the M Protein comprises the amino acid sequence set forth in SEQ ID NO:21.

[0072] In one specific embodiment, the M Protein comprises the amino acid sequence set forth in SEQ ID NO:22.

[0073] In one specific embodiment, the M Protein comprises the amino acid sequence set forth in SEQ ID NO:23.

[0074] In one specific embodiment, the M Protein comprises the amino acid sequence set forth in SEQ ID NO:24.

[0075] In one specific embodiment, the M Protein comprises the amino acid sequence set forth in SEQ ID NO:25.

[0076] In one specific embodiment, the M Protein comprises the amino acid sequence set forth in SEQ ID NO:26.

[0077] In one specific embodiment, the M Protein comprises the amino acid sequence set forth in SEQ ID NO:27.

[0078] In one specific embodiment, the M Protein comprises the amino acid sequence set forth in SEQ ID NO:28.

[0079] In one specific embodiment, the M Protein comprises the amino acid sequence set forth in SEQ ID NO:29.

[0080] In one specific embodiment, the M Protein comprises the amino acid sequence set forth in SEQ ID NO:30.

[0081] An oncolytic virus comprising the above-mentioned M protein, wherein the oncolytic virus further comprises a G protein, wherein the G protein comprises amino acid substitutions at one or more of positions 438, 453, 471 and 487 compared to the amino acid sequence shown in SEQ ID NO: 31.

[0082] In some specific embodiments, the G protein further comprises an amino acid substitution at one or more of positions 53, 141, 172, 217, 232, 331, 371, or 436.

[0083] In some specific embodiments, the amino acid substitution in the G protein is a valine to isoleucine mutation at position 53 (V53I), and / or an alanine to valine mutation at position 141 (A141V), and / or an aspartic acid to tyrosine mutation at position 172 (D172Y), and / or a lysine to glutamic acid mutation at position 217 (K217E), and / or an aspartic acid to glycine mutation at position 232 (D232G), and / or a valine to alanine mutation at position 331. (V331A), and / or a valine to glutamic acid mutation at position 371 (V371E), and / or a glycine to aspartic acid mutation at position 436 (G436D), and / or a threonine to serine mutation at position 438 (T438S), and / or a phenylalanine to leucine mutation at position 453 (F453L), and / or a threonine to isoleucine mutation at position 471 (T471I), and / or a tyrosine to histidine mutation at position 487 (Y487H).

[0084] In one specific embodiment, the amino acid substitution in the G protein comprises a valine to isoleucine mutation at position 53 (V53I).

[0085] In one specific embodiment, the amino acid substitution in the G protein comprises an alanine to valine mutation at position 141 (A141V).

[0086] In one specific embodiment, the amino acid substitution in the G protein comprises an aspartic acid to tyrosine mutation at position 172 (D172Y).

[0087] In one specific embodiment, the amino acid substitution in the G protein comprises a lysine to glutamic acid mutation at position 217 (K217E).

[0088] In one specific embodiment, the amino acid substitution in the G protein comprises an aspartic acid to glycine mutation at position 232 (D232G).

[0089] In one specific embodiment, the amino acid substitution in the G protein comprises a valine to alanine mutation at position 331 (V331A).

[0090] In one specific embodiment, the amino acid substitution in the G protein comprises a valine to glutamic acid mutation at position 371 (V371E).

[0091] In one specific embodiment, the amino acid substitution in the G protein comprises a glycine to aspartic acid mutation at position 436 (G436D).

[0092] In one specific embodiment, the amino acid substitution in the G protein comprises a threonine to serine mutation at position 438 (T438S).

[0093] In one specific embodiment, the amino acid substitution in the G protein comprises a phenylalanine to leucine mutation at position 453 (F453L).

[0094] In one specific embodiment, the amino acid substitution in the G protein comprises a threonine to isoleucine mutation at position 471 (T471I).

[0095] In one specific embodiment, the amino acid substitution in the G protein comprises a tyrosine to histidine mutation at position 487 (Y487H).

[0096] In one specific embodiment, the G protein has an amino acid substitution of V53I.

[0097] In one specific embodiment, the G protein has the following amino acid substitutions: V53I, A141V.

[0098] In one specific embodiment, the G protein has the following amino acid substitutions: V53I, A141V, D172Y.

[0099] In one specific embodiment, the G protein has the following amino acid substitutions: V53I, A141V, D172Y, K217E.

[0100] In one specific embodiment, the G protein has the following amino acid substitutions: V53I, A141V, D172Y, K217E, D232G.

[0101] In one specific embodiment, the G protein has the following amino acid substitutions: V53I, A141V, D172Y, K217E, D232G, V331A.

[0102] In one specific embodiment, the G protein has the following amino acid substitutions: V53I, A141V, D172Y, K217E, D232G, V331A, V371E.

[0103] In a specific embodiment, the G protein has the following amino acid substitutions: V53I, A141V, D172Y, K217E, D232G, V331A, V371E, G436D.

[0104] In a specific embodiment, the G protein has the following amino acid substitutions: V53I, A141V, D172Y, K217E, D232G, V331A, V371E, G436D, T438S.

[0105] In a specific embodiment, the G protein has the following amino acid substitutions: V53I, A141V, D172Y, K217E, D232G, V331A, V371E, G436D, T438S, F453L.

[0106] In a specific embodiment, the G protein has the following amino acid substitutions: V53I, A141V, D172Y, K217E, D232G, V331A, V371E, G436D, T438S, F453L, T471I.

[0107] In a specific embodiment, the G protein has the following amino acid substitutions: V53I, A141V, D172Y, K217E, D232G, V331A, V371E, G436D, T438S, F453L, T471I, Y487H.

[0108] In a specific embodiment, the G protein has the following amino acid substitutions: A141V, D172Y, K217E, D232G, V331A, V371E, G436D, T438S, F453L, T471I, Y487H.

[0109] In a specific embodiment, the G protein has the following amino acid substitutions: D172Y, K217E, D232G, V331A, V371E, G436D, T438S, F453L, T471I, Y487H.

[0110] In a specific embodiment, the G protein has the following amino acid substitutions: K217E, D232G, V331A, V371E, G436D, T438S, F453L, T471I, Y487H.

[0111] In a specific embodiment, the G protein has the following amino acid substitutions: D232G, V331A, V371E, G436D, T438S, F453L, T471I, Y487H.

[0112] In a specific embodiment, the G protein has the following amino acid substitutions: V331A, V371E, G436D, T438S, F453L, T471I, Y487H.

[0113] In one specific embodiment, the G protein has the following amino acid substitutions: V371E, G436D, T438S, F453L, T471I, Y487H.

[0114] In one specific embodiment, the G protein has the following amino acid substitutions: G436D, T438S, F453L, T471I, Y487H.

[0115] In one specific embodiment, the G protein has the following amino acid substitutions: T438S, F453L, T471I, Y487H.

[0116] In one specific embodiment, the G protein has the following amino acid substitutions: F453L, T471I, Y487H.

[0117] In one specific embodiment, the G protein has the following amino acid substitutions: T471I, Y487H.

[0118] In one specific embodiment, the G protein has an amino acid substitution of Y487H.

[0119] In one specific embodiment, the G protein comprises the amino acid sequence set forth in SEQ ID NO:32.

[0120] In one specific embodiment, the G protein comprises the amino acid sequence set forth in SEQ ID NO:33.

[0121] In one specific embodiment, the G protein comprises the amino acid sequence set forth in SEQ ID NO:34.

[0122] In one specific embodiment, the G protein comprises the amino acid sequence set forth in SEQ ID NO:35.

[0123] In one specific embodiment, the G protein comprises the amino acid sequence set forth in SEQ ID NO:36.

[0124] In one specific embodiment, the G protein comprises the amino acid sequence set forth in SEQ ID NO:37.

[0125] In one specific embodiment, the G protein comprises the amino acid sequence set forth in SEQ ID NO:38.

[0126] In one specific embodiment, the G protein comprises the amino acid sequence set forth in SEQ ID NO:39.

[0127] In one specific embodiment, the G protein comprises the amino acid sequence set forth in SEQ ID NO:40.

[0128] In one specific embodiment, the G protein comprises the amino acid sequence set forth in SEQ ID NO:41.

[0129] In one specific embodiment, the G protein comprises the amino acid sequence set forth in SEQ ID NO:42.

[0130] In one specific embodiment, the G protein comprises the amino acid sequence set forth in SEQ ID NO:43.

[0131] In one specific embodiment, the G protein comprises the amino acid sequence set forth in SEQ ID NO:44.

[0132] In one specific embodiment, the G protein comprises the amino acid sequence set forth in SEQ ID NO:45.

[0133] In one specific embodiment, the G protein comprises the amino acid sequence set forth in SEQ ID NO:46.

[0134] In one specific embodiment, the G protein comprises the amino acid sequence set forth in SEQ ID NO:47.

[0135] In one specific embodiment, the G protein comprises the amino acid sequence set forth in SEQ ID NO:48.

[0136] In one specific embodiment, the G protein comprises the amino acid sequence set forth in SEQ ID NO:49.

[0137] In one specific embodiment, the G protein comprises the amino acid sequence set forth in SEQ ID NO:50.

[0138] In one specific embodiment, the G protein comprises the amino acid sequence set forth in SEQ ID NO:51.

[0139] In one specific embodiment, the G protein comprises the amino acid sequence set forth in SEQ ID NO:52.

[0140] In one specific embodiment, the G protein comprises the amino acid sequence set forth in SEQ ID NO:53.

[0141] In one specific embodiment, the G protein comprises the amino acid sequence set forth in SEQ ID NO:54.

[0142] An oncolytic virus comprising the above-mentioned M protein, or the M protein and the G protein, and further comprising an N protein, wherein the N protein comprises amino acid substitutions at one or more of positions 14, 155, and 353 compared to the amino acid sequence shown in SEQ ID NO: 55.

[0143] In some specific embodiments, the amino acid substitution in the N protein comprises an isoleucine to valine mutation at position 14 (I14V), and / or an arginine to lysine mutation at position 155 (R155K), and / or a serine to asparagine mutation at position 353 (S353N).

[0144] In one specific embodiment, the amino acid substitution in the N protein comprises an isoleucine to valine mutation at position 14 (I14V).

[0145] In one specific embodiment, the amino acid substitution in the N protein comprises an arginine to lysine mutation at position 155 (R155K).

[0146] In one specific embodiment, the amino acid substitution in the N protein comprises a serine to asparagine mutation at position 353 (S353N).

[0147] In one specific embodiment, the N protein has the amino acid substitution I14V.

[0148] In one specific embodiment, the N protein has the following amino acid substitutions: I14V, R155K.

[0149] In one specific embodiment, the N protein has the following amino acid substitutions: I14V, R155K, S353N.

[0150] In one specific embodiment, the N protein has the following amino acid substitutions: R155K, S353N.

[0151] In one specific embodiment, the N protein has an amino acid substitution of S353N.

[0152] In one specific embodiment, the N protein comprises the amino acid sequence set forth in SEQ ID NO:56.

[0153] In one specific embodiment, the N protein comprises the amino acid sequence set forth in SEQ ID NO:57.

[0154] In one specific embodiment, the N protein comprises the amino acid sequence set forth in SEQ ID NO:58.

[0155] In one specific embodiment, the N protein comprises the amino acid sequence set forth in SEQ ID NO:59.

[0156] In one specific embodiment, the N protein comprises the amino acid sequence set forth in SEQ ID NO:60.

[0157] An oncolytic virus comprising the above-mentioned M protein, or the M protein and the G protein, or the M protein, the G protein and the N protein, or the M protein and the N protein, and further comprising a P protein, wherein the P protein comprises amino acid substitutions at one or more of positions 50, 76, 99, 126, 140, 151, 168, 170, 189, and 237 compared to the amino acid sequence shown in SEQ ID NO: 61.

[0158] In some specific embodiments, the amino acid substitution in the P protein is an arginine to lysine mutation at position 50 (R50K), and / or a valine to alanine mutation at position 76 (V76A), and / or an asparagine to glutamic acid mutation at position 99 (D99E), and / or a leucine to serine mutation at position 126 (L126S), and / or a leucine to serine mutation at position 140 (L126S). 140S), and / or a histidine to tyrosine mutation at position 151 (H151Y), and / or an isoleucine to methionine mutation at position 168 (I168M), and / or a lysine to glutamic acid mutation at position 170 (K170E), and / or a tyrosine to serine mutation at position 189 (Y189S), and / or an asparagine to aspartic acid mutation at position 237 (N237D).

[0159] In one specific embodiment, the amino acid substitution in the P protein comprises an arginine to lysine mutation at position 50 (R50K).

[0160] In one specific embodiment, the amino acid substitution in the P protein comprises a valine to alanine mutation at position 76 (V76A).

[0161] In one specific embodiment, the amino acid substitution in the P protein comprises an asparagine to glutamic acid mutation at position 99 (D99E).

[0162] In one specific embodiment, the amino acid substitution in the P protein comprises a leucine to serine mutation at position 126 (L126S).

[0163] In one specific embodiment, the amino acid substitution in the P protein comprises a leucine to serine mutation at position 140 (L140S).

[0164] In one specific embodiment, the amino acid substitution in the P protein comprises a histidine to tyrosine mutation at position 151 (H151Y).

[0165] In one specific embodiment, the amino acid substitution in the P protein comprises an isoleucine to methionine mutation at position 168 (I168M).

[0166] In one specific embodiment, the amino acid substitution in the P protein comprises a lysine to glutamic acid mutation at position 170 (K170E).

[0167] In one specific embodiment, the amino acid substitution in the P protein comprises a tyrosine to serine mutation at position 189 (Y189S).

[0168] In one specific embodiment, the amino acid substitution in the P protein comprises an asparagine to aspartic acid mutation at position 237 (N237D).

[0169] In one specific embodiment, the P protein has an amino acid substitution of R50K.

[0170] In one specific embodiment, the P protein has the following amino acid substitutions: R50K, V76A.

[0171] In one specific embodiment, the P protein has the following amino acid substitutions: R50K, V76A, D99E.

[0172] In a specific embodiment, the P protein has the following amino acid substitutions: R50K, V76A, D99E, L126S.

[0173] In a specific embodiment, the P protein has the following amino acid substitutions: R50K, V76A, D99E, L126S, L140S.

[0174] In one specific embodiment, the P protein has the following amino acid substitutions: R50K, V76A, D99E, L126S, L140S, H151Y.

[0175] In a specific embodiment, the P protein has the following amino acid substitutions: R50K, V76A, D99E, L126S, L140S, H151Y, I168M.

[0176] In a specific embodiment, the P protein has the following amino acid substitutions: R50K, V76A, D99E, L126S, L140S, H151Y, I168M, K170E.

[0177] In a specific embodiment, the P protein has the following amino acid substitutions: R50K, V76A, D99E, L126S, L140S, H151Y, I168M, K170E, Y189S.

