RU2562115C1 - Newcastle disease virus strain for studying oncolytic properties and mechanisms of oncolysis to create prototype anticancer preparation - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: characterised strain is isolated from cloacal swab of clinically healthy rock pigeon. The isolated strain passed 3 passages on developed chicken embryos and is deposited at the State collection of viruses of the Research institute of virology n.a. D.I. Ivanovsky of the Russian Ministry of Health under the registration number 2778.

EFFECT: strain is capable to lyze selectively cancer cells, without causing infectious disease and death of mammals and can be used to study oncolytic properties and mechanisms of oncolysis with prospect of creation of a prototype anticancer preparation used in virology, microbiology, veterinary medicine and oncology.

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Description

Technical field

The present invention relates to oncolytic strains of the Newcastle disease virus and can be used in virology, microbiology, veterinary medicine and oncology to study the biological, virological and oncolytic properties in order to create a prototype anticancer drug.

State of the art

According to the World Health Organization, more than five million people die from malignant neoplasms annually in the world and about three million cases of new diseases are recorded. According to forecasts, the annual number of deaths from cancer will only increase and by 2030 will reach 13 million cases per year (WHO (World Health Organization) (World Health Statictics, 2012 - 2012. - p. 176. http://www.who.int /gho/publications/world_health_statistics/EN_WHS2012_Part2.pdf) [1].

To date, the cure rate of cancer is only 26%. Oncology mortality rates in the Novosibirsk region are among the highest in Siberia and exceed the average level in Russia. In the dynamics of the detectability and mortality of the population from cancer in Novosibirsk, there has been a steady increase.

The development of highly effective methods of treating malignant neoplasms remains an urgent task of modern medicine. Despite the significant advances in modern therapies in this area, the ability to detect tumors in the early stages and selectively affect them remains extremely limited.

The main disadvantage of the currently used methods of chemotherapy and radiotherapy is their low therapeutic index (TI). This indicator characterizes the difference in the degree of effect of the corresponding therapeutic agent on tumor and normal cells of the body (in a simplified form, the ratio of the number of killed cancer cells to the number of killed normal cells). The therapeutic index of modern chemotherapeutic agents does not exceed 6: 1.

According to the opinion adopted by the international scientific community, TIs of drugs targeted by bacterial vectors and oncolytic viruses can reach 100,000: 1, which is due to their high specificity for tumor cells.

The idea of the possibility of using viruses for the treatment of tumors arose in the first half of the 20th century. However, the date of birth of modern "virotherapy" is considered to be 1991, when the journal "Science" published the results of a study of the oncolytic potential of genetically modified herpes simplex virus type 1 (Martuza RL et al. Experimental therapy of human glioma by means of a geneticaly engineered virus mutant // Science. - 1991. - Vol. 252 (5007). - Pages. 854-856) [2]. To date, in the world, as a result of large-scale studies, a number of promising drugs based on various oncolytic viruses have been obtained. Nevertheless, to date, the receipt of such drugs is associated with complex manipulations at the genetic level. We should not forget that to create existing anticancer drugs based on viruses, mammalian viruses were used, which are conditionally pathogenic for humans and can significantly increase their pathogenicity during evolution or due to spontaneous mutagenesis.

Description of analogues

Currently, the presence of oncolytic activity in the Newcastle disease virus (hereinafter - VBI) is known. It is able to selectively lyse cancer cells, while remaining safe for normal mammalian tissue cells. Several studies have demonstrated the ability of this virus to specific oncolysis. A significant difference was found in the rate of virus replication in tumor and normal mammalian cells (Zhan X. et al. Sendai virus recombinant vaccine expressing hPIV-3 HN or Flicits protective immunity and combines with a second recombinant to prevent hPIV-1, hPIV-3 and RSV infections // Vaccine. - 2008. - Vol. 26 (27-28). - Pages. 3480-3488. Doi: 10.1016 / j.vaccine.2008.04.022.) [3].

There is an assumption that the high tropism of VBI to tumor cells is due to impaired synthesis of proteins responsible for the elimination of VBI (Elankumaran S. et al. Newcastle disease virus exerts oncolysis by both intrinsic and extrinsic caspase-dependent pathways of cell death // J Virol. - 2006 . - Vol. 80 (15). - P. 7522-7534. Doi: 10.1128 / JVI.00241-06.) [4].

There are analogues in America, however, information about these strains, their properties and the possibility of use is currently not in the public domain. Nevertheless, antitumor drugs based on the VBI line PV1703 and Cassell73 undergo the first stage of clinical trials (Ravindra PV et al. Newcastle disease virus as an oncolytic agent // Indian J Med Res. - 2009. - Vol. 130 (5). - Pages . 507-513) [5].

The mechanism of the oncolytic action of this virus is still not fully understood, although in recent years new works have appeared that describe the mechanisms of the virus entering the cell (Cantin C, Holguera J., Ferreira L., Villar,., Munoz-Barroso IJ Gen. Virol. 2007 Vol. 88. Pt. 2. P. 559-569) [6], induction of tumor cell death in response to virus infection (Fabian ian., Csatary CM., Szeberenyi J., Csatary LK p53-independent endoplasmic reticulum stress- mediated cytotoxicity of a Newcastle disease virus strain in tumor cell lines // J. Virol. 2007. Vol. 81. N. 6. P. 2817-2830) [7], replication of VBI (Puhlmann J., Puehler F., Mumberg D., Boukamp P., Beier R. Racl is required for oncolytic NDV replication in human cancer cells and establishes a link between tumorigenesis and se nsitivity to oncolytic virus // Oncogene. 2010. Vol. 29. N. 15. P. 2205-2216) [8].

At present, some vaccines attenuated by selection of VBI strains have passed several phases of clinical trials in the USA, Canada, China and European countries (Germany, Hungary). (Bai FL, Yu YH, Tian Η., Ren GP, Wang Η., Zhou B., Han XH, Yu QZ, Li DS Genetically engineered Newcastle disease virus expressing interleukin-2 and TNF-related apoptosis-inducing ligand for cancer therapy // Cancer Biol. Ther. 2014. Vol. 15. N. 9. P. 1226-1238) [9]; (Zamarin D., Palese P. Oncolytic Newcastle disease virus for cancer therapy: old challenges and new directions // Future Microbiol. 2012. Vol. 7. N. 3. P. 347-367) [10].

They demonstrated the safety of the studied strains for humans and the ability to suppress the development of tumors and prolong the life of tumor patients. On the other hand, based on the high preclinical efficacy of oncolytic viruses against tumor cell cultures in vitro and in vivo, scientists expect much more results from clinical trials.

Thus, obtaining a domestic producer strain of VBN to create a drug with oncolytic activity on its basis is promising. The relevance of this is confirmed by the absence in Russia of commercial analogues of such strains.

The objective of the invention is to obtain a new strain of Newcastle disease virus that is able to selectively lyse cancer cells of mammals, without causing an infectious disease and death of mammals to study the oncolytic properties and mechanisms of oncolysis to create a prototype antitumor drug.

The solution to this problem is achieved by obtaining a strain of Newcastle disease virus NDV / Altai / 770/2011.

The technical result of the invention is the establishment of oncolytic properties and oncolysis mechanisms of the obtained new strain NDV / Altai / pigeon / 770/2011 of the Newcastle disease virus under conditions of rejection of excessive attenuation and the use of a natural type of strain VBN with the prospect of creating a prototype anticancer drug used in virology, microbiology , veterinary medicine and oncology.

Disclosure of invention

Characterization of the claimed strain.

Strain NDV / Altai / pigeon / 770/2011 is a representative of the Newcastle disease virus, genus Avulavirus, family Paromyxoviridae. It belongs to the group of pigeon paramyxovirus type 1 strains (GPMV-1) associated with pigeon infection. The strain is deposited in the State virus collection Research Institute of Virology. D.I. Ivanovsky of the Ministry of Health of Russia under registration number 2778 (July 7, 2014).

Source of receipt. The Newcastle disease virus isolate was originally isolated from the cloacal flush of a clinically healthy gray pigeon (Columba livid). Material was collected in the fall of 2011 in the Altai Territory, Russian Federation. The Newcastle disease virus isolate was named NDV / Altai / pigeon / 770/2011.

The isolated strain passed 3 passages on developing chicken embryos (RCE). Indication of the virus was carried out in a hemagglutination reaction (RGA) with rooster erythrocytes: the hemagglutinating activity indicator was 128 GAU / 50 μl.

Virus identification. The primary identification of isolated Newcastle disease virus was carried out by RT-PCR with the detection of the results in a closed tube in real time using primers and a probe specific to the conserved regions of the M gene of Newcastle disease virus, according to (Miller PJ et al. Newcastle disease: evolution of genotypes and the related diagnostic challenges // Infect. Genet. Evol. 2010. Vol. 10. N. LP. 26-35) [11].

Full genome sequencing and phylogenetic analysis allowed the VBN strain to be assigned to class IIb VIb genotype (GenBank: KJ920204). Appendix I.

Cultural properties. Strain NDV / Altai / pigeon / 770/2011 during cultivation of a primary culture of chicken embryo fibroblast cells (FEC) on a monolayer causes a cytopathic effect (CPD) for 2-3 days (cultivation temperature 37 ° C, in an atmosphere of 5% CO ₂ ).

Pathogenicity for humans. The strains of the Newcastle disease virus are not pathogenic for humans (Cassel W. A., Garrett R. E. Newcastle disease virus as an antineoplastic agent // Cancer. - 1965. - Vol. 18. - P. 863-868) [12], Laurie S.A. et al. A phase 1 clinical study of intravenous administration of PV701, anoncolytic virus, using two-step desensitization // Clin Cancer Res. - 2006. - Vol. 12 (8). - P. 2555-2562) [13].

Pathogenicity for poultry. Newcastle disease virus strains are classified as lentogenic (weakly pathogenic), mesogenic and velogenic (highly pathogenic) (ΟΙΕ, World Organization for Animal Health, Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, Paris, France, 7th edition, 2012, Vol. 1-2 , Ch. 2.03.14. Available at http://ww.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.03.14_NEWCASTLE_DIS.pdf) [14]. Strain NDV / Altai / pigeon / 770/2011 refers to the mid-pathogenic (mesogenic) variant of the Newcastle disease virus. Infectious activity of the isolated virus was 7.2 ± 0.09 lg EID ₅₀ / ml.

The pathogenicity of the strain was determined according to standard methods in tests for determining the average life time (MDT) of SPF-developing chicken embryos and the intracerebral index of pathogenicity of day old chicks [14].

The average lifetime of MDT = 76 hours. The index of intracerebral pathogenicity ICRI = 0.675.

Pathogenicity for mammals. The strain does not show pathogenicity for mammals (Alexander D.J. Newcastle disease virus and other avian paramyxoviruses // Rev Sci Tech. - 2000. - Vol. 19 (2). - P. 443-462) [15].

For long-term storage, the strain must be lyophilized using a solution of gelatin and sucrose (SAG) as a protective medium. Lyophilization with the addition of SAGE in a ratio of 1: 4 allows you to maintain a stable infectious activity of the virus for at least 2 years when stored at -70 ° C.

The following nutrient media are used to cultivate the strain in cell culture: MEM needle, DMEM containing 2 μg / ml trypsin, 2 mmol / l glutamine, 100 μg / ml penicillin and 100 IU / ml streptomycin.

List of figures, drawings and other materials

Fig. 1. Magnetic resonance imaging. A series of frontal sections on day 28 from the start of the in vivo experiment on BALB / C mice. Arrows indicate the location of the tumor. 1. Experimental mice that received intratumoral injections of the NDV / Altai / pigeon / 770/2011 virus with a maximum concentration of 5 days: 1a - complete tumor regression compared to control animals; 16 - significant reduction of the tumor compared with control animals. 2. Control mice receiving injections of saline: 1a, 2b.

Fig. 2. A fragment of the central part of the tumor KREBS-2 on the 20th day after a series of intratumoral injections of strain NDV / Altai / 770/2011. Vast fields of necrosis of the tumor tissue (arrows) are visible. Hematoxylin and eosin stain.

Fig. 3. Segmented neutrophils near a blood vessel in a small lesion of KREBS-2 tumor tissue.

Fig. 4. The individual arrows indicate the activated macrophage and lymphocyte. Segmented neutrophils, a lymphocyte were isolated. Hematoxylin and eosin stain.

Fig. 5. KREBS-2 tumor islet in muscle tissue. Hematoxylin and eosin stain.

Fig. 6. Oncolytic effect of VBN strain NDV / Altai / pigeon / 770/2011 on human tumor cell lines and untransformed human peripheral blood mononuclear cells in the in vitro system on the third day after infection.

Fig. 7. A. Immunostaining of control non-virus infected tumor cells of the human colorectal cancer line HCT116. Confocal microscopy, 100x magnification. The nuclei are stained with DAPI. B. Immunostaining of human colorectal cancer cell line HCT116 cells infected with strain NDV / Altai / pigeon / 770/2011 (dilution of 8 GAE / 10,000 cells). The second day after infection with the virus. Confocal microscopy, 100x magnification. The nuclei are stained with DAPI, secondary antibodies are Alex546.

Examples of using the virus to study the possibility of creating on its basis a prototype oncolytic drug.

To identify the oncolytic properties of the NDV / Altai / pigeon / 770/2011 strain, experiments were carried out on the infection of mice with tumor cells, followed by intratumoral injections of the NDV virus strain NDV / Altai / pigeon / 770/2011. The results of this experiment revealed the presence of the proposed VBI strain the ability to neutralize tumor cells, preventing the formation of the tumor. This can occur under the influence of the immune system induced by the presence of the virus, or, more likely, by the direct lysis mechanism of the tumor cells NDV / Altai / pigeon / 770/2011 proposed by the authors. In favor of the second hypothesis is the fact that with the introduction of strain NDV / Altai / pigeon / 770/2011 in an already formed tumor, a decrease in the dynamics of tumor growth was observed. Morphological examination revealed the destruction of tumor tissue by the studied strain.

The obtained data reveal the presence of strain NDV / Altai / pigeon / 770/2011 the ability to specifically destroy mammalian neoplasms in the in vivo system without prior adaptation to the tumor cell culture. This makes this strain a promising agent for creating anticancer drugs based on it through preliminary adaptation with a wide spectrum of interaction and a high therapeutic index.

Example 1. The study of the oncolytic properties of the strain NDV / Altai / pigeon / 770/2011 on a model of a solid tumor of KREBS-2 mice with the introduction of the virus into the formed tumor in vivo.

To assess the effect of VBI on an already formed tumor, the following pilot experiment was performed. Two groups of immunocompetent BALB / C (♂) mice were grafted intramuscularly into the thigh of KREBS-2 ascites carcinoma cells at a concentration of 500,000 cells in 100 μl of phosphate buffer. On the third day after intramuscular inoculation of tumor cells in the thighs of all mice, a seal appeared that was characteristic of the onset of the formation of a solid tumor node. 3 days after infection, with the onset of tumor formation, the control group was started to inject 100 μl of physiological saline solution intramuscularly into the tumor localization area, and 100 μl of live NDV / Altai / pigeon / 770/2011 virus at a concentration of 256 GAE was introduced intratumorally 100 μl Injections of saline and virus continued daily for 5 days. During this period of the experiment, it is difficult to talk about changes in the size of the tumor, its development or the beginning of regression, since the inflammatory process, characterized by swelling, develops in the area of the tumor in the first stages of the introduction of the virus, which prevents a visual assessment of possible changes. However, at later stages, tumors of experimental mice were compared with tumors of control mice. So on the 8th – 9th day of the experiment, it became visually noticeable that the thigh with the tumor of the control mice had a more pronounced spherical shape than the experimental ones. During the experiment, the mice of both groups actively consumed food and water. However, already on days 14-16, control animals became less mobile. On the 28th day from the beginning of the experiment, the prevalence of the intramuscular tumor process was estimated using the method of magnetic resonance imaging (MRI) and tumor material was collected from all mice.

Figure 1 shows a series of frontal sections obtained by MRI, in which the tumor is clearly visualized against a background of healthy tissues. According to the obtained images of sections of the tumor sites, it was possible to identify significant differences in the development of the tumor process between control and experimental animals. Series of frontal sections of the tumor node of the control animals are presented in blocks 1A and 1B of Figure 1. In the case of one experimental mouse that received intratumoral injections of the NDV / Altai / pigeon / 770/2011 virus, almost complete tumor regression was observed compared to the tumor sizes of the control animals. On the block of sections 2A, it can be seen that the hip into which the tumor tissues were grafted does not differ from the healthy one in size and the presence of a tumor node in the MRI image series is not detected. The tumor of another experimental mouse injected with the virus underwent significant reduction compared with the tumors of the control animals that received injections of saline. Images of the tumor node in the thigh of the second experimental mouse are presented on the block section 2B.

On the 28th day after infection, a biomaterial of tumor tissue was collected from mice from each group. Structural analysis at the light-optical level showed the presence of numerous necrosis in the tumor node in mice receiving injections of the NDV / Altai / Pigeon / 770/2011 virus. Figure 2 shows a fragment of the preparation of the tumor node from the experimental mouse. In order to identify histological structures, sections were stained according to the generally accepted method of review staining with hematoxylin-eosin. On the 20th day after the completion of the course of virus injections, in the central part of the KREBS-2 tumor node, extensive fields of tumor tissue necrosis are indicated by arrows.

In the area of blood vessels granulocytes - segmented neutrophils are observed. Between muscle fibers lymphocytes and activated macrophages are also found (see Fig. 3 and Fig. 4). Figure 3 shows segmented neutrophils near a blood vessel in a small lesion of KREBS-2 tumor tissue. In Figure 4, the activated macrophage and lymphocyte are indicated by separate arrows. Segmented neutrophils, a lymphocyte were isolated.

The connective capsule is poorly expressed, small tumor invasions into the underlying tissue are visible. Apoptotic bodies are often found.

A morphological study of tumor material from a mouse, in which almost complete regression of the tumor was observed according to the MRI image, revealed almost complete destruction of the tumor. Separate tumor islands were preserved in muscle tissue, which is shown in Figure 5 (see Fig. 5).

The results of this experiment show the presence of a VBN strain of moderate pathogenicity for birds that can neutralize mammalian tumor cells and prevent tumor formation. This process can occur under the influence of the immune system induced by the presence of the virus, or by the mechanism of direct lysis of tumor cells by viral particles. If the second assumption is considered correct, the presence of oncolytic activity in a strain pathogenic for birds and the absence of it on a non-pathogenic one is explained by the fact that apatogenic strains of VBI are not able to lyse cells, while the studied strain, on the contrary, has this ability, which allows it to lyse cancerous mammalian cells, preventing tumor formation.

Example 2. Assessment of the viability of human tumor cell lines after infection with strain NDV / Altai / pigeon / 770/2011 in the in vitro system.

To assess the cytopathic effect of the virus strain, four human tumor lines were used: HCT116 + / + (colorectal cancer cell line), MCF7 (mammary adenocarcinoma cell line with no caspase-3 expression), Η1299 (non-small cell lung carcinoma cell line), U87 (cell line glioblastomas). Mononuclear cells of human peripheral blood served as a control. Cells were incubated with different dilutions of the virus from 2 to 16 GAE / 10,000 cells. The concentration of the virus was increased in increments of 2 times. Virus cells were incubated for 1 hour at 37 ° C. Control tumor cells were incubated in growth medium without virus for 1 hour.

3 days after the infection of the cell lines with VBI strains, the cell viability analysis was performed by the colometric method using the commercially available CellTiter 96®Aueous One Solution Cell Proliferation Assay kit (Promega, USA). The optical density was measured at a wavelength of 490 nm.

The results of assessing the viability of tumor cell lines are presented as a histogram in Figure 6 as the average relative values of the proportion of living cells on the third day after treatment with virus strains to the fraction of control untreated cells taking into account standard deviation (mean relative value ± standard deviation).

The natural strain NDV / Altai / pigeon / 770/2011 showed a high oncolytic potential relative to all tumor cell lines in humans. The viability of cell lines — colorectal cancer HCT116, glioblastoma U87 and lung cancer H1299 — varies from 40% to 60%. On the third day of infection with this strain, 45% of tumor cells remain viable in colorectal cancer culture, and about 40% in glioblastoma cell culture. For the line of non-small cell lung carcinoma Η1299, this value is 60%. The oncolytic effect of the strain relative to MCF7 breast adenocarcinoma cells was less pronounced, since the viability of this cell line was about 80%. In this case, human peripheral blood mononuclear cells (PBMCs) were practically not susceptible to the action of this strain. Their viability is about 100% (see Fig. 6).

Earlier it was noted that pigeon paramyxovirus type-1 strains, which include NDV / Altai / pigeon / 770/2011, have well-defined oncolytic properties due to the structure of their replicative complex (Dortmans JC et al. The viral replication complex is associated with the virulence of Newcastle disease virus // J Virol. - 2010. - Vol. 84 (19). - P. 10113-10120) [16], which is confirmed by our experiment. The virus safety against healthy mammalian cells has been tested on human peripheral blood mononuclear cells. The results of assessing cell viability after infection with selected VBI strains have confirmed the literature that VBI is not pathogenic for human cells (Schirrmacher V., Fournier P. Newcastle disease virus: a promising vector for viral therapy, immune therapy, and gene therapy of cancer // Gene Therapy of Cancer. - 2009. - Vol. 542. - P. 565-605) [17]; (Zemp FJ et al. Oncolytic viruses as experimental treatments for malignant gliomas: using a scourge to treat a devil // Cytokine Growth Factor Rev. - 2010. - Vol. 21 (2-3). - P. 103-117) [ eighteen].

The presence of the virus in the cells of human tumor lines was confirmed using the technique of immunocytochemical staining with fluorescent antibodies on the second day after infection with the virus using confocal microscopy (magnification 100x). Immunostaining of HCT116 colorectal cancer control cells not infected with the virus gave a negative result. In the obtained images, which are shown in Figure 7, the nuclei of cells stained with DAPI are clearly visible.

Figure 7A shows a preparation of immunocytochemically stained control non-virus infected human colorectal cancer cell line HCT116 cells. No other stained fluorescence accumulations and aggregations in the cytoplasm and nuclei other than stained DAPI nuclei were found on the preparation of control tumor cells. In Figure 7B, in colorectal cancer cells HCT116 infected with the NDV / Altai / pigeon / 770/2011 strain (dilution of 8 GAE / 10,000 cells), after immunocytochemical staining, in addition to the nuclei, a bright fluorescent staining of filamentous structures located in the cytoplasm of the cells is revealed. These structures correspond to the position of the viral particles of strain NDV / Altai / pigeon / 770/2011 in tumor cells. In colorectal cancer cells HCT 116, which are sensitive to the oncolytic effect of strain NDV / Altai / pigeon / 770/2011, stained viral filamentous structures were detected in the cytoplasm of all cells and are located mainly uniformly across all cytoplasms.

When infected with strain NDV / Altai / pigeon / 770/2011 of the tumor cell line HCT116, active replication of the virus is observed in all infected cells. This probably explains the high sensitivity of this cell line to the effects of the virus. And, as a result, the more effective the infection of the tumor cells with the virus, the correspondingly more of them undergo oncolysis and die.

The unique ability of strain NDV / Altai / pigeon / 770/2011 was shown to exhibit selective lytic activity against mammalian tumor cells, as well as actively replicate and persist in these cells for a long time without preliminary adaptation, destroying the neoplasm.

Thus, the unique properties of the proposed strain allow it to be used to study the mechanisms of destruction of mammalian cancer cells at the ultrastructural level using light-optical and electron microscopy methods. These properties are promising for basic and applied medical research.

These properties make the strain NDV / Altai / pigeon / 770/2011 promising for the further study of oncolytic activity and the development of candidate antitumor drugs.

Information Sources Used

1. World Health Statictics, 2012. 2012. P. 176. http://www.who.int/gho/publications/world health statistics / EN WHS2012 Part2.pdf

2. Martuza R.L. et al. Experimental therapy of human glioma by means of a geneticaly engineered virus mutant // Science. 1991. Vol. 252. N. 5007. P. 854-856.

3. Zhan X. et al. Sendai virus recombinant vaccine expressing hPIV-3 HN or Flicits protective immunity and combines with a second recombinant to prevent hPIV-1, hPIV-3 and RSV infections // Vaccine. 2008. Vol. 26. N. 27-28. P. 3480-3488.

4. Elankumaran S. et al. Newcastle disease virus exerts oncolysis by both intrinsic and extrinsic caspase-dependent pathways of cell death // J. Virol. 2006. Vol. 80. N. 15. P. 7522-7534.

5. Ravindra P.V. et al. Newcastle disease virus as an oncolytic agent // Indian J Med Res. 2009. Vol. 130. N. 5. P. 507-513.

6. Cantin C, Holguera J., Ferreira L., Villar E., Muñoz-Barroso I. http://www.ncbi.nlm.nih.gOv/pubmed/17251575// J. Gen. Virol. 2007. Vol. 88. Pt. 2. P. 559-569.

7. Fabian Z., Csatary S.M., Szeberenyi J., Csatary L.K. p53-independent endoplasmic reticulum stress-mediated cytotoxicity of a Newcastle disease virus strain in tumor cell lines // J. Virol. 2007. Vol. 81. N. 6. P. 2817-2830.

8. Puhlmann J., Puehler F., Mumberg D., Boukamp P., Beier R. Racl is required for oncolytic NDV replication in human cancer cells and establishes a link between tumorigenesis and sensitivity to oncolytic virus // Oncogene. 2010. Vol. 29. N.15. P. 2205-2216.

9. Bai F.L., Yu Y.H., Tian H., Ren G.P., Wang H., Zhou B., Han X.H., Yu Q.Z., Li D.S. Genetically engineered Newcastle disease virus expressing interleukin-2 and TNF-related apoptosis-inducing ligand for cancer therapy // Cancer Biol. Ther. 2014. Vol. 15. N. 9. P. 1226-1238.

10. Zamarin D., Palese P. Oncolytic Newcastle disease virus for cancer therapy: old challenges and new directions // Future Microbiol. 2012. Vol. 7. N. 3. P. 347-367.

11. Miller P.J. et al. Newcastle disease: evolution of genotypes and the related diagnostic challenges // Infect. Genet. Evol. 2010. Vol. 10. N. 1. P. 26-35.

12. Cassel W.A., Garrett R.E. Newcastle disease virus as an antineoplastic agent // Cancer. 1965. Vol. 18. P. 863-868.

13. Laurie S.A. et al. A phase 1 clinical study of intravenous administration of PV701, anoncolytic virus, using two-step desensitization // Clin. Cancer Res. 2006. Vol. 12. N. 8. P. 2555-2562.

14. ΟΙΕ, World Organization for Animal Health, Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, Paris, France, 7th edition. 2012. Vol. 1-2. Ch.2.03.14. http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.03.14_NEWCASTLE_DIS.pdf.

15. Alexander D.J. Newcastle disease virus and other avian paramyxoviruses // Rev. Sci. Tech. 2000. Vol. 19. N. 2. P. 443-462.

16. Dortmans J.C. et al. The viral replication complex is associated with the virulence of Newcastle disease virus // J. Virol. 2010. Vol. 84. N. 19. P. 10113-10120.

17. Schirrmacher V., Fournier P. Newcastle disease virus: a promising vector for viral therapy, immune therapy, and gene therapy of cancer // Gene Therapy of Cancer. 2009. Vol. 542. P. 565-605.

18. Zemp F.J. et al. Oncolytic viruses as experimental treatments for malignant gliomas: using a scourge to treat a devil // Cytokine Growth Factor Rev. 2010. Vol. 21. N. 2-3. P. 103-117.

Newcastle disease virus strain NDV / Altai / pigeon / 770/2011

for studying oncolytic properties and mechanisms of oncolysis

to create a prototype antitumor drug

Nucleotide sequence

        1 accaaacaga gaatctgtga gttacgataa agggcgaaga agcaatcgaa atcgtacggg

       61 tagaaggtgt gaacctcgag tgcgaggccg aagctcaaac tcgagggagc cttctgccgg

      121 tatgtcatcc gtctttgacg agtacgagca actccttgct gctcaaaccc gccccaacgg

      181 agcccacgga ggaggagaaa aagggagcac cttaaaagtt gaggtcccag tattcactct

      241 taacagcgat gatccagaag atagatggaa ttttgcggta ttttgtcttc ggattgctgt

      301 tagcgaggac gccaacaaac cactcagaca aggtgctctc atatctctct tatgctccca

      361 ttctcaagtg atgagaaatc atgttgccct tgcaggaagg cagaacgagg ccacactggc

      421 tgttcttgag atcgatggtt ttaccgacaa tgtgccccag ttcaacaaca ggagcggagt

      481 gtctgaagag agggcacaga gattcatggt gatagcaggg tctcttcccc gggcatgcag

      541 taacagtact ccattcatta cggctggagt tgaagatgat gcgccagaag atatcactga

      601 cactctggaa agaatcctat ctatccaggc tcaggtatgg gtcacggtag caaaggccat

      661 gactgcatat gagacagcag atgagtcaga aacaaggagg attaataaat atatgcagca

      721 aggtagagtc caaaagaagt atatcctcca ccccgtatgc aggagtgcaa ttcaactcac

      781 aatcagacat tccctggcag tccgcatttt cttagttagt gagcttaaga ggggccgcaa

      841 tacggcaggt gggagctcta catattacaa cttagttggg gatgtggact catacataag

      901 gaacaccggg ctgactgcgt tcttcctgac actcaagtat ggaatcaata ccaagacgtc

      961 agcccttgca ctcagcagcc tcacaggtga tatccaaaaa atgaaacagc tcatgcgtct

     1021 atatcggatg aaaggagaaa atgcaccgta catgacattg ttaggcgaca gtgaccagat

     1081 gagcttcgca ccagctgaat atgcacaact ctattctttt gccatgggca tggcatcggt

     1141 attagataag ggaactagta aataccaatt cgccagagac tttatgagca cgtcattttg

     1201 gagactcgga gtggagtatg cccaggcgca gggaagtagc atcaatgagg atatggctgc

     1261 tgaactgaag ctaactccag cagcaaggag gggcttggca gctgctgccc aacgcgtatc

     1321 cgaggaaatt ggcagtgtgg acattcccac tcaacaggct ggggtcctca ctgggctcag

     1381 tgacgaaggc ccccgaggct cacagggtgg attaagcaag tcgcagggac aatcggatgc

     1441 cggggatgga gaggcccaat tcctggacct gatgagagca gtggcaaaca gcatgcgaga

     1501 atcaccaaac tccacacaga gcaccactca ccaggagccc cccccgactc ctgggccaac

     1561 ccaagacaac gacatcgact gggggtactg atcaacaaca cccagcctgc ccccgccgag

     1621 ccgccccaaa ccctccgccc aaacctcccc caaactcccc aacctaccgc cccacacggc

     1681 caaaccggca ggagcacccc cnnnnnnnnnn nnnnnncccc cnnnnnnncg atcccgcccg

     1741 tccaaggcaa cacaggcgca actcggtcca ccggcaatca gcgcagagcc caaagtatta

     1801 gaaaaaaata cgggtagaag agagacacgt agaggtcagg acaattcacc ggggtctcta

     1861 cctcttctctc tactcagtgg actagggtga agatggccac ctttacagat gctgagatag

     1921 aggatatatt agagaccagt ggaaccgtca ttgacagcat aattacggcc cagggcaaat

     1981 cagcagagaa tgtcgggagg agcataaccc cacaaggcaa gaccaaggct ccgaacacgg

     2041 cacgagataa acatgggaac acccagccac cagctggcca ggacacctcc gaacaacaag

     2101 acaggccaga caaacagcca tccatacctg aacaggcgac ccaatacgac aacctgccga

     2161 ccgcatccaa cgaaccgctt cccactcagg ccgcaggtga ggctggcgac acacagctca

     2221 agacaggagc aagcaattct cttctgtcta tgcttgacaa gctgagcagt aaatcgtcta

     2281 atgctaaaaa ggggccacgg tcaaaccctc aggaaggcca tcatcaacct ctggcccaac

     2341 aacacgggag tcagccaagc cgtggaaaca accaggagag accacagcac caggccaaag

     2401 ccacccctgg aagccagggc acagacgcga acacagcata tcatggacaa cggaaggagt

     2461 cacaaccatc agctggtgca acccctcatg tgccccagtc agggcaaagc caagacagta

     2521 ctcctgcatc tgcggatcat gtccaaccac ctgtcgactt tgtgcaagcg atgatgtcta

     2581 tgatggaggc attatcacag aaggtaagta aagttgacta tcagttagac ctagtcttaa

     2641 agcagacatc ctccatcccc atgatgcgat ctgaaatcca acagcttaaa acatctgtag

     2701 cgatcatgga agctaattta ggtatgatga agattctgga ccctggttgt gctaacgttt

     2761 catctttgag tgacctacga gcagttgccc gatcccaccc agttttaatt tcgggccccg

     2821 gagatccatc tccttatgtg acgcaagggg gtgaaatgat actcaataaa ctctcgcaac

     2881 cggtcaagaa cccttctgag tcaatcaaat ctgctatgac gagcggatcc gatatcggag

     2941 tggagaagga cacagtccgt gcattaatta cctcacgccc gatgcatcca aactcctcaa

     3001 ccaagctcct gaataagctg gatgcagcca agtcgatcga agagatcaga aagatcaaac

     3061 gccttgcgct gaatggctga ttaccgtcac aactcacaac aggctcctgc cccattagtg

     3121 tcacaaggaa tctttcttga gccttccctc acaaacccat gcttcagcac ctcaagcaac

     3181 aattccctcc cttccctccc acttccctga atgacagcac aactgcaatt aatccaacag

     3241 cattagagat taagaaaaaa tacgggtaga atcaaagtac cctgactgtg ccaaaatgga

     3301 ctcatccaga acaatcgggc tgtactttga ttctgccctc ccctctagca gcctgttagc

     3361 attcccgatc gtcttacaag acacaggaga tgggaagaag aaaatcaccc cacaatacag

     3421 aatccagcgt cttgactcat ggacagacag taaggaagac tcggtattca tcaccaccta

     3481 cggatttatc ttccaaatag ggaatgaaga ggtcactgtc ggtatgatta atgacaatcc

     3541 cagacatgag ttactttcct ctgcaatgct ctgcctagga agtgttccaa atgacggaga

     3601 tcttgttaaa ttggcgaggg cctgcctcac tatggtaata acttgcaaga agagtgcaac

     3661 taatactgag agaatagtct tctcagtaat gcaggcacct caggtgctgc aaagctgtat

     3721 ggttgtggca aataagtact cgtcagtgaa tgcagtgaag catgttaaag cacccgagaa

     3781 gatccctgga agcggaaccc tagagtataa agtgaacttt gtctctttga ctgtggtgcc

     3841 gaggaaggat gtctacaaga tcccaactgc agcattgaaa atatctggct caagcctata

     3901 taatctcgca ctcaatgtca ctattgatgt ggaggtagat ccgaagagcc cgctagtcaa

     3961 atctctttct aagtctgaca gtggatacta tgctaatctt tttttgcata tcgggcttat

     4021 gtccactgta gacaagaagg gaaagaaagt gacatttgac aagatagagg aaaagataag

     4081 aagactcaat ctatctgtcg ggctcagtga tgtgctcgga ccttctgtgc ttgtgaaggc

     4141 gagaggtgca cggactaagc tactggcacc ttttttctcc agcagcggga cggcctgcta

     4201 tcctatagca aatgcctccc ctcaggttgc taagatactc tggagtcaaa ccgcgcacct

     4261 gcggagtgta aaggtcatca ttcaagctgg cacccaacgt gctgttgcag tgactgccga

     4321 tcatgaagtc acctctacta agatagagaa gaggcacacc attgccaaat acaatccttt

     4381 caagaagtaa gctgcatccc tgaaactgcg acccacccac ctctctcaat caccgtgaca

     4441 ctagatgatg atctatcttg attgcttatg gttagttcac ctgtctatct aattaagaaa

     4501 aacacgggta gaagagtctg gatctcggct agcacattca aggcgcaaca tgagctccaa

     4561 atcttccacc aggatcccag cacccttgat gttgatcgcc cgaatcatgc tgatactgag

     4621 ctgcattcgt ctgacaagct ctcttgatgg caggcccctc gcagctgcgg ggattgtggt

     4681 aacaggagat aaagcagtca acatatacac ctcatctcag acagggtcaa tcataatcaa

     4741 gttgctcccg aatatgccca aggataaaga ggcgtgtgca aaagccccgt tagaagcata

     4801 caacagaaca ctgactgctt tactcaaccc ccttggtgat tccatccgca agatacaagg

     4861 atctgtatcc acaccaggag gaaagagaca gaagcgcttt ataggtgcca ttattggcag

     4921 tatagctcta ggggttgcga catcggcaca gataacagca gctgcggccc tgatacaagc

     4981 caaccagaat gccgccaaca tcctccggct taaggagagc attgctgcaa ccaatgaagc

     5041 tgtgcatgag gtcaccgatg gattatcgca actagcagtg gcagttggga agatgcagca

     5101 gttcgttaat gaccaattta ataatacggc acgagaattg gactgtataa aagttacaca

     5161 acaagtcggt atagaactca acctatacct aactgaattg actacggtgt tcgggccaca

     5221 aatcacttcc cctgccctaa ctcagctgac catccaggcg ctttataatt tagctggtgg

     5281 caatatggat tacttattga ccaagttagg tatagggaac aatcagctca gctcattaat

     5341 tggtagtggc ttgatcacag gccaccctat attgtatgac tcacagactc aactcctggg

     5401 catacaggta aatttacctt cagtcgggaa ccttaataat atgcgtgcca cctatttggg

     5461 gactttatct gtaagcacaa ccaaagggtt tgcctcagca cttgtcccga aggtagtgac

     5521 acaagtcggc tccgtgatag aagaacttga cacctcacac tgtatagagt ctgatcttga

     5581 tttatattgc acaagaatag tgacattccc catgtctcca ggcatttatt cctgtctgag

     5641 cggcaacacg tcagcttgca tgtattcaaa gactgcaggt gcactcacca cgccatatat

     5701 ggccctcaaa ggctcagtta ttgccaattg caaaataaca acatgtaggt gtgtagaccc

     5761 cccgggtatc atatcacaaa actatggaga agctgtatct ctgatagata gacattcatg

     5821 caatgtctta tcattagatg ggataactct gaggctcagt ggagagtttg atgtaactta

     5881 ccaaaagaat atctcaatac tagattctca agtcatcgtg acaggcaacc ttgatatatc

     5941 aaccgagctt ggaaatgtca acaattcaat aagcagtgcc ctggacaggt tagcggaaag

     6001 caacaacaag ttagacaaag taaatgtcaa ccttaccagc acacctgctc tcattaccta

     6061 tatcgttcta actgtcatat ctcttgtttt cggtgcactt agcctggttt tagcatgcta

     6121 cctgatgtat aaacaaaagg cacaacaaaa gactttgata tggcttggga ataataccct

     6181 tgatcagatg agagccacta caagaacatg aatgcaggcg agatgcggat gtgtctccag

     6241 cggtgacctg tgtgtcaact ttgacaatct gttcaataga agacttaaga aaaaattact

     6301 ggatatacgc aacccaaaag caatacacgg gtagaacggt tgaagaagct gtctttcaat

     6361 caggaactag gcttcacaac atccgctcta ccacatcacc tacagcagac ctcacccatg

     6421 gacagagtag ttcgcagggt tgcgctagag aatgaagaaa gggaagcaaa gaacacatgg

     6481 cgtttggttt tccggattgc agttttactt ctagtagtaa tgaccttagc tatctctgcg

     6541 gctgtcctgg tgcatagcat gagggccagt acgccgcgcg atattgtaag catatcgacc

     6601 gtgatctcaa agacggagga gaagattact tccttactca gttcaaatca agatgtagta

     6661 gataggatat ataagcaggt ggctcttgaa tctccgctgg cactgctaaa tactgaatct

     6721 ataattatga atgcaataac gtctctatct catcaaatta acggggctgc gaataatagc

     6781 ggttgtgggg cacctgttca tgacccagat tatatcgggg ggataggcaa agaactcata

     6841 atagacgaca ccagtgatgt cacatcattc tatccatctg catatcaaga acacctgaat

     6901 tttatcccgg cgcctactac aggatcaggt tgcactcgga taccctcatt tgacatgagt

     6961 gctacccact actgttatac tcacaatgtg atattgtctg gttgcagaga tcactcccac

     7021 tcacatcaat acttagcact cggtgtgctt cggatatctg caacaggaag ggtcttcttc

     7081 tcgactctgc gttccatcaa tttggatgat acccaaaatc gaaagtcttg cagtgtgagt

     7141 gcaactccta tgggttgtga tatactgtgc tccaaagtca cagagactga agaagaggac

     7201 tacaagtcaa ttacccccac atcaatggtg cacggaaggt taggttttga cggtcaatat

     7261 catgagaagg acttagacgt cacagtctta tttaaggatt gggtggcaaa ttacccggga

     7321 gtgggaagtg gatcttttat tgatgaccgt gtatggttcc caatttacgg agggctcaaa

     7381 cctaattcgc ctagcgacac cgcacaagaa gggaattatg taatatacaa gcgttataat

     7441 aacacatgcc ctgatgaaca aagttatcaa atccggatgg ctaaagcttc gtacaagcct

     7501 gggcggttcg gtggaaagcg cgtgcagcag gccatcttat cgatcaaagt gtcaacatct

     7561 ttaggtgagg acccggtgtt gaccgttccg cctaatacaa ttacgctcat gggggccgaa

     7621 ggcagaattc ttacaatagg gacatctcat ttcttgtatc aacgtggatc ttcatatttc

     7681 tctcccgcct tattatatcc tatgacagtc tataataaaa cagctactct tcatagtcct

     7741 tatacattta acgctttcac ccggccaggt agtgttcctt gtcaggcatc agcaagatgt

     7801 cccaactcat gtatcactgg agtctatact gatccgtacc ccttaatctt ccatagaaac

     7861 cacaccttga gaggggtctt cgggacaatg cttgatgata tacaagcaag actcaaccct

     7921 gtgtctgcag tattcgacaa tatatcccgc agtcgtgtaa cccgagtaag ttcaagcagc

     7981 accaaggcgg catacacgac atcgacatgt tttaaagttg tcaagactaa taaaacttat

     8041 tgtcttagca ttgcagaaat atccaatact ctttttggag aattcaggat cgttccttta

     8101 ctagttgaga ttcttaaggg tgataaggtt taagaagcca gatctgacca gctgagtcga

     8161 acataggatt actcggaaaa tggtgctgtg ccacctatct ctcacaacac caaggatcaa

     8221 actagatatc actgcaagcc caactctcat actaccgggc agccacaatc tgacaatgct

     8281 aatgtgatca gtctgaattt ttcctatagt agattcattt agaaaaaaca tgaatggtag

     8341 caagatataa ggggaaacaa cgtacaggag atagcacggg taggaaatgg cgggctccgg

     8401 tcctgaaagg gcagagcatc aaattatcct accagagtca catctatcct cgccattagt

     8461 caagcacaaa ttgctctatt actggaaatt aactgggcta ccgctccctg acgaatgcga

     8521 ctttgatcac ctcattatca gcaggcaatg gaagaaaata cttgaatctg ccactcctga

     8581 cattgagagg atgataaaac ttgggcgggc ggtgcatcag actctcaacc acaattccaa

     8641 gataaccgga gtactccatc ccaggtgttt ggaagaacta tctagtattg agatctctga

     8701 ttcaactaac aaattccgga agattgaaaa gaagatccag attcacaaca caaggtatgg

     8761 agacctgttc gcaaagctgt gtacgcacgt tgaaaagaaa ttgctgggat catccaggtc

     8821 taacaatgtc tcacgatcag aggagttcag cagcatccgt acagatccgg cattctggtt

     8881 ccactcaaaa tggtccaaag ccaaatttgc atggctccat ataaaacagg cccagaggca

     8941 tctgattgta gcagcaagaa caaggtccgc agtcaacaag ttagtgacat tgacccataa

     9001 gacaggacaa gtctttgtta ctcctgagct tgtcattgtg acacatacag atgagaacaa

     9061 gttcacatgc ctcactcaag aacttgtatt aatgtatgca gatatgatgg agggcagaga

     9121 catggtcaac ataatatcat ctacggcgac acatctcaga agtctatcag agaaaattga

     9181 tgatattctg cggttaatag atgctctgtc aaaggatttg ggcaatcaag tctatgacgt

     9241 tgtatcattg atggagggat ttgcgtacag tgctgttcag ctgcttgaac catcaggtac

     9301 atttgcagga gatttctttg cattcaactt acaagaacta aaaaacactc taactgagct

     9361 cctcccaaat gatatagccg aatcagtaac tcacgcaatc gcctccatat tctctggttt

     9421 agaacagaat caagcagctg agatgttgtg cttactgcgt ttgtggggtc acccattgct

     9481 tgagtctcgt agtgcagcaa gggcagtcag gagccagatg tgcgcaccta aaatggtaga

     9541 tttcgatatg atcctccaag tattatcttt ctttaaggga acaatcatca atggatacag

     9601 gaagaagaac tcaggtgtgt ggccacgagt taaggtagat acgatatacg ggaaagtcat

     9661 tgggcagcta cacgctgatt cagcggagat ttcgcatgat atcatgctga gggagtacaa

     9721 gagtttatct gcccttgaat tcgagccatg tatagaatat gaccctgtca ccaatctaag

     9781 tatgttctta aaggacaagg caattgcaca tccgagagat aactggcttg cttcttttag

     9841 gcggaacctt ctctctgagg accagaggaa acaggtaaag gaggcaacct ctactaaccg

     9901 cctcttgata gagttcttag aatcaaatga ttttgatccg tataaggaga tggaatattt

     9961 gacaaccctt gagtacctaa gagacgacag cgtggcagta tcgtactcac tcaaagagaa

    10021 ggaggtgaag gttaatgggc ggatttttgc taagttgaca aagaaattaa ggaactgcca

    10081 ggtaatggca gaagggattc tagctgacca gattgcgcct tttttccagg ggaatggggt

    10141 cattcaagat agcatatcct tgaccaagag tatgttagcg atgagtcaac tgtcttttaa

    10201 cagcaacaag aaacgtatca ctgactgcaa agagagggtt tcctcaaaac gcaatcatga

    10261 cttgaagagc aagaatcgtc gaagagttgc tactttcatc acaacggacc tgcaaaagta

    10321 ttgtcttaac tggagatacc agacagttaa actcttcgca catgccatca atcagctgat

    10381 gggcctacct cacttcttcg aatggattca tcttagactg atggatacta cgatgtttgt

    10441 aggagaccct tttaatcctc caagtgaccc gactgactgc gatctatcca gagtcccaaa

    10501 tgatgatata tatattgtca gtgctagagg gggcattgag ggattatgcc agaaactatg

    10561 gacaatgatc tcaattgctg caattcaact cgctgcagca aggtcgcatt gtcgggttgc

    10621 ctgcatggta caaggtgaca atcaagtaat agccgtaaca aaagaagtaa gatcagatga

    10681 ctccccagat atggttttga cacaactgca tcaagctagt gataatttct tcaaagaact

    10741 gattcacgtt aatcatctga ttggccacaa cctgaaagat cgtgaaacca tcaggtcaga

    10801 cacattcttc atatacagca agcggatatt caaagacgga gcaatactca gtcaggtcct

    10861 caaaaattca tcaaaattgg tgctaatatc aggcgatctt ggtgaaaata ctgtaatgtc

    10921 ctgtgccaac attgcatcca ctgtagcacg gctatgcgag aacgggcttc ctaaagattt

    10981 ctgttattat ttaaattacc taatgagctg tgtgcagaca tactttgatt ctgagttttc

    11041 tattactcat agctcacaac cagattcaaa ccagccctgg atcgaggaca tttcttttgt

    11101 acactcatat gttttgactc ctgctcagct gggaggactg agtaaccttc aatactcaag

    11161 gctctacaca aggaatattg gtgacccggg gaccactgct tttgcggagg taaagcgatt

    11221 agaaacagtt gggttgctga atcctagcat tatgaccaac atcttgactc ggccacctgg

    11281 caatggagac tgggctagcc tatgcaacga tccttattct tttaattttg agactgtcgc

    11341 aagcccaaat attgtcctta agaaacatac ccaaaaagtc ttatttgaga cttgttcaaa

    11401 ccctctacta tccggagtac atacagagga caatgaggca gaggagaagg cattggctga

    11461 attcctactc aatcaagaag cagttcaccc acgtgtcgca catgctatca tggaagcaag

    11521 ctctgtaggt aggagaaagc aaattcaagg gcttgttgac actacaaaca ctgtgatcaa

    11581 gattgcgctg accaggaggc ccctcggtat caagaggctg acacggataa tcaattactc

    11641 gagcatgcat gcaatgctat tcagagatga tgttttcttt tctagtcgat ccaaccaccc

    11701 attagtctct tctactatgt gttcactgac actagcagat tatgcccgga acagaagctg

    11761 gtcacctttg acagggggta ggaaaatact gggtgtgtct aaccctgata ccatagaact

    11821 tgtggaggga gagattctta gtgtcagtgg ggggtgcaca aaatgtgaca gcggagatga

    11881 acagtttact tggtttcatc ttccaagcaa tatagagttg actgatgaca ccagcaagaa

    11941 tcccccgatg agagtgccgt atctcgggtc aaaaacccaa gagaggaggg ctgcttcgct

    12001 tgcaaaaata gctcatatgt cgccacatgt gaaggcagca ctaagagcat catctgtgtt

    12061 gatctgggct tatggggaca acgaagtaaa ctggactgct gcacttaaga ttgcacggtc

    12121 tcgatgtaat ataagctcag agtatcttcg gctattatca cccctgccca cagctgggaa

    12181 tctccaacat agattggatg atggcataac ccagatgaca tttacccctg catctctcta

    12241 cagagtatcc ccttatgttc acatatctaa tgattctcaa agactattta ccgaagaagg

    12301 agtcaaagag gggaatgttg tttatcaaca aattatgctc ctgggcctat ctctcattga

    12361 atcactcttc ccaatgacaa caaccaagac gtatgatgag atcacattac acctccacag

    12421 caaatttagc tgctgcatca gggaagcgcc tgttgctgtc cctttcgagc tacttgggtt

    12481 ggcaccagaa ctaaggatgg taacctcaaa taagttcatg tatgatccca gccctatatc

    12541 agagagagat tttgcaagac ttgacttgac catattcaag agttatgagc tcaatttaga

    12601 gtcatactct acaatggagc taatgaacat tctctcaata tctagtggga agttgattgg

    12661 ccagtctgtg gtctcctatg atgaagatac ctctataaag aatgatgcca taatagtgta

    12721 cgacaacaca cgaaattgga tcagtgaagc ccagaattca gatgtggtcc gcttgttcga

    12781 atatgcagca ctcgaggtcc tcctcgactg ttcttatcaa ctctactatc tgagggtaag

    12841 aggtctcaac aacattgtcc tatacatgag tgacttatac aagaatatgc cagggattct

    12901 actctctaat attgcggcca caatatctca ccccatcatt cactcaaggt tgaatgcagt

    12961 gggtctaatc aaccatgatg ggtcacacca gcttgctgac atagatttca tcgaaatgtc

    13021 cgcgaagctg ttagtctctt gcactcggcg tgtagtctca ggcttatacg cggggaataa

    13081 atacgatctg ttgttccctt ccgttttaga cgacaacctg agtgaaaaga tgcttcagct

    13141 gatttcccgg ttatgctgtc tgtatacagt gctttttgct acgacaagag aaatcccaaa

    13201 aataaggggc ctatccgcag aagaaaaatg ctcagtactc actgagtacc tactgtcaga

    13261 tgctgtgaag ccattgctta ggtctgagca agtgaactct atcacgtctc ccaatataat

    13321 tacgttcccg gccaatctat actacatgtc taggaagagc cttaatttaa tcagagaacg

    13381 agaggacaga gatactatct tggcgttgtt attcccccag gaaccgctgc ttgagctctg

    13441 tccagtacaa gacattggtg ctcgagtgag agatcctttt actcgaaaac ctgcatcatt

    13501 catacaagag ttaaatctga gtgctccagc aagatacgac gcatttaccc ttaatgaggc

    13561 ttgctttgag catacattgc cgaacccgga agaagattac ctagtacggt atttgtttag

    13621 aggaataggg actgcttcat cttcttggta taaggtatct catcttcttt ctgtacctga

    13681 ggtcagatgt gcgagacgtg ggaactcctt atacttggca gaggggagtg gagccatcat

    13741 gagtcttctt gaactgcata taccacatga gaccatctat tataatacgc ttttctcgaa

    13801 tgagatgaac ccaccacaac gacatttcgg acctacccca acacagtttc taaattccgt

    13861 agtttatagg aatctacaag cggaagtgcc gtgtaaagac ggatttgtcc aggagttctg

    13921 cccactatgg agagagaatt cagaagaaag tgacctgacc tcagataagg cagtgggata

    13981 tatcacatct gtggtgccct acaggtctgt atcattacta cattgtgaca ttgaaatccc

    14041 tcctgggtcc aaccaaagct tgttagatca actagctact aatttatctt tgattgccat

    14101 gcattctgta agggaaggcg gagtggtaat catcaaagta ctgtatgtaa tgggatacta

    14161 cttccattta ctcatgaatt tattcactcc atgttccaca aaaggataca ttctctctaa

    14221 tggctatgcc tgcagagggg atatggagtg ctacttgata tttgtcatgg gctatttagg

    14281 cgggcccaca tttgtgcatg aggtggtgag gatggcaaaa actctaatac aacggcatgg

    14341 tacgcttctg tctaaatcag atgagattac gttaactagg ctgtttacct cacaacggca

    14401 tcgtgtaata gacatcctgt ctagcccttt accgagacta atgaagttct tgagagaaaa

    14461 tattgatgct gcgttgattg aagccggggg acagcctgtc cgtccattct gtgcagagag

    14521 tttagtgtgc acactaacag atatgactca gatgactcag attattgcca gccacattga

    14581 cacagtcatt cgatctgtga tttatatgga ggctgagggt gacctcgctg acacagtgtt

    14641 cttgtttact ccttacaatc tctctgcaga cggtaaaaag aggacatcac ttaaacagtg

    14701 tacgaggcag atcctagagg tcacaatact gggtctcaga gtcagagatc tcaataaagt

    14761 aggtgacata ataagcttgg tactcagagg tatggtttct ctagaggacc tcatcccact

    14821 aaggacatac ctgaagcgca gtacttgccc gaagtacttg aagacagtct tagggattac

    14881 taaactcaaa gagatgttca cagatacctc cttgctatac ctgactcgtg ctcaacaaaa

    14941 attctacatg aaaaccatag gtaatgcagc caagggatat tacagtaaca atgactccta

    15001 aaggcaatca cacattaatg acctatcttc ctaatcaatt gttctctcac tgacttgatt

    15061 atactgtatt agaaaaaagt taaattctga ttctttgaaa ctcgtgctcg gattcaatta

    15121 attatcctga agcagaattg aacgtagtta tccttaatta tagtcctgtc attcaccaaa

    15181 tctttgttt

//

Claims (1)

The strain of the virus of Newcastle disease to study the oncolytic properties and mechanisms of oncolysis to create a prototype antitumor drug, deposited in the State virus collection of the Research Institute of Virology. DI. Ivanovo Ministry of Health of Russia under registration number 2778.

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