RU2496873C1 - STRAIN OF ENTEROVIRUS COXSACKIE B6 SELECTIVELY INFECTING AND LYSING TUMOROUS HUMAN CELLS in vitro - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: described strain is produced by performance of a series of adaptation passages of a parent strain of the virus ZhEV-15 Coxsackie B6 on the culture of cells HEK293 highly sensitive to this virus and the neoplastic cell line C33A (HPV-negative carcinoma of human uterine neck), with production of a new strain ZhEV-15^L of the virus Coxsackie B6. The strain selectively infects and lyses tumorous human cells and has a fragment of a genome sequence represented in the dwg. 2, being its marker criterion. The strain is deposited in the State Collection of the Federal Service for Oversight of Consumer Protection and Welfare of agents of viral infections and rickettsial diseases of the Federal Budget Institution of Science State Science Centre of Virusology and Biotechnology "Vector" under the registration number V-576.

EFFECT: invention provides for the possibility of promising development of a viral oncolytic preparation on its basis for therapy of malignant human tumours and definition of a full nucleotide sequence of its genome.

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Description

The invention relates to strains of the Coxsackie B6 virus, namely, strain JEW-15 ^L , selectively infecting and lysing human tumor cells in vitro, promising for the development on its basis of a viral oncolytic drug for the treatment of human malignant neoplasms and determining the complete nucleotide sequence of its genome, and can be used in medical virology, biotechnology and microbiology.

In modern society, the problem of cancer incidence is becoming more acute. According to the World Health Organization, more than five million people die every year from malignant neoplasms in the world. Despite the significant successes achieved in the diagnosis and treatment of cancer, malignant neoplasms occupy second place in developed countries among the causes of deaths, following cardiovascular diseases. Currently, surgical methods, chemo- and radiation therapy are the main ways to treat cancer. Many forms of tumors are inoperable, resistant to chemotherapy and radiotherapy. In addition, a significant disadvantage of chemotherapy and radiotherapy is their low therapeutic index. Therefore, increasing the dose or a combination of therapies to overcome resistance or enhance the destruction of cancer cells is limited by toxicity to normal tissues.

The above proves the need to develop new effective and safe cancer therapies, this is one of the main tasks of modern world science. In recent decades, there has been a rapid development of research aimed at the development of tumor-specific therapeutic agents based on various viruses.

The parent strain of the inventive strain JEV-15, along with other enterovirus strains of the ZhEV series (live enterovirus vaccine), was isolated at the Institute of Poliomyelitis and Viral Encephalitis of the Academy of Medical Sciences of the USSR [1] during mass examinations of children in institutions. All enteroviral strains (10 different immunological types) of the ZHEV series were isolated from clinically healthy children carriers of these viruses, which remained healthy during the period of the presence of these enteroviruses in their digestive tract and later during periodic examinations for three years (observation period). After conducting safety and areactogenicity studies of these strains [2, 3, 4] ZHEV enterovirus strains (ZHEV-4, ZHEV-7, ZHEV-8, ZHEV-11, ZHEV-15, etc.) were used for oral vaccination of children for the purpose of prevention and stopping outbreaks of enterovirus infections in institutions. Large-scale studies have shown the high interfering activity of live enterovirus vaccines and the resulting vaccination efficacy against enterovirus infections, as well as influenza outbreaks and acute respiratory viral infections among children and adults [5, 6, 7].

To date, several non-modified genetic engineering strains of enterovirus strains with selective oncolytic activity have been described.

The viral preparation Rigvir is known, which is an ECHO7 enterovirus (analogue), registered with the Latvian State Drug Agency on 04/28/2004. Reg. No. 04-0229 and recommended for systemic and local use by patients with melanoma in combination with chemo-and surgical therapy. The oncotropism of Rigvir virus in relation to melanoma cells is due to the adaptation of the original ECHO7 strain to replication in human melanoma cell cultures in vitro. Recently, ECHO1 virus has a high tropism for neoplastic cells of the human ovary [10].

However, the above analogues, including the prototype strain, provide lysis of a narrow spectrum of sensitive human tumor cells and have insufficient ability to selectively infect cells.

The technical result of the invention is the creation of such a non-pathogenic strain of the virus, which is able to selectively infect and lyse a wider range of human neoplastic cells of different tissue affiliation and provide the possibility of creating on its basis an oncolytic viral preparation for the treatment of human malignant neoplasms.

The indicated technical result is achieved by carrying out a series of adaptive passages of the parental Coxsackie B6 virus strain on a HEK293 cell culture highly sensitive to this virus and the neoplastic cell line C33A (HPV-negative carcinoma of the human cervix uteri), to obtain a new strain of JEW-15 ^L Coxsackie B6 virus, which selectively infects and lysing human tumor cells in vitro to create, on its basis, an antitumor drug having a fragment of the genomic nucleotide and amino acid sequence ION 2, which is a marker of his signs, and deposited in the State Collection of Rospotrebnadzor pathogens of viral infections, and rickettsial Federal State Institution of Science State Scientific Center of Virology and Biotechnology "Vector" (FBUN SSC "Vector") under the registration number V-576.

The main differences between the proposed strain JEV-15 ^L from the above enterovirus strains are its ability to selectively infect and lysis a wider spectrum of sensitive human tumor cells and the parent strain of the inventive JEV-15 (along with other strains of the ZHEV series) was used as live vaccine preparation.

Characterization of the claimed strain. Strain JEV-15 ^L is a typical representative of enteroviruses - the Picornaviridae family, the genus Enterovirus, the species Human enterovirus B, and the serotype Coxsackievirus B6. The inventive strain JEW-15 ^{L was} obtained by serial passaging of the parent strain JEV-15 of the Coxsackie B6 virus on transformed and tumor cell lines of humans, HEK293 and C33A, respectively. Strain JEW-15, along with other viral strains of the JEW series, efficiently propagated in the kidney cell cultures of green monkeys and rhesus monkeys, causing typical manifestations of the cytopathic effect [1]. In order to adapt the parental strain ZHEV-15 to replication in a tumor culture of human cells, the methodology described in detail in the following works [11, 12] was used; In order to accumulate genetically different variants, three consecutive passages were carried out on a HEK293 cell culture, followed by five adaptation passages on a tumor culture of C33A cells, with a gradually decreasing infection rate. After the cloning of the viral population by the method of limiting dilutions [11] and cultivation in cell culture HEK293, in order to obtain preparative amounts of viral material, the resulting enterovirus variant was named pcs. ZHEV-15 ^L.

Table 1.

A comparative study of the lytic activity of the strains JEW-15 ^L and JEW-15 against cell cultures of VERO, HEK293 and C33A

Strain VERO culture titer (TTCP ₅₀ / ml) Title on culture HEK293 (TTsPD ₅₀ / ml) The titer on the culture C33A (TTsPD ₅₀ / ml) ZHEV-15 5 × 10 ⁸ 5 × 10 ⁶ 5 × 10 ⁵ ZHEV-15 ^L 10 ⁹ 2 × 10 ⁹ 2 × 10 ⁹

The initial characterization of the infectious (lytic) properties of the strain ЖЭВ-15 ^L was carried out in comparison with the parent ЖЭВ-15 on VERO, HEK293, and C33A cell cultures (Table 1).

As follows from the presented results, the ZhEV-15 ^L strain has significantly higher lytic activity against HEK293 and C33A cells compared to the parent strain, despite the fact that both strains infect and lyse VERO cell culture with almost the same efficiency (transplanted green kidney cell culture monkeys).