[0178] In a specific embodiment, the P protein has the following amino acid substitutions: R50K, V76A, D99E, L126S, L140S, H151Y, I168M, K170E, Y189S, N237D.

[0179] In a specific embodiment, the P protein has the following amino acid substitutions: V76A, D99E, L126S, L140S, H151Y, I168M, K170E, Y189S, N237D.

[0180] In a specific embodiment, the P protein has the following amino acid substitutions: D99E, L126S, L140S, H151Y, I168M, K170E, Y189S, N237D.

[0181] In a specific embodiment, the P protein has the following amino acid substitutions: L126S, L140S, H151Y, I168M, K170E, Y189S, N237D.

[0182] In one specific embodiment, the P protein has the following amino acid substitutions: L140S, H151Y, I168M, K170E, Y189S, N237D.

[0183] In one specific embodiment, the P protein has the following amino acid substitutions: H151Y, I168M, K170E, Y189S, N237D.

[0184] In one specific embodiment, the P protein has the following amino acid substitutions: I168M, K170E, Y189S, N237D.

[0185] In one specific embodiment, the P protein has the following amino acid substitutions: K170E, Y189S, N237D.

[0186] In one specific embodiment, the P protein has the following amino acid substitutions: Y189S, N237D.

[0187] In one specific embodiment, the P protein has the amino acid substitution N237D.

[0188] In one specific embodiment, the P protein comprises the amino acid sequence set forth in SEQ ID NO:62.

[0189] In one specific embodiment, the P protein comprises the amino acid sequence set forth in SEQ ID NO:63.

[0190] In one specific embodiment, the P protein comprises the amino acid sequence set forth in SEQ ID NO:64.

[0191] In one specific embodiment, the P protein comprises the amino acid sequence set forth in SEQ ID NO:65.

[0192] In one specific embodiment, the P protein comprises the amino acid sequence set forth in SEQ ID NO:66.

[0193] In one specific embodiment, the P protein comprises the amino acid sequence set forth in SEQ ID NO:67.

[0194] In one specific embodiment, the P protein comprises the amino acid sequence set forth in SEQ ID NO:68.

[0195] In one specific embodiment, the P protein comprises the amino acid sequence set forth in SEQ ID NO:69.

[0196] In one specific embodiment, the P protein comprises the amino acid sequence set forth in SEQ ID NO:70.

[0197] In one specific embodiment, the P protein comprises the amino acid sequence set forth in SEQ ID NO:71.

[0198] In one specific embodiment, the P protein comprises the amino acid sequence set forth in SEQ ID NO:72.

[0199] In one specific embodiment, the P protein comprises the amino acid sequence set forth in SEQ ID NO:73.

[0200] In one specific embodiment, the P protein comprises the amino acid sequence set forth in SEQ ID NO:74.

[0201] In one specific embodiment, the P protein comprises the amino acid sequence set forth in SEQ ID NO:75.

[0202] In one specific embodiment, the P protein comprises the amino acid sequence set forth in SEQ ID NO:76.

[0203] In one specific embodiment, the P protein comprises the amino acid sequence set forth in SEQ ID NO:77.

[0204] In one specific embodiment, the P protein comprises the amino acid sequence set forth in SEQ ID NO:78.

[0205] In one specific embodiment, the P protein comprises the amino acid sequence set forth in SEQ ID NO:79.

[0206] In one specific embodiment, the P protein comprises the amino acid sequence set forth in SEQ ID NO:80.

[0207] An oncolytic virus comprising the above-mentioned M protein, or M protein and G protein, or M protein, G protein and N protein, or M protein, G protein, N protein and P protein, or M protein and N protein, or M protein and P protein, or M protein, G protein and P protein, or M protein, N protein and P protein, wherein the oncolytic virus further comprises an L protein, wherein the L protein comprises amino acid substitutions at one or more of positions 87 and 487 compared to the amino acid sequence shown in SEQ ID NO: 81.

[0208] In some specific embodiments, the amino acid substitution in the L protein comprises a serine to proline mutation at position 87 (S87P) and / or an isoleucine to threonine mutation at position 487 (I487T).

[0209] In one specific embodiment, the amino acid substitution in the L protein comprises a serine to proline mutation at position 87 (S87P).

[0210] In one specific embodiment, the amino acid substitution in the L protein comprises an isoleucine to threonine mutation at position 487 (I487T).

[0211] In one specific embodiment, the L protein has an amino acid substitution of S87P.

[0212] In one specific embodiment, the L protein has the following amino acid substitutions: S87P, I487T.

[0213] In one specific embodiment, the L protein has an amino acid substitution of I487T.

[0214] In one specific embodiment, the L protein comprises the amino acid sequence set forth in SEQ ID NO:82.

[0215] In one specific embodiment, the L protein comprises the amino acid sequence set forth in SEQ ID NO:83.

[0216] In one specific embodiment, the L protein comprises the amino acid sequence set forth in SEQ ID NO:84.

[0217] In some specific embodiments, the oncolytic virus is Rhabdovirus It was obtained by site-directed mutagenesis based on the above.

[0218] In some specific embodiments, the oncolytic virus is obtained by site-directed mutagenesis based on the Vesicular Stomatitis Virus (abbreviated as "VSV").

[0219] In some specific embodiments, the oncolytic virus is derived from the Indiana MuddSummer subtype of VSV virus by site-directed mutagenesis.

[0220] In some specific embodiments, the oncolytic virus contains or expresses an exogenous target protein.

[0221] In some specific embodiments, the oncolytic virus comprises a nucleic acid molecule, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding an M protein having the amino acid substitution, and / or a nucleic acid sequence encoding a G protein having the amino acid substitution, and / or a nucleic acid sequence encoding an N protein having the amino acid substitution, and / or a nucleic acid sequence encoding a P protein having the amino acid substitution, and / or a nucleic acid sequence encoding an L protein having the amino acid substitution.

[0222] In some specific embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding the exogenous target protein.

[0223] In some specific embodiments, the nucleic acid sequence of the nucleic acid molecule encoding the exogenous target protein is located between the nucleic acid sequence encoding the M protein having the amino acid substitution, and / or the nucleic acid sequence encoding the G protein having the amino acid substitution, and / or the nucleic acid sequence encoding the N protein having the amino acid substitution, and / or the nucleic acid sequence encoding the P protein having the amino acid substitution, and / or the nucleic acid sequence encoding the L protein having the amino acid substitution.

[0224] In a second aspect, the present application provides an oncolytic virus expression vector, which employs the following technical solution:

[0225] An oncolytic virus expression vector, wherein said oncolytic virus expression vector is capable of producing any one of the oncolytic viruses described in the present application.

[0226] In a third aspect, the present application provides a virus-producing cell, which employs the following technical solution:

[0227] A virus-producing cell, said virus-producing cell being capable of producing any one of the oncolytic viruses described in the present application.

[0228] In a fourth aspect, the present application provides a pharmaceutical composition, which employs the following technical solutions:

[0229] A pharmaceutical composition comprising any one of the oncolytic viruses described herein and optionally a pharmaceutically acceptable carrier.

[0230] In a fifth aspect, the present application provides methods for preparing the oncolytic viruses, oncolytic virus expression vectors, virus-producing cells and / or pharmaceutical compositions described above.

[0231] In a sixth aspect, the present application provides the use of the above-described oncolytic virus, oncolytic virus expression vector, virus-producing cell and / or pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of a disease and / or condition.

[0232] In some specific embodiments, the oncolytic viruses, oncolytic virus expression vectors, virus-producing cells and / or pharmaceutical compositions are used in methods for the slow and continuous killing of abnormally proliferating cells.

[0233] In some specific embodiments, the disease and / or condition comprises abnormally proliferating cells from tumor cells or tumor tissue-associated cells, preferably the tumor cells are cancer cells, more preferably the cancer cells are metastatic cancer cells.

[0234] In some specific embodiments, the tumor comprises a solid tumor and / or a blood tumor.

[0235] In summary, the present application has the following beneficial effects:

[0236] The oncolytic viruses provided herein all have good infectivity and in vitro killing ability in abnormally proliferating (tumor) LLC cells, 4T1 cells, MC38 cells, and HeLa cells, and are difficult to eliminate in LLC cells, 4T1 cells, MC38 cells, and HeLa cells. Furthermore, the oncolytic viruses provided herein all have low infectivity and in vitro killing ability in normal MEF cells, and are easily eliminated in normal MEF cells. Therefore, the oncolytic viruses provided herein are commonly used to infect and kill tumor, cancer, and other cells, and are easily eliminated in tumor, cancer, and other cells, further improving the cure rate of oncolytic viruses in tumor, cancer, and other cells. At the same time, the oncolytic viruses provided above do not damage normal cells and are easily eliminated in normal cells, further ensuring the safety of normal cells. [Brief explanation of the drawings]

[0237] [Figure 1] FIG. 1 shows the results of detecting the ability of the oncolytic viruses prepared in the present application and wild-type oncolytic viruses to infect LLC cells. [Figure 2] FIG. 2 shows the results of detecting the infectivity of the oncolytic viruses prepared in the present application and wild-type oncolytic viruses to 4T1 cells. [Figure 3] FIG. 3 shows the results of detecting the ability of the oncolytic viruses prepared in the present application and wild-type oncolytic viruses to infect MC38 cells. [Figure 4] FIG. 4 shows the results of detecting the infectivity of the oncolytic viruses prepared in the present application and wild-type oncolytic viruses to Hela cells. [Figure 5] FIG. 5 shows the results of detecting the ability of the oncolytic viruses prepared in the present application and wild-type oncolytic viruses to infect MEF cells. [Figure 6] FIG. 6 shows the results of detecting the in vitro killing ability of the oncolytic virus prepared in the present application and the wild-type oncolytic virus on LLC cells. [Figure 7]FIG. 7 shows the results of detecting the in vitro killing ability of the oncolytic virus prepared in the present application and the wild-type oncolytic virus on 4T1 cells. [Figure 8] FIG. 8 shows the results of detecting the in vitro killing ability of the oncolytic virus prepared in the present application and the wild-type oncolytic virus on MC38 cells. [Figure 9] FIG. 9 shows the results of detecting the in vitro killing ability of the oncolytic virus prepared in the present application and the wild-type oncolytic virus on Hela cells. [Figure 10] FIG. 10 shows the results of detecting the in vitro killing ability of the oncolytic viruses prepared in the present application and wild-type oncolytic viruses on MEF cells. [Figure 11] FIG. 11 shows the state in which the oncolytic virus prepared in the present application and the wild-type oncolytic virus induce the expression of IFN-β in LLC cells. [Figure 12] FIG. 12 shows the state in which the oncolytic virus prepared in the present application and the wild-type oncolytic virus induce the expression of IFN-β in 4T1 cells. [Figure 13] FIG. 13 shows the state in which the oncolytic virus prepared in the present application and the wild-type oncolytic virus induce the expression of IFN-β in MC38 cells. [Figure 14] FIG. 14 shows the state in which the oncolytic virus prepared in the present application and the wild-type oncolytic virus induce the expression of IFN-β in Hela cells. [Figure 15] FIG. 15 shows the state in which the oncolytic virus prepared in the present application and the wild-type oncolytic virus induce the expression of IFN-β in MEF cells.

[0238] In the above-mentioned figure, No. 0 on the horizontal axis represents the wild-type oncolytic virus, and Nos. 1 to 79 on the horizontal axis represent the oncolytic viruses prepared in Preparation Examples 1 to 79, respectively.

[0239] Vertical axis Log 10 TCID50 represents the TCID50 value calculated by the Karber method, and Log10 The higher the TCID50 value, the better the oncolytic virus's ability to infect the cells. Log 10 A smaller TCID50 value indicates a lower ability of the oncolytic virus to infect the cells.

[0240] Vertical axis OD 570 represents the OD value of the cells, and OD 570 The larger the OD value, the lower the killing ability of the oncolytic virus to the cell. 570 The smaller the value, the better the killing ability of the oncolytic virus to the cell.

[0241] The IFN-β level on the vertical axis represents the expression status of the IFN-β gene; a higher IFN-β level indicates a weaker ability of the oncolytic virus to reproduce in the cell and a greater likelihood of removal; a lower IFN-β level indicates a stronger ability of the oncolytic virus to reproduce in the cell and a greater likelihood of removal.

[0242] Those skilled in the art can readily understand other aspects and advantages of the present application from the following detailed description. In the following detailed description, only exemplary embodiments of the present application are described and explained. As those skilled in the art will understand, depending on the content of the present application, those skilled in the art may modify the specific embodiments disclosed without departing from the spirit and scope of the invention of the present application. Correspondingly, the drawings and description of the present application are illustrative only and not restrictive. DETAILED DESCRIPTION OF THE INVENTION

[0243] Hereinafter, the embodiments of the present invention will be described with specific examples, but those skilled in the art will easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Term definition

[0244] As used herein, the term "oncolytic virus" generally refers to a virus capable of replicating in and killing tumor cells. Oncolytic viruses include, but are not limited to, vesicular stomatitis virus (VSV), poxvirus, herpes simplex virus, measles virus, Semliki Forest virus, poliovirus, reovirus, Seneca Valley virus, echotype enterovirus, coxsackievirus, Newcastle disease virus, and Malabar virus. In some embodiments, the oncolytic virus is modified to enhance selectivity for tumor cells. In some embodiments, the oncolytic virus is modified to reduce immunogenicity. In some embodiments, the oncolytic virus of the present application is a VSV virus. In some embodiments, the VSV virus is a mutant of the Indiana MuddSummer subtype strain of VSV virus. In some embodiments, site-specific gene mutations may be performed on the M protein, and / or G protein, and / or N protein, and / or P protein, and / or L protein of the VSV virus.

[0245] In some embodiments, the oncolytic viruses described herein are genetically modified oncolytic viruses, e.g., modified in one or more genes to improve their tumor selectivity and / or preferentially replicate in dividing cells. The genetic modifications may be modifications of genes involved in DNA replication, nucleic acid metabolism, host tropism, surface attachment, virulence, lysis, and spread, or modifications incorporating exogenous genes. The exogenous genes may include exogenous immunomodulatory genes, exogenous screening genes, exogenous reporter genes, etc. The modified oncolytic viruses are modified at the amino acid level, e.g., by inserting, deleting, or substituting one or more amino acids.

[0246] In the present application, the term "M protein" generally refers to the VSV virus matrix protein. The M protein is an important virulence factor of the VSV virus and is also a protein known to interfere with the innate immune response of mice to the VSV virus. The term "M protein" further includes its homologs, orthologs, variations, functionally active fragments, etc. In the present application, the wild-type VSV virus Indiana MuddSummer subtype M protein may comprise the amino acid sequence set forth in SEQ ID NO: 1. In the present application, the M protein of the oncolytic virus may comprise the amino acid sequence set forth in SEQ ID NOs: 2 to 29.

