RU2225440C2 - Strain of ebol virus "zair ch-5" for carrying out model experiments and preparing diagnostic and vaccine preparations - Google Patents

Abstract

FIELD: biotechnology, medicinal virology. SUBSTANCE: the strain "Zair CH-5" is obtained in laboratory of extra- -dangerous infections of the State scientific center of virology and biotechnology "Vector" and deposited 20.03.2001 in NII Collection of cultures of microorganisms of the State scientific center of virology and biotechnology "Vector" (Novosibirsk district, s. Koltsovo GNTS VB "Vector") at registration number V-337. The strain of Ebol virus is adapted to broad spectrum of biological objects and used for carrying out model experiments in different biological objects and animal species for investigation of molecular-biological mechanism of alteration of Ebol virus virulence and the development of diagnostic and vaccine preparations. EFFECT: valuable properties of virus strain. 1 tbl

Description

 The invention relates to biotechnology and medical virology and can be used in healthcare and scientific research in the study of molecular biological mechanisms of changing the virulence of the Ebola virus.

 The Ebola virus was isolated in 1976 and assigned to the Filiviridae family. Filoviruses, being the causative agents of hemorrhagic fevers, belong to the group of viruses that cause especially dangerous diseases. Means of specific prophylaxis and effective treatment of diseases caused by the Ebola virus have not yet been developed (Ebola hemorrhagic fever in Zaire in 1976, Report of the International Commission. - WHO Bulletin 1998, 56, 2, pp. 213-237).

 Under these conditions, the question of obtaining new strains of the Ebola virus for conducting model experiments and preparing diagnostic and vaccine preparations is very relevant.

 Known strains of the Ebola virus Zaire 76 (Connolly BM, Steele KE, Davis KJ, Geisbert TW, Kell WM, Jaax NK, Jarling PB Pathogenesis of experimental Ebola virus invection in guinea pigs / J. Infect. Dis, 1999; 179 (Suppl): S203-17) and strain Zaire 95 (Bray M., Davis K., Geisbert T., Schmaljohn C., Huggins J. A mouse model for evaluation of prophylaxis and therapy of Ebola hemorrhagic fever / J. Infect Dis, 1999; 179 (Suppl): S. 248-218). These strains were used to conduct model experiments to study the pathogenesis of Ebola hemorrhagic fever and ways of its prevention and treatment in newborn ICR mice.

 However, to create diagnostic and vaccine preparations, it is required to obtain new strains adapted to different biological objects and animal species.

 The closest analogue (prototype) used for the selection of the new inventive virus strain Zaire Ch-15 is a strain of the Ebola virus Zaire Mayinga (Chepurnov AA, Tuzova MN, Temovoy VA, Chernukhin IV Suppressive effect of Ebola virus on T cell proliferation in vivo is provided by a 125-kDa GP viral protein / Immunology Letters 68 (1999) 257-261). Strain Zaire Mayinga obtained from the Belarusian Research Institute of Epidemiology and Microbiology, is a 10% homogenate of the liver of infected monkeys M. Rhesus.

The main characteristics of the prototype strain:
The name of the virus in accordance with the rules of nomenclature and position in the modern classification system: Mononegavirales order; family Filoviridae; genus Ebolavirus; view Zaire virus; Zaire Mayinga strain.

 Field of use of the virus: study of the pathogenesis of Ebola hemorrhagic fever and ways of its prevention and treatment in newborn ICR mice.

 Nucleic acid type: RNA. Nucleic acid structure: one non-infectious negative linear single-stranded RNA molecule.

Molecular mass of nucleic acid: 4.2 • 10 ⁶ Yes. Virion mass: 4.64 • 10 ⁸ Yes. Virion morphology: long filamentous forms, sometimes with branches, sometimes U-shaped or circular. Sizes of the virion: diameter - about 80 nm, particle length can reach 14000 nm. Capsid symmetry type: spiral.

 Physico-chemical properties of the virion: sedimentation coefficient of about 1400 S, density of virions of about 1.14 g / ml, which corresponds to the density of structures containing RNA, protein and lipids. Virions contain 8 proteins: 2 glycoproteins (GP1 and GP2, transmembrane proteins with a molecular weight of 120 and 26 kDa, respectively), 4 ribonucleocapsid complex proteins (NP with a molecular weight of 104 kDa and an encapsidation function; VP35 with a molecular weight of 35 kDa and a phosphoprotein analogue function; VP 30 with a molecular weight of 30 kDa and the function of encapsidation and binding; L with a molecular weight of 180 kDa and the function of RNA polymerase), as well as 2 proteins associated with the membrane (VP24 with a molecular weight of 24 kDa and an unknown function; VP40 with a molecular weight of 40 kDa and the function ma riksnogo protein).

 Cultural traits: in the Vero cell culture, the virus does not cause clear cytopathic changes, although signs of cellular changes are noted - foci of rounded cells with increased optical density appear. In the culture fluid and cells by electron microscopy, on the 3rd day after infection, characteristic virus particles consisting of a nucleocapsid mass are detected; the final budding of virions occurs on cell membranes.

Pathogenicity for the body: the virus in large concentrations can accumulate in the blood (10 ^5.5 -10 ^6.5 PFU / ml, tissue of the liver, spleen, lungs and pancreas). Transmission mechanism, source of infection, geographical distribution: contact spread of infection from person to person, infection is carried out by direct contact with infected blood, urine, nasopharyngeal discharge of a sick person; the source of infection is a sick person; no carriers of the Ebola virus have been found in nature. Geographical distribution: Central African States (Zaire). Contagiousness: high.

 Genetic interactions: the structure of the genome of the Ebola virus indicates kinship with the families Paramyxoviridae and Rhabdoviridae.

Method and conditions for long-term storage: stored in the form of a 10% suspension of the liver of infected monkeys M. Rhesus, prepared on yolk-sucrose medium at a temperature of minus 70 ^o C.

 The disadvantage of the prototype strain is that it is adapted to a narrow range of biological objects, which does not allow it to be used to create diagnostic and vaccine preparations.

 The technical result of the invention is to obtain a domestic author's strain of Ebola virus, adapted to a wider range of biological objects and used to conduct model experiments on different biological objects and animal species in order to study the molecular biological mechanism of changing the virulence of the Ebola virus and creating diagnostic and vaccine preparations.

 The Ebola virus strain Zaire Ch-15 (the author's name) was obtained at the Vector laboratory of the highly dangerous viral infections laboratory of the State Scientific Center for Virology and Biotechnology and was deposited on March 20, 2001 at the Collection of Microorganism Cultures Scientific Research Institute of the State Scientific Center for Virology and Biotechnology Vector "(Novosibirsk region, Koltsovo, State Research Center of the World Bank" Vector ") under registration number V-337.

The name of the virus in accordance with the rules of nomenclature and position in the modern classification system:
Order: Mononegavirales
Family: Filoviridae
Genus: Ebolavirus
View: Zaire vims
Strain: Zaire Ch-15
The new Zaire Ch-15 virus strain was obtained from the original Zaire Mayinga strain by 15 consecutive passages in a diploid cell culture of human lung embryo L-68.

MORPHOLOGY (architecture)
Virion shape: long filamentous forms, sometimes with branches, sometimes U-shaped or circular.

 Virion size: diameter - about 80 nm, particle length can reach 14000 nm.

 Capsid (shape, size): diameter - about 80 nm, particle length can reach 14000 nm.

Capsid Symmetry Type: Spiral
GENO AND CHEMOTAXONOMIC SIGNS
Nucleic acid (NK) type: RNA
Molecular Weight of NK: 4.64 • 10 ⁶ Yes
NK form: one non-infectious linear negative single-stranded RNA molecule.

 Restriction characteristics: (8 proteins) 2 glycoproteins (GP1 and GP2), 4 proteins of the ribonucleocapsid complex (NP; VP35; VP; L), 2 proteins associated with the membrane (VP24, VP40).

Carbohydrates: glycoproteins GP1 and GP2
Enzymes: RNA polymerase; transcriptase
The presence of mutations in the gene: partially studied; nucleotide substitutions were found in the gene encoding the Vp24 protein His> TYR ¹⁸⁶ , Asp> Gly ¹⁶⁵ .

 Viral genome strategy (reproduction method): genomic RNA performs two matrix functions: transcription and replication. General replication scheme: parent RNA negative chain> RNA plus chain> offspring RNA negative chain> offspring virions.

 Other features: the virion contains the transcriptase enzyme; the synthesized mRNAs have the length of one gene encoding a single polypeptide, which leads to the control of the relative amount of individual proteins.

 Physico-chemical properties of the virion: sedimentation coefficient of about 1400 S, floating density of the virions of about 1.14 g / ml, which corresponds to the density of structures containing RNA, protein and lipids. Virions contain 8 proteins: 2 glycoproteins (GP1 and GP2, transmembrane proteins with a molecular weight of 120 and 26 kDa, respectively), 4 ribonucleocapsid complex proteins (NP with a molecular weight of 104 kDa and an encapsidation function; VP35 with a molecular weight of 35 kDa and a phosphoprotein analogue function; VP 30 with a molecular weight of 30 kDa and the function of encapsidation and binding; L with a molecular weight of 180 kDa and the function of RNA polymerase), as well as 2 proteins associated with the membrane (VP24 with a molecular weight of 24 kDa and an unknown function; VP40 with a molecular weight of 40 kDa and the function ma riksnogo protein).

BIOTECHNICAL CHARACTERISTICS:
Cultivation conditions (medium, T ^o , time,): medium MEM needle with the addition of 2% cattle serum and antibiotics at the rate of 100 units / ml. Cultivation is carried out at a temperature of 37 ^o C for 5 days on cell cultures of L-68 or Vero. Final virus titer: 4.2 • 10 ⁴ PFU / ml.

Method and conditions for long-term storage: stored in the form of a 10% suspension of the liver of infected monkeys M. Rhesus, prepared on yolk-sucrose medium at a temperature of minus 70 ^o C.

 Method for determination (testing): titration by the method of formation of negative colonies under agar coating on a Vero cell culture.

 Safety: refers to the 1st group of biological hazard, all work is carried out in conditions of maximum biological safety.

CULTURAL PROPERTIES
In vitro cultivation conditions: nutrient medium MEM needle with the addition of 2% cattle serum and antibiotics at the rate of 100 units / ml, sensitive Vero or L-68 cells, cultivation temperature (37.0 ± 0.5) ^o С, infectious dose of the virus 0.01 PFU / ml, cultivation time 120 ± 12 hours. The number of passages is 15.

 Cytopathic effect: in the Vero cell culture, the virus does not cause clear cytopathic changes, although signs of cellular changes are noted - foci of rounded cells with increased optical density appear.

 Specific inclusions (cytoplasmic, nuclear): in the culture fluid and cells using electron microscopy already on the 3rd day after infection, characteristic virus particles consisting of a nucleocapsid mass are detected; the final budding of virions occurs on cell membranes.

Virus titer (under certain conditions): 4.2 • 10 ⁴ PFU / ml; accumulation time - 72 hours.

Resistance to external factors and chemicals. Viruses are inactivated by various detergents (tween-80, ND-40), ether, chlorine-containing disinfectants, 1% formalin, beta-propiolactone, as well as ultraviolet and gamma radiation. Viruses are completely inactivated at a temperature of (58 ± 2) ^o С and exposure for 2 hours.

ANTIGENIC PROPERTIES
Antigenic structure: antigenic properties are preserved, there are no serological differences between the original virus strain Zaire Mayinga and Zaire Ch-15. The virus neutralization constant by homologous serum: at least 1000. The claimed strain does not possess hemagglutinating activity.

ADDITIONAL CHARACTERISTICS AND PROPERTIES
Immunogenicity: causes the production of antiviral antibodies, including virus neutralizing ones. Virulence: virulence is preserved for guinea pigs, monkeys, reduced for newborn mice ICR from 7.45 to 2.25 lg LD ₅₀ . Contagiousness: high in direct contact with infectious material, the possibility of infection by airborne droplets is allowed. Attenuation stability: retains its stability after passages on Vero cell culture.

MARKER SIGNS OF THE STRAIN AND METHODS FOR THEIR IDENTIFICATION: There are no genetic, biochemical, and immunological signs. Other signs: reduced virulence for newborn ICR mice from 7.45 to 2.25 lg LD ₅₀ .

 Genetic interactions: the structure of the genome of the Ebola virus indicates kinship with the families Paramyxoviridae and Rhabdoviridae.

 Characterization of the life cycle of the virus: penetration through microtraumas of the skin and mucous membranes, primary replication occurs most likely in macrophages, after which the virus enters the cells of the liver (the main target organ), spleen, kidneys, and adrenal glands with blood flow. Viral replication is characteristic of viruses with a negative genome. Viral nucleocapsid mass accumulates in the cytoplasm in the form of inclusion bodies. The final budding of virions occurs on cell membranes.

 Stability of biological properties: retains acquired properties after passages on Vero cell culture.

Method and conditions for long-term storage: stored in the form of a virus-containing culture fluid at a temperature of minus 70 ^o C.

Passage history of the Ebola virus strain "Zaire Ch-15"
Eibola virus Mayinga strain obtained from the Belarusian Research Institute of Epidemiology and Microbiology was used as a starting material for selection.

According to the passage story, the Mainga strain passed 24 passages on a Vero E6 cell culture. It was highly virulent for newborn mice (7.45 lg LD ₅₀ ) and somewhat infectious (4.5 lg ID ₅₀ ) for guinea pigs. As a result of the studies, 15 consecutive passages of the initial virus were carried out in a diploid culture of L-68 cells. Passaging on the cell culture was performed by infecting the formed monolayer of human lung embryo cells (L-68) with a virus at a dose of 0.1-0.01 PFU / cell. Harvest was taken on the 5-6th day and a fresh monolayer of cell culture was infected with the obtained virus.

 Virus titration was performed by negative colony agar-coated colonies on a Vero cell culture and by lethal effect upon intracerebral infection of newborn white mice.

 The experiments used guinea pigs weighing 200-300 g, outbred white mice (newborns and adults), BALB / c mice and rabbits weighing 1-1.5 kg. Animals were kept on a standard diet. All painful procedures were performed using analgesics.

The harvest of each passage, collected on the 5th day after infection, was titrated on suction mice and a monolayer of Vero cells under agar coating. As can be seen from the table, the biological titer of the virus harvest in PFU decreased from the 1st to the 3rd passage, and then increased, reaching a plateau by the 12th passage, after which it remained unchanged. The virulence for newborn mice was constantly decreasing, reaching a plateau with a residual virulence of 2.25 lg LD ₅₀ to the 12th passage, and further also remained unchanged. Infections for guinea pigs during the passage of the virus on the cell culture L-68 has not changed.

 Thus, the inventive strain provides an increase in pathogenicity in the considered types of biological objects, which allows it to be used to conduct model experiments on these biological objects and animal species in order to study the molecular biological mechanism of changing the virulence of the Ebola virus and developing approaches to the creation of diagnostic and vaccine preparations.

Claims (1)

The strain of the Ebola virus "Zaire Ch-15", a collection of SSC WB "Vector", registration number V-337, used for model experiments and the preparation of diagnostic and vaccine preparations.

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