Novel severe fever with thrombocytopenia syndrome virus

Abstract

To provide a severe fever with thrombocytopenia syndrome virus of a novel genomic type, and immunogenic compositions comprising the virus.SOLUTION: The invention provides a novel genotype of severe fever with thrombocytopenia syndrome virus (SFTSV) and immunogenic composition thereof, where the SFTSV comprises L gene, M gene, NP gene and NS gene each having a specific base sequence in which a 1447-th amino acid of a protein expressed in ORF (6255 bp) of an L gene thereof is valine or a 1913-rd amino acid thereof is lysine, where a 83-rd amino acid of a protein expressed in ORF (3222 bp) of an M gene thereof is tyrosine, or a 404-th amino acid thereof is threonine or a 904-th amino acid thereof is valine.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a severe fever with thrombocytopenia syndrome virus of a novel genotype and its use as an immunogenic composition. [Background technology]

[0002] Severe fever with thrombocytopenia syndrome (SFTS) is a serious disease characterized by high fever, vomiting, diarrhea, thrombocytopenia, leukopenia, and multiple organ failure, with a mortality rate of 6-30% (Yu XJ et al., N.Engl.J.Med.2011;364:1523-32; Ding F et al Clin Infect Dis 2013;56:1682-3).

[0003] The causative agent of SFTS is the severe fever thrombocytopenia syndrome virus (SFTSV), which belongs to the family Bunyaviridae. Bunyaviruses are negative-strand RNA viruses that contain three segments, and consist of five genera, including Orthobunyavirus, Hantavirus, Nairovirus, Phlebovirus, and Tospovirus. SFTSV belongs to the Phlebovirus genus, which also includes Rift valley fever virus. SFTSV was first reported in China in 2011 (Yu XJ et al., 2013). al.ibid), a new mutant virus that has been persistently occurring not only in China but also in Korea and Japan. SFTSV is a spherical virus with a diameter of 80-100 nm. This virus is a single-stranded negative sense It has three genes, which are the RNA segments: large (L) segment, medium (M) segment, and small (S) segment (NP, NS).

[0004] The SFTS virus is transmitted by the tick Haemaphysalis longicornis, which is known to be widely distributed in Korea (Chae JS et al. J Vet Sci 2008;9:285-93; Kim CM et al. Appl Environ Microbiol 2006;72:5766-76). Seroconversion and viremia of the SFTS virus have been found in domestic animals such as goats, sheep, cattle, pigs, and dogs, and these animals are also thought to have acted as intermediate vectors in areas where the SFTS virus has spread (Zhao L et al. Emerg Infect Dis 2013;18:963-5; Niu G et al. Emerg Infect Dis 2013;19:756-63). SFTSV has been detected in the blood of patients, and the concentration of SFTSV is particularly high in the blood of severely ill patients, making human-to-human transmission possible via blood (Tang X, Wu W, Wang H, et al. J Infect Dis 2013;207:736-739.).

[0005] Since antiviral drugs against SFTSV have not yet been developed, conservative treatment for organ failure, such as blood transfusions and neoalgesia, is the basis of SFTS treatment. In China, the combination of ribavirin has been introduced into treatment guidelines since 2012, but the results of treatment recently published showed no difference in mortality between the ribavirin-treated and non-treated groups. Therefore, a vaccine against SFTSV is needed, but the reality is that no vaccine has yet been developed. Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a severe fever with thrombocytopenia virus of a novel genotype and an immunogenic composition using the same. [Means for solving the problem]

[0007] To achieve the above object, the present invention provides a novel severe fever with thrombocytopenia virus.

[0008] The present invention also provides an immunogenic composition for preventing or treating severe fever with thrombocytopenia.

[0009] The present invention also provides an antibody against severe fever with thrombocytopenia virus or an antigen thereof.

[0010] The present invention also provides a diagnostic kit for severe fever with thrombocytopenia virus.

[0011] The present invention also provides a method for detecting severe fever with thrombocytopenia virus antibodies.

[0012] The present invention also provides a method for producing an antiserum against severe fever with thrombocytopenia virus.

[0013] Additionally, the present invention provides methods for providing information regarding the diagnosis of severe fever with thrombocytopenia. Effect of the Invention

[0014] The severe fever thrombocytopenia virus of the present invention is a novel virus that is genetically different from conventional thrombocytopenia viruses and is phylogenetically subdivided into the B genotype. Therefore, due to the characteristics of vaccines that exhibit protective ability limited to viruses of a certain genotype, the novel virus of the present invention can be usefully used as a vaccine with excellent cross-immunogenicity against SFTSV. [Brief description of the drawings]

[0015] [Figure 1]FIG. 1 shows the phylogenetic tree of the three novel viruses of the present invention and the SFTSV L gene isolated in China, Japan, and Korea. [Diagram 2] FIG. 1 shows the phylogenetic tree of the three novel viruses of the present invention and the SFTSV M gene isolated in China, Japan, and Korea. [Diagram 3] FIG. 1 shows the phylogenetic tree of the S(NP) genes of the three novel viruses of the present invention and SFTSV isolated in China, Japan, and Korea. [Figure 4] FIG. 1 shows a phylogenetic tree of the S (NS) genes of the three novel viruses of the present invention and SFTSV isolated in China, Japan, and Korea. [Diagram 5] This figure shows the amino acid mutation sites in the L and M genes of three novel viruses of the B-1 (CB3 / 2016), B-2 (CB7 / 2017) and B-3 (CB6 / 2016) genotypes of the present invention. [Figure 6] This figure confirms the effectiveness of vaccines against three novel viruses of the B-1 (CB3 / 2016), B-2 (CB7 / 2017), and B-3 (CB6 / 2016) genotypes of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Hereinafter, the present invention will be described in detail with reference to the accompanying drawings. However, the following examples are presented as examples of the present invention, and if it is determined that a detailed description of a well-known technology or configuration may unnecessarily obscure the gist of the present invention, the detailed description may be omitted and the present invention is not limited thereby. The present invention is subject to various modifications and applications within the scope of the claims and the scope of equivalents interpreted therefrom.

[0017] In addition, the terminology used in this specification is used to appropriately express the preferred embodiment of the present invention, and may vary depending on the intention of the user or operator, or the practice of the field to which the present invention belongs. Therefore, the definition of the term should be determined based on the entire contents of this specification. Throughout the specification, when a part "includes" a certain element, this does not exclude other elements, but means that other elements may be further included, unless otherwise specified to the contrary.

[0018] In one aspect, the present invention relates to a severe fever thrombocytopenia syndrome virus (SFTSV) in which the 1447th amino acid of the protein expressed by the L gene ORF (6255 bp) is valine or the 1913th amino acid is lysine, and the 83rd amino acid of the protein expressed by the M gene ORF (3222 bp) is tyrosine, the 404th amino acid is threonine, or the 904th amino acid is valine. In one embodiment of the present invention, the virus is designated B-1(CB3).

[0019] In one embodiment, the severe fever with thrombocytopenia virus belongs to genotype B-1 and may be a virus that is conventionally classified as genotype B, in which the 1447th amino acid, isoleucine, of the protein expressed in the ORF (6255 bp) of the L gene is replaced with valine, the 1913th amino acid, arginine, is replaced with lysine (SEQ ID NO: 13), and the 904th amino acid, isoleucine, of the M gene is replaced with valine (SEQ ID NO: 14).

[0020] In one embodiment, the severe fever with thrombocytopenia virus may include an L gene having a base sequence of SEQ ID NO: 1, an M gene having a base sequence of SEQ ID NO: 2, and an S gene having NP having a base sequence of SEQ ID NO: 3 and NS having a base sequence of SEQ ID NO: 4.

[0021] In one embodiment, the severe fever with thrombocytopenia virus may include the amino acid sequence of SEQ ID NO: 13 expressed in the ORF of the L gene (6255 bp), the amino acid sequence of SEQ ID NO: 14 expressed in the ORF of the M gene (3222 bp), the amino acid sequence of SEQ ID NO: 15 expressed in the ORF of the NP gene, and the amino acid sequence of SEQ ID NO: 16 expressed in the ORF of the NS gene.

[0022] In one aspect, the present invention relates to a severe fever with thrombocytopenia virus, in which the amino acid at position 1447 of the protein expressed in the ORF of the L gene (6255 bp) is isoleucine or the amino acid at position 1913 is arginine, and the amino acid at position 83 of the protein expressed in the ORF of the M gene (3222 bp) is phenylalanine, the amino acid at position 404 is threonine, or the amino acid at position 904 is isoleucine. In one embodiment of the present invention, the virus is designated B-2 (CB4).

[0023] In one embodiment, the severe fever with thrombocytopenia virus may belong to genotype B-2 and may be a virus in which the 83rd amino acid, tyrosine, in the M gene of a virus previously classified as genotype B is replaced with phenylalanine.

[0024] In one embodiment, the severe fever with thrombocytopenia virus may include an L gene having a base sequence of SEQ ID NO:5, an M gene having a base sequence of SEQ ID NO:6, an NP having a base sequence of SEQ ID NO:7, and an S gene having an NS having a base sequence of SEQ ID NO:8.

[0025] In one embodiment, the severe fever with thrombocytopenia virus may include the amino acid sequence of SEQ ID NO: 17 expressed in the ORF of the L gene, the amino acid sequence of SEQ ID NO: 18 expressed in the ORF of the M gene, the amino acid sequence of SEQ ID NO: 19 expressed in the ORF of the NP gene, and the amino acid sequence of SEQ ID NO: 20 expressed in the ORF of the NS gene.

[0026] In one aspect, the present invention relates to a severe fever with thrombocytopenia virus, in which the amino acid at position 1447 of the protein expressed in the ORF (6255 bp) of the L gene is isoleucine or the amino acid at position 1913 is arginine, and the amino acid at position 83 of the protein expressed in the ORF (3222 bp) of the M gene is tyrosine, the amino acid at position 404 is alanine, or the amino acid at position 904 is isoleucine. In one embodiment of the present invention, the virus is designated B-3 (CB1).

[0027] In one embodiment, the severe fever with thrombocytopenia virus may belong to genotype B-3 and may be a virus in which the 404th amino acid, threonine, in the M gene of a virus previously classified as genotype B is replaced with alanine.

[0028] In one embodiment, the severe fever with thrombocytopenia virus may include an L gene having a base sequence of SEQ ID NO:9, an M gene having a base sequence of SEQ ID NO:10, and an S gene having an NP having a base sequence of SEQ ID NO:11 and an NS having a base sequence of SEQ ID NO:12.

[0029] In one embodiment, the severe fever with thrombocytopenia virus may include the amino acid sequence of SEQ ID NO: 21 expressed in the ORF of the L gene, the amino acid sequence of SEQ ID NO: 22 expressed in the ORF of the M gene, the amino acid sequence of SEQ ID NO: 23 expressed in the ORF of the NP gene, and the amino acid sequence of SEQ ID NO: 24 expressed in the ORF of the NS gene.

[0030] In one embodiment of the present invention, genetic analysis of the isolated severe fever with thrombocytopenia virus revealed that the gene sequences were different from those of known severe fever with thrombocytopenia virus genes, and phylogenetic classification revealed that each gene was subdivided into at least three genotypes that were not part of the known B genotype group and were separable genotypes.

[0031] The term "substitution" as used herein means the replacement of one or more amino acids or nucleotides by other amino acids or nucleotides, respectively.

[0032] The severe fever with thrombocytopenia virus of the present invention is a negative-stranded RNA virus belonging to the genera Bunyaviridae and Phlebovirus. It is a spherical virus with a diameter of 80 to 100 nm, transmitted by Haemaphysalis longicornis, and has a dielectric constant consisting of large (L), medium (M), and small (S) segments. It is also involved in the transcriptional regulation of RNA-dependent RNA polymerase (RdRp), glycoprotein preproteinase (GPRP), and cytoplasmic RNA (PCR). cursor(M), glycoprotein N(Gn), glycoprotein C(Gc), nucleocapsid protein(NP) and non-s The negative or antisense strand (the antisense of the sense or plus strand that codes for the viral proteins) encodes the proteins or genes as antisense, and after the sense or plus strand RNA is generated for the expression of the genes into proteins, translation is carried out from this, and the proteins are produced.

[0033] In one aspect, the present invention relates to an immunogenic composition for preventing or treating severe fever with thrombocytopenia, comprising a severe fever with thrombocytopenia virus or an antigen thereof as an active ingredient.

[0034] In one embodiment, the immunogenic composition of the present invention can include an inactivated severe fever with thrombocytopenia virus and a pharma- ceutically acceptable carrier or adjuvant.

[0035] In one embodiment, the immunogenic composition may be a vaccine composition, the form of which may be selected from the group consisting of live vaccines, inactivated vaccines, subunit vaccines produced using attenuated severe fever with thrombocytopenia virus genes, vector vaccines, chimeric vaccines, DNA and RNA vaccines.

[0036] In one embodiment, the immunogenic composition may contain as an active ingredient a severe fever with thrombocytopenia virus comprising an L gene having a base sequence of SEQ ID NO: 1, an M gene having a base sequence of SEQ ID NO: 2, and an S gene having a base sequence of SEQ ID NO: 3, a severe fever with thrombocytopenia virus comprising an L gene having a base sequence of SEQ ID NO: 4, an M gene having a base sequence of SEQ ID NO: 5, and an S gene having a base sequence of SEQ ID NO: 6, a severe fever with thrombocytopenia virus comprising an L gene having a base sequence of SEQ ID NO: 7, an M gene having a base sequence of SEQ ID NO: 8, and an S gene having a base sequence of SEQ ID NO: 9, or an antigen of these.

[0037] To produce the immunogenic composition (i.e. vaccine) of the invention, the virus according to the invention or its antigens are converted into a physiologically acceptable form. This can be done on the basis of experience in the production of vaccines used for influenza vaccination (as disclosed by Stickl, H. et al.

[1974] Dtsch. med. Wschr. 99, 2386-2392). For the production of the vaccination, for example, the virus particles are freeze-dried in ampoules, preferably glass ampoules, in 100 ml of phosphate-buffered saline (PBS) in the presence of 1% human albumin and 2% peptone. Alternatively, the vaccination can be produced by sequential freeze-drying of the virus in a formulation. This formulation can contain additional additives such as mannitol, dextran, sugars, glycine, lactose or polyvinylpyrrolidone or other auxiliary substances such as antioxidants or inert gases, stabilizers or recombinant proteins suitable for in vivo administration (e.g. human serum albumin). The glass ampoules can then be sealed and stored between 4°C and room temperature for several months. However, unless other requirements exist, the ampoules can be stored preferably at below -20°C.

[0038] For vaccination or treatment, the lyophilisate is dissolved in 0.1-0.5 ml of an aqueous solution, preferably saline or Tris buffer, and can be administered systemically or locally, i.e. parenterally, subcutaneously, intramuscularly or by any other administration route known to those skilled in the art. The administration form, dose and number of administrations can be optimized by those skilled in the art in known manner. However, most commonly, patients receive a second vaccination about one month to six weeks after the first vaccination.

[0039] In the present invention, the term "prevention" refers to all actions that suppress or delay the occurrence, spread, and recurrence of severe fever with thrombocytopenia by administering the immunogenic composition according to the present invention.

[0040] The term "treatment" as used in the present invention means any action of improving or favorably modifying the symptoms of severe fever with thrombocytopenia and its complications by administering the immunogenic composition according to the present invention. Those skilled in the art to which the present invention pertains will be able to know the exact criteria for diseases for which the composition of the present application is effective and judge the degree of improvement, enhancement, and treatment by referring to materials presented by the Korean Medical Association, etc.

[0041] The term "therapeutically effective amount" used in combination with an active ingredient in the present invention means an amount effective for preventing or treating a disease caused by transplant rejection, and the therapeutically effective amount of the composition of the present invention may vary depending on various factors, such as the administration method, the target site, and the condition of the patient. Therefore, the dosage when used in the human body should be determined to be an appropriate amount, taking into consideration both safety and efficacy. It is also possible to estimate the amount used in humans from the effective amount determined through animal experiments. These matters to be considered when determining the effective amount are described, for example, in Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed.(2001), Pergamon Press, and EW Martin ed., Remington's Pharmaceutical Sciences, 18th ed.(1990), Mack Publishing Co.

[0042] The pharmaceutical composition of the present invention is administered in a pharmacologic effective amount. The term "pharmacologic effective amount" as used herein means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment and without causing side effects, and the effective dose level can be determined based on factors including the patient's health condition, type of transplant, severity, drug activity, drug sensitivity, administration method, administration time, administration route, and excretion rate, treatment duration, co-administered or co-used drugs, and other factors well known in the medical field. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered in single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that provides maximum effect at a minimum amount without side effects, which can be easily determined by one of ordinary skill in the art.

[0043] The pharmaceutical composition of the present invention may contain carriers, diluents, excipients, or combinations of two or more thereof that are commonly used in biological preparations. The term "pharmaceutical acceptable" as used in the present invention means that the composition exhibits the property of being non-toxic to cells or humans exposed to the composition. The carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, and may be, for example, a compound listed in the Merck Index, 13th ed., Merck & Co. Inc., saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, or a mixture of one or more of these components, and other common additives such as antioxidants, buffers, bacteriostatic agents, etc. may be added as necessary. In addition, the composition may be formulated into injection forms such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, or tablets by additionally adding diluents, dispersants, surfactants, binders, and lubricants. Furthermore, the composition may be formulated into injection forms such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, or tablets by appropriate methods in the art or in Remington's Pharmaceutical The composition can be suitably formulated according to each disease or ingredient using the methods disclosed in Science (Mack Publishing Company, Easton PA, 18th, 1990).

[0044] In one embodiment, the pharmaceutical composition may be in one or more dosage forms selected from the group including oral dosage forms, topical preparations, suppositories, sterile injection solutions and sprays, with oral or injection dosage forms being more preferred.

[0045] The term "administration" as used in the present invention means providing a given substance to an individual or patient in any suitable manner, and can be administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally or locally in the form of an injection) or orally depending on the intended method, and the dosage range varies depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and disease severity. Liquid preparations for oral administration of the composition of the present invention include suspensions, oral liquids, emulsions, syrups, etc., which may contain various excipients such as wetting agents, sweeteners, flavoring agents, preservatives, etc. in addition to water and liquid paraffin, which are commonly used simple diluents. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, etc. The pharmaceutical composition of the present invention can also be administered by any device that can transfer the active substance to target cells. Preferred administration methods and preparations are intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections, etc. Injectables can be prepared using aqueous solvents such as physiological saline and Ringer's solution, or non-aqueous solvents such as vegetable oils, higher fatty acid esters (e.g., ethyl oleate, etc.), and alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.), and can contain pharmaceutical carriers such as stabilizers to prevent deterioration (e.g., ascorbic acid, sodium bisulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers for adjusting pH, and preservatives to prevent microbial growth (e.g., phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.).

[0046] The term "individual" as used in the present invention means any animal, including monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, or guinea pigs, including humans, that have or may have the above-mentioned cancers, and the term "specimen" can be whole blood, plasma, serum, urine, or saliva separated therefrom.

[0047] The pharmaceutical composition of the present invention may further include a pharma- ceutically acceptable additive, and examples of the pharma-ceutically acceptable additive include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, candy, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, sucrose, dextrose, sorbitol, and talc. The pharma-ceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 to 90 parts by weight based on the composition, but is not limited thereto.

[0048] In one aspect, the invention relates to antibodies generated in response to immunization with the viruses of the invention or antigens thereof.

[0049] The antibody includes not only the form of a whole antibody but also functional fragments of an antibody molecule. A whole antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is linked to a heavy chain by a disulfide bond. A functional fragment of an antibody molecule means a fragment that retains an antigen-binding function, and examples of antibody fragments include (i) a Fab fragment consisting of a light chain variable region (VL), a heavy chain variable region (VH), a light chain constant region (CL) and the first heavy chain constant region (CH1); (ii) an Fd fragment consisting of a VH and CH1 domain; (iii) an Fv fragment consisting of a single antibody VL and VH domain; and (iv) a dAb fragment consisting of a VH domain (Ward ES et al., Nature 2003, 144:131-132, 2003). 341:544-546 (1989)]; (v) isolated CDR regions; (vi) a F(ab')2 fragment, which is a bivalent fragment comprising two linked Fab fragments; (vii) a single chain Fv molecule (scFv) in which the VH and VL domains are joined by a peptide linker to form an antigen-binding site; (viii) bispecific single chain Fv dimers (PCT / US92 / 09965) and (ix) a diabody, which is a multivalent or multispecific fragment produced by gene fusion (WO94 / 13804).

[0050] In one aspect, the present invention relates to a diagnostic kit for severe fever with thrombocytopenia virus, comprising the severe fever with thrombocytopenia virus or an antigen thereof, or an antibody thereto.

[0051] In one embodiment, the kit comprises a virus sample containing the virus according to the present invention and a reagent for detecting the antigen-antibody complex, the reagent for detecting the antigen-antibody complex comprising a radial immunoassay, an ELISA (Enzyme linked immunosorbent assay) or an immunofluorescence assay.

[0052] In one embodiment, the detection of the antigen-antibody complex can be achieved by immunoelectrophoresis such as Ouchterlony plate, Western blot, Crossed IE, Rocket IE, Fused Rocket IE, Affinity IE, which can easily detect the antibody and / or antigen by antigen-antibody binding. The reagent or substance used in such a method is known, and can be detected, for example, through an antigen-antibody reaction, a substrate that specifically binds to the antigen, a nucleic acid or peptide aptamer, a reaction with a receptor, ligand, or cofactor that interacts with the complex, or a mass spectrometer. The reagent or substance that specifically interacts with or binds to the antigen-antibody complex of the present application can be used in a chip format or together with nanoparticles. The immunoanalysis or immunostaining method is described in Enzyme Immunoassay, ET Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzyme-linked immunosorbent assay (ELISA), in Methods in Molecular Biology, Vol. 1, Walker, JM ed., Humana Press, NJ, 1984, etc. The presence or absence of infection can be diagnosed by analyzing the final signal intensity from the above-mentioned immunoanalysis process, i.e., by performing a signal comparison with a normal sample.

[0053] In one aspect, the invention relates to a diagnostic composition comprising severe fever with thrombocytopenia virus or an antigen thereof, or an antibody thereto.

[0054] The compounds of the invention used in diagnostic compositions are preferably detectably labeled. A variety of methods that can be used to label biomolecules are known to those skilled in the art and are contemplated within the scope of the present invention. Such methods are described in detail in: Tijssen, "Practice and theory of enzyme immunoassays", Burden, RHand von Knippenburg (Eds), Volume 15 (1985), "Basic methods in molecular biology'; Davis et al., "Labeling of biological molecules using enzyme immunoassays", Vol. 15 (1985), ... LG, Dibmer MD; Battey Elsevier (1990), Mayer et al., (Eds)” Immunochemical methods in "cell and molecular biology" Academic Press, London (1987), or in the series" Methods in Enzymology, Academic Press, Inc.

[0055] There are many alternative labeling methods known to those of ordinary skill in the art. Examples of the types of labels that can be used in the present invention include enzymes, radioisotopes, colloidal metals, fluorescent compounds, chemiluminescent compounds, and bioluminescent compounds.

[0056] Commonly used labels include fluorescent substances (e.g., fluorescein, rhodamine, Texas Red, etc.), enzymes (e.g., wasabi peroxidase, β-galactosidase, alkaline phosphatase), radioisotopes (e.g., 32P or 125I), biotin, digoxigenin, colloidal metals, chemiluminescent or bioluminescent compounds (e.g., dioxygenase, luminol or acridinium). Labeling methods such as covalent attachment of enzymes or biotinyl groups, iodination, phosphorylation, biotinylation, etc. are well known in the art.

[0057] Detection methods include, but are not limited to, autoradiography, fluorescence microscopy, direct and indirect enzyme reactions. Commonly used detection analysis methods include radioisotope or non-radioisotope methods. These include Western blotting, overlay analysis, RIA (Radioimmuno Assay) and IRMA (Immune Radioimmunometric Assay), EIA (Enz There are several types of immunoassays, including ELISA (Enzyme Linked Immuno Assay), ELISA (Enzyme Linked Immuno Sorbent Assay), FIA (Fluorescent Immuno Assay) and CLIA (Chemioluminescent Immune Assay).

[0058] In one aspect, the present invention relates to a method for detecting severe fever with thrombocytopenia virus antibodies, comprising the steps of contacting a sample isolated from a specimen with a virus of the present invention or an antigen thereof under conditions allowing the formation of an antigen / antibody complex, and detecting the formation of an antigen / antibody complex.

[0059] In one aspect, the invention relates to a method for producing antisera against severe fever with thrombocytopenia virus from a non-human animal, comprising administering to the non-human animal a virus of the invention or an antigen thereof in an amount effective to induce an immune response, and collecting antisera or plasma containing antibodies against said severe fever with thrombocytopenia virus.

[0060] In one aspect, the present invention relates to a method for providing information regarding the diagnosis of severe fever with thrombocytopenia, comprising the steps of contacting a sample isolated from a specimen with the virus of the present invention or an antigen thereof to form an antigen-antibody complex, and detecting the formation of said complex.

[0061] The present invention will be described in more detail with reference to the following examples, which are merely for illustrative purposes and are not intended to limit the scope of the present invention.

[0062] Example 1. Virus isolation

[0063] We confirmed positive / negative results for severe fever thrombocytopenia syndrome virus (SFTSV) using real-time PCR, PCR and ELISA analysis using blood from patients with suspected severe fever thrombocytopenia symptoms among patients visiting a university hospital, blood from animals (goats and abandoned dogs) and wild tick homogenates. Specifically, on the day before virus infection, VeroE6 cells were dispensed into 12-well plates and cultured to a cell density of over 60%, then the cells were washed with PBS and infected with 300 μl of serum from a patient suspected of infection (serum obtained by centrifuging the whole blood at 3000 rpm for 20 minutes). After infection, the serum was removed and the cells were washed with PBS, replaced with 1% FBS DMEM medium and cultured for 2 weeks. After 2 weeks, the presence or absence of virus isolation was confirmed by RT-PCR (confirmed by real time PCR after reverse transcription) and immunofluorescence assay (using a laboratory-produced mouse SFTSV NP antibody as the primary antibody and a FITC-counjugated antibody as the secondary antibody). If the virus was not isolated, the virus was isolated by infecting another VeroE6 cell line with the supernatant from the first infection. The isolated viruses were named CB3 / 2016, CB7 / 2017, and CB6 / 2016.

[0064] Example 2. Genetic analysis of isolated viruses

[0065] The viruses CB3 / 2016, CB7 / 2017, and CB6 / 2016 isolated using Vero E6 cells were subjected to reverse transcription, PCR, and next generation sequencing (NGS) to confirm the full-length sequences of their L, M, and S (NP, NS) genes. Specifically, RNA was extracted from each virus, and cDNA was prepared using reverse transcription PCR. Then, PCR was performed on the L, M, and S genes of each SFTS virus to obtain their full-length genes. For gene sequence analysis, the NGS method was used to tagment and index PCR the L, M, and S genes of each virus using the Illumina Nextera XT kit according to the protocol provided by Illumina. Then, the final samples were used with the Illumina miniseq instrument to generate Fasta Q files, and the gene sequences of the generated files were analyzed using the CLC main workbench program to analyze the full-length gene sequences of each virus. The confirmed gene sequence was integrated with the genes of conventional viruses isolated in Korea, China, and Japan, and genetic analysis was performed. As a result, it was confirmed that the SFTSVs CB1, CB3, and CB4 isolated in the present invention are SFTSVs of a new genotype that is genetically different from the genes of viruses currently isolated in China and Korea (YuXJ et al., N.Engl.J.Med.2011) and the viruses first isolated in Korea (Gangwon / 2012). In addition, phylogenetic genetic analysis of the L, M, and S (NP, NS) genes of the virus of the present invention and the viruses isolated in Korea, China, and Japan was performed using the MEGA 7.0 program. As a result, it was confirmed that the virus of the present invention was close to group B, to which the majority of isolates in Korea belong, but showed differences from conventional viruses in the genes L, M, and S (NP and NS) genes and was subdivided into at least three or more groups (Figures 1 to 4). Therefore, the three SFTSV genotypes isolated in this invention were further subdivided and named B-1 (CB3 / 2016), B-2 (CB7 / 2017), and B-3 (CB6 / 2016).

[0066] Example 3. Identifying differences in the amino acid sequences of the L, M, and S genes of different viruses

[0067] 3-1. Differences in the amino acid sequence of the L gene

[0068] The amino acid sequences of the new genotype B-1, B-2 and B-3 viruses isolated in the present invention were analyzed based on the ORF (open reading frame) of the L, M and S genes, and the results showed differences from the L and M genes. Specifically, among the B genotypes, the ORF (6255 bp) of the L gene encodes RdRp, and in the genotype B-1 virus of the present invention, the amino acid at position 1447 of RdRp is represented as valine or the amino acid at position 1913 is represented as lysine, while in the genotype B-2 virus and B-3 virus, the amino acid at position 1447 of RdRp is represented as isoleucine or the amino acid at position 1913 is represented as arginine (FIG. 5A and Table 1).

[0069] [Table 1]

[0070] 3-2. Differences in the amino acid sequence of the M gene

[0071] Analysis of the amino acid sequences of the L, M and S genes of the new detailed genotype B-1, B-2 and B-3 viruses isolated in the present invention showed differences in the L and M genes. Specifically, the ORF (3222 bp) of the M gene of the B genotype encodes Gn and Gc proteins, and the genotype B-1 virus of the present invention shows that the 83rd amino acid of the ORF of the M gene is tyrosine, the 404th amino acid is threonine, or the 904th amino acid is valine, the genotype B-2 virus shows that the 83rd amino acid of the ORF of the M gene is phenylalanine, the 404th amino acid is threonine, or the 904th amino acid is isoleucine, and the genotype B-3 virus shows that the 83rd amino acid of the ORF of the M gene is tyrosine, the 404th amino acid is alanine, or the 904th amino acid is isoleucine (Figure 5B and Table 1).

[0072] Example 4. Comparison of gene homology by genotype

[0073] Currently, SFTSV isolated in Korea were subjected to genetic analysis, including the detailed genotypes B-1, B-2, and B-3 of the present invention, and the homology of the L, M, and S genes of each genotype was compared and analyzed. As a result, viruses belonging to the same genotype showed about 96-100% genetic homology (nucleotide level), whereas viruses of different genotypes showed relatively low genetic homology of 91-97% (Tables 2-5). In addition, in the case of the genotype B-3 group of the present invention, the homology within the same group was relatively low at about 95-100%, and it was confirmed that it is possible to subdivide other genotype groups even within the B-3 group.

[0074] [Table 2]

[0075] [Table 3]

[0076] [Table 4]

[0077] [Table 5]

[0078] Example 5. Analysis of genotype-specific cross-immunogenicity and confirmation of vaccine efficacy

[0079] 5-1. Analysis of genotype-specific cross-immunogenicity To compare the cross-immunogenicity of each genotype, FRNT50 (fifty Specifically, the new genotypes B-1, B-2 and B-3 viruses isolated in the present invention were grown in large quantities, inactivated by adding formalin (0.05%), and the presence or absence of inactivation was confirmed by virus isolation three times. Proteins were produced by ultracentrifugation of the 20% sucrose vaccine, and ferrets were immunized with the protein. Two weeks later, the ferrets were boosted (2 weeks after the second immunization), and blood was collected and serum was separated. The separated serum was inactivated at 56°C for 30 minutes, diluted 1:1, and then serially diluted 2-fold. The virus diluted to 200 FFU / ml was reacted with the serially diluted virus at 1:1 at 37°C. VeroE6 cells dispensed into a 6-well plate were washed and infected with the reacted virus, and after 1 hour, 0.8% DMEM agarose gel containing 1% FBS was poured onto the cells. Five days after infection, the cells were fixed using formalin, and after 3 hours, they were washed three times, and then treated with 10% triton x-100 for 5 minutes at room temperature. They were then washed three times and blocked with 5% BSA. The produced polyclonal NP antibody was used as the primary antibody, and after incubation, the wells were washed three times, and then reacted with HRP-conjugated antibody as the secondary antibody for 1 hour, washed, and then colored with DAB to confirm the results. A value up to 50% reduction in focus formation from wells infected with the virus alone was interpreted as effective.

[0080] [Table 6]

[0081] As a result of examining the cross-immunogenicity of B-1, B-2 and B-3, the highest titers were observed against each virus as shown in Table 6. Therefore, it was found that high cross-neutralizing reactions were observed between viruses belonging to the same genotype, but relatively low neutralizing reactions were observed between viruses of different genotypes.

[0082] 5-2. Confirmation of vaccine effectiveness

[0083] The new genotypes B-1, B-2 and B-3 viruses isolated in this invention were mass-produced and inactivated by adding formalin (0.05%), and the inactivation was confirmed through virus isolation three times. The inactivated whole vaccine was administered at 2-week intervals. Five ferrets were immunized twice with each virus, and then 1 × 10 7.6 The challenge inoculation was 1 ml / ml.

[0084] As a result of the challenge, all animals in the control group died within 10 days of infection with the challenge virus, but all ferrets in the vaccinated group survived, and increases in body temperature and weight loss were observed 2-8 days after infection, but they were subsequently confirmed to recover (Figure 6).

[0085] To summarize the above results, SFTSV exists as a genotype with various genes, and viruses belonging to the same genotype show relatively high genetic homology and high cross-immune reactivity, but show relatively low genetic homology and low cross-immunogenicity with viruses of other genotypes. Therefore, it can be inferred that in order to show cross-immunogenicity against various genotypes, protective ability is limited to viruses of a certain genotype.

[0086] Therefore, the novel viruses CB3 / 2016, CB7 / 2017 and CB6 / 2016 belonging to the subgenotypes B-1, B-2 and B-3 of the present invention are considered to be useful as vaccines with excellent cross-immunogenicity against SFTSV of genotype B.

Claims

1. the 1447th amino acid of the protein expressed by the ORF (6255 bp) of the L gene is valine or the 1913th amino acid is lysine, the 83rd amino acid of the protein expressed by the ORF (3222 bp) of the M gene is tyrosine, the 404th amino acid is threonine, or the 904th amino acid is valine; A severe fever thrombocytopenia syndrome virus (SFTSV), comprising an L gene comprising the base sequence of SEQ ID NO:1, an M gene comprising the base sequence of SEQ ID NO:2, an NP gene comprising the base sequence of SEQ ID NO:3, and an NS gene comprising the base sequence of SEQ ID NO:

4.

2. An immunogenic composition for use in preparing a vaccine for preventing or treating severe fever with thrombocytopenia, comprising the severe fever with thrombocytopenia virus according to claim 1 as an active ingredient, A composition exhibiting enhanced cross-immunogenicity against seroconverting thrombocytopenia virus.

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