[0247] In the present application, the term "G protein" generally refers to the glycoprotein of the VSV virus, also referred to as the envelope protein. The term "G protein" further includes its homologs, orthologs, variations, functionally active fragments, etc. In the present application, the wild-type VSV virus Indiana MuddSummer subtype G protein may comprise the amino acid sequence set forth in SEQ ID NO: 31. In the present application, the G protein of the oncolytic virus may comprise the amino acid sequence set forth in SEQ ID NOs: 32 to 54.

[0248] In the present application, the term "N protein" generally refers to the nucleocapsid protein of the VSV virus. The term "N protein" further includes its homologs, orthologs, variations, functionally active fragments, etc. In the present application, the wild-type VSV virus Indiana MuddSummer subtype N protein may comprise the amino acid sequence set forth in SEQ ID NO: 55. In the present application, the N protein of the oncolytic virus may comprise the amino acid sequence set forth in SEQ ID NOs: 56 to 60.

[0249] In the present application, the term "P protein" generally refers to the phosphoprotein of the VSV virus. The term "P protein" further includes its homologs, orthologs, variations, functionally active fragments, etc. In the present application, the wild-type VSV virus Indiana MuddSummer subtype P protein may comprise the amino acid sequence set forth in SEQ ID NO: 61. In the present application, the P protein of the oncolytic virus may comprise the amino acid sequence set forth in SEQ ID NOs: 62 to 80. In the present application, the term "L protein" generally refers to the VSV virus RNA polymerase protein. The L gene of the VSV virus encodes the RNA poly E protein. The term "L protein" further includes its homologs, orthologs, variations, functionally active fragments, etc. In the present application, the wild-type VSV virus Indiana MuddSummer subtype L protein may comprise the amino acid sequence set forth in SEQ ID NO: 81. In the present application, the L protein of the oncolytic virus may comprise the amino acid sequence set forth in SEQ ID NOs: 82 to 84.

[0250] In the present application, the mutation site of a protein is usually expressed as "amino acid + amino acid site number + (mutated amino acid)". In the present application, the mutation includes, but is not limited to, addition, substitution, deletion, and / or removal of amino acids. For example, the term "M51R" usually means a mutation from methionine M to arginine R at position 51.

[0251] In the present application, the term "amino acid substitution" generally refers to the replacement of one amino acid residue present in a parent sequence with another amino acid residue. The amino acid in the parent sequence may be replaced by, for example, chemical peptide synthesis or recombinant methods known to those skilled in the art. Thus, "substituting at position xx" generally refers to the replacement of the amino acid present at position xx with an alternative amino acid residue. In the present application, the amino acid substitution may also include amino acid mutation.

[0252] In this application, the term "mutation" generally refers to a change in the nucleotide or amino acid sequence of a wild-type molecule. Amino acid changes may include amino acid substitutions, deletions, deletions, insertions, additions, truncations, or processing or truncation of proteins.

[0253] In this application, the term "nucleic acid molecule" generally refers to any length of nucleotides. In this application, the term "nucleic acid molecule" can encode a protein contained in the oncolytic virus. In this application, the nucleic acid molecule may comprise DNA and / or RNA. In some cases, the RNA may comprise single-stranded RNA (ssRNA) or double-stranded RNA (dsRNA), and the single-stranded RNA may comprise sense RNA, antisense RNA, or ambisense RNA.

[0254] In this application, the term "expression vector" generally refers to a nucleic acid carrier. Under appropriate conditions, it is typically capable of expressing a target gene and / or target protein. In some embodiments of this application, the expression vector contains one or more component nucleic acid molecules for expressing a virus (e.g., an oncolytic virus). For example, the expression vector contains at least one viral genomic element and can be packaged into a virus or as a viral particle.

[0255] In the present application, the term "virus-producing cell" generally refers to a cell, cell line, or cell culture that may contain, or already contains, a nucleic acid molecule or expression vector described herein, or that is capable of expressing an oncolytic virus described herein. The cell may comprise the progeny of a single host cell. The cell may be obtained by using in vitro transfection of an expression vector described herein.

[0256] In this application, the term "pharmaceutical composition" refers to a formulation that is in a form that is acceptable for effecting the biological activity of the active ingredient and that does not contain additional ingredients that are unacceptably toxic to the subject to which it is administered. In certain embodiments, these formulations may include an active pharmaceutical ingredient and a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical product includes a pharmaceutical product for parenteral, transdermal, intraluminal, intraarterial, intrathecal, and / or intranasal administration or for direct injection into tissue. The pharmaceutical product may be administered in different ways, such as, for example, intravenous, intraperitoneal, subcutaneous, intramuscular, topical, or intradermal administration.

[0257] As used herein, the term "prevention" generally refers to preventing the occurrence, development, recurrence, and / or spread of a disease or one or more symptoms thereof by taking certain measures in advance. As used herein, the term "treatment" generally refers to eliminating or ameliorating a disease or one or more symptoms associated with a disease. In some embodiments, treatment generally refers to the administration of one or more drugs to a patient suffering from the disease to eliminate or remit the disease. In some embodiments, "treatment" may refer to the pharmaceutical composition and / or drug product being administered after the onset of symptoms of a particular disease, with or without other drugs. For example, the pharmaceutical composition and / or drug product described herein may be used to prevent the occurrence, development, recurrence, and / or progression of a tumor.

[0258] In this application, the term "tumor" generally refers to any new pathological tissue growth. Tumors can be benign or malignant. In this application, the tumor may be a solid tumor and / or a blood tumor. For research purposes, these tissues can be isolated from readily available sources by methods well known to those skilled in the art.

[0259] The present application provides oncolytic viruses obtained by mutating sites in the amino acid sequences of the M protein, G protein, N protein, P protein, and L protein of a wild-type VSV virus, specifically the Indiana strain of VSV or the Indiana MuddSummer subtype strain of VSV. The amino acid sequence of the M protein is set forth in SEQ ID NO: 1, the amino acid sequence of the G protein is set forth in SEQ ID NO: 31, the amino acid sequence of the N protein is set forth in SEQ ID NO: 55, the amino acid sequence of the P protein is set forth in SEQ ID NO: 61, and the amino acid sequence of the L protein is set forth in SEQ ID NO: 81. In the present application, the M protein, G protein, N protein, P protein, and L protein can all be modified. The present application modifies the VSV virus as follows to obtain an oncolytic virus.

[0260] An oncolytic virus, the oncolytic virus comprising an M protein, the M protein comprising amino acid substitutions at one or more of positions 32, 33, 49, 54, 133, and 225 compared to the amino acid sequence set forth in SEQ ID NO: 1. The M protein further comprises amino acid substitutions at one or more of positions 21, 51, 111, 221, and 226.

[0261] The amino acid substitutions in the M protein include an asparagine to serine mutation at position 32 (N32S), and / or a methionine to alanine mutation at position 33 (M33A), and / or an asparagine to aspartic acid mutation at position 49 (N49D), and / or a histidine to tyrosine mutation at position 54 (H54Y), and / or an alanine to threonine mutation at position 133 (A133T), and / or a valine to isoleucine mutation at position 225 (V225I), and the amino acid substitutions in the M protein The amino acid substitution further comprises a glycine to glutamic acid mutation at position 21 (G21E), and / or the amino acid substitutions in the M protein comprise a methionine to arginine mutation at position 51 (M51R), and / or a methionine to alanine mutation at position 51 (M51A), and / or a leucine to alanine mutation at position 111 (L111A), and / or a valine to phenylalanine mutation at position 221 (V221F), and / or a serine to arginine mutation at position 226 (S226R).

[0262] In the present application, the M protein may comprise an amino acid mutation at position 21.

[0263] In the present application, the M protein may contain amino acid mutations at positions 21 and 32.

[0264] In the present application, the M protein may comprise amino acid mutations at positions 21, 32 and 33.

[0265] In the present application, the M protein may comprise amino acid mutations at positions 21, 32, 33 and 49.

[0266] In the present application, the M protein may contain amino acid mutations at positions 21, 32, 33, 49 and 54.

[0267] In the present application, the M protein may contain amino acid mutations at positions 21, 32, 33, 49, 54 and 111.

[0268] In the present application, the M protein may contain amino acid mutations at positions 21, 32, 33, 49, 54, 111, and 133.

[0269] In the present application, the M protein may contain amino acid mutations at positions 21, 32, 33, 49, 54, 111, 133, and 225.

[0270] In the present application, the M protein may contain amino acid mutations at positions 21, 32, 33, 49, 51, 54, 111, 133, and 225.

[0271] In the present application, the M protein may contain amino acid mutations at positions 21, 32, 33, 49, 51, 54, 111, 133, 221, and 225.

[0272] In the present application, the M protein may contain amino acid mutations at positions 21, 32, 33, 49, 51, 54, 111, 133, 221, 225, and 226.

[0273] In the present application, the M protein may contain amino acid mutations at positions 32, 33, 49, 51, 54, 111, 133, 221, 225, and 226.

[0274] In the present application, the M protein may contain amino acid mutations at positions 33, 49, 51, 54, 111, 133, 221, 225, and 226.

[0275] In the present application, the M protein may contain amino acid mutations at positions 49, 51, 54, 111, 133, 221, 225, and 226.

[0276] In the present application, the M protein may contain amino acid mutations at positions 51, 54, 111, 133, 221, 225, and 226.

[0277] In the present application, the M protein may contain amino acid mutations at positions 54, 111, 133, 221, 225, and 226.

[0278] In the present application, the M protein may contain amino acid mutations at positions 111, 133, 221, 225, and 226.

[0279] In the present application, the M protein may contain amino acid mutations at positions 133, 221, 225 and 226.

[0280] In the present application, the M protein may contain amino acid mutations at positions 221, 225 and 226.

[0281] In the present application, the M protein may contain amino acid mutations at positions 225 and 226.

[0282] In the present application, the M protein may comprise an amino acid mutation at position 226.

[0283] In the present application, the M protein may contain amino acid mutations at positions 32, 49, 54 and 225.

[0284] In the present application, the M protein may contain amino acid mutations at positions 32, 49, 54, 225 and 226.

[0285] In the present application, the M protein may contain amino acid mutations at positions 32, 49, 51, 54, 221, 225, and 226.

[0286] In the present application, the M protein may contain amino acid mutations at positions 32, 33, 49, 51, 54, 221, 225, and 226.

[0287] In the present application, the M protein may contain amino acid mutations at positions 32, 49, 51, 54, 133, 221, 225, and 226.

[0288] In the present application, the M protein may contain amino acid mutations at positions 32, 33, 49, 51, 54, 133, 221, 225, and 226.

[0289] In the present application, the M protein may contain amino acid mutations at positions 21, 32, 49, 51, 54, 111, 225, and 226.

[0290] In this application, the M Protein may also contain amino acid substitutions at other positions.

[0291] The oncolytic virus further comprises a G protein, which comprises amino acid substitutions at one or more of positions 438, 453, 471 and 487 compared to the amino acid sequence set forth in SEQ ID NO: 31.

[0292] In the present application, the amino acid substitution in the G protein is a valine to isoleucine mutation at position 53 (V53I), and / or an alanine to valine mutation at position 141 (A141V), and / or an aspartic acid to tyrosine mutation at position 172 (D172Y), and / or a lysine to glutamic acid mutation at position 217 (K217E), and / or an aspartic acid to glycine mutation at position 232 (D232G), and / or a valine to alanine mutation at position 331 (V331A). ), and / or a valine to glutamic acid mutation at position 371 (V371E), and / or a glycine to aspartic acid mutation at position 436 (G436D), and / or a threonine to serine mutation at position 438 (T438S), and / or a phenylalanine to leucine mutation at position 453 (F453L), and / or a threonine to isoleucine mutation at position 471 (T471I), and / or a tyrosine to histidine mutation at position 487 (Y487H). For example, the G protein comprises the amino acid sequence set forth in SEQ ID NO: 43.

[0293] In the present application, the G protein may comprise an amino acid mutation at position 53.

[0294] In the present application, the G protein may comprise amino acid mutations at positions 53 and 141.

[0295] In the present application, the G protein may comprise amino acid mutations at positions 53, 141, and 172.

[0296] In the present application, the G protein may comprise amino acid mutations at positions 53, 141, 172, and 217.

[0297] In the present application, the G protein may comprise amino acid mutations at positions 53, 141, 172, 217, and 232.

[0298] In the present application, the G protein may include amino acid mutations at positions 53, 141, 172, 217, 232, and 331.

[0299] In the present application, the G protein may contain amino acid mutations at positions 53, 141, 172, 217, 232, 331, and 371.

[0300] In the present application, the G protein may include amino acid mutations at positions 53, 141, 172, 217, 232, 331, 371, and 436.

[0301] In the present application, the G protein may include amino acid mutations at positions 53, 141, 172, 217, 232, 331, 371, 436, and 438.

[0302] In the present application, the G protein may include amino acid mutations at positions 53, 141, 172, 217, 232, 331, 371, 436, 438, and 453.

[0303] In the present application, the G protein may include amino acid mutations at positions 53, 141, 172, 217, 232, 331, 371, 436, 438, 453, and 471.

[0304] In the present application, the G protein may include amino acid mutations at positions 53, 141, 172, 217, 232, 331, 371, 436, 438, 453, 471, and 487.

[0305] In the present application, the G protein may include amino acid mutations at positions 141, 172, 217, 232, 331, 371, 436, 438, 453, 471, and 487.

[0306] In the present application, the G protein may include amino acid mutations at positions 172, 217, 232, 331, 371, 436, 438, 453, 471, and 487.

[0307] In the present application, the G protein may include amino acid mutations at positions 217, 232, 331, 371, 436, 438, 453, 471, and 487.

[0308] In the present application, the G protein may include amino acid mutations at positions 232, 331, 371, 436, 438, 453, 471, and 487.

[0309] In the present application, the G protein may include amino acid mutations at positions 331, 371, 436, 438, 453, 471, and 487.

[0310] In the present application, the G protein may include amino acid mutations at positions 371, 436, 438, 453, 471, and 487.

[0311] In the present application, the G protein may contain amino acid mutations at positions 436, 438, 453, 471, and 487.

[0312] In the present application, the G protein may contain amino acid mutations at positions 438, 453, 471, and 487.

[0313] In the present application, the G protein may contain amino acid mutations at positions 453, 471, and 487.

[0314] In the present application, the G protein may contain amino acid mutations at positions 471 and 487.

[0315] In the present application, the G protein may comprise an amino acid mutation at position 487.

[0316] In the present application, the G protein may also contain amino acid substitutions at other positions.

[0317] In one embodiment, the G protein contains at least one or more amino acid substitutions in a conserved area. For example, the conserved area may include amino acids 437 to 461 of the G protein. In one embodiment, the G protein contains at least one or more amino acid substitutions in a cleavage area of ​​the cytoplasmic domain. For example, the cleavage area of ​​the cytoplasmic domain may include amino acids 483 to 511 of the G protein.

[0318] In the present application, the G protein may comprise at least amino acid substitutions at positions 438, 453, 471 and 487.

