Immortalized potamochoerus porcus macrophage

Abstract

Provided are immortalized Potamochoerus porcus macrophage cells obtained by causing Potamochoerus porcus macrophages to express at least one protein selected from the group consisting of an SV40 large T antigen and a telomerase reverse transcriptase.

Description

Immortalized river boar macrophages

[0001] The present invention relates to immortalized wild boar macrophages and a method for producing the macrophages. The present invention also relates to a method for producing boar-infectious viruses or vaccines using the macrophages. Furthermore, the present invention relates to a method for detecting boar-infectious viruses or a method for detecting neutralizing antibodies against boar-infectious viruses using the macrophages.

[0002] African swine fever (ASF) is a highly fatal infectious disease that occurs when the ASF virus (ASFV) infects pigs and other animals, causing fever and hemorrhagic lesions. Effective preventive and therapeutic methods for ASF, such as vaccines, have not yet been established. Macrophages are the target cells for infection by ASFV in the body, and currently, immortalized porcine macrophage cell lines have been developed by the present inventors (Patent Documents 1 and 2, Non-Patent Documents 1 to 3), which are being utilized in the research and development of ASF vaccines.

[0003] Furthermore, the natural host of ASFV in Africa is the red river boar (Potamochoerus porcus). The red river boar, which belongs to the genus Sus, is distinct from pigs, which belong to the genus Sus (Sus). Immortalized river boar macrophage cells are expected to be useful for characterizing ASF virus strains and for research and development of ASF vaccines. However, such immortalized river boar macrophages have not yet been established, and their creation has been desired.

[0004] JP 2021-61772 A International Publication No. 2022 / 107793

[0005] Takato Takenouchi et al. , Front Vet Sci. 2017 Aug 21:4:132. Takato Takenouchi et al. , Front Vet Sci. 2022 Jul 18:9:919077. Takato Takenouchi et al. , Front Vet Sci. 2022 Nov 18:9:1058124.

[0006] The present invention has been made in view of the problems associated with the prior art, and an object of the present invention is to provide immortalized river boar macrophages.

[0007] The present inventors have developed a method for producing immortalized cells by introducing two genes (SV40 large T antigen gene and porcine-derived telomerase reverse transcriptase gene) into primary cultured porcine macrophages (Patent Documents 1 and 2, Non-Patent Documents 1 to 3). They have also demonstrated that immortalized porcine macrophage cell lines are highly susceptible to ASF virus (Patent Documents 1 and 2, Non-Patent Documents 2 and 3).

[0008] To solve this problem, we attempted to apply the above-mentioned immortalized cell production method to macrophages from river boars, which are different from pigs at the genus level. More specifically, we isolated and cultured macrophages from the blood of red river boars and attempted to produce an immortalized cell line using the above-mentioned method.

[0009] As a result, analysis of the mitochondrial DNA base sequence confirmed that the resulting cell line was derived from red river boar. Furthermore, it was found that this cell line had the ability to divide at least 50 times. Furthermore, immunostaining revealed the expression of at least three macrophage markers. Furthermore, induction of inflammatory cytokine production in response to bacterial cell wall components (lipopolysaccharide (LPS) and muramyl dipeptide), a characteristic of macrophages, was also detected. The cells were also found to have phagocytic ability. Thus, we successfully produced an immortalized red river boar blood macrophage cell line (Red River Hog, Zoorasia, Japan / Immortalized Blood Macrophage, RZJ / IBM cells).

[0010] Furthermore, the susceptibility of the cells to various ASF virus strains was evaluated by a cytopathic effect (CPE) test and a hemadsorption (HAD) test, which measures the hemadsorption reaction specifically observed in ASFV-infected cells, and compared with that of immortalized porcine kidney macrophage (IPKM) cells (Patent Document 1, Non-Patent Document 1).

[0011] As a result, for the highly virulent strains (Arm07, Ken05, and E75) that cause severe symptoms in pigs and the attenuated strain (L60V) that causes no symptoms in pigs, CPE was detected in all four strains in IPKM cells, but CPE was detected only in the attenuated strain (L60V) in RZJ / IBM cells. Thus, it was found that IPKM cells and RZJ / IBM cells have significantly different responses to ASF virus strains.

[0012] On the other hand, the results of the HAD test showed that when RZJ / IBM cells were inoculated with each of the four ASF virus strains, HAD characteristic of ASF virus infection was detected, indicating that all tested ASF virus strains were propagated. Therefore, it was revealed that the three highly virulent strains of virus did not exhibit CPE despite propagating in RZJ / IBM cells. In other words, compared to immortalized macrophages derived from pigs, those derived from river boars were found to be superior in that they suppressed cell death while allowing the infected virus to propagate.

[0013] Furthermore, attempts were made to infect RZJ / IBM cells with various strains of classical swine fever (CSF) virus (a strain prevalent in Japan, a highly virulent strain, and a vaccine strain), and it was found that all of the CSF virus strains were able to infect and grow in RZJ / IBM cells.

[0014] The present invention is based on the first successful production of immortalized river boar macrophages and the discovery that the produced immortalized river boar macrophages are susceptible to viral infection, and is specifically as follows.

[0015] That is, the present invention provides the following aspects.

[0016] [1] Immortalized wild boar macrophage cells.

[0017] [2] The cell described in [1], which is obtained by expressing at least one protein selected from the group consisting of SV40 large T antigen and telomerase reverse transcriptase in a macrophage of a river boar.

[0018] [3] The cell described in [2], wherein the expression is from a lentivirus encoding the protein.

[0019] [4] The cell described in [2] or [3], wherein the telomerase reverse transcriptase is porcine-derived telomerase reverse transcriptase.

[0020] [5] A method for producing immortalized macrophages, comprising a step of expressing at least one protein selected from the group consisting of SV40 large T antigen and telomerase reverse transcriptase in macrophages of river boars.

[0021] [6] The method according to [5], wherein the step comprises introducing a lentivirus encoding the protein into macrophages of the river boar to express the protein.

[0022] [7] The method according to [5] or [6], wherein the telomerase reverse transcriptase is porcine-derived telomerase reverse transcriptase.

[0023] [8] A method for producing a boar-infective virus, comprising the steps of contacting the cell according to any one of [1] to [4] with a boar-infective virus and allowing the virus to grow in the cell.

[0024] [9] A method for producing a vaccine containing a boar-infectious virus, comprising the steps of: contacting the cell according to any one of [1] to [4] with a boar-infectious virus and propagating the virus in the cell; isolating the propagated boar-infectious virus; and mixing the isolated boar-infectious virus with a pharmacologically acceptable carrier or medium.

[0025]

[10] The method of producing a boar-infecting virus according to [8] or [9], wherein the boar-infecting virus is African swine fever virus.

[0026]

[11] The cell according to any one of [1] to [4], which has DNA in which a reporter gene is functionally linked downstream of the promoter region of a gene derived from a boar-infectious virus.

[0027]

[12] The cell described in

[11] , wherein the boar-infecting virus is African swine fever virus.

[0028]

[13] A method for detecting boar infectious viruses, comprising the steps of: culturing the cells according to

[11] or

[12] in the presence of a test sample; detecting expression of the reporter gene in the cells; and determining that the test sample contains boar infectious viruses when expression of the reporter gene is detected.

[0029]

[14] A method for detecting a neutralizing antibody against a boar-infecting virus, the method comprising the steps of: contacting the cell according to any one of [1] to [4] with the boar-infecting virus in the presence of a biological sample isolated from a test boar, and allowing the virus to grow in the cell; detecting the number of the grown virus; and determining that the biological sample contains a neutralizing antibody against the virus when the number of the virus detected in the step is lower than the number of the virus grown in the cell according to any one of [1] to [4] in the absence of the biological sample.

[0030]

[15] The method according to

[13] or

[14] , wherein the boar-infectious virus is African swine fever virus.

[0031] According to the present invention, it is possible to provide a cell line of river boar macrophages that can be passaged. Furthermore, since the immortalized river boar macrophages are susceptible to boar-infecting viruses, it is possible to grow various viruses, enabling the production and development of vaccines against these viruses. In particular, since the immortalized river boar macrophages can grow the infected viruses while suppressing their cell death, it is possible to provide these viruses with high productivity. Furthermore, because they are susceptible to infection, it is also possible to detect (test, diagnose) the viruses.

[0032] This is a micrograph showing the results of a mixed culture of red river boar blood and feeder pig kidney cells. Approximately two weeks after the start of the mixed culture, spherical cells (red river boar blood macrophages) appeared, weakly adhering and growing on the sheet of pig kidney cells (indicated by the arrow in the figure). This is a micrograph showing the results of observing cells recovered from the culture supernatant of the mixed culture and attached to a suspension culture dish. This is a micrograph showing the results of immunostaining to detect macrophage markers (Iba1, CD172a, CD203a, MHC-II) for the cells attached to the suspension culture dish. In the figure, "NC" indicates a negative control. In the color representation, brown represents antibody staining and blue represents nuclear staining. This is a micrograph showing the results of observing immortalized macrophages (RZJ / IBM cells) isolated from red river boar blood. 1 is a micrograph showing the results of immunostaining of macrophage markers (Iba1, CD172a, CD203a) in RZJ / IBM cells. In the color representation, brown represents antibody staining. This is a graph showing the results of testing the cell proliferation of RZJ / IBM cells. This is an electrophoresis photograph showing the results of PCR detection of the insertion of SV40LT and pTERT genes into the genomic DNA of RZJ / IBM cells. This is a graph showing the results of comprehensive analysis by RNAseq of gene expression changes in RZJ / IBM cells treated with LPS and untreated cells. In the figure, the vertical axis shows the expression level (TPM: transcripts per million) of the inflammatory cytokine (IL1A). This is a graph showing the results of comprehensive analysis by RNAseq of gene expression changes in RZJ / IBM cells treated with LPS and untreated cells. In the figure, the vertical axis shows the expression level (TPM) of an inflammatory cytokine (IL1B). This is a graph showing the results of a comprehensive analysis by RNAseq of gene expression changes in RZJ / IBM cells treated with LPS and untreated cells. In the figure, the vertical axis shows the expression level (TPM) of an inflammatory cytokine (IL18). This is a graph showing the results of a comprehensive analysis by RNAseq of gene expression changes in RZJ / IBM cells treated with LPS and untreated cells.In the figure, the vertical axis shows the expression level (TPM) of inflammatory cytokines (TNF). This graph shows the results of a comprehensive analysis of gene expression changes in RZJ / IBM cells treated with LPS and untreated cells using RNA-seq. In the figure, the vertical axis shows the expression level (TPM) of interferon (INFB1). This graph shows the results of a comprehensive analysis of gene expression changes in RZJ / IBM cells treated with LPS and untreated cells using RNA-seq. In the figure, the vertical axis shows the expression level (TPM) of an internal standard (GAPDH). These are micrographs showing the results of detecting cytopathic effect (CPE) or hemadsorption (HAD) reaction to evaluate the susceptibility of immortalized porcine kidney macrophage (IPKM) cells to the African swine fever virus (ASFV) European / China epidemic strain Armenia 07 (genotype: type II). In the figure, "Control" indicates a non-virus-infected control. These are micrographs showing the results of detecting cytopathic effect (CPE) or hemadsorption (HAD) reaction to evaluate the susceptibility of RZJ / IBM cells to ASFV Armenia 07 (genotype: II) or the Vero cell-adapted strain Lisbon 60 / V. In the figure, "Control" indicates a control not infected with virus. This is a graph showing the growth efficiency of ASFV Armenia 07 (genotype: II) in IPKM cells or RZJ / IBM cells. In the figure, the vertical axis represents virus titer (Log). １０ T.C.I.D. ５０ / mL), and the horizontal axis represents the number of days after inoculation of the virus. This is a graph showing the growth efficiency of Kenya05 / Tk-1 (genotype: I) strain of ASFV isolated from soft ticks in IPKM cells or RZJ / IBM cells. In the figure, the vertical axis represents the virus titer (Log １０ T.C.I.D. ５０ / mL), and the horizontal axis represents the number of days after inoculation of the virus. This is a graph showing the growth efficiency of the ASFV standard strain Espana75 (genotype: I) in IPKM cells or RZJ / IBM cells. In the figure, the vertical axis represents the virus titer (Log １０ T.C.I.D. ５０ / mL), and the horizontal axis represents the number of days after inoculation of the virus. This is a graph showing the growth efficiency of ASFV Lisbon 60 / V in IPKM cells or RZJ / IBM cells. In the figure, the vertical axis represents the virus titer (Log １０ T.C.I.D. ５０ / mL), and the horizontal axis represents the number of days after inoculation of the virus. These are micrographs showing the results of fluorescent immunostaining of infection of RZJ / IBM cells with various classical swine fever virus (CSFV) strains. In the figure, "Mock" represents a control not infected with the virus. These are graphs showing the growth efficiency of each virus strain in the culture supernatant of RZJ / IBM cells after inoculation of various CSFV strains. In the figure, the vertical axis represents the virus titer (Log １０ T.C.I.D. ５０ / mL), and the horizontal axis indicates the number of days after virus inoculation.

[0033] The present invention relates to immortalized river boar macrophages (immortalized river boar macrophages). In the present invention, "wild boar" refers to an animal belonging to the family Suidae, suborder Suidae, order Cetacea, class Mammalia, and unless otherwise specified, refers to wild boar in this broad sense. "River boar" refers to an animal belonging to the genus Potamochoerus, one of the genera in the family Suidae. Examples of such animals include the red river boar (Potamochoerus porcus) and the river boar (Potamochoerus larvatus). "Pig" refers to a livestock species belonging to the genus Sus, one of the genera in the family Suidae.

[0034] In the present invention, the "macrophages" to be immortalized may be activated macrophages (inflammatory M1 type macrophages or anti-inflammatory M2 type macrophages) or resting macrophages. Furthermore, there are no particular limitations on the tissues in which the macrophages are present. Examples of macrophages (so-called tissue macrophages) according to the present invention include blood macrophages, kidney macrophages, alveolar macrophages, intestinal macrophages, liver macrophages, brain macrophages, and osteoclasts.

[0035] "Blood macrophages" can be prepared, for example, as shown in the Examples below, by co-culturing blood collected from river boars with feeder cells and isolating spherical cells that adhere to and grow on the feeder cells. The "blood" to be subjected to the co-cultivation is not particularly limited in its form, and examples thereof include anticoagulant-treated blood (e.g., heparin-treated peripheral blood), coagulated blood, and hemolyzed blood.

[0036] The "feeder cells" used in the mixed culture are not particularly limited as long as they can maintain blood macrophages, and examples thereof include kidney-derived cells, lung-derived cells, intestine-derived cells, liver-derived cells, brain-derived cells, and bone-derived cells. More preferred examples include monolayer cell sheets obtained by culturing cells derived from each tissue (primary culture systems (mixed culture systems) obtained by culturing cells derived from each tissue from which macrophage-like cells have been removed), as described below. The origin of the feeder cells is also not particularly limited, but is preferably wild boar (an animal belonging to the genus Sus), more preferably an animal belonging to the Suidae or Suidae families, and even more preferably a pig.

[0037] "Other tissue macrophages" can be prepared, for example, as follows. First, tissues (e.g., kidney (renal cortex, etc.), lung (pulmonary parenchyma, etc.), intestine (small intestine, etc.), liver (hepatic parenchyma, etc.), brain (cerebrum, etc.), bone (bone marrow, etc.)) collected from river boar are minced, washed with a buffer solution, subjected to enzymatic treatment, and then cultured. Macrophage-like cells can be prepared by isolating them from the mixed culture system (a monolayer cell sheet and macrophage-like cells adhering thereto) that arises during the culture process. The "buffer" used to wash the minced tissue is not particularly limited, and examples include Dulbecco's phosphate-buffered saline (DPBS), phosphate-buffered saline (PBS), Tris-HCl buffer (TBS), and HEPES buffer. The enzymes used in the "enzyme treatment" are not particularly limited, as long as they can separate and disperse cells from the tissue. Examples include collagenase, dispase, DNase (e.g., DNase I), trypsin, hyaluronidase, elastase, and pronase, with a preferred combination being collagenase, dispase, and DNase I. The treatment temperature and time are also not limited and can be adjusted appropriately depending on the type of enzyme used and the degree of cell separation and dispersion. To produce the mixed culture system, the cells cultured after the enzyme treatment may be further treated with the enzyme to detach them from the culture vessel, disperse them, and re-seede them. The enzyme treatment here can also be carried out using the enzymes described above, but a preferred combination is a protease (an enzyme having at least the activity of degrading non-collagenous proteins), collagenase, and DNase (e.g., product name: Accumax (registered trademark), manufactured by Sigma-Aldrich).

[0038] The medium used for isolating and growing the above-mentioned "blood macrophages" and "other tissue macrophages" (hereinafter also referred to as "medium for primary culture of tissue macrophages") is not particularly limited as long as it can maintain each tissue macrophage, and can be prepared by appropriately adding well-known and commonly used medium additives to a known basal medium. Examples of "basal media" include DMEM medium, DMEM medium (high glucose), DMEM medium (low glucose), RPMI 160 medium, RPMI 1640 medium, Ham's F12 medium, KSOM medium, Eagle's MEM medium, Glasgow MEM medium, αMEM medium, Ham's medium, Fisher's medium, BME medium, BGJb medium, CMRL 1066 medium, MEM Zinc Option Improved Medium, IMDM medium, Medium 199 medium, and any mixture thereof. Examples of "medium additives" include antibiotics (penicillin, streptomycin, gentamicin, vancomycin, etc.), antifungal agents (pimaricin, amphotericin B, etc.), functional proteins (insulin, transferrin, lactoferrin, etc.), reducing agents (monothioglycerol, 2-mercaptoethanol, catalase, superoxide dismutase, N-acetylcysteine, etc.), lipids other than fatty acids (cholesterol, etc.), amino acids (alanine, L-glutamine, non-essential amino acids, etc.), peptides (glutathione, reduced glutathione, etc.), nucleotides, etc. (nucleosides, cytidine, adenosine 5'-isothiazolinone, etc.), and the like. Examples of suitable organic compounds include, but are not limited to, inorganic salts (e.g., iron(III) nitrate, iron(II) sulfate, copper sulfate, zinc sulfate), inorganic salts (e.g., sodium, potassium, calcium, magnesium, phosphorus, chlorine), carbon sources (e.g., glucose, galactose, fructose, sucrose), vitamins, inorganic compounds (e.g., selenious acid), organic compounds (e.g., para-aminobenzoic acid, ethanolamine, corticosterone, progesterone, lipoic acid, putrescine, pyruvic acid, lactic acid, triiodothyronine), buffer compounds (e.g., HEPES, sodium bicarbonate), and pH indicators (e.g., phenol red).

[0039] The culture conditions using such a medium are not particularly limited, but the culture temperature is usually 30 to 40° C., preferably 37° C. The carbon dioxide concentration in the gas in contact with the medium is usually 1 to 10% by volume, preferably 2 to 5% by volume.

[0040] By culturing cells derived from each tissue under these culture conditions, primary cultured macrophages derived from each tissue can be obtained. The culture time required to obtain the cells is not particularly limited, but is usually one week to two months, preferably two to three weeks. Furthermore, because the adhesion between the primary cultured macrophages and the feeder cells or cell sheet is loose, the macrophages can be collected by centrifuging the culture supernatant. Furthermore, because macrophages readily adhere to culture vessels for suspension cell culture or non-tissue culture, more selective isolation from other cells is possible by seeding the primary cultured macrophages back onto such a culture vessel.

[0041] The method for immortalizing the wild boar macrophages thus obtained is not particularly limited, and can be carried out by introducing at least one immortalization gene. Examples of immortalization genes include SV40 large T antigen (SV40 T antigen), telomerase reverse transcriptase (TERT), Myc, and Ras. However, it is preferable to introduce SV40 T antigen and TERT, and from the viewpoint of increasing the efficiency of immortalization of wild boar macrophages, it is more preferable to introduce SV40 T antigen and pig-derived TERT.

[0042] The introduction of an immortalizing gene can be carried out by using a vector encoding the gene. The vector may be linear or circular, and examples thereof include viral vectors, plasmid vectors, episomal vectors, artificial chromosome vectors, and transposon vectors.

[0043] Examples of viral vectors include retroviral vectors such as lentivirus, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, herpes virus vectors, vaccinia virus vectors, poxvirus vectors, poliovirus vectors, Silvis virus vectors, rhabdovirus vectors, paramyxovirus vectors, and orthomyxovirus vectors. Examples of plasmid vectors include plasmid vectors for expression in animal cells, such as pcDNA3.1, pA1-11, pXT1, pRc / CMV, pRc / RSV, and pcDNAI / Neo. Of these vectors, retroviral vectors are preferred, and lentiviruses are more preferred, from the viewpoint of increasing the efficiency of gene transfer into river boar macrophages.

[0044] In addition to the immortalizing gene, the vector of the present invention may contain expression control sequences such as a promoter, an enhancer, a poly A addition signal, a terminator, etc.; a replication origin or a nucleotide sequence encoding a protein that binds to the replication origin and controls replication; a 5' cap structure, a 5' untranslated region including a Shine-Dalgarno sequence, a Kozak sequence, etc.; a polyadenylation signal, a 3' untranslated region including an AU-rich element, a GU-rich element, etc.; nucleotides encoding other proteins, etc.

[0045] The immortalization gene can be operably positioned downstream of a promoter to efficiently transcribe each polynucleotide. Examples of such "promoters" include the EF1α promoter, CMV promoter, SRα promoter, SV40 early promoter, LTR promoter, RSV promoter, HSV-TK promoter, MSCV promoter, hTERT promoter, β-actin promoter, CAG promoter, metallothionein promoter, and heat shock promoter.

[0046] Examples of "nucleotides encoding other proteins" include marker genes such as reporter genes and drug resistance genes.

[0047] When multiple immortalization genes are introduced, these genes may be incorporated into a single vector or into separate vectors, but from the viewpoint of increasing expression efficiency, it is preferable to incorporate them into separate vectors. When incorporating them into a single vector, for example, by inserting an IRES, a 2A peptide sequence, or the like into the vector, it becomes possible to express multiple immortalization genes in a polycistronic manner.

[0048] Methods for introducing the vector into cells include lipofection, microinjection, calcium phosphate, DEAE-dextran, electroporation, and particle gun methods. Furthermore, when the vector of the present invention is a retroviral vector, appropriate packaging cells may be selected based on the LTR sequence and packaging signal sequence contained in the vector, and retroviral particles may be prepared using these cells. Examples of packaging cells include PG13, PA317, GP+E-86, GP+envAm-12, and Psi-Crip. Furthermore, 293 cells and 293T cells, which have high transfection efficiency, can also be used as packaging cells. Furthermore, viral particles prepared in this manner can be introduced into cells using the Polybrene method, the Protamine method, the RetroNectin method, and the like.

[0049] Such gene introduction and subsequent maintenance culture can be performed using the above-mentioned tissue macrophage primary culture medium and culture conditions using the medium. Furthermore, immortalized river boar macrophages established by introducing an immortalizing gene in this manner exhibit a proliferative capacity of at least one month, preferably two months or more, more preferably three months or more, even more preferably four months or more, and even more preferably five months or more. The doubling time of immortalized river boar macrophages is at least four days, preferably three days, more preferably two days, and even more preferably one day. Furthermore, it is preferable that the immortalized river boar macrophages maintain the characteristics of macrophages. For example, at least one gene selected from macrophage-specific genes Iba1, CD172a, CD203a, CD16, and CD204 (MSR-A) is expressed, preferably two or more genes, more preferably three or more genes, even more preferably four or more genes, and particularly preferably all of these genes are expressed. Furthermore, the immortalized river boar macrophage may express at least one gene selected from the group consisting of CD163 and CD169, which are marker genes for specific macrophage subpopulations, and MHC-II, which is an antigen-presenting cell marker gene. Furthermore, the immortalized river boar macrophage according to the present invention retains at least one of the following macrophage characteristics: the ability to produce inflammatory cytokines in response to stimulation with bacterial cell wall components (such as LPS), phagocytic ability, and the ability to mature IL-18 associated with inflammasome activity, and preferably retains two or more of these functions.

[0050] Unlike primary culture macrophages, immortalized river boar macrophages grow in a densely spread (sheet-like) state. Therefore, since morphological changes in immortalized river boar macrophages are easy to detect, they are also useful in African swine fever virus infection tests (CPE test, HAD test, etc.) that use the degeneration of infected cells as an indicator, as described below.

[0051] (Wild Boar-Infectious Virus) In the present invention, the "wild boar-infectious virus (a virus that infects animals belonging to the Suidae family)" may be a virus that can infect at least wild boars, but may also be a virus that can infect wild boars, or may be a virus that can infect livestock species (pigs). Furthermore, the wild boar-infectious virus may be a DNA virus (double-stranded (ds) DNA virus, single-stranded (ss) DNA virus, or DNA virus containing both ss and ds DNA regions), or an RNA virus (single-stranded (ss) RNA virus (positive-strand RNA virus or negative-strand RNA virus), double-stranded (ds) RNA virus).

[0052] Examples of "wild boar-infectious viruses" include arboviruses such as African swine fever virus (ASFV) and Japanese encephalitis virus, and classical swine fever virus (CSFV). In Japan, on February 5, 2020, the name "African swine fever" defined in the Livestock Infectious Disease Prevention Act was revised to "African swine fever."

[0053] (Method for producing boar-infectious virus) The method for producing (propagation method, amplification method) the boar-infectious virus of the present invention is a method comprising the steps of contacting immortalized river boar macrophages with the boar-infectious virus and propagating the virus in the immortalized river boar macrophages.

[0054] The boar-infectious virus to be contacted with the immortalized river boar macrophages is as described above, but it may be not only the isolated virus itself, but also a sample that may contain the virus. Examples of such "samples" include boar-derived tissues, cells, cultures thereof, washings, or extracts, as well as samples collected from the habitat or rearing environment of wild boars, washings, or cultures thereof.

[0055] The "contact" is usually carried out by adding the boar-infecting virus to a culture medium for culturing the immortalized river boar macrophages. Such a "culture medium" is not particularly limited, but may be the above-mentioned culture medium for primary culture of tissue macrophages.

[0056] The "propagation" of the boar-infectious virus can be carried out by contacting and culturing immortalized river boar macrophages infected with the virus. The culture temperature is not particularly limited, but is usually 30 to 40°C, preferably 37°C. The carbon dioxide concentration in the gas contacting the culture medium is not particularly limited, but is usually 1 to 10% by volume, preferably 2 to 5% by volume. The culture period after contact with the boar-infectious virus is not particularly limited, but is usually 1 to 10 days, preferably 2 to 7 days, and more preferably 3 to 5 days.

[0057] Those skilled in the art can determine whether boar-infectious viruses have proliferated using known methods. Examples of such methods include the cytopathic effect (CPE) test, which uses CPE as an indicator, as described in the Examples below, and methods for detecting the degree of intracellular ATP depletion associated with CPE (e.g., the Viral ToxGlo assay provided by Promega). Methods for detecting genes derived from boar-infectious viruses or their expression can also be used. Gene expression may be at the transcription level (RNA level) or the translation level (protein level). Methods for detecting genes (genomic DNA, genomic RNA) or RNA include, for example, PCR (RT-PCR, real-time PCR, quantitative PCR), sequencing, DNA microarray analysis, Northern or Southern blotting, in situ hybridization, dot blot, RNase protection assay, and mass spectrometry. Furthermore, gene or RNA levels can be quantitatively detected by counting the number of reads in so-called next-generation sequencing. Methods for detecting proteins include, for example, methods of detection using antibodies (immunological methods) such as ELISA, antibody array, immunoblotting, imaging cytometry, flow cytometry, radioimmunoassay, immunoprecipitation, and immunohistochemical staining, as well as mass spectrometry.

[0058] (Method for producing a vaccine) The method for producing a vaccine containing the wild boar-infectious virus of the present invention comprises the steps of: contacting immortalized river boar macrophages with the wild boar-infectious virus and propagating the virus in the immortalized river boar macrophages; isolating the propagated wild boar-infectious virus; and mixing the isolated wild boar-infectious virus with a pharmacologically acceptable carrier or vehicle.

[0059] The step of propagating the virus in the immortalized river boar macrophages is as described above. "Isolation" of the propagated wild boar-infectious virus means separation, purification, and / or concentration from the culture medium of the immortalized river boar macrophages and / or the cells. Methods for isolating the virus include, for example, filtration of the culture medium, cell disruption (sonication, hypotonic solution treatment, freeze-thawing, etc.), centrifugation (ultracentrifugation, density gradient centrifugation, etc.), and concentration (ammonium sulfate, resin column, polyethylene glycol salting-out, etc.).

[0060] The boar-infectious virus isolated in this manner may be used as a vaccine as it is (a so-called live vaccine), or in a live-attenuated form (a so-called live-attenuated virus), or in an inactivated form. Furthermore, a part of the isolated boar-infectious virus (protein, polypeptide, sugar, glycoprotein, lipid, nucleic acid, etc.) may also be used as a vaccine as long as it has immunogenicity.

[0061] A live attenuated virus is a virus that has a reduced level of toxicity compared to a virus isolated from the field. Attenuated viruses can be obtained by known methods, such as growth in the presence of mutagens, adaptation to cultured cells by serial (long-term) passage in vitro, or growth of a boar-infecting virus under conditions that deviate from its natural growth environment (e.g., high temperature conditions). Live attenuated viruses can also be obtained by deleting or recombining specific viral genes using genome editing, gene modification techniques, etc.

[0062] Those skilled in the art can inactivate viruses using known methods, including formaldehyde treatment, UV irradiation, X-ray irradiation, electron beam irradiation, gamma ray irradiation, alkylation treatment, ethyleneimine treatment, thimerosal treatment, β-propiolactone treatment, and glutaraldehyde treatment.

[0063] Examples of the "pharmacologically acceptable carrier" to be mixed with the isolated boar infectious virus include stabilizers, excipients, preservatives, surfactants, chelating agents, and binders. Examples of the "pharmacologically acceptable medium" include water, physiological saline, phosphate buffer, and Tris-HCl buffer. Those skilled in the art can select and use these carriers and media appropriately or in combination from known carriers and media used in the art, depending on the vaccine formulation and method of use. Furthermore, the form of the vaccine is not particularly limited, and may be, for example, a suspension or a freeze-dried form.

[0064] To enhance the vaccine effect, an adjuvant may be further mixed in. Examples of adjuvants include inorganic substances such as aluminum gel adjuvant, microorganisms or substances derived from microorganisms (e.g., BCG, muramyl dipeptide, Bordetella pertussis, pertussis toxin, cholera toxin), surfactants (e.g., saponin, deoxycholic acid), emulsions of oily substances (e.g., mineral oil, vegetable oil, animal oil), and alum.

[0065] (Method for detecting boar-infectious virus) The present invention provides immortalized river boar macrophages having DNA in which a reporter gene is functionally linked downstream of the promoter region of a gene derived from boar-infectious virus.

[0066] The present invention also provides a method for detecting boar-infectious viruses, the method comprising the steps of culturing immortalized river boar macrophages carrying the DNA in the presence of a test sample, detecting expression of the reporter gene in the immortalized river boar macrophages, and determining that the test sample contains the boar-infectious virus when expression of the reporter gene is detected.

[0067] The "immortalized river boar macrophages" into which the DNA is introduced are as described above. The DNA may be in the form of a vector as explained above in the "immortalizing gene." Furthermore, the DNA can be introduced into immortalized river boar macrophages by those skilled in the art using the methods listed above in the explanation of the "immortalizing gene."

[0068] The "promoter region of a gene derived from a boar-infectious virus" in the DNA is not particularly limited as long as it is derived from a boar-infectious virus and is a region that can activate the expression of a gene located downstream thereof in response to infection and proliferation of the virus, and may be any of an immediate-early gene, an early gene, a late-early gene, and a very-late gene.

[0069] Those skilled in the art can select a gene derived from a boar-infectious virus to use by referring to publicly known information, as appropriate. For example, a gene derived from ASFV can be selected by referring to the list of "Functions of Proteins Encoded by ASFV" in Table 1 of Virus Taxonomy, 9th Edition, pp. 155-157, 2012, International Committee on Taxonomy of Viruses (ICTV). The promoter regions of the p72, U104L, CD2v, DNA polymerase, or p30 genes are preferably used (see Portugal R.S. et al., Virology, August 2017, Vol. 508, pp. 70-80).

[0070] The "reporter gene" functionally (operably) linked downstream of the promoter region is not particularly limited, and known genes can be used as appropriate. Examples include fluorescent protein genes, luciferase genes, and chromogenic enzyme genes. Specific examples of fluorescent protein genes include the GFP (green fluorescent protein) gene, the YFP (yellow fluorescent protein) gene, and the RFP (red fluorescent protein) gene. Specific examples of luciferase genes include the aequorin gene and the luciferase gene. Specific examples of chromogenic enzyme genes include the chloramphenicol acetyltransferase (CAT) gene, the β-glucuronidase (GUS) gene, the β-galactosidase gene, the alkaline phosphatase gene, and the SEAP gene.

[0071] In the detection method of the present invention, the fluorescence, luminescence, color development, etc. that are generated in response to the expression of these reporter genes can be used as an indicator to detect whether or not immortalized river boar macrophages are infected with boar infectious virus, and ultimately the presence of boar infectious virus in the test sample.

[0072] The test sample is not particularly limited as long as it is a sample in which boar-infectious viruses may be present, and examples include tissues, cells, cultures thereof, washings, or extracts derived from wild boars, or samples collected from the habitat or rearing environment of wild boars, washings, or cultures thereof.

[0073] Furthermore, the "culture medium" used for culture in the detection method of the present invention is not particularly limited, and examples thereof include the above-mentioned culture medium for primary culture of tissue macrophages. The culture temperature is not particularly limited, but is usually 30 to 42°C, preferably 37°C. The carbon dioxide concentration in the gas in contact with the culture medium is not particularly limited, but is usually 1 to 10% by volume, preferably 2 to 5% by volume. The culture period in the presence of a test sample until reporter gene expression is detected is not particularly limited, but is usually 1 to 10 days, preferably 2 to 7 days, and more preferably 2 to 5 days.

[0074] (Method for detecting neutralizing antibodies) The method of the present invention for detecting neutralizing antibodies against boar-infecting viruses comprises the steps of: bringing immortalized river boar macrophages into contact with the boar-infecting virus in the presence of a biological sample isolated from a test boar, and allowing the virus to grow in the immortalized river boar macrophages; detecting the number of the grown viruses; and determining that the biological sample contains a neutralizing antibody against the virus when the number of viruses detected in the step is lower than the number of viruses grown in the immortalized river boar macrophages in the absence of the biological sample.

[0075] The term "neutralizing antibody" used in the present invention refers to an antibody that inhibits infection or proliferation of boar-infectious viruses. Such antibodies include all classes and subclasses of immunoglobulins. The "subject boar" is not particularly limited as long as it is a boar, regardless of whether it has been infected with a boar-infectious virus. "Biological samples" isolated from subject boars include boar-derived samples (for example, blood (serum, plasma, etc.), mucus (saliva, nasal secretions, milk, gastrointestinal secretions, etc.), and antibodies purified therefrom).

[0076] "Contact" is as described above. Regarding growth conditions, the culture temperature is not particularly limited, but is typically 30 to 40°C, preferably 37°C. The carbon dioxide concentration in the gas contacting the culture medium is not particularly limited, but is typically 1 to 10% by volume, preferably 2 to 5% by volume. The culture period in the presence or absence of a biological sample is not particularly limited, but is typically 1 to 10 days, preferably 2 to 7 days, and more preferably 3 to 5 days. Furthermore, as described above, the propagated virus can be detected by a CPE test or the like, or by detecting a gene derived from a boar-infecting virus or its expression. Thus, in the detection method of the present invention, the presence or absence of a neutralizing antibody may be determined using not only the virus count itself, but also the gene (e.g., genomic DNA amount) or its expression level that reflects the virus count as an indicator.

[0077] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples. The examples were carried out using the materials and methods shown below.

[0078] <Materials and Methods> (Immunostaining) Cells were placed in an 8-well chamber slide (Asahi Glass) at 2 × 10 ５ Cells were seeded at 1 / well and cultured. After washing once with DPBS, the cells were fixed with 4% paraformaldehyde phosphate buffer (Nacalai Tesque), permeabilized with 1% Triton X-100 / PBS solution, and blocked with Blocking One Histo (Nacalai Tesque). Next, the cells were incubated with primary antibodies at room temperature for 1 hour and then stained using the EnVision system (DAKO). Cell nuclei were counterstained with Mayer's hematoxylin solution (Fujifilm Wako). