Recombinant virus against japanese encephalitis virus genotype 5 domestic isolate MK541529.1, and use thereof

Abstract

The present invention relates to a recombinant virus against Japanese encephalitis virus type 5 domestic isolate MK541529.1 and a use thereof, and provides a composition for producing the recombinant Japanese encephalitis virus, a method for producing the recombinant Japanese encephalitis virus, the recombinant Japanese encephalitis virus produced by the method, and a vaccine composition against Japanese encephalitis, the vaccine composition comprising the recombinant virus as an active ingredient.

Description

Recombinant virus for Japanese encephalitis virus genotype 5 domestic isolate MK541529.1 and its use

[0001] This relates to a recombinant virus for Japanese encephalitis virus type 5 domestically isolated strain MK541529.1 and its use.

[0002] Japanese encephalitis is a representative infectious disease transmitted by mosquitoes. It is a fatal zoonotic disease that causes encephalitis in humans accompanied by high fever and coma, and eventually leads to death. In Korea and Japan, the small red house mosquito (Culex tritaeniorhynchus) is known to be the main vector of Japanese encephalitis. However, in Southeast Asia, including China and India, Culex vishnui, Culex pipiens, Culex annulirostris, and Anopheles spp. are also known to transmit the Japanese encephalitis virus. Unlike adults who show asymptomatic infection, children under 14 years of age or the elderly who are infected with Japanese encephalitis experience high fever, headache, paresthesia, impaired consciousness, coma, and death. During the recovery period, aftereffects such as speech impairment and impaired judgment appear. Domestic animals, including pigs, horses, cattle, sheep, and goats, as well as wild birds such as pigeons and herons, bats, and reptiles, are susceptible to Japanese encephalitis. Pigs, including wild boars, are particularly known as amplification hosts for the Japanese encephalitis virus, making them an important public health target for immunization. Mosquito-borne Japanese encephalitis virus infection in adult and fattening pigs causes viremia with asymptomatic clinical symptoms. However, Japanese encephalitis infection in pregnant sows can cause reproductive disorders, including premature birth, mummified fetuses, and the delivery of weak piglets with neurological symptoms. In boars, the virus invades the reproductive tract, inhibiting spermatogenesis and reducing the number of spermatozoa and the number of malformed spermatozoa, which lowers the conception rate.

[0003] Meanwhile, Japanese encephalitis virus, the causative agent of Japanese encephalitis, is a positive single-stranded RNA virus belonging to the genus Flavivirus. The RNA genome is composed of a single open reading frame (ORF) into a polyprotein, which consists of three structural proteins and seven nonstructural proteins. The polyprotein, composed of [C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5], is cleaved at the NS2B-NS3 region by a viral protease. Phylogenetically, Japanese encephalitis virus is divided into five genotypes, which are classified based on sequence analysis of the envelope (E) gene. Most Japanese encephalitis virus strains isolated in Korea have been identified as genotypes I and III. However, since genotype V was identified in mosquitoes in Paju, Gyeonggi Province in 2010, genotype V was also identified in Gangwon Province and southern Gyeonggi Province in 2012. Moreover, genotype V was detected in one domestic human specimen in 2015 and in two domestic human specimens in 2018. Furthermore, it has been reported that the existing Japanese encephalitis virus vaccine has a reduced efficacy against infection with Japanese encephalitis virus genotype V. Therefore, it is necessary to prevent and prepare for infection with not only Japanese encephalitis virus genotypes I and III, which were previously prevalent in Korea, but also Japanese encephalitis virus genotype V.

[0004] Under this technical background, the inventors of the present invention produced a recombinant virus for the domestically isolated strain MK541529.1 of Japanese encephalitis virus type 5, which has recently been prevalent in Korea, and completed the present invention based on this.

[0005] One aspect provides a composition for producing a recombinant Japanese encephalitis virus, comprising a cDNA comprising a combination of a first polynucleotide comprising a base sequence of SEQ ID NO: 1; a second polynucleotide comprising a base sequence of SEQ ID NO: 2; and a third polynucleotide comprising a base sequence of SEQ ID NO: 3, or an expression vector comprising the cDNA.

[0006] Another aspect provides a method for producing a recombinant Japanese encephalitis virus using the above composition.

[0007] Another aspect provides a recombinant Japanese encephalitis virus produced by the above method.

[0008] Another aspect provides a Japanese encephalitis vaccine composition comprising the recombinant Japanese encephalitis virus as an active ingredient.

[0009] Another aspect provides a diagnostic composition for Japanese encephalitis comprising the recombinant Japanese encephalitis virus as an active ingredient.

[0010]

[0011] Other purposes and advantages of this application will be further clarified by the detailed description below, taken in conjunction with the appended claims and drawings. Any details not described herein are readily apparent and inferable to those skilled in the technical field of this application or similar technical fields, and therefore, their description is omitted.

[0012] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein is well known and commonly used in the art.

[0013]

[0014] One aspect provides a composition for producing a recombinant Japanese encephalitis virus, comprising a cDNA comprising a combination of a first polynucleotide comprising a base sequence of SEQ ID NO: 1; a second polynucleotide comprising a base sequence of SEQ ID NO: 2; and a third polynucleotide comprising a base sequence of SEQ ID NO: 3, or an expression vector comprising the cDNA.

[0015] The term "Japanese encephalitis virus (JEV)" used herein refers to a positive single-stranded RNA virus belonging to the genus Flavivirus, which has a genome comprising a long open reading frame (ORF) and a 5' and 3' nontranslated region (NTR) at both ends of the ORF, which have cis-acting elements that play an important role in viral self-replication. The ORF is translated into a single large polyprotein, which is processed co-translationally or post-translationally to produce three structural proteins and seven nonstructural proteins. At this time, the genes corresponding to the proteins have the following arrangement in the JEV RNA genome: C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5. In addition, the Japanese encephalitis virus is divided into five genotypes in terms of phylogenetic classification, and most Japanese encephalitis viruses isolated in Korea have been mainly of genotypes I and III, but recently, genotype V has been frequently detected. Under this technical background, there is a need for development of Japanese encephalitis virus genotype V, specifically, a recombinant Japanese encephalitis virus genotype V (hereinafter referred to as Japanese encephalitis virus genotype 5) with effective infectivity, but it is difficult to secure a cDNA clone with effective infectivity due to the genetic instability of the cDNA molecule.

[0016] As used herein, the term "recombinant Japanese encephalitis virus" refers to a Japanese encephalitis virus that is artificially synthesized or non-naturally occurring or engineered, wherein "non-naturally occurring or engineered" means a state that is not in a natural state but is created by artificial modification. The recombinant Japanese encephalitis virus may be obtained, for example, through the construction of an infectious cDNA clone and an in vitro transcription process, and for the purposes of the present invention, may have the genetic characteristics of Japanese encephalitis virus genotype 5.

[0017] In one specific example, the recombinant Japanese encephalitis virus may be derived from the Japanese encephalitis virus genotype 5 domestic isolate MK541529.1, and more specifically, may be produced through a process of securing a cDNA clone having infectivity based on the genetic information of virus RNA isolated from transfected cells transfected with the domestic isolate MK541529.1. Here, the Japanese encephalitis virus genotype 5 domestic isolate MK541529.1 can be obtained from a known database such as the Korea Disease Control and Prevention Agency (KDCA) or the GenBank of the National Institutes of Health (NIH).

[0018] In one specific example, the recombinant Japanese encephalitis virus cDNA may include G1031C and T10043A mutations compared to cDNA obtained from Japanese encephalitis virus genotype 5 domestic isolate MK541529.1.

[0019] In one specific example, the composition for producing the recombinant Japanese encephalitis virus may include a cDNA comprising a combination of a first polynucleotide comprising the base sequence of SEQ ID NO: 1; a second polynucleotide comprising the base sequence of SEQ ID NO: 2; and a third polynucleotide comprising the base sequence of SEQ ID NO: 3.

[0020] As used herein, the term "polynucleotide" refers to a polymeric material comprising a plurality of nucleotide units, specifically, a polymer in which a plurality of nucleotide units are linked to each other by phosphodiester bonds of a sugar / phosphate backbone, and may be used interchangeably with the terms "nucleic acid" and "nucleic acid molecule". The polynucleotide is a biopolymer essential to living organisms, and may be RNA or DNA that encodes genetic information through a unique base sequence. The polynucleotide may be isolated, artificially synthesized, or non-naturally occurring or engineered. Here, the artificial modification may include cDNA that mimics the genetic characteristics and structure of natural RNA, specifically, Japanese encephalitis virus genotype 5 RNA, and has effective infectivity.

[0021] The first to third polynucleotides may include the full-length of Japanese encephalitis virus genotype 5 domestic isolate MK541529.1, which exhibits effective infectivity, and additionally includes G1031C and T10043A mutations. Each of the polynucleotides may include a base sequence having a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% with the base sequence of SEQ ID NO: 1, 2, or 3, respectively, under the premise that they exhibit the same functionality. The "% of sequence homology" for the base sequences can be determined by comparing the comparison region with two optimally aligned sequences.

[0022] In one specific example, the composition for producing the recombinant Japanese encephalitis virus may include an expression vector comprising a cDNA comprising a combination of a first polynucleotide comprising the base sequence of SEQ ID NO: 1; a second polynucleotide comprising the base sequence of SEQ ID NO: 2; and a third polynucleotide comprising the base sequence of SEQ ID NO: 3.

[0023] As used herein, the term "vector" refers to a means for expressing or transfecting a target gene in a host cell, including a plasmid vector; a cosmid vector; a viral vector such as a bacteriophage vector, an adenovirus vector, a retrovirus vector, and an adeno-associated virus vector. In the vector, a nucleic acid encoding an antigen-binding fragment or a recombinant fusion protein is operably linked to a promoter. In one embodiment, the expression vector may be a pACYC177 vector containing a T7 promoter at the 5' end and an HDVr at the 3' end.

[0024] As used herein, the term "operably linked" means a functional association between a nucleic acid expression regulatory sequence (e.g., a promoter, a signal sequence, or an array of transcription factor binding sites) and another nucleic acid sequence, whereby the regulatory sequence regulates transcription and / or translation of the other nucleic acid sequence.

[0025] In the case of a prokaryotic cell as a host, it is common to include a strong promoter that can drive transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, and T7 promoter), a ribosome binding site for translation initiation, and a transcription / translation termination sequence. Also, for example, when a eukaryotic cell is used as a host, promoters derived from the genome of mammalian cells (e.g., metallothionine promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Rous sarcoma virus (RSV) promoter) can be used, and generally have a polyadenylation sequence as a transcription termination sequence. In some cases, the vector may be fused with other sequences to facilitate purification of the recombinant fusion protein expressed therefrom. The sequences to be fused include, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), and 6xHis (hexahistidine; Quiagen, USA). The vector includes antibiotic resistance genes commonly used in the art as selectable markers, for example, resistance genes for ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0026] As used herein, the term "transfection" refers to a molecular biological technique in which a DNA chain fragment or plasmid containing a different type of foreign gene than that originally contained in the cell is introduced between cells and binds with the DNA present in the original cell, thereby changing the genetic properties of the cell. The transformation may refer to the insertion of an expression vector containing the antigen-binding fragment or recombinant fusion protein gene into a host cell.

[0027] In one embodiment, based on the genetic information of viral RNA isolated from transfected cells transfected with Japanese encephalitis virus genotype 5 domestic isolate MK541529.1, a cDNA clone having infectivity was secured, and then cDNA containing G1031C and T10043A mutations and an expression vector pACYC177 JYJEV5F containing the same were constructed. Thereafter, in vitro transcription was performed using the linearized expression vector pACYC177 JYJEV5F as a template to obtain viral RNA, and the effective cell pathological effect of the viral RNA was confirmed.

[0028]

[0029] Another aspect provides a method for producing a recombinant Japanese encephalitis virus, comprising: linearizing an expression vector or plasmid comprising a combination of a first polynucleotide comprising a base sequence of SEQ ID NO: 1; a second polynucleotide comprising a base sequence of SEQ ID NO: 2 and a third polynucleotide comprising a base sequence of SEQ ID NO: 3; and performing in vitro transcription (IVT) using the linearized expression vector as a template.

[0030] Among the terms or elements mentioned in the method for producing the above recombinant Japanese encephalitis virus, those mentioned in the description of the composition for producing the above recombinant Japanese encephalitis virus are as described above.

[0031] As used herein, the term "plasmid" refers to a vector capable of expressing a target protein in a suitable host cell, and a genetic construct comprising regulatory elements operably linked to enable expression of the gene insert. In one embodiment, the plasmid may comprise expression regulatory elements such as a promoter, an operator, an initiation codon, a termination codon, a polyadenylation signal, and / or an enhancer. The plasmid may be a linearized plasmid, which may be obtained by contacting plasmid DNA with a restriction enzyme under suitable conditions where the restriction enzyme cleaves the plasmid DNA at the recognition site and destroys the plasmid structure. In addition, elements such as the type of plasmid and the restriction enzyme recognition site in the plasmid may be applied without limitation using techniques known in the art. In one embodiment, the plasmid may be a pACYC177 vector (pACYC177 JYJEV5F) having a cleavage map of FIG. 2, which includes a T7 promoter at the 5' end and HDVr at the 3' end, and includes the cDNA described above.

[0032] As used herein, the term “in vitro transcription (IVT)” refers to a process in which a target mRNA molecule is synthesized in a cell-free system (in vitro), preferably wherein DNA constituting a plasmid can be used as a template for generating mRNA transcripts, and RNA polymerase can be used to control the in vitro transcription process. Typically, a DNA template for RNA in in vitro RNA transcription can be obtained by cloning a specific cDNA corresponding to each RNA to be transcribed in vitro and introducing it into a suitable vector for RNA in vitro transcription, such as a plasmid.

[0033]

[0034] Another aspect is to provide a recombinant Japanese encephalitis virus comprising the base sequence of sequence number 4.

[0035] Among the terms or elements mentioned in the above recombinant Japanese encephalitis virus, those mentioned in the description of the method for producing the recombinant Japanese encephalitis virus and the composition for producing the recombinant Japanese encephalitis virus are as described above.

[0036] In one specific example, the recombinant Japanese encephalitis virus may mimic the genetic characteristics and structure of Japanese encephalitis virus genotype 5 as described above. In addition, the recombinant virus may be derived from the domestic isolate MK541529.1 of Japanese encephalitis virus genotype 5, and specifically, may be produced through a composition for producing the recombinant Japanese encephalitis virus, and may be composed of the base sequence of SEQ ID NO: 4. The recombinant Japanese encephalitis virus may include a base sequence having a sequence homology of 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more to the base sequence of SEQ ID NO: 4, under the premise that it exhibits the same functionality.

[0037]

[0038] Another aspect is to provide a vaccine composition for Japanese encephalitis comprising the recombinant Japanese encephalitis virus as an active ingredient.

[0039] The term "vaccine" as used herein refers to a biological preparation containing an antigen that provides immunity to the body, and refers to an immunogen or antigenic substance that creates immunity in the body by injection or oral administration to humans or animals to prevent infectious diseases. In vivo immunity is broadly divided into autoimmunity, in which immunity is automatically acquired after infection with a pathogen, and passive immunity, which is acquired through an externally administered vaccine. While autoimmunity is characterized by a long period of antibody production related to immunity and persistent immunity, passive immunity by vaccines has the disadvantage of having an immediate effect on the treatment of infectious diseases but having a short duration.

[0040] As used herein, the term “immunogen” or “antigenic substance” may be any one selected from the group consisting of a peptide derived from the virus, a polypeptide, a lactic acid bacteria expressing the polypeptide, a protein, a lactic acid bacteria expressing the protein, an oligonucleotide, a polynucleotide, and a recombinant virus. For example, the antigenic substance may be in the form of an inactivated whole or partial virus preparation, an attenuated virus preparation, or an antigen molecule obtained by conventional protein purification, genetic engineering techniques, or chemical synthesis.

[0041] The vaccine composition may be characterized in that it further contains at least one second adjuvant selected from the group consisting of a stabilizer, an emulsifier, aluminum hydroxide, aluminum phosphate, a pH adjuster, a surfactant, a liposome, an iscom adjuvant, a synthetic glycopeptide, a bulking agent, a carboxypolymethylene, a bacterial cell wall, a derivative of a bacterial cell wall, a bacterial vaccine, an animal poxvirus protein, a subviral particle adjuvant, cholera toxin, N,N-dioctadecyl-N',N'-bis(2-hydroxyethyl)-propanediamine, monophosphoryl lipid A, dimethyldioctadecyl-ammonium bromide, and mixtures thereof.

[0042] The above vaccine composition may include a veterinarily acceptable carrier. As used herein, the term "veterinarily acceptable carrier" includes any and all solvents, dispersion media, coatings, adjuvants, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, and the like. Carriers, excipients, and diluents that may be included in the vaccine composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, glycerin, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0043] In addition, the vaccine composition can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. and sterile injection solutions according to conventional methods. When formulating, diluents or excipients such as commonly used fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants can be prepared. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations can be prepared by mixing the lecithin-like emulsifier with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous preparations, suspensions, emulsions, and lyophilized preparations. Non-aqueous preparations and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0044] The above vaccine may be administered to a subject in various forms. "Injection" may be performed by any one method selected from the group consisting of subcutaneous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, nasal administration, oral administration, transdermal administration, and oral administration.

[0045] In addition, the terms "injection" or "administration" in this specification may vary in dosage depending on the age, sex, weight, etc. of the subject of administration, and the dosage of the vaccine may also vary depending on the route of administration, the degree of disease, sex, weight, age, etc. The vaccine adjuvant of the present invention may be used together when administering the vaccine by parenteral, mucosal (oral and nasal, etc.) and transdermal routes.

[0046] The above vaccine composition may include one or more adjuvants to improve or enhance the immune response. Suitable adjuvants include compositions comprising peptides, aluminum hydroxide, aluminum phosphate, aluminum oxide, and mineral or vegetable oils such as Marcol 52 and one or more emulsifiers, or surface-active substances such as lysoxazole, polycations, and polyanions.

[0047] The above vaccine composition may additionally include one or more immunostimulants, preferably cholera toxin (CT), aluminum hydroxide, carbopol, mineral oil, or biodegradable oil.

[0048] The term "adjuvant" as used herein generally refers to any substance that enhances the humoral and / or cellular immune response to an antigen. Traditional vaccines are composed of unprocessed preparations of killed pathogenic microorganisms, and impurities associated with the pathogenic microorganism culture can act as adjuvants to enhance the immune response. However, when a homogenous preparation of purified protein subunits is used as an antigen for vaccination, the immunity induced by such antigens is insufficient, necessitating the addition of some foreign substance as an adjuvant. By using an adjuvant, a smaller dose of antigen may be required to stimulate the immune response, thereby reducing the cost of vaccine production. These adjuvants are classified according to their source material (mineral, bacteria, plant) and composition (emulsion suspension). Commercially available adjuvants include aluminum hydroxide, carbopol, mineral oil, or biodegradable oil.

[0049] The above vaccine may be a live vaccine or an inactivated vaccine. The live vaccine refers to a virus that has its pathogenicity weakened, so that when administered to an animal, clinical symptoms of the disease are absent or reduced. The attenuated strain of the present invention can be isolated by methods known in the art, for example, through virus subculture. The inactivated vaccine is a virus that has its pathogenicity inactivated without losing its immunogenicity by heating (56-60°C, 30-60 minutes) or using chemicals such as formalin or phenol. The inactivation of the virus can be performed by methods known in the art.

[0050]

[0051] Another aspect provides a diagnostic composition for Japanese encephalitis virus comprising the recombinant Japanese encephalitis virus or an antigen thereof, or a preparation capable of detecting the same, as an active ingredient. The details regarding the Japanese encephalitis virus and the like are as described above.

[0052] In this specification, the term "diagnosis" means confirming a pathological condition, and may mean confirming whether a disease caused by Japanese encephalitis virus has developed by confirming the presence or absence of expression of a gene specific to Japanese encephalitis virus, and the prognosis during antiviral drug treatment after development.

[0053] The agent capable of detecting the virus, a culture of the virus or an antigen thereof may preferably be an antibody specific for the virus, a culture of the virus or an antigen thereof.

[0054] As used herein, the term "antibody" refers to a substance produced in the immune system by antigen stimulation, also called an immunoglobulin, that specifically binds to a specific antigen, circulates in the lymph and blood, and causes an antigen-antibody response. While antibodies exhibit specificity for a specific antigen, immunoglobulins include both antibodies and antibody-like substances that lack antigen specificity. The latter polypeptide is produced at low levels, for example, in the lymphatic system, and at increased levels by myeloma. In the present invention, the antibody may be the PEDV itself, the spike protein, or an epitope contained therein. The antibody may be a monoclonal or polyclonal antibody.

[0055] The recombinant Japanese encephalitis virus genotype 5 according to the daily aspect has the structural or genetic characteristics of the original Japanese encephalitis virus and possesses effective cell infectivity, and can be used to develop an antigen that can effectively diagnose Japanese encephalitis virus genotype 5, which is currently prevalent in Korea, or a vaccine that can effectively prevent it.

[0056] Figure 1 is a schematic diagram showing the RNA genome structure of Japanese encephalitis virus.

[0057] FIG. 2 is a schematic diagram illustrating first to third polynucleotides (JEV5 Fragement1 to JEV5 Fragement3) according to one embodiment. In FIG. 2, A represents a capsid protein (C), B represents a peptide precursor (prM), C represents an envelope (E), D represents nonstructural protein 1 (NS1), E represents nonstructural protein 2A (NS2A), F represents nonstructural protein 2B (NS2B), and G represents nonstructural protein 3 (NS3). H represents nonstructural proteins 4A, 4B, and 5 (NS4A, NS4B, and NS5).

[0058] Figure 3 is a cleavage map of the expression vector pACYC177 JYJEV5F for the production of recombinant Japanese encephalitis virus genotype 5 according to one embodiment.

[0059] Figure 4 shows the results of confirming the recombinant Japanese encephalitis virus genotype 5 by blotting according to one embodiment.

[0060] Figure 5 shows the results of confirming cell infection by recombinant Japanese encephalitis virus genotype 5 according to one embodiment.

[0061] Figure 6 shows the results of confirming cell infection after transfecting Vero cells with recombinant Japanese encephalitis virus genotype 5 and Japanese encephalitis virus genotype 5 MK541529.1 strain according to one embodiment.

[0062] Figure 7 shows the results of confirming the infection of recombinant Japanese encephalitis virus genotype 5 according to one embodiment using purified PCR products and AgeI restriction enzyme.

[0063] The present invention will be described in more detail through the following examples. However, these examples are provided for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0064]

[0065] Example 1: Production of recombinant Japanese encephalitis virus genotype 5

[0066] In this example, a recombinant virus for Japanese encephalitis virus genotype 5 was produced.

[0067] 1-1. Creation of expression vector

[0068] The Japanese encephalitis virus genotype 5 MK541529.1 strain, provided by the Korea Disease Control and Prevention Agency, was infected in C6 / 36 cells, which are mosquito-derived cells, and the virus titer was increased. Thereafter, viral RNA was extracted from the C6 / 36 cells, and cDNA was synthesized by RT-PCR using reverse transcriptase. Thereafter, to produce a recombinant virus for Japanese encephalitis virus genotype 5, the full-length Japanese encephalitis virus gene was inserted into the pACYC177 vector, which is known as a low copy number. Specifically, PCR was performed by dividing it into a total of eight parts including the 5'UTR and 3'UTR, and cloning was performed using the first to third polynucleotides (JEV5 Fragement 1 to JEV5 Fragement 3) using an infusion cloning kit. Meanwhile, information on the first to third polynucleotides is as shown in FIG. 2 and Table 1.

[0069] [Table 1]

[0070]

[0071] Afterwards, the pACYC177 vector containing the T7 promoter at the 5' end and the HDVr at the 3' end was infusion cloned to complete the pACYC177 JYJEV5F clone, as shown in Fig. 3. In addition, to distinguish the parent virus from the recombinant virus, silent mutations were made, such as G at nucleotide 1,031 belonging to the Envelope gene to C and T at nucleotide 10,043 belonging to the NS5 gene to A, as shown in Table 2.

[0072] [Table 2]

[0073]

[0074] 1-2. Production of viral RNA using in vitro transcription

[0075] To produce viral RNA from the recombinant virus clone, the pACYC177 JYJEV5F expression vector of 1-1 above was linearized by treatment with the AvrII restriction enzyme. Subsequently, in vitro transcription was performed using the T7 promoter located 5' forward, thereby obtaining a recombinant Japanese encephalitis virus comprising the base sequence of SEQ ID NO: 4.

[0076] Thereafter, as shown in Fig. 4, the produced viral RNA was confirmed on a 0.7% agarose gel through blotting.

[0077]

[0078] Example 2: Confirmation of cell infectivity of recombinant Japanese encephalitis virus genotype 5.

[0079] In this example, the cell infectivity of recombinant Japanese encephalitis virus genotype 5 RNA was examined. To this end, 8 μg of the prepared viral RNA was mixed with Lipofectamine 2000, incubated at room temperature for 5 minutes, and then transfected into Vero cells, monkey kidney cells. The typical cytopathic effect (CPE) of viral infection in Vero cells was then observed microscopically. A non-transfected group served as a negative control.

[0080] As a result, as shown in Fig. 5, a cell lesion effect was confirmed in Vero cells on days 4 and 5 after transfection. From the above results, it was confirmed that the recombinant Japanese encephalitis virus according to one embodiment possesses effective infectious ability.

[0081]

[0082] Example 3: Reconfirmation of the cell infectivity of recombinant Japanese encephalitis virus genotype 5.

[0083] In this example, we attempted to re-verify whether the cell infectivity confirmed in Example 2 was due to the recombinant Japanese encephalitis virus genotype 5 according to one embodiment. To this end, Japanese encephalitis virus genotype 5 MK541529.1 strain (parent, JEV5) and the recombinant virus (JYJEV5F) were transfected into Vero cells in the same manner as in Example 2, and the cell lesion effect was confirmed under a microscope. Thereafter, the supernatant of the infected Vero cells was harvested and viral RNA was extracted. Thereafter, cDNA was synthesized through RT-PCR, and a portion of the NS5 gene, which is the silent mutation site mentioned above, was PCR-treated, and the PCR product was purified. The purified PCR product was cut with AgeI restriction enzyme, and the resulting product was confirmed.

[0084] As a result, as shown in Fig. 6, the cell lesion effect was confirmed in Vero cells on the 4th day after transfection. In addition, as shown in Fig. 7, the band according to the treatment with AgeI restriction enzyme was confirmed to distinguish the original virus from the recombinant virus, and through this, the cell infection of the recombinant Japanese encephalitis virus genotype 5 RNA was confirmed again. The above results indicate that the recombinant virus according to one embodiment possesses effective infectious ability, and thus can be utilized in the development of a vaccine or therapeutic agent for Japanese encephalitis virus genotype 5.

[0085]

[0086] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. A composition for producing a recombinant Japanese encephalitis virus, comprising a cDNA comprising a combination of a first polynucleotide comprising a base sequence of sequence number 1; a second polynucleotide comprising a base sequence of sequence number 2; and a third polynucleotide comprising a base sequence of sequence number 3, or an expression vector comprising the cDNA.

2. A composition according to claim 1, wherein the recombinant Japanese encephalitis virus is derived from Japanese encephalitis virus genotype 5 domestic isolate MK541529.

1.

3. A composition according to claim 1, wherein the recombinant Japanese encephalitis virus cDNA comprises G1031C and T10043A mutations compared to the cDNA obtained from the Japanese encephalitis virus genotype 5 domestic isolate MK541529.

1.

4. A method for producing a recombinant Japanese encephalitis virus, comprising: linearizing an expression vector comprising a combination of a first polynucleotide comprising a base sequence of SEQ ID NO: 1; a second polynucleotide comprising a base sequence of SEQ ID NO: 2; and a third polynucleotide comprising a base sequence of SEQ ID NO: 3; and performing in vitro transcription (IVT) using the linearized expression vector as a template.

5. A method for producing a recombinant Japanese encephalitis virus according to claim 4, wherein the expression vector is pACYC177 JYJEV5F having the cleavage map of FIG.

2.

6. A recombinant Japanese encephalitis virus consisting of the base sequence of sequence number 4.

7. A recombinant Japanese encephalitis virus according to claim 6, wherein the recombinant Japanese encephalitis virus is produced by a method comprising: linearizing an expression vector comprising a combination of a first polynucleotide comprising the base sequence of SEQ ID NO: 1; a second polynucleotide comprising the base sequence of SEQ ID NO: 2 and a third polynucleotide comprising the base sequence of SEQ ID NO: 3; and performing in vitro transcription using the linearized expression vector as a template.

8. In claim 7, the recombinant Japanese encephalitis virus cDNA comprises G1031C and T10043A mutations compared to the cDNA obtained from the Japanese encephalitis virus genotype 5 domestic isolate MK541529.

1.

9. A vaccine composition for Japanese encephalitis comprising the recombinant Japanese encephalitis virus of claim 6 as an active ingredient.

10. A vaccine composition according to claim 9, wherein the Japanese encephalitis is caused by infection with Japanese encephalitis virus genotype 5.

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