[0319] The oncolytic virus further comprises an N protein, which comprises amino acid substitutions at one or more of positions 14, 155, and 353 compared to the amino acid sequence set forth in SEQ ID NO: 55.

[0320] In the present application, the amino acid substitutions in the N protein include an isoleucine to valine mutation at position 14 (I14V), and / or an arginine to lysine mutation at position 155 (R155K), and / or a serine to asparagine mutation at position 353 (S353N). For example, the N protein comprises the amino acid sequence set forth in SEQ ID NO:58.

[0321] In the present application, the N protein may comprise an amino acid mutation at position 14.

[0322] In the present application, the N protein may contain amino acid mutations at positions 14 and 155.

[0323] In the present application, the N protein may contain amino acid mutations at positions 14, 155 and 353.

[0324] In the present application, the N protein may contain amino acid mutations at positions 155 and 353.

[0325] In the present application, the N protein may comprise an amino acid mutation at position 353.

[0326] In the present application, the N protein may also contain amino acid substitutions at other positions.

[0327] The oncolytic virus further comprises a P protein, which comprises amino acid substitutions at one or more of positions 50, 76, 99, 126, 140, 151, 168, 170, 189, and 237 compared to the amino acid sequence set forth in SEQ ID NO: 61.

[0328] In the present application, the amino acid substitutions in the P protein include an arginine to lysine mutation at position 50 (R50K), and / or a valine to alanine mutation at position 76 (V76A), and / or an asparagine to glutamic acid mutation at position 99 (D99E), and / or a leucine to serine mutation at position 126 (L126S), and / or a leucine to serine mutation at position 140 (L140S), and / or a histidine to tyrosine mutation at position 151 (H151Y), and / or an isoleucine to methionine mutation at position 168 (I168M), and / or a lysine to glutamic acid mutation at position 170 (K170E), and / or a tyrosine to serine mutation at position 189 (Y189S), and / or an asparagine to aspartic acid mutation at position 237 (N237D). For example, the P protein comprises the amino acid sequence set forth in SEQ ID NO:71.

[0329] In the present application, the P protein may comprise an amino acid mutation at position 50.

[0330] In the present application, the P protein may contain amino acid mutations at positions 50 and 76.

[0331] In the present application, the P protein may contain amino acid mutations at positions 50, 76 and 99.

[0332] In the present application, the P protein may contain amino acid mutations at positions 50, 76, 99, and 126.

[0333] In the present application, the P protein may contain amino acid mutations at positions 50, 76, 99, 126, and 140.

[0334] In the present application, the P protein may contain amino acid mutations at positions 50, 76, 99, 126, 140, and 151.

[0335] In the present application, the P protein may include amino acid mutations at positions 50, 76, 99, 126, 140, 151, and 168.

[0336] In the present application, the P protein may include amino acid mutations at positions 50, 76, 99, 126, 140, 151, 168, and 170.

[0337] In the present application, the P protein may include amino acid mutations at positions 50, 76, 99, 126, 140, 151, 168, 170, and 189.

[0338] In the present application, the P protein may include amino acid mutations at positions 50, 76, 99, 126, 140, 151, 168, 170, 189, and 237.

[0339] In the present application, the P protein may include amino acid mutations at positions 76, 99, 126, 140, 151, 168, 170, 189, and 237.

[0340] In the present application, the P protein may include amino acid mutations at positions 99, 126, 140, 151, 168, 170, 189, and 237.

[0341] In the present application, the P protein may include amino acid mutations at positions 126, 140, 151, 168, 170, 189, and 237.

[0342] In the present application, the P protein may include amino acid mutations at positions 140, 151, 168, 170, 189, and 237.

[0343] In the present application, the P protein may contain amino acid mutations at positions 151, 168, 170, 189, and 237.

[0344] In the present application, the P protein may contain amino acid mutations at positions 168, 170, 189, and 237.

[0345] In the present application, the P protein may contain amino acid mutations at positions 170, 189, and 237.

[0346] In the present application, the P protein may contain amino acid mutations at positions 189 and 237.

[0347] In the present application, the P protein may comprise an amino acid mutation at position 237.

[0348] In the present application, the P protein may also contain amino acid substitutions at other positions.

[0349] The oncolytic virus further comprises an L protein, which comprises amino acid substitutions at one or more of positions 87 and 487 compared to the amino acid sequence set forth in SEQ ID NO: 81. For example, the L protein comprises the amino acid sequence set forth in SEQ ID NO: 83.

[0350] In the present application, the amino acid substitutions in the L protein include a serine to proline mutation at position 87 (S87P) and / or an isoleucine to threonine mutation at position 487 (I487T).

[0351] In the present application, the P protein may comprise an amino acid mutation at position 87.

[0352] In the present application, the P protein may contain amino acid mutations at positions 87 and 487.

[0353] In the present application, the P protein may comprise an amino acid mutation at position 487.

[0354] In the present application, the P protein may also contain amino acid substitutions at other positions.

[0355] The oncolytic virus may comprise a nucleic acid molecule and an exogenous target protein, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding an M protein having the amino acid substitution, and / or a nucleic acid sequence encoding a G protein having the amino acid substitution, and / or a nucleic acid sequence encoding an N protein having the amino acid substitution, and / or a nucleic acid sequence encoding a P protein having the amino acid substitution, and / or a nucleic acid sequence encoding an L protein having the amino acid substitution.

[0356] Furthermore, the nucleic acid molecule comprises a nucleic acid sequence encoding the exogenous target protein, wherein the nucleic acid sequence encoding the exogenous target protein is located between the nucleic acid sequence encoding the M protein having the amino acid substitution, and / or the nucleic acid sequence encoding the G protein having the amino acid substitution, and / or the nucleic acid sequence encoding the N protein having the amino acid substitution, and / or the nucleic acid sequence encoding the P protein having the amino acid substitution, and / or the nucleic acid sequence encoding the L protein having the amino acid substitution.

[0357] In the present application, the oncolytic virus described in the present application can be obtained through a virus packaging process and a virus rescue process. A specific process may include infecting and inoculating BSR-T7 cells with poxvirus vTF7-3 expressing T7 RNA polymerase, and then performing lipofectamine transfection with expression plasmids and backbone plasmids cloning VSV N, VSV P, and VSV L genes, respectively, to obtain the target oncolytic virus.

[0358] The present application further provides oncolytic virus expression vectors, virus-producing cells and pharmaceutical compositions.

[0359] The oncolytic virus expression vector may comprise a nucleic acid sequence encoding the M protein and G protein of the oncolytic virus, and the oncolytic virus expression vector may further comprise a nucleic acid sequence encoding the N protein, P protein, and L protein of the oncolytic virus.

[0360] The virus-producing cells are capable of producing the oncolytic viruses described above, and may include BSR-T7 cells, Vero cells, 293 cells, MRC-5 cells, and WI38 cells.

[0361] The pharmaceutical composition comprises the oncolytic virus described above and, optionally, a pharmaceutically acceptable carrier.

[0362] In certain embodiments, the pharmaceutical composition may include an appropriate formulation of one or more (pharmaceutically effective) adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. Acceptable components of pharmaceutical compositions are preferably non-toxic to subjects at the dosages and concentrations used. Pharmaceutical compositions of the present application include, but are not limited to, liquid, frozen, and lyophilized compositions.

[0363] In certain embodiments, the pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents compatible with pharmaceutical administration, and is generally safe and non-toxic.

[0364] The pharmaceutical composition comprises the oncolytic virus described above and, optionally, other pharmaceutically acceptable agents.

[0365] The pharmaceutical compositions may be used in combination treatment of diseases including, but not limited to, the treatment of tumors.

[0366] In some embodiments, the pharmaceutical composition comprises a drug product for parenteral, transdermal, intraluminal, intraarterial, intravenous, intrathecal, and / or intranasal administration or direct injection into tissue. For example, the pharmaceutical composition can be administered to a patient or subject by infusion or injection. In some embodiments, the pharmaceutical composition can be administered in different ways, such as intravenously, intraperitoneally, subcutaneously, intramuscularly, topically, or intradermally. In some embodiments, the pharmaceutical composition can be administered without interruption. The uninterrupted (or continuous) administration can be achieved by metering the flow of therapeutic agent into the patient's body using a miniature pump system worn by the patient, as described in WO 2015 / 036583.

[0367] The present application also provides a method for preparing the above-mentioned oncolytic virus, which may include a method for preparing an oncolytic virus expression vector, a virus-producing cell, and / or a pharmaceutical composition. Any method suitable for producing oncolytic viruses can be used to produce the oncolytic virus of the present application. For example, cells can be transfected with a poxvirus expressing T7 RNA polymerase, and then transfected with a plasmid expressing the oncolytic virus N protein, L protein, and P protein and a backbone plasmid, and the oncolytic virus of the present application can be obtained through a virus rescue process.

[0368] The present application further provides the use of the above-mentioned oncolytic viruses, oncolytic virus expression vectors, virus-producing cells and / or pharmaceutical compositions in the preparation of a medicament for preventing and / or treating a disease and / or condition.

[0369] The oncolytic virus provided in the present application has specific mutations at amino acid sites on the M protein, G protein, N protein, P protein, and L protein of the oncolytic virus, thereby further improving the oncolytic virus's ability to infect abnormally proliferating (tumor) LLC cells, 4T1 cells, MC38 cells, and HeLa cells. At the same time, the oncolytic virus prepared above has low infectivity for both normal cells and normal MEF cells, indicating that the oncolytic virus prepared in the present application can be effectively used to infect cells such as tumors and cancers without damaging normal cells, and has the potential for wide application.

[0370] The oncolytic viruses provided herein have specific mutations at amino acid sites in the M, G, N, P, and L proteins of the oncolytic viruses, thereby further improving the oncolytic viruses' ability to infect abnormally proliferating (tumor) LLC, 4T1, MC38, and HeLa cells. The oncolytic viruses also have improved in vitro killing abilities of LLC, 4T1, MC38, and HeLa cells. At the same time, the prepared oncolytic viruses have little effect on normal MEF cells, indicating that the prepared oncolytic viruses can be effectively used to damage and kill abnormal cells, such as tumors and cancers, without damaging normal cells.

[0371] The oncolytic virus provided in the present application is difficult to eliminate in abnormally proliferating (tumor) LLC cells, 4T1 cells, MC38 cells, and Hela cells. Relatively speaking, wild-type oncolytic viruses are more easily eliminated in LLC cells, MC38 cells, and Hela cells. The oncolytic virus provided in the present application has site-specific mutations in the amino acids of the M protein, G protein, N protein, P protein, and L protein of the oncolytic virus, respectively, making the oncolytic virus more difficult to eliminate in LLC cells, 4T1 cells, MC38 cells, and Hela cells, further ensuring that the oncolytic virus can better exert its infection and killing ability in LLC cells, 4T1 cells, MC38 cells, and Hela cells. At the same time, the oncolytic virus provided in the present application is more easily eliminated in normal MEF cells, further ensuring the safety of normal MEF cells and thereby improving the safety of the oncolytic virus.

[0372] The present application will be described in more detail below with reference to Preparation Examples 1 to 80, Examples 1 to 3, and Figures 1 to 15. Preparation example Preparation Examples 1 to 29

[0373] Oncolytic viruses are provided in Preparation Examples 1 to 29, and the main difference between them is the site of amino acid site-specific mutation on the M protein of the wild-type oncolytic virus. The specific methods for constructing the oncolytic viruses corresponding to each Preparation Example are as follows. (1) Plasmid construction

[0374] Using the pRV-core plasmid (BioVector NTCC Plasmid Carrier Bacterial Cell Genetic Stock Center) as a template, PCR was used to introduce the mutation sites shown in Table 1. The pRV-core plasmid was then subjected to PCR with primers carrying each mutation site. The PCR products were then subjected to 1% agarose gel electrophoresis and gel recovery using a gel recovery kit to obtain plasmids with different mutation sites in the M protein. This resulted in the construction of the plasmid pRV-core Mut.

[0375] JPEG2023143023000001.jpg208149 (2) Virus rescue

[0376] The constructed plasmid pRV-core Mut was transfected into BSR-T7 cells (purchased from the American Type Culture Collection (ATCC)) using a calcium phosphate transfection kit (Thermo Fisher Scientific).

[0377] The four plasmids were mixed in a mass ratio of 10:5:4:1 for pRV-core Mut, pP, pN, and pL, resulting in a total of 5 μg of plasmid. The plasmids were diluted with 200 μl of opti-MEM medium (Thermo Fisher Scientific) and 7.5 μl of Transfection Reagent Plus Reagent (Life Technologies) were added to obtain a transfection plasmid premix solution, where pP( Rhabdovirus Plasmid carrying the phosphoprotein gene), pN( Rhabdovirus Plasmid carrying a nucleoprotein gene), pL( Rhabdovirus The parent carrier of the three plasmids pN, pP, and pL is pCAGGS (purchased from ATCC). Dilute 10 μl of Lipofectamine LTX (Thermo Fisher Scientific) with 200 μl of opti-MEM medium to obtain the LTX mixture. Plasmid transfection was performed according to the method described in the Lipofectamine LTX instruction manual. After 6 hours, BSR-T7 cells were washed twice with PBS and then inoculated into DMEM medium (Thermo Fisher Scientific) containing 10% fetal bovine serum and cultured for 3 days. The cell supernatant obtained by culturing BSR-T7 cells was transferred to Vero cells (Thermo Fisher Scientific), which were then cultured at 37°C for 3 days. The green fluorescence within the cells was observed under a fluorescence microscope to identify the status of virus rescue, and the rescued mutations were then analyzed. Rhabdovirus The library is passaged through Vero cells and monoclonal virus stocks are picked from the constructed plaque screening system.

[0378] (3) M protein gene sequencing: Viral genomic RNA was extracted using a Trizol kit, reverse transcription was performed using random primers, and PCR was performed on the reverse-transcribed cDNA using primers designed for the M protein gene sequence. The primer sequences are as follows: JPEG2023143023000002.jpg1043

[0379] The product was recovered by 1% agarose gel electrophoresis and sent to a sequencing company for sequencing. The sequencing results are shown in Table 1. Preparation Examples 30-52

[0380] Oncolytic viruses are provided in Preparation Examples 30 to 52, and the main difference between them is the amino acid site-specific mutation sites on the M protein and G protein of the wild-type oncolytic virus. Here, the mutation site in the M protein is the same as the corresponding mutation site in Preparation Example 24, and the mutation site in the G protein is shown in Table 2.

[0381] The construction method of the oncolytic virus provided in the above Preparation Example differs from the construction method of Preparation Example 11 in the following points: introducing the mutation sites shown in Table 2 into the constructed plasmid of step (1) using PCR techniques; Step (3) is the determination of the G protein gene sequence. Viral genomic RNA is extracted using a Trizol kit, reverse-transcribed using random primers, and PCR is performed on the reverse-transcribed cDNA using primers designed for the G protein gene sequence. The primer sequences are as follows: JPEG2023143023000003.jpg1053

[0382] The product was recovered by 1% agarose gel electrophoresis and sent to a sequencing company for sequencing. The sequencing results are shown in Table 2.

[0383] JPEG2023143023000004.jpg169149 Preparation example 53~57

[0384] Preparative Examples 53 to 57 provide oncolytic viruses, each differing mainly in the amino acid site-specific mutations in the M protein, G protein, and N protein of the wild-type oncolytic virus. The mutation sites in the M protein and G protein are the same as the corresponding mutation sites in Preparative Example 41, and the mutation site in the N protein is shown in Table 3.

[0385] The construction method of the oncolytic virus provided in the above Preparation Example differs from the construction method of Preparation Example 41 in the following respects: introducing the mutation sites shown in Table 3 into the constructed plasmid of step (1) using PCR techniques; Step (3) is sequencing the N protein gene. Viral genomic RNA is extracted using a Trizol kit, reverse-transcribed using random primers, and PCR is performed on the reverse-transcribed cDNA using primers designed for the N protein gene sequence. The primer sequences are as follows: JPEG2023143023000005.jpg1050

[0386] The product was recovered by 1% agarose gel electrophoresis and sent to a sequencing company for sequencing. The sequencing results are shown in Table 3.

[0387] JPEG2023143023000006.jpg37149 Preparation example 58~76

[0388] Preparation Examples 58 to 76 provide oncolytic viruses, each differing mainly in the amino acid site-specific mutations in the M protein, G protein, N protein, and P protein of the wild-type oncolytic virus. The mutation sites in the M protein, G protein, and N protein are the same as the corresponding mutation sites in Preparation Example 55, and the mutation site in the P protein is shown in Table 3.

[0389] The construction method of the oncolytic virus provided in the above Preparation Example differs from the construction method of Preparation Example 55 in the following respects: introducing the mutation sites shown in Table 4 into the constructed plasmid of step (1) using PCR techniques; Step (3) is P protein gene sequencing: viral genomic RNA is extracted using a Trizol kit, reverse transcription is performed using random primers, and PCR is performed on the reverse-transcribed cDNA using primers designed for the P protein gene sequence. The primer sequences are as follows: JPEG2023143023000007.jpg1054

[0390] The product was recovered by 1% agarose gel electrophoresis and sent to a sequencing company for sequencing. The sequencing results are shown in Table 4.

[0391] JPEG2023143023000008.jpg130150 Preparation example 77~79

[0392] Preparative Examples 77 to 79 each provide an oncolytic virus, and the difference between them is mainly the amino acid site-specific mutation sites on the M protein, G protein, N protein, P protein, and L protein of the wild-type oncolytic virus. Here, the mutation sites of the M protein, G protein, N protein, and P protein are the same as the corresponding mutation sites in Preparative Example 67, and the mutation site of the L protein is shown in Table 5.

[0393] The construction method of the oncolytic virus provided in the above Preparation Example differs from the construction method of Preparation Example 67 in the following respects: introducing the mutation sites shown in Table 5 into the constructed plasmid of step (1) using PCR techniques; Step (3) is sequencing the L protein gene. Viral genomic RNA is extracted using a Trizol kit, reverse-transcribed using random primers, and PCR is performed on the reverse-transcribed cDNA using primers designed for the L protein gene sequence. The primer sequences are as follows: JPEG2023143023000009.jpg1053

[0394] The product was recovered by 1% agarose gel electrophoresis and sent to a sequencing company for sequencing. The sequencing results are shown in Table 5.

[0395] JPEG2023143023000010.jpg27149 Preparation example 80

[0396] This preparation example provides a packaging process for an oncolytic virus prepared by any one of the above preparation examples 1 to 79, and specifically includes the following steps: 1) BSR-T7 cells (purchased from ATCC) were infected and inoculated with the poxvirus vTF7-3 (BioVector NTCC Plasmid Carrier Cell Gene Storage Center) expressing T7 RNA polymerase.

[0397] Specific process: BSR-T7 cells were plated in a 6-well plate, with the number of cells per well set at 3 × 10 5 The cells were then transfected with the poxvirus vTF7-3, which expresses T7 RNA polymerase, 14-16 hours after transfection. After 6 hours of infection, the BSR-T7 cells were rinsed once with DPBS buffer (Thermo Fisher Scientific) and transfection was performed. 2) Transfection process

[0398] Specifically, the process involves the following steps: pRV-core Mut, pP, pN, and pL are mixed in a mass ratio of 10:5:4:1, with a total of 5 μg of plasmid. The plasmids are diluted with 200 μl of opti-MEM medium (Thermo Fisher Scientific) and 7.5 μl of Transfection Reagent Plus Reagent (Life Technologies) are added to obtain a transfection plasmid premix solution, where pP( Rhabdovirus Plasmid carrying the phosphoprotein gene), pN( Rhabdovirus Plasmid carrying a nucleoprotein gene), pL( Rhabdovirus The parent carrier of the three plasmids pN, pP, and pL is pCAGGS (purchased from ATCC). Dilute 10 μl of Lipofectamine LTX (Thermo Fisher Scientific) with 200 μl of opti-MEM medium to obtain the LTX mixture. 200 μl of the LTX mixture and 200 μl of the transfection plasmid premix were mixed and incubated at room temperature for 15 minutes to obtain an LTX-DNA mixture. In step 1), the DPBS buffer in the 6-well plate was replaced with Opti-MEM medium, and the LTX-DNA mixture was added to the 6-well plate containing the BSR-T7 cells. The 6-well plate was gently shaken to distribute the LTX-DNA mixture evenly throughout the plate. After 6-8 hours of transfection, the transfection reagent was removed and 3 ml of fresh complete medium (Thermo Fisher Scientific) was added. After 72 hours, the BSR-T7 cell supernatant was collected and filtered through a 0.22 μm filter to obtain the corresponding oncolytic viruses in Preparations 1-79. Example Example 1

[0399] In this example, the oncolytic viruses and wild-type oncolytic viruses prepared in Preparation Examples 1 to 79 were used to test and detect their infectivity to different cells.

[0400] The detection method was the TCID50 detection method, that is, 200 pfu each of Preparations 1 to 79 and wild-type oncolytic viruses was added to the culture medium of different cells, and half of the tissue culture infective dose (TCID50) produced by each oncolytic virus was detected.

[0401] The detected cells include LLC cells (mouse lung cancer cell line), 4T1 cells (mouse breast cancer cell line), MC38 cells (mouse colon cancer cell line), Hela cells (human cervical cancer cell line), and MEF cells (human fibroblast cell line).

[0402] Specific detection methods include: (1) Add 3 mL of Vero (LLC / 4T1 / MC38 / Hela / MEF) cell suspension to a 6-well culture plate, and adjust the cell volume to 4 × 10 5 The cells / well were then cultured in a 6-well culture plate at 37°C and 5% CO2 for 16 hours, with a total of 6 wells, including 2 wells for MEF cells. (2) 200 pfu of the oncolytic virus prepared in the Preparation Example was added to each well of a 6-well culture plate. After 24 hours, 100 μl of supernatant from MEF cells and each Vero cell was collected and added to the wells of a 96-well culture plate. The cell volume of each cell was adjusted to 1 × 10 4 The cells / ml were cultured in a 96-well culture plate at 37°C and 5% CO2 for 16 hours. (3) In a 1.5 ml EP tube, the supernatant obtained in step (2) was serially diluted 10-fold to obtain 10 -1 From 10 -11 There were a total of 11 titers, and the diluted supernatants were inoculated into 96-well culture plates, one column per dilution, for a total of 8 wells, with 100 μl inoculated per well. (4) After 48 hours, the fluorescence state of the cells in each well is observed. If fluorescence is detected, the well is recorded as infected, and TCID50 is calculated according to the Karber method.

[0403] The detection results are shown in Figures 1 to 5. Here, the horizontal axis 0 represents the wild-type oncolytic virus, the horizontal axes 1 to 79 represent the oncolytic viruses prepared in Preparation Examples 1 to 79, respectively, and the vertical axis Log 10 TCID50 represents the TCID50 value calculated by the Karber method, and Log 10 The higher the TCID50 value, the better the oncolytic virus's ability to infect the cells. Log 10 A smaller TCID50 value indicates a lower ability of the oncolytic virus to infect the cells.

[0404] FIG. 1 shows the results of detecting the ability of the oncolytic viruses prepared in the present application and wild-type oncolytic viruses to infect LLC cells. FIG. 2 shows the results of detecting the infectivity of the oncolytic viruses prepared in the present application and wild-type oncolytic viruses to 4T1 cells. FIG. 3 shows the results of detecting the ability of the oncolytic viruses prepared in the present application and wild-type oncolytic viruses to infect MC38 cells. FIG. 4 shows the results of detecting the infectivity of the oncolytic viruses prepared in the present application and wild-type oncolytic viruses to Hela cells. FIG. 5 shows the results of detecting the ability of the oncolytic viruses prepared in the present application and wild-type oncolytic viruses to infect MEF cells.

[0405] As can be seen from the figures, the oncolytic viruses prepared in Preparation Examples 1 to 79 of the present application all have good infectivity for LLC cells, 4T1 cells, MC38 cells, and HeLa cells, and in particular, the infectivity of the oncolytic viruses provided in Preparation Examples 77 to 79 for LLC cells, 4T1 cells, MC38 cells, and HeLa cells is comparable to that of wild-type oncolytic viruses. At the same time, the oncolytic viruses prepared above, particularly those provided in Preparation Examples 77 to 79, all have low infectivity for MEF cells, indicating that the oncolytic viruses prepared in the present application can be effectively used to infect cells of tumors, cancers, etc., without damaging normal cells, and have broad potential for application. Example 2

[0406] In this example, the oncolytic viruses and wild-type oncolytic viruses prepared in Preparation Examples 1 to 79 were used to perform in vitro killing tests on different cells.

[0407] The detection method was the MTT detection method, that is, 200 pfu each of Preparation Examples 1 to 79 and wild-type oncolytic viruses was added to the culture medium of different cells, and after 24 hours, cell activity was detected by the MTT detection method.

[0408] The cells detected include LLC cells, 4T1 cells, MC38 cells, Hela cells, and MEF cells.

[0409] Specific detection methods include: (1) Add 100 μl of Vero (LLC / 4T1 / MC38 / Hela / MEF) cell suspension to a 96-well culture plate to obtain a cell volume of 1 × 10 4The cells / well were allowed to reach 100 cells per well, and the 96-well culture plate was incubated at 37°C and 5% CO2 for 16 hours. (2) The oncolytic viruses prepared in the Preparation Examples were diluted to MOIs (multiplicity of infection) of 0.001, 0.01, 0.1, and 1.0, and the oncolytic viruses of each dilution gradient were inoculated into the 96-well culture plate of step (1), with 4 wells per dilution gradient, and 100 μl per well. The 96-well culture plate was then cultured at 37°C and 5% CO2 for 40 hours under circulating conditions. (3) Remove the cell supernatant from the 96-well culture plate in step (2), add fresh medium and MTT solution to the 96-well culture plate at a volume of 20 μL / well, and culture the 96-well culture plate at 37°C and 5% CO2 for 4 hours. (4) Centrifuge the 96-well culture plate at room temperature for 5 minutes, set the rotation speed to 2500 rpm / min, gently aspirate the supernatant using a 1 mL disposable sterile syringe, and add DMSO to each well of the 96-well culture plate at a volume of 100 μl per well. Place the plate at 37°C for 10 minutes. Shake the plate for 2 minutes using a multi-function microplate reader and measure the OD value of each well on the 96-well culture plate at a wavelength of 570 nm or 490 nm.

[0410] The detection results are shown in Figures 6 to 10. Here, the horizontal axis 0 represents the wild-type oncolytic virus, the horizontal axes 1 to 79 represent the oncolytic viruses prepared in Preparation Examples 1 to 79, respectively, and the vertical axis represents OD 570 represents the OD value of the cells, and OD 570 The larger the OD value, the lower the killing ability of the oncolytic virus to the cell. 570 The smaller the value, the better the killing ability of the oncolytic virus to the cell.

[0411] FIG. 6 shows the results of detecting the in vitro killing ability of the oncolytic virus prepared in the present application and the wild-type oncolytic virus on LLC cells. FIG. 7 shows the results of detecting the in vitro killing ability of the oncolytic virus prepared in the present application and the wild-type oncolytic virus on 4T1 cells. FIG. 8 shows the results of detecting the in vitro killing ability of the oncolytic virus prepared in the present application and the wild-type oncolytic virus on MC38 cells. FIG. 9 shows the results of detecting the in vitro killing ability of the oncolytic virus prepared in the present application and the wild-type oncolytic virus on Hela cells. FIG. 10 shows the results of detecting the in vitro killing ability of the oncolytic viruses prepared in the present application and wild-type oncolytic viruses on MEF cells.

[0412] As can be seen from the figures, the oncolytic viruses prepared in Preparation Examples 1 to 79 of the present application all have good in vitro killing ability against LLC cells, 4T1 cells, MC38 cells, and HeLa cells. In particular, the oncolytic viruses provided in Preparation Examples 77 to 79 have in vitro killing ability against LLC cells, 4T1 cells, MC38 cells, and HeLa cells that exceeds that of wild-type oncolytic viruses. At the same time, the above-prepared oncolytic viruses have almost no killing effect on MEF cells, indicating that the oncolytic viruses prepared in the present application can be effectively used to damage and kill abnormal cells such as tumors and cancers without damaging normal cells. Although wild-type oncolytic viruses have good in vitro killing ability against LLC, MC38, and 4T1 cells, they also significantly damage and kill MEF cells, limiting their clinical application. Therefore, the wild-type oncolytic virus modification proposed in this application ensures the safety of oncolytic viruses in normal cells while also ensuring the killing ability of oncolytic viruses in tumor and cancer cells, potentially opening up a wide range of clinical applications. Example 3

[0413] In this example, the oncolytic viruses prepared in Preparation Examples 1 to 79 and wild-type oncolytic viruses were tested to detect the induction of IFN-β expression in different cells.

[0414] The detection indicator is the expression of the IFN-β gene in different cells. The IFN-β gene is a soluble glycoprotein gene produced by cells that has a wide range of antiviral, antitumor, and immunomodulatory effects. The expression of the IFN-β gene can be used to determine the cell's ability to eliminate oncolytic viruses. High expression of the IFN-β gene indicates that oncolytic viruses are easily eliminated within the cell, while low expression of the IFN-β gene indicates that oncolytic viruses are difficult to eliminate within the cell.

[0415] The cells detected include LLC cells, 4T1 cells, MC38 cells, Hela cells, and MEF cells.

[0416] Specific detection methods include: (1) Add 100 μl of Vero (LLC / 4T1 / MC38 / Hela / MEF) cell suspension to a 96-well culture plate to obtain a cell volume of 1 × 10 4 The cells / well were allowed to reach 100 cells per well, and the 96-well culture plate was incubated at 37°C and 5% CO2 for 16 hours. (2) The oncolytic viruses prepared in the Preparation Examples were diluted to MOIs (multiplicities of infection) of 0.001, 0.01, 0.1, and 1.0, and the oncolytic viruses of each dilution gradient were inoculated into the 96-well culture plate of step (1), with 4 wells per dilution gradient, and 100 μl per well. The 96-well culture plate was then cultured at 37°C in a 5% CO2 atmosphere for 40 hours. (3) Each set of cells cultured and obtained in step (2) was disrupted, total RNA was extracted from each cell using TRIzol (Invitrogen), reverse transcribed into cDNA using the PrimeScript RT Reagent Kit with DNA Eraser (Takara) reverse transcription kit, stained with LightCycler 480 SYBR Green I Master (Roche) dye, and the Ct value of each gene was detected using a LightCycler 480 quantitative PCR instrument. The relative expression level of the target gene IFN-β was calculated using the ΔΔCt method.

[0417] The detection results are shown in Figures 11 to 15. Here, the horizontal axis 0 represents the wild-type oncolytic virus, the horizontal axes 1 to 79 represent the oncolytic viruses prepared in Preparation Examples 1 to 79, respectively, and the vertical axis, IFN-β level, represents the expression status of the IFN-β gene, with a higher IFN-level value indicating a weaker reproduction ability of the oncolytic virus within the cell and easier elimination, and a lower IFN-β level value indicating a stronger reproduction ability of the oncolytic virus within the cell and harder elimination.

[0418] FIG. 11 shows the state in which the oncolytic virus prepared in the present application and the wild-type oncolytic virus induce the expression of IFN-β in LLC cells. FIG. 12 shows the state in which the oncolytic virus prepared in the present application and the wild-type oncolytic virus induce the expression of IFN-β in 4T1 cells. FIG. 13 shows the state in which the oncolytic virus prepared in the present application and the wild-type oncolytic virus induce the expression of IFN-β in MC38 cells. FIG. 14 shows the state in which the oncolytic virus prepared in the present application and the wild-type oncolytic virus induce the expression of IFN-β in Hela cells. FIG. 15 shows the state in which the oncolytic virus prepared in the present application and the wild-type oncolytic virus induce the expression of IFN-β in MEF cells.

[0419] As can be seen from the above figures, the oncolytic viruses and wild-type oncolytic viruses provided in Preparation Examples 1 and 21 of the present application have poor reproduction ability in LLC cells, 4T1 cells, MC38 cells, and HeLa cells, and are easily eliminated. Relatively speaking, the oncolytic viruses provided in Preparation Examples 2-20 and 22-79 are less likely to be eliminated in LLC cells, 4T1 cells, MC38 cells, and HeLa cells. In particular, the oncolytic viruses provided in Preparation Examples 77-79 are less likely to be eliminated in LLC cells, 4T1 cells, MC38 cells, and HeLa cells, further ensuring that the oncolytic viruses can better exert their infection and killing abilities in LLC cells, 4T1 cells, MC38 cells, and HeLa cells. At the same time, the oncolytic viruses provided in the present application are more likely to be eliminated in MEF cells, further ensuring the safety of MEF cells and thereby improving the safety of the oncolytic viruses.

[0420] The specific examples are merely illustrative of the present application and do not limit the present application. After reading this specification, a person skilled in the art may make amendments to the examples as necessary without making any creative contribution, but all such amendments within the scope of the claims of the present application shall be protected by the Patent Law.

Claims

1. an oncolytic virus, The oncolytic virus is a vesicular stomatitis virus Indiana MudSummer subtype and comprises an M protein, wherein the M protein comprises an amino acid substitution selected from any one of the following amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO: 1: 1) the amino acid substitutions in the M protein are G21E and N32S; 2) the amino acid substitutions in the M protein are G21E, N32S, and M33A; 3) the amino acid substitutions in the M protein are G21E, N32S, M33A, and N49D; 4) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, and H54Y; 5) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, H54Y, and L111A; 6) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, H54Y, L111A, and A133T; 7) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, H54Y, L111A, A133T, and V225I; 8) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, M51R, H54Y, L111A, A133T, and V225I; 9) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, M51R, H54Y, L111A, A133T, V221F, and V225I; 10) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 11) The amino acid substitutions in the M protein are N32S, M33A, N49D, M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 12) The amino acid substitutions in the M protein are M33A, N49D, M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 13) The amino acid substitutions in the M protein are N49D, M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 14) The amino acid substitutions in the M protein are M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 15) The amino acid substitutions in the M protein are H54Y, L111A, A133T, V221F, V225I, and S226R; 16) The amino acid substitutions in the M protein are L111A, A133T, V221F, V225I, and S226R; 17) The amino acid substitutions in the M protein are A133T, V221F, V225I, and S226R; 18) The amino acid substitutions in the M protein are V221F, V225I, and S226R; 19) The amino acid substitutions in the M protein are V225I and S226R; 20) The amino acid substitution in the M protein is S226R; 21) The amino acid substitutions in the M protein are N32S, N49D, H54Y, V225I, and S226G; 22) The amino acid substitutions in the M protein are N32S, N49D, M51R, H54Y, V221F, V225I, and S226R; 23) The amino acid substitutions in the M protein are N32S, M33A, N49D, M51R, H54Y, V221F, V225I, and S226R; 24) The amino acid substitutions in the M protein are N32S, N49D, M51R, H54Y, A133T, V221F, V225I, and S226R; 25) The amino acid substitutions in the M protein are N32S, M33A, N49D, M51R, H54Y, A133T, V221F, V225I, and S226R; 26) The amino acid substitutions in the M protein are G21E, N32S, N49D, M51A, H54Y, L111A, V225I, and S226R. An oncolytic virus characterized by:

2. The amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, M51R, H54Y, L111A, A133T, V221F, V225I, and S226R. The oncolytic virus according to claim 1 .

3. The M protein comprises the amino acid sequence shown in SEQ ID NO:

12. The oncolytic virus according to claim 1 .

4. an oncolytic virus, The oncolytic virus is a vesicular stomatitis virus Indiana MudSummer subtype and comprises an M protein, wherein the M protein comprises an amino acid substitution selected from any one of the following amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO: 1: 1) the amino acid substitutions in the M protein are G21E and N32S; 2) the amino acid substitutions in the M protein are G21E, N32S, and M33A; 3) the amino acid substitutions in the M protein are G21E, N32S, M33A, and N49D; 4) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, and H54Y; 5) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, H54Y, and L111A; 6) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, H54Y, L111A, and A133T; 7) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, H54Y, L111A, A133T, and V225I; 8) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, M51R, H54Y, L111A, A133T, and V225I; 9) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, M51R, H54Y, L111A, A133T, V221F, and V225I; 10) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 11) The amino acid substitutions in the M protein are N32S, M33A, N49D, M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 12) The amino acid substitutions in the M protein are M33A, N49D, M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 13) The amino acid substitutions in the M protein are N49D, M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 14) The amino acid substitutions in the M protein are M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 15) The amino acid substitutions in the M protein are H54Y, L111A, A133T, V221F, V225I, and S226R; 16) The amino acid substitutions in the M protein are L111A, A133T, V221F, V225I, and S226R; 17) The amino acid substitutions in the M protein are A133T, V221F, V225I, and S226R; 18) The amino acid substitutions in the M protein are V221F, V225I, and S226R; 19) The amino acid substitutions in the M protein are V225I and S226R; 20) The amino acid substitution in the M protein is S226R; 21) The amino acid substitutions in the M protein are N32S, N49D, H54Y, V225I, and S226G; 22) The amino acid substitutions in the M protein are N32S, N49D, M51R, H54Y, V221F, V225I, and S226R; 23) The amino acid substitutions in the M protein are N32S, M33A, N49D, M51R, H54Y, V221F, V225I, and S226R; 24) The amino acid substitutions in the M protein are N32S, N49D, M51R, H54Y, A133T, V221F, V225I, and S226R; 25) The amino acid substitutions in the M protein are N32S, M33A, N49D, M51R, H54Y, A133T, V221F, V225I, and S226R; 26) The amino acid substitutions in the M protein are G21E, N32S, N49D, M51A, H54Y, L111A, V225I, and S226R; The oncolytic virus further comprises a G protein, and the G protein comprises amino acid substitutions at one or more of positions 438, 453, 471, and 487 compared to the amino acid sequence set forth in SEQ ID NO:

31. An oncolytic virus characterized by:

5. The G protein further comprises an amino acid substitution at one or more of positions 53, 141, 172, 217, 232, 331, 371, and 436. The oncolytic virus according to claim 4 .

6. The amino acid substitution in the G protein is a valine to isoleucine mutation at position 53 (V53I), and / or an alanine to valine mutation at position 141 (A141V), and / or an aspartic acid to tyrosine mutation at position 172 (D172Y), and / or a lysine to glutamic acid mutation at position 217 (K217E), and / or an aspartic acid to glycine mutation at position 232 (D232G), and / or a valine to alanine mutation at position 331 (V331A), and / or a valine to glutamic acid mutation at position 371 (V371E), and / or a glycine to aspartic acid mutation at position 436 (G436D), and / or a threonine to serine mutation at position 438 (T438S), and / or a phenylalanine to leucine mutation at position 453 (F453L), and / or a threonine to isoleucine mutation at position 471 (T471I), and / or a tyrosine to histidine mutation at position 487 (Y487H). The oncolytic virus according to claim 4 or 5.

7. The G protein-like amino acid substitution is selected as any one of the following: 1) the amino acid substitution in the G protein comprises V53I; 2) the amino acid substitutions in the G protein include V53I and A141V; 3) the amino acid substitutions in the G protein include V53I, A141V, and D172Y; 4) the amino acid substitutions of the G protein include V53I, A141V, D172Y, and K217E; 5) the amino acid substitutions of the G protein include V53I, A141V, D172Y, K217E, and D232G; 6) the amino acid substitutions of the G protein include V53I, A141V, D172Y, K217E, D232G, and V331A; 7) The amino acid substitutions of the G protein include V53I, A141V, D172Y, K217E, D232G, V331A, and V371E; 8) The amino acid substitutions of the G protein include V53I, A141V, D172Y, K217E, D232G, V331A, V371E, and G436D; 9) The amino acid substitutions of the G protein include V53I, A141V, D172Y, K217E, D232G, V331A, V371E, G436D, and T438S; 10) The amino acid substitutions of the G protein include V53I, A141V, D172Y, K217E, D232G, V331A, V371E, G436D, T438S, and F453L; 11) The amino acid substitutions of the G protein include V53I, A141V, D172Y, K217E, D232G, V331A, V371E, G436D, T438S, F453L, and T471I; 12) The amino acid substitutions of the G protein include V53I, A141V, D172Y, K217E, D232G, V331A, V371E, G436D, T438S, F453L, T471I, and Y487H; 13) The amino acid substitutions of the G protein include A141V, D172Y, K217E, D232G, V331A, V371E, G436D, T438S, F453L, T471I, and Y487H; 14) The amino acid substitutions of the G protein include D172Y, K217E, D232G, V331A, V371E, G436D, T438S, F453L, T471I, and Y487H; 15) The amino acid substitutions of the G protein include K217E, D232G, V331A, V371E, G436D, T438S, F453L, T471I, and Y487H; 16) The amino acid substitutions of the G protein include D232G, V331A, V371E, G436D, T438S, F453L, T471I, and Y487H; 17) The amino acid substitutions of the G protein include V331A, V371E, G436D, T438S, F453L, T471I, and Y487H; 18) The amino acid substitutions of the G protein include V371E, G436D, T438S, F453L, T471I, and Y487H; 19) The amino acid substitutions of the G protein include G436D, T438S, F453L, T471I, and Y487H; 20) The amino acid substitutions of the G protein include T438S, F453L, T471I, and Y487H; 21) The amino acid substitutions of the G protein include F453L, T471I, and Y487H; 22) The amino acid substitutions of the G protein include T471I and Y487H; 23) The amino acid substitution of the G protein includes Y487H. The oncolytic virus according to claim 6 .

8. The amino acid substitutions in the G protein include V53I, A141V, D172Y, K217E, D232G, V331A, V371E, G436D, T438S, F453L, T471I, and Y487H. The oncolytic virus according to claim 7 .

9. The G protein comprises the amino acid sequence set forth in SEQ ID NO:

43. The oncolytic virus according to claim 7 .

10. an oncolytic virus, The oncolytic virus is a vesicular stomatitis virus Indiana MudSummer subtype and comprises an M protein, wherein the M protein comprises an amino acid substitution selected from any one of the following amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO: 1: 1) the amino acid substitutions in the M protein are G21E and N32S; 2) the amino acid substitutions in the M protein are G21E, N32S, and M33A; 3) the amino acid substitutions in the M protein are G21E, N32S, M33A, and N49D; 4) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, and H54Y; 5) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, H54Y, and L111A; 6) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, H54Y, L111A, and A133T; 7) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, H54Y, L111A, A133T, and V225I; 8) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, M51R, H54Y, L111A, A133T, and V225I; 9) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, M51R, H54Y, L111A, A133T, V221F, and V225I; 10) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 11) The amino acid substitutions in the M protein are N32S, M33A, N49D, M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 12) The amino acid substitutions in the M protein are M33A, N49D, M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 13) The amino acid substitutions in the M protein are N49D, M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 14) The amino acid substitutions in the M protein are M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 15) The amino acid substitutions in the M protein are H54Y, L111A, A133T, V221F, V225I, and S226R; 16) The amino acid substitutions in the M protein are L111A, A133T, V221F, V225I, and S226R; 17) The amino acid substitutions in the M protein are A133T, V221F, V225I, and S226R; 18) The amino acid substitutions in the M protein are V221F, V225I, and S226R; 19) The amino acid substitutions in the M protein are V225I and S226R; 20) The amino acid substitution in the M protein is S226R; 21) The amino acid substitutions in the M protein are N32S, N49D, H54Y, V225I, and S226G; 22) The amino acid substitutions in the M protein are N32S, N49D, M51R, H54Y, V221F, V225I, and S226R; 23) The amino acid substitutions in the M protein are N32S, M33A, N49D, M51R, H54Y, V221F, V225I, and S226R; 24) The amino acid substitutions in the M protein are N32S, N49D, M51R, H54Y, A133T, V221F, V225I, and S226R; 25) The amino acid substitutions in the M protein are N32S, M33A, N49D, M51R, H54Y, A133T, V221F, V225I, and S226R; 26) The amino acid substitutions in the M protein are G21E, N32S, N49D, M51A, H54Y, L111A, V225I, and S226R; The oncolytic virus further comprises an N protein, wherein the N protein comprises amino acid substitutions at one or more of positions 14, 155, and 353 compared to the amino acid sequence set forth in SEQ ID NO:

55. An oncolytic virus characterized by:

11. an oncolytic virus, The oncolytic virus is a vesicular stomatitis virus Indiana MudSummer subtype and comprises an M protein, wherein the M protein comprises an amino acid substitution selected from any one of the following amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO: 1: 1) the amino acid substitutions in the M protein are G21E and N32S; 2) the amino acid substitutions in the M protein are G21E, N32S, and M33A; 3) the amino acid substitutions in the M protein are G21E, N32S, M33A, and N49D; 4) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, and H54Y; 5) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, H54Y, and L111A; 6) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, H54Y, L111A, and A133T; 7) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, H54Y, L111A, A133T, and V225I; 8) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, M51R, H54Y, L111A, A133T, and V225I; 9) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, M51R, H54Y, L111A, A133T, V221F, and V225I; 10) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 11) The amino acid substitutions in the M protein are N32S, M33A, N49D, M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 12) The amino acid substitutions in the M protein are M33A, N49D, M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 13) The amino acid substitutions in the M protein are N49D, M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 14) The amino acid substitutions in the M protein are M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 15) The amino acid substitutions in the M protein are H54Y, L111A, A133T, V221F, V225I, and S226R; 16) The amino acid substitutions in the M protein are L111A, A133T, V221F, V225I, and S226R; 17) The amino acid substitutions in the M protein are A133T, V221F, V225I, and S226R; 18) The amino acid substitutions in the M protein are V221F, V225I, and S226R; 19) The amino acid substitutions in the M protein are V225I and S226R; 20) The amino acid substitution in the M protein is S226R; 21) The amino acid substitutions in the M protein are N32S, N49D, H54Y, V225I, and S226G; 22) The amino acid substitutions in the M protein are N32S, N49D, M51R, H54Y, V221F, V225I, and S226R; 23) The amino acid substitutions in the M protein are N32S, M33A, N49D, M51R, H54Y, V221F, V225I, and S226R; 24) The amino acid substitutions in the M protein are N32S, N49D, M51R, H54Y, A133T, V221F, V225I, and S226R; 25) The amino acid substitutions in the M protein are N32S, M33A, N49D, M51R, H54Y, A133T, V221F, V225I, and S226R; 26) The amino acid substitutions in the M protein are G21E, N32S, N49D, M51A, H54Y, L111A, V225I, and S226R; The oncolytic virus further comprises a G protein, which comprises amino acid substitutions at one or more of positions 438, 453, 471, and 487 compared to the amino acid sequence set forth in SEQ ID NO: 31, or at one or more of positions 53, 141, 172, 217, 232, 331, 371, and 436; and the oncolytic virus further comprises an N protein, which comprises amino acid substitutions at one or more of positions 14, 155, and 353 compared to the amino acid sequence set forth in SEQ ID NO:

55. An oncolytic virus characterized by:

12. The amino acid substitutions in the N protein include an isoleucine to valine mutation at position 14 (I14V), and / or an arginine to lysine mutation at position 155 (R155K), and / or a serine to asparagine mutation at position 353 (S353N).

12. The oncolytic virus according to claim 10 or 11.

13. The amino acid substitution in the N protein is selected as any one of the following: 1) the amino acid substitution in the N protein comprises I14V; 2) the amino acid substitutions in the N protein include I14V and R155K; 3) the amino acid substitutions in the N protein include I14V, R155K, and S353N; 4) the amino acid substitutions of the N protein include R155K and S353N; 5) The amino acid substitution in the N protein includes S353N. The oncolytic virus according to claim 10 or 11.

14. The amino acid substitutions in the N protein include I14V, R155K, and S353N. The oncolytic virus of claim 13 .

15. The N protein comprises the amino acid sequence set forth in SEQ ID NO:58 The oncolytic virus of claim 13 .

16. an oncolytic virus, The oncolytic virus is a vesicular stomatitis virus Indiana MudSummer subtype and comprises an M protein, wherein the M protein comprises an amino acid substitution selected from any one of the following amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO: 1: 1) the amino acid substitutions in the M protein are G21E and N32S; 2) the amino acid substitutions in the M protein are G21E, N32S, and M33A; 3) the amino acid substitutions in the M protein are G21E, N32S, M33A, and N49D; 4) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, and H54Y; 5) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, H54Y, and L111A; 6) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, H54Y, L111A, and A133T; 7) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, H54Y, L111A, A133T, and V225I; 8) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, M51R, H54Y, L111A, A133T, and V225I; 9) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, M51R, H54Y, L111A, A133T, V221F, and V225I; 10) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 11) The amino acid substitutions in the M protein are N32S, M33A, N49D, M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 12) The amino acid substitutions in the M protein are M33A, N49D, M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 13) The amino acid substitutions in the M protein are N49D, M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 14) The amino acid substitutions in the M protein are M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 15) The amino acid substitutions in the M protein are H54Y, L111A, A133T, V221F, V225I, and S226R; 16) The amino acid substitutions in the M protein are L111A, A133T, V221F, V225I, and S226R; 17) The amino acid substitutions in the M protein are A133T, V221F, V225I, and S226R; 18) The amino acid substitutions in the M protein are V221F, V225I, and S226R; 19) The amino acid substitutions in the M protein are V225I and S226R; 20) The amino acid substitution in the M protein is S226R; 21) The amino acid substitutions in the M protein are N32S, N49D, H54Y, V225I, and S226G; 22) The amino acid substitutions in the M protein are N32S, N49D, M51R, H54Y, V221F, V225I, and S226R; 23) The amino acid substitutions in the M protein are N32S, M33A, N49D, M51R, H54Y, V221F, V225I, and S226R; 24) The amino acid substitutions in the M protein are N32S, N49D, M51R, H54Y, A133T, V221F, V225I, and S226R; 25) The amino acid substitutions in the M protein are N32S, M33A, N49D, M51R, H54Y, A133T, V221F, V225I, and S226R; 26) The amino acid substitutions in the M protein are G21E, N32S, N49D, M51A, H54Y, L111A, V225I, and S226R; The oncolytic virus further comprises a P protein, and the P protein comprises amino acid substitutions at one or more of positions 50, 76, 99, 126, 140, 151, 168, 170, 189, and 237 compared to the amino acid sequence set forth in SEQ ID NO:

61. An oncolytic virus characterized by:

17. an oncolytic virus, The oncolytic virus is a vesicular stomatitis virus Indiana MudSummer subtype and comprises an M protein, wherein the M protein comprises an amino acid substitution selected from any one of the following amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO: 1: 1) the amino acid substitutions in the M protein are G21E and N32S; 2) the amino acid substitutions in the M protein are G21E, N32S, and M33A; 3) the amino acid substitutions in the M protein are G21E, N32S, M33A, and N49D; 4) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, and H54Y; 5) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, H54Y, and L111A; 6) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, H54Y, L111A, and A133T; 7) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, H54Y, L111A, A133T, and V225I; 8) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, M51R, H54Y, L111A, A133T, and V225I; 9) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, M51R, H54Y, L111A, A133T, V221F, and V225I; 10) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 11) The amino acid substitutions in the M protein are N32S, M33A, N49D, M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 12) The amino acid substitutions in the M protein are M33A, N49D, M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 13) The amino acid substitutions in the M protein are N49D, M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 14) The amino acid substitutions in the M protein are M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 15) The amino acid substitutions in the M protein are H54Y, L111A, A133T, V221F, V225I, and S226R; 16) The amino acid substitutions in the M protein are L111A, A133T, V221F, V225I, and S226R; 17) The amino acid substitutions in the M protein are A133T, V221F, V225I, and S226R; 18) The amino acid substitutions in the M protein are V221F, V225I, and S226R; 19) The amino acid substitutions in the M protein are V225I and S226R; 20) The amino acid substitution in the M protein is S226R; 21) The amino acid substitutions in the M protein are N32S, N49D, H54Y, V225I, and S226G; 22) The amino acid substitutions in the M protein are N32S, N49D, M51R, H54Y, V221F, V225I, and S226R; 23) The amino acid substitutions in the M protein are N32S, M33A, N49D, M51R, H54Y, V221F, V225I, and S226R; 24) The amino acid substitutions in the M protein are N32S, N49D, M51R, H54Y, A133T, V221F, V225I, and S226R; 25) The amino acid substitutions in the M protein are N32S, M33A, N49D, M51R, H54Y, A133T, V221F, V225I, and S226R; 26) The amino acid substitutions in the M protein are G21E, N32S, N49D, M51A, H54Y, L111A, V225I, and S226R; The oncolytic virus further comprises a G protein, which comprises amino acid substitutions at one or more of positions 438, 453, 471, and 487 compared to the amino acid sequence set forth in SEQ ID NO: 31, or at one or more of positions 53, 141, 172, 217, 232, 331, 371, and 436; and the oncolytic virus further comprises a P protein, which comprises amino acid substitutions at one or more of positions 50, 76, 99, 126, 140, 151, 168, 170, 189, and 237 compared to the amino acid sequence set forth in SEQ ID NO:

61. An oncolytic virus characterized by:

18. an oncolytic virus, The oncolytic virus is a vesicular stomatitis virus Indiana MudSummer subtype and comprises an M protein, wherein the M protein comprises an amino acid substitution selected from any one of the following amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO: 1: 1) the amino acid substitutions in the M protein are G21E and N32S; 2) the amino acid substitutions in the M protein are G21E, N32S, and M33A; 3) the amino acid substitutions in the M protein are G21E, N32S, M33A, and N49D; 4) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, and H54Y; 5) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, H54Y, and L111A; 6) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, H54Y, L111A, and A133T; 7) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, H54Y, L111A, A133T, and V225I; 8) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, M51R, H54Y, L111A, A133T, and V225I; 9) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, M51R, H54Y, L111A, A133T, V221F, and V225I; 10) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 11) The amino acid substitutions in the M protein are N32S, M33A, N49D, M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 12) The amino acid substitutions in the M protein are M33A, N49D, M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 13) The amino acid substitutions in the M protein are N49D, M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 14) The amino acid substitutions in the M protein are M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 15) The amino acid substitutions in the M protein are H54Y, L111A, A133T, V221F, V225I, and S226R; 16) The amino acid substitutions in the M protein are L111A, A133T, V221F, V225I, and S226R; 17) The amino acid substitutions in the M protein are A133T, V221F, V225I, and S226R; 18) The amino acid substitutions in the M protein are V221F, V225I, and S226R; 19) The amino acid substitutions in the M protein are V225I and S226R; 20) The amino acid substitution in the M protein is S226R; 21) The amino acid substitutions in the M protein are N32S, N49D, H54Y, V225I, and S226G; 22) The amino acid substitutions in the M protein are N32S, N49D, M51R, H54Y, V221F, V225I, and S226R; 23) The amino acid substitutions in the M protein are N32S, M33A, N49D, M51R, H54Y, V221F, V225I, and S226R; 24) The amino acid substitutions in the M protein are N32S, N49D, M51R, H54Y, A133T, V221F, V225I, and S226R; 25) The amino acid substitutions in the M protein are N32S, M33A, N49D, M51R, H54Y, A133T, V221F, V225I, and S226R; 26) The amino acid substitutions in the M protein are G21E, N32S, N49D, M51A, H54Y, L111A, V225I, and S226R; The oncolytic virus further comprises a G protein, wherein the G protein comprises amino acid substitutions at one or more of positions 438, 453, 471, and 487, or at one or more of positions 53, 141, 172, 217, 232, 331, 371, and 436, compared to the amino acid sequence set forth in SEQ ID NO: 31; the oncolytic virus further comprises an N protein; The N protein comprises amino acid substitutions at one or more of positions 14, 155, and 353 compared to the amino acid sequence set forth in SEQ ID NO: 55, and the oncolytic virus further comprises a P protein, wherein the P protein comprises amino acid substitutions at one or more of positions 50, 76, 99, 126, 140, 151, 168, 170, 189, and 237 compared to the amino acid sequence set forth in SEQ ID NO:

61. An oncolytic virus characterized by:

19. The amino acid substitutions in the P protein include an arginine to lysine mutation at position 50 (R50K), and / or a valine to alanine mutation at position 76 (V76A), and / or an asparagine to glutamic acid mutation at position 99 (D99E), and / or a leucine to serine mutation at position 126 (L126S), and / or a leucine to serine mutation at position 140 (L140S), and and / or a histidine to tyrosine mutation at position 151 (H151Y), and / or an isoleucine to methionine mutation at position 168 (I168M), and / or a lysine to glutamic acid mutation at position 170 (K170E), and / or a tyrosine to serine mutation at position 189 (Y189S), and / or an asparagine to aspartic acid mutation at position 237 (N237D).

19. An oncolytic virus according to any one of claims 16 to 18.

20. The amino acid substitution in the P protein is selected as any one of the following: 1) the amino acid substitution in the P protein comprises R50K; 2) the amino acid substitutions of the P protein include R50K and V76A; 3) the amino acid substitutions of the P protein include R50K, V76A, and D99E; 4) the amino acid substitutions of the P protein include R50K, V76A, D99E, and L126S; 5) the amino acid substitutions of the P protein include R50K, V76A, D99E, L126S, and L140S; 6) the amino acid substitutions of the P protein include R50K, V76A, D99E, L126S, L140S, and H151Y; 7) The amino acid substitutions of the P protein include R50K, V76A, D99E, L126S, L140S, H151Y, and I168M; 8) The amino acid substitutions of the P protein include R50K, V76A, D99E, L126S, L140S, H151Y, I168M, and K170E; 9) The amino acid substitutions of the P protein include R50K, V76A, D99E, L126S, L140S, H151Y, I168M, K170E, and Y189S; 10) The amino acid substitutions of the P protein include R50K, V76A, D99E, L126S, L140S, H151Y, I168M, K170E, Y189S, and N237D; 11) The amino acid substitutions of the P protein include V76A, D99E, L126S, L140S, H151Y, I168M, K170E, Y189S, and N237D; 12) The amino acid substitutions of the P protein include D99E, L126S, L140S, H151Y, I168M, K170E, Y189S, and N237D; 13) The amino acid substitutions of the P protein include L126S, L140S, H151Y, I168M, K170E, Y189S, and N237D; 14) The amino acid substitutions of the P protein include L140S, H151Y, I168M, K170E, Y189S, and N237D; 15) The amino acid substitutions of the P protein include H151Y, I168M, K170E, Y189S, and N237D; 16) The amino acid substitutions of the P protein include I168M, K170E, Y189S, and N237D; 17) The amino acid substitutions of the P protein include K170E, Y189S, and N237D; 18) The amino acid substitutions of the P protein include Y189S and N237D; 19) The amino acid substitution of the P protein includes N237D.

19. An oncolytic virus according to any one of claims 16 to 18.

21. The amino acid substitutions in the P protein include R50K, V76A, D99E, L126S, L140S, H151Y, I168M, K170E, Y189S, and N237D. The oncolytic virus of claim 20.

22. The P protein comprises the amino acid sequence set forth in SEQ ID NO:

71. The oncolytic virus of claim 20.

23. an oncolytic virus, The oncolytic virus is a vesicular stomatitis virus Indiana MudSummer subtype and comprises an M protein, wherein the M protein comprises an amino acid substitution selected from any one of the following amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO: 1: 1) the amino acid substitutions in the M protein are G21E and N32S; 2) the amino acid substitutions in the M protein are G21E, N32S, and M33A; 3) the amino acid substitutions in the M protein are G21E, N32S, M33A, and N49D; 4) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, and H54Y; 5) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, H54Y, and L111A; 6) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, H54Y, L111A, and A133T; 7) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, H54Y, L111A, A133T, and V225I; 8) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, M51R, H54Y, L111A, A133T, and V225I; 9) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, M51R, H54Y, L111A, A133T, V221F, and V225I; 10) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 11) The amino acid substitutions in the M protein are N32S, M33A, N49D, M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 12) The amino acid substitutions in the M protein are M33A, N49D, M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 13) The amino acid substitutions in the M protein are N49D, M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 14) The amino acid substitutions in the M protein are M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 15) The amino acid substitutions in the M protein are H54Y, L111A, A133T, V221F, V225I, and S226R; 16) The amino acid substitutions in the M protein are L111A, A133T, V221F, V225I, and S226R; 17) The amino acid substitutions in the M protein are A133T, V221F, V225I, and S226R; 18) The amino acid substitutions in the M protein are V221F, V225I, and S226R; 19) The amino acid substitutions in the M protein are V225I and S226R; 20) The amino acid substitution in the M protein is S226R; 21) The amino acid substitutions in the M protein are N32S, N49D, H54Y, V225I, and S226G; 22) The amino acid substitutions in the M protein are N32S, N49D, M51R, H54Y, V221F, V225I, and S226R; 23) The amino acid substitutions in the M protein are N32S, M33A, N49D, M51R, H54Y, V221F, V225I, and S226R; 24) The amino acid substitutions in the M protein are N32S, N49D, M51R, H54Y, A133T, V221F, V225I, and S226R; 25) The amino acid substitutions in the M protein are N32S, M33A, N49D, M51R, H54Y, A133T, V221F, V225I, and S226R; 26) The amino acid substitutions in the M protein are G21E, N32S, N49D, M51A, H54Y, L111A, V225I, and S226R; The oncolytic virus further comprises an L protein, and the L protein comprises an amino acid substitution at one or more of positions 87 and 487 compared to the amino acid sequence set forth in SEQ ID NO:

81. An oncolytic virus characterized by:

24. An oncolytic virus comprising: The oncolytic virus is a vesicular stomatitis virus Indiana MudSummer subtype and comprises an M protein, wherein the M protein comprises an amino acid substitution selected from any one of the following amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO: 1: 1) the amino acid substitutions in the M protein are G21E and N32S; 2) the amino acid substitutions in the M protein are G21E, N32S, and M33A; 3) the amino acid substitutions in the M protein are G21E, N32S, M33A, and N49D; 4) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, and H54Y; 5) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, H54Y, and L111A; 6) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, H54Y, L111A, and A133T; 7) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, H54Y, L111A, A133T, and V225I; 8) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, M51R, H54Y, L111A, A133T, and V225I; 9) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, M51R, H54Y, L111A, A133T, V221F, and V225I; 10) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 11) The amino acid substitutions in the M protein are N32S, M33A, N49D, M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 12) The amino acid substitutions in the M protein are M33A, N49D, M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 13) The amino acid substitutions in the M protein are N49D, M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 14) The amino acid substitutions in the M protein are M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 15) The amino acid substitutions in the M protein are H54Y, L111A, A133T, V221F, V225I, and S226R; 16) The amino acid substitutions in the M protein are L111A, A133T, V221F, V225I, and S226R; 17) The amino acid substitutions in the M protein are A133T, V221F, V225I, and S226R; 18) The amino acid substitutions in the M protein are V221F, V225I, and S226R; 19) The amino acid substitutions in the M protein are V225I and S226R; 20) The amino acid substitution in the M protein is S226R; 21) The amino acid substitutions in the M protein are N32S, N49D, H54Y, V225I, and S226G; 22) The amino acid substitutions in the M protein are N32S, N49D, M51R, H54Y, V221F, V225I, and S226R; 23) The amino acid substitutions in the M protein are N32S, M33A, N49D, M51R, H54Y, V221F, V225I, and S226R; 24) The amino acid substitutions in the M protein are N32S, N49D, M51R, H54Y, A133T, V221F, V225I, and S226R; 25) The amino acid substitutions in the M protein are N32S, M33A, N49D, M51R, H54Y, A133T, V221F, V225I, and S226R; 26) The amino acid substitutions in the M protein are G21E, N32S, N49D, M51A, H54Y, L111A, V225I, and S226R; The oncolytic virus further comprises a G protein, which comprises amino acid substitutions at one or more of positions 438, 453, 471, and 487 compared to the amino acid sequence set forth in SEQ ID NO: 31, or at one or more of positions 53, 141, 172, 217, 232, 331, 371, and 436; and the oncolytic virus further comprises an L protein, which comprises amino acid substitutions at one or more of positions 87 and 487 compared to the amino acid sequence set forth in SEQ ID NO:

81. An oncolytic virus characterized by:

25. An oncolytic virus comprising: The oncolytic virus is a vesicular stomatitis virus Indiana MudSummer subtype and comprises an M protein, wherein the M protein comprises an amino acid substitution selected from any one of the following amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO: 1: 1) the amino acid substitutions in the M protein are G21E and N32S; 2) the amino acid substitutions in the M protein are G21E, N32S, and M33A; 3) the amino acid substitutions in the M protein are G21E, N32S, M33A, and N49D; 4) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, and H54Y; 5) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, H54Y, and L111A; 6) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, H54Y, L111A, and A133T; 7) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, H54Y, L111A, A133T, and V225I; 8) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, M51R, H54Y, L111A, A133T, and V225I; 9) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, M51R, H54Y, L111A, A133T, V221F, and V225I; 10) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 11) The amino acid substitutions in the M protein are N32S, M33A, N49D, M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 12) The amino acid substitutions in the M protein are M33A, N49D, M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 13) The amino acid substitutions in the M protein are N49D, M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 14) The amino acid substitutions in the M protein are M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 15) The amino acid substitutions in the M protein are H54Y, L111A, A133T, V221F, V225I, and S226R; 16) The amino acid substitutions in the M protein are L111A, A133T, V221F, V225I, and S226R; 17) The amino acid substitutions in the M protein are A133T, V221F, V225I, and S226R; 18) The amino acid substitutions in the M protein are V221F, V225I, and S226R; 19) The amino acid substitutions in the M protein are V225I and S226R; 20) The amino acid substitution in the M protein is S226R; 21) The amino acid substitutions in the M protein are N32S, N49D, H54Y, V225I, and S226G; 22) The amino acid substitutions in the M protein are N32S, N49D, M51R, H54Y, V221F, V225I, and S226R; 23) The amino acid substitutions in the M protein are N32S, M33A, N49D, M51R, H54Y, V221F, V225I, and S226R; 24) The amino acid substitutions in the M protein are N32S, N49D, M51R, H54Y, A133T, V221F, V225I, and S226R; 25) The amino acid substitutions in the M protein are N32S, M33A, N49D, M51R, H54Y, A133T, V221F, V225I, and S226R; 26) The amino acid substitutions in the M protein are G21E, N32S, N49D, M51A, H54Y, L111A, V225I, and S226R; The oncolytic virus further comprises a G protein, which comprises amino acid substitutions at one or more of positions 438, 453, 471, and 487 compared to the amino acid sequence set forth in SEQ ID NO: 31, or at one or more of positions 53, 141, 172, 217, 232, 331, 371, and 436; the oncolytic virus further comprises an N protein, which comprises amino acid substitutions at one or more of positions 14, 155, and 353 compared to the amino acid sequence set forth in SEQ ID NO: 55; and the oncolytic virus further comprises an L protein, which comprises amino acid substitutions at one or more of positions 87 and 487 compared to the amino acid sequence set forth in SEQ ID NO:

81. An oncolytic virus characterized by:

26. An oncolytic virus comprising: The oncolytic virus is a vesicular stomatitis virus Indiana MudSummer subtype and comprises an M protein, wherein the M protein comprises an amino acid substitution selected from any one of the following amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO: 1: 1) the amino acid substitutions in the M protein are G21E and N32S; 2) the amino acid substitutions in the M protein are G21E, N32S, and M33A; 3) the amino acid substitutions in the M protein are G21E, N32S, M33A, and N49D; 4) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, and H54Y; 5) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, H54Y, and L111A; 6) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, H54Y, L111A, and A133T; 7) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, H54Y, L111A, A133T, and V225I; 8) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, M51R, H54Y, L111A, A133T, and V225I; 9) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, M51R, H54Y, L111A, A133T, V221F, and V225I; 10) the amino acid substitutions in the M protein are G21E, N32S, M33A, N49D, M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 11) The amino acid substitutions in the M protein are N32S, M33A, N49D, M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 12) The amino acid substitutions in the M protein are M33A, N49D, M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 13) The amino acid substitutions in the M protein are N49D, M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 14) The amino acid substitutions in the M protein are M51R, H54Y, L111A, A133T, V221F, V225I, and S226R; 15) The amino acid substitutions in the M protein are H54Y, L111A, A133T, V221F, V225I, and S226R; 16) The amino acid substitutions in the M protein are L111A, A133T, V221F, V225I, and S226R; 17) The amino acid substitutions in the M protein are A133T, V221F, V225I, and S226R; 18) The amino acid substitutions in the M protein are V221F, V225I, and S226R; 19) The amino acid substitutions in the M protein are V225I and S226R; 20) The amino acid substitution in the M protein is S226R; 21) The amino acid substitutions in the M protein are N32S, N49D, H54Y, V225I, and S226G; 22) The amino acid substitutions in the M protein are N32S, N49D, M51R, H54Y, V221F, V225I, and S226R; 23) The amino acid substitutions in the M protein are N32S, M33A, N49D, M51R, H54Y, V221F, V225I, and S226R; 24) The amino acid substitutions in the M protein are N32S, N49D, M51R, H54Y, A133T, V221F, V225I, and S226R; 25) The amino acid substitutions in the M protein are N32S, M33A, N49D, M51R, H54Y, A133T, V221F, V225I, and S226R; 26) The amino acid substitutions in the M protein are G21E, N32S, N49D, M51A, H54Y, L111A, V225I, and S226R; The oncolytic virus further comprises a G protein, wherein the G protein comprises amino acid substitutions at one or more of positions 438, 453, 471, and 487 compared to the amino acid sequence set forth in SEQ ID NO: 31, or amino acid substitutions at one or more of positions 53, 141, 172, 217, 232, 331, 371, and 436; the oncolytic virus further comprises an N protein, wherein the N protein comprises amino acid substitutions at positions 14, 155, 353, and 487 compared to the amino acid sequence set forth in SEQ ID NO:

55. the oncolytic virus further comprises a P protein, wherein the P protein comprises amino acid substitutions at one or more of positions 50, 76, 99, 126, 140, 151, 168, 170, 189, and 237 compared to the amino acid sequence set forth in SEQ ID NO:61; and the oncolytic virus further comprises an L protein, wherein the L protein comprises amino acid substitutions at one or more of positions 87 and 487 compared to the amino acid sequence set forth in SEQ ID NO:

81. An oncolytic virus characterized by:

27. The amino acid substitutions in the L protein include a serine to proline mutation at position 87 (S87P) and / or an isoleucine to threonine mutation at position 487 (I487T).

27. An oncolytic virus according to any one of claims 23 to 26.

28. The amino acid substitution in the L protein is selected from any one of the following: 1) the amino acid substitution in the L protein comprises S87P; 2) the amino acid substitutions in the L protein include S87P and I487T; 3) The amino acid substitution in the L protein includes I487T.

27. An oncolytic virus according to any one of claims 23 to 26.

29. The amino acid substitutions in the L protein include S87P and I487T. The oncolytic virus of claim 28.

30. The N protein comprises the amino acid sequence set forth in SEQ ID NO:

83. The oncolytic virus of claim 28.

31. The oncolytic virus contains or expresses an exogenous nucleic acid, target gene or protein.

27. An oncolytic virus according to any one of claims 1 to 5, 10 to 11, 16 to 18, and 23 to 26.

32. The oncolytic virus comprises a nucleic acid molecule, the nucleic acid molecule comprising a nucleic acid sequence encoding an M protein having the amino acid substitution, and / or a nucleic acid sequence encoding a G protein having the amino acid substitution, and / or a nucleic acid sequence encoding an N protein having the amino acid substitution, and / or a nucleic acid sequence encoding a P protein having the amino acid substitution, and / or a nucleic acid sequence encoding an L protein having the amino acid substitution.

27. The oncolytic virus of claim 26.

33. The nucleic acid molecule comprises an exogenous nucleic acid, a nucleic acid sequence encoding a target gene or a protein. The oncolytic virus of claim 32.

34. Among the nucleic acid molecules, the nucleic acid sequence encoding the exogenous nucleic acid, target gene, or protein is located between the nucleic acid sequence encoding the M protein having the amino acid substitution, and / or the nucleic acid sequence encoding the G protein having the amino acid substitution, and / or the nucleic acid sequence encoding the N protein having the amino acid substitution, and / or the nucleic acid sequence encoding the P protein having the amino acid substitution, and / or the nucleic acid sequence encoding the L protein having the amino acid substitution.

34. The oncolytic virus of claim 33.

35. 1. An oncolytic virus expression vector, comprising: The oncolytic virus expression vector is capable of expressing the oncolytic virus of claim 1. An oncolytic virus expression vector characterized by:

36. A virus-producing cell, The virus-producing cells are capable of producing the oncolytic virus of claim 1. A virus-producing cell characterized by:

37. 1. A pharmaceutical composition comprising: The pharmaceutical composition comprises the oncolytic virus of claim 1 and, optionally, a pharmaceutically acceptable carrier. A pharmaceutical composition comprising:

38. A method for preparing an oncolytic virus described in any one of claims 1 to 5, 10 to 11, 16 to 18, and 23 to 26, an oncolytic virus expression vector described in claim 35, a virus-producing cell described in claim 36, and / or a pharmaceutical composition described in claim 37.

39. A method for producing a drug for preventing and / or treating tumors or cancer using an oncolytic virus described in any one of claims 1 to 5, 10 to 11, 16 to 18, and 23 to 26, an oncolytic virus expression vector described in claim 35, a virus-producing cell described in claim 36, and / or a pharmaceutical composition described in claim 37.

40. The oncolytic virus, the oncolytic virus expression vector, the virus-producing cell and / or the pharmaceutical composition are used in a method for continuously killing abnormally proliferating cells.

40. The method of claim 39.

41. The abnormally proliferative cells are selected from tumor cells or tumor tissue-associated cells.

41. The method of claim 40.

42. The tumor may be a solid tumor or a hematologic tumor.

42. The method of claim 41 .

43. The pharmaceutical composition described in claim 37, which is a pharmaceutical composition for preventing and / or treating tumors or cancer.

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