When studying the infectious (cytolytic) properties of the strain ЖЕВ-15 ^L on a panel of tumor and normal human cells (see Example 1), it was shown that it has a high degree of selectivity of lytic activity in vitro with respect to the used tumor cell cultures. Strain ZHEV-15 ^L does not infect and does not lyse normal diploid cells FEC-15 and MRC-5 (infectious titer below 10 ² TPPD ₅₀ / ml), and at the same time, it effectively lyses neoplastic cell cultures. The highest values of selectivity indices (IP) pcs. ZHEV-15 ^L , the ratio of the lytic activity of the virus (infectious titer) for the tumor cells used in the study to normal human diploid cells, is marked for tumor cells C33A, HepG2 and DU-145 and is not less than 10 ⁷ , 5 × 10 ⁶ and 5 × 10 ⁵ , respectively. The IP for tumor cell cultures SW480 and A549 is not lower than 2 × ^{10 3} . Used as strains of comparison pcs. ZHEV-8 (Koksaki A7 virus) and pcs. JEV-10 (Coxsackie B1 virus) did not show selective lytic activity against tumor cell cultures in vitro. As mentioned above, the Coxsackie A21 virus strain (prototype), on the basis of which the oncolytic drug Cavatak ^tm is developed, which is currently undergoing Phase II clinical trials, has a pronounced selective in vitro lytic activity against human malignant melanoma cells. According to the literature data, the IC of this strain for cell cultures of Sk-Mel-28 (melanoma) and MRC-5 is 5 × 10 ⁴ , for DU-145 (prostate carcinoma) and MRC-5 - 10 ³ , and the cell culture of pulmonary adenocarcinoma A549 even less sensitive to this strain than normal diploid culture of lung cells MRC-5 [8, 9, 13]. The data indicate that the inventive strain ZHEV-15 ^L effectively infects and lyses a wider range of tumor cultures of human cells in vitro and has a more pronounced degree of selective lytic activity against prostate carcinoma and pulmonary adenocarcinoma cells compared to the prototype strain.

Thus, the non-pathogenic enterovirus strain ZHEV-15 ^L has a pronounced ability to selectively infect and lyse tumor cultures of cells of various tissue in vitro, which indicates the prospect of further studies of the oncolytic activity of this enterovirus strain in order to develop an effective and safe oncolytic viral preparation based on it for the treatment of human malignant neoplasms.

To establish the genotype of the strains of the inventive JEW-15 ^L and parent JEW-15, samples of the viral material were examined by RT-PCR using a set of oligonucleotide primers on 5'UTR for the diagnosis and genotyping of human enteroviruses of types A, B, C, D. According to a specific nucleotide sequence 5'UTR of the genome, it was established that the strains JEV-15 ^L and JEV-15 belong to the Koksaki B6 enterovirus. Using virus-specific primers designed for the entire genome of the Koksaki B6 enterovirus, the complete nucleotide sequences of the genomic RNAs of strains JEV-15 and JEV-15 ^L were determined (see Example 2). Compared with the parent strain pcs. ZHEV-15 ^L has three nucleotide substitutions: C ₂₆₇₉ → A, A ₄₄₀₃ → G and A ₆₇₉₂ → T; the first two of which lead to amino acid substitutions - Thr → Lys (protein VP1) and Ser → Gly (protein 2C) (Figure 1, Table 2). Thus, the structural differences of the genome pcs were identified. ZHEV-15 ^L from the parent strain, acquired in the process of adaptation of the latter to HEK293 and C33A cell cultures, providing replicative benefits pcs. ZHEV-15 ^L with infection of these cells (figure 1).

The complete nucleotide sequence of the genome of strain JEV-15 ^{L was} deposited in the international GenBank database under the number JQ041368 (http://www.ncbi.nlm.nih.gov/genbank/) . When analyzing the complete nucleotide sequence of the genome of strain ЖЕВ-15 ^L in comparison with published sequences of various enteroviruses, it was shown that the closest strain to the present strain is the Schmitt strain of the Coxsackie V6 virus ( Coxsackievirus B6 strain Schmitt , GenBank AF105342, AF114384) (Fig. .3), the homology percentage was 99% for the nucleotide (14 nucleotide differences) and 99% for the amino acid sequences (8 amino acid differences). At the same time, with other enterovirus genotypes, the percentage of homology in both the nucleotide and amino acid sequences is lower than 91% (Table 3).

Marker genetic traits. Strain ZHEV-15 ^L has three nucleotide substitutions with respect to the parent strain ZHEV-15: C ₂₆₇₉ → A, A ₄₄₀₃ → G and A ₆₇₉₂ → T; the first two of which lead to amino acid substitutions - Thr → Lys (protein VP1) and Ser → Gly (protein 2C). Marker features of the claimed strain contain a fragment of the genomic nucleotide and amino acid sequence shown in figure 2.

Cultural properties. Strain JEV-15 ^{L is} cultivated on a monolayer of HEK293 cell culture (human embryo kidney cells transformed with genes E1A and E1B of the 5th serotype adenovirus), which is approved by GISK named after L.A. Tarasevich (protocol No. 14 dated 10/28/03 of the meeting of the Scientific Council of the GISK named after L. Tarasevich; protocol No. 9 dated 11/20/03 of the MIBP Committee) for use as a substrate for the production of the anticancer virus drug “Cancerolysin” (a mutant variant of human adenovirus 5 serotype defective in the gene of the E1B55K protein). When a cell monolayer is infected, the strain ZHEV-15 ^L induces a cytopathic effect (CPP) typical of enteroviruses after 16-48 hours depending on the multiplicity of infection (cultivation temperature 37 ° C, in an atmosphere with 5% CO ₂ ). The infectious titer of the virus is 10 ⁸ - 10 ⁹ TTCP ₅₀ / ml (50% tissue cytopathogenic dose).

The HEK293 cell culture was grown as a monolayer on DMEM medium (Gibco BRL, Germany) containing 10% fetal calf serum (ETF) (Gibco BRL, Germany) with 80 μg / ml gentamicin sulfate. A subconfluent monolayer of HEK293 cells infects pcs. ZHEV-15 ^L with an optimal multiplicity of 0.1 - 0.01 TCPD ₅₀ / cell, after adsorption for 1 h at 37 ° C, the infected cell monolayer is incubated in DMEM support medium containing 2% ETS until 75-90% of the CPPs are reached. After that, the culture medium together with the cells was selected and the virus-containing suspension was clarified by low-speed centrifugation (5000 rpm, 10 min). Obtaining preparative quantities of purified pieces. ZHEV-15 ^{L is} carried out according to the following methodology: concentration is carried out with polyethylene glycol (PEG) 6000 (7%, 4 ⁰ С, 12 h), after which the suspension is centrifuged 2000g, 4 ⁰ С, 2 h. The supernatant is drained and the precipitate is resuspended in PBS, suspension homogenize by ultrasound. After that, purification is carried out in the density gradient of sucrose or cesium chloride. When a sucrose density gradient is used, a 15-45% linear gradient of 30 ml is usually used in test tubes with a volume of 35 - 40 ml (for example, from a SW28 rotor, Beckman). 1/10 volume of 10% NP-40 is added to the sample to be cleaned, the sample volume should not exceed 6 ml per tube, and carefully layered on the previously obtained gradient. The tubes are centrifuged at 80000g, 4 ^° C for 4 hours (for example, in the SW28 rotor on an L8 centrifuge, Beckman at 25,000 rpm). The gradient is fractionated, the infectious virus should peak at a distance of 2/3 of the length of the tube from the meniscus. If it is necessary to obtain a higher degree of purification, additional centrifugation is carried out in a preformed density gradient of cesium chloride.

The conditions of preservation and storage of the strain. Like other enteroviruses, the strain ZHEV-15 ^L is quite resistant to the external environment; storage in frozen form at -70 ⁰ С (virus-containing culture medium clarified from cell debris or purified virus in PBS) during the year does not lead to a significant loss of infectivity. MgCl ₂ (1M) has a pronounced stabilizing effect with respect to the preservation of the infectivity of enteroviruses. For long-term storage, the strain is recommended to be lyophilized using a solution of gelatin and sucrose (SAG) as a protective medium. Lyophilization with the addition of SAS allows you to maintain a stable infectious activity of the virus for an indefinite time when stored at -20 ° C.

For large-scale production and purification of strain ZHEV-15 ^L, it is possible to use the methodology of roller monolayer cultivation and cultivation on microcarriers under the conditions of a bioreactor, purification by zonal flow centrifugation or ultrafiltration and chromatography.

Pathogenicity for humans. Like other enteroviral strains of the ZHEV series, the parent piece. ZHEV-15 and the claimed strain ZHEV-15 ^L are non-pathogenic for humans [2, 3, 4].

Pathogenicity for laboratory animals. Strain ZHEV-15 ^{L is} not pathogenic for sucker mice with intracerebral administration. Intracerebral infection of newborn mice (two-day suckers) with a dose of 2x10 ⁷ TPCP ₅₀ pcs. ZHEV-15 ^L does not cause signs of neurovirulence, myopathic and other pathological manifestations throughout the observation period (3 months). In this regard, it should be noted that the Koksaki A21 virus strain (prototype), on the basis of which the oncolytic drug Cavatak ^tm was developed, causes lethal myositis with paralytic manifestations in mice [14].

Strain ZHEV-15 ^{L was} deposited in the State collection of Rospotrebnadzor of causative agents of viral infections and rickettsioses of the Federal Budget Institution of Science State Scientific Center for Virology and Biotechnology "Vector" (FBSI SSC WB "Vector") under registration number V-576 (certificate of deposit is attached).

The application contains the following graphic materials.

Figure 1 shows the nucleotide and amino acid differences between the strains of HEV-15 (prototype) and HEV-15 ^L. Figure 2 and the appendix shows a fragment of the nucleotide and amino acid sequence of the inventive strain ZHEV-15 ^L , with marker features that distinguish it from the strain of the prototype ZHEV-15. Figure 3 shows the phylogenetic tree of enterovirus strains constructed from the nucleotide sequences of full-sized genomic RNAs (Neighbor Joining method).

Example 1. The definition and comparative analysis of the complete nucleotide sequence of the genome of strain JEV-15 ^L .

To establish the genotype of strains of ЖЕВ-15 ^L and parent ЖЕВ-15, samples of viral material after passaging on a HEK293 cell culture were studied by RT-PCR using a set of 5'UTR oligonucleotide primers for the diagnosis and genotyping of human enteroviruses of types A, B, C, D. The resulting DNA fragments were sequenced. According to the specific nucleotide sequence of the 5'UTR genome, the affiliation of strains JEV-15 ^L and JEV-15 to the Koksaki B6 enterovirus was established. After establishing the genotype of the strains, primers for the entire genome of the Koksaki B6 enterovirus were calculated and the complete nucleotide sequence of the genomic RNA of strains JEV-15 ^L and JEV-15 was determined. After PCR using virus-specific primers, the samples were purified using a Wizard SV Gel and PCR Clean-Up System kit manufactured by Promega (USA) and sequenced on the forward and reverse DNA chains. The nucleotide sequences were determined using an ABI 3130xl Genetic Analyzer (Applied Biosystems) automated sequencer (USA). All sequences were determined twice in independent experiments. The complete nucleotide sequence of the genome of strain JEV-15 ^{L was} deposited in the international GenBank database under the number JQ041368 (http://www.ncbi.nlm.nih.gov/genbank/) .

When analyzing the complete nucleotide sequences of genomes, strains JEV-15^L and JEV-15 are typed as Coxsackie B6 enterovirus. The specific nucleotide sequence of strains JEV-15^L and JEW-15 of the Coxsackie B6 virus was 7340 nucleotides. Compared with the parent strain pcs. ZHEV-15^L has three nucleotide substitutions: C₂₆₇₉→ A, A₄₄₀₃→ G and A₆₇₉₂→ T; the first two of which lead to amino acid substitutions - Thr → Lys (VP1 protein) and Ser → Gly (2C protein) (Table 2). This table also presents the identified differences in the genomic sequences of pieces. ZHEV-15^L and ZHEV-15 from the strainSchmittCoxsackie B6 virus (GenBank AF105342). Thus, the structural differences of the genome pcs were identified. ZHEV-15^L from the parent strain, acquired during the adaptation of the latter to HEK293 and C33A cell cultures.

When comparing a sequence of pcs. ZHEV-15^Lwith the full nucleotide sequence of the genomic RNA strainSchmittCoxsackie B6 virus (Canada, 1999, GenBank AF105342) the homology percentage was 99% for the nucleotide (14 nucleotide differences) and 99% for the amino acid sequences (8 amino acid differences) (Table 3). Strain ZHEV-15^L in comparison with the strainSchmitthas amino acid differences in protein VP1: Thr₆₅₄→ Lys, Ile₆₉₉→ Val and Ala₇₂₇→ Thr are inrhv-like region. In addition, there is a Glu difference in VP1₅₉₄→ Val. At the same time, with other enterovirus genotypes, the percentage of homology in both the nucleotide and amino acid sequences is lower than 91% (Table 3).

table 2

Revealed structural differences of genomic RNA of strains ZHEV-15 ^L , ZHEV-15 and Schmitt virus Koksaki V6

Gene Position
(nt.) Shmitt ZHEV-15 ZHEV-15 ^L nt. A.K. nt. A.K. nt. A.K. 5 'UTR 218 C - T - T - VP4 777 A Gln G Arg G Arg 818 A Lys C Gln C Gln VP2 1369 A Ala G Ala G Ala VP1 2499 A Glu T Val T Val 2679 C Thr C Thr A Lys 2813 A Ile G Val G Val 2897 G Ala A Thr A Thr 3024 C Ser T Leu T Leu 2B 3757 T Tyr C Tyr C Tyr 2C 4403 A Ser A Ser G Gly 3D 6605 C Phe T Phe T Phe 6792 C Thr A Thr T Thr 6905 T Ile C Ile C Ile

Phylogenetic analysis of enterovirus strains by the nucleotide sequence of the complete genomes (Neighbor Joining method [16]) showed that the closest strain to the present strain of enterovirus ZHEV-15 ^L is Schmitt strain Coxsackie B6 virus ( Coxsackievirus B6 strain Schmitt , GenBank AF115342, GenBank AF105342 ) (Figure 3). The inventive strain JEV-15 ^L meets the English transliteration as LEV-15 ^L (live enterovirus vaccine-15 ^L ). The full-length genomic sequence of the JEV-15 ^L strain was deposited in GenBank under the name LEV-15 ^L (JQ041368).

Table 3

The percentage of homology of the strain JEV-15 ^L with other strains of enteroviruses

Virus Place of selection Year allocated % homology
nucleotides % homology
amino acids Coxsackievirus B6 strain Schmitt Canada 1999 99 99 Human echovirus 27 strain Bacon USA 2003 84 91 Human echovirus 11 Turkey 2007 82 89 Human coxsackievirus B4 strain Tuscany Italy 2007 80 88 Human echovirus 26 strain Coronel USA 2003 79 88 Human coxsackievirus B5 isolate 2000 / CSF / KOR Korea 2000 79 88 Human echovirus 13 strain Del Carmen USA 2003 79 88 Human echovirus 25 strain JV-4 USA 2003 79 88 Echovirus type 12 strain Travis Germany 1994 79 88 Human coxsackievirus B3 strain 28 USA 1994 79 88 Coxsackievirus B2 strain Ohio-1 Sweden 1999 78 88 Human echovirus 6 strain D'Amori USA 2003 78 88 Human echovirus 4 strain AUS250G Australia 2007 77 88 Human echovirus 14 strain Tow USA 2003 77 88

Example 2. The study of the lytic activity of strain JEV-15 ^L  Coxsackie B6 virus in a panel of normal (non-tumorigenic) diploid and tumor cultures of human cells.

A study was made of the infectious (cytolytic) properties of non-pathogenic enterovirus strains ZHEV-15 ^L , ZHEV-8 (Koksaki virus A7) and ZHEV-10 (Koksaki virus V1) on a panel of tumor and normal human cells. Infectious (lytic) activity of enterovirus strains was determined in vitro on cultures of human tumor cells of various tissue affiliation: A549 (human lung adenocarcinoma), SW480 (human colon adenocarcinoma), C33A (cervical HPV-negative carcinoma), DU-145 (carcinoma prostate gland), HepG2 (hepatocellular carcinoma) and on normal diploid cultures of human cells: FEC-15 (diploid culture of the musculoskeletal tissue of a human embryo), MRC-5 (diploid culture of a human embryo). The lytic activity of the studied strains for the respective cell types was determined by the micromethod according to Chanas et al. [15] on 96-well culture plates (Costar) and expressed through TCPD ₅₀ (50% tissue cytopathogenic dose). The results of the study are presented in Table 4. It was shown that strain ЖЕВ-15 ^L practically does not infect used normal human diploid cells (infectious titer less than 10 ² TCPD ₅₀ / ml) and effectively lyses tumor cultures of SW480, А549, С33А, DU-145 and HepG2. The selectivity index - the ratio of the lytic activity of the virus (infectious titer) for the tumor cells used in the study to normal human diploid cells - varies from 2 × 10 ³ (for pairs of A549 / diploid cell cultures) to more than 10 ⁷ (for pairs of cell cultures C33A / diploid). Thus, the enterovirus strain ZHEV-15 ^L has a pronounced ability to selectively infect and lyse tumor cultures of cells of various tissue in vitro. Other investigated enterovirus strains JEV-8 (Coxsackie A7 virus) and JEV-10 (Coxsackie B1 virus) exhibit similar infectious activity against both diploid cells and tumor cells of human cells.

Table 4

Infectious titers of enterovirus strains for various normal and tumor cults of human cells

Strain Caption on the culture FECh-15 (TTsPD ₅₀ / ml) The titer on the culture of MRC-5 (TTCP ₅₀ / ml) Caption on culture SW480 (TTsPD ₅₀ / ml) The titer on the culture A549 (TTCP ₅₀ ) / ml) C-33A culture titer (TCPD ₅₀ / ml) The titer on the culture DU-145 (TTsPD ₅₀ / ml) HepG2 culture titer (TTCP ₅₀ / ml) ZHEV-8
(Koksaki A7) 2 × 10 ⁶ 5 × 10 ⁸ 4 × 10 ⁶ 10 ⁶ 10 ⁹ 4 × 10 ⁶ n.i. ZHEV-10 (Koksaki V1) 2 × 10 ⁴ 10 ⁵ 4 × 10 ⁵ 2 × 10 ⁵ 10 ⁷ 10 ⁶ n.i. ZHEV-15 ^L <10 ² <10 ² 5 × 10 ⁵ 2 × 10 ⁵ 10 ⁹ 5 × 10 ⁷ 5 × 10 ⁸

Note: n.i. - not investigated.

Information sources

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2. Vintovkina I.S., Aliev A.A., Milner B.I. and others. The experience of using the phenomenon of interference for sonation of the intestines of children from pathogenic enteroviruses. - Enterovirus infections. MB IPVE AMS USSR. 1970, p. 54-58.

3. Galko N.V. The results of the study of live enterovirus vaccine ZHEV-4 in experiments on immunization of children. - Enterovirus infections. L., 1971, p. 194-202.

4. Shvetskaya B.D., Nosatenko A.I., Gruntovskaya L.G. and others. Immunological changes in vaccinated with live enteroviral vaccine ZHEV6 and children in contact with them. - Eteroviral infections. M., 1970, p. 32-35.

5. Voroshilova M.K., Koroleva G.A., Tolskaya N.A. and others. The main results of studies conducted in the laboratory of enteroviruses IPVE AMS USSR - Proceedings IPVE AMS USSR, M., 1973, v.21, issue 2, pp. 7-18.

6. Chumakov M.P., Voroshilova M.K., Boyko V.M. et al. The results of extensively controlled trials of the epidemiological efficacy of live enterovirus vaccines for the emergency prevention of influenza and viral acute respiratory infections. Proceedings of IPVE AMS USSR. 1973, v.21, issue 2, pp. 19-28.

7. Tamm O.M., Kuslat T.R., Kutsar K.K. The use of live polio vaccine and enterovirus circulation in the Estonian SSR in 1965-1970 Zhurn. microbiol., 1973, No. 7, p. 76-81.

8. Au G., Lindberg A., Barry R., Shafren D. Oncolysis of vascular malignant human melanoma tumors by Coxsackievirus A21. Int. J. Oncol. 2005, 26, 1471-1476.

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11. Pan J., Narayanan B., Shah S., Yoder J., Cifuente J., Hafenstein S., and Bergelson J. Single amino acid changes in the virus capsid permit Coxsackievirus B3 to bind decay-accelerating factor. J.of Virol., 2011, 85 (14), p. 7436-7443.

12. Reagan K. J., Goldberg B., and Crowell R. L. 1984. Altered receptor specificity of coxsackievirus B3 after growth in rhabdomyosarcoma cells.J. Virol. 49:635-640.

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14. Kelly E., Hadac E., Greiner S., Russell S. Engineering microRNA responsiveness to decrease virus pathogenicity. Nat Med. 2008; 14: 1278-83.

15. Chanas A., Johnson B., Simpson D. et al. Antigenic relationships of alphaviruses by a simple microculture cross-neutralization method. J. of Gen. Virol., 1976, Vol. 32, p. 295-300.

16. Saiton N. and Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 1987, 4, 406-425.

Claims (1)

Koksaki B6 enterovirus strain, selectively infecting and lysing human tumor cells in vitro and deposited in the State collection of Rospotrebnadzor of causative agents of viral infections and rickettsioses of the Federal Budgetary Institution of Science, State Vector Center for Virology and Biotechnology "Vector" under registration number V-576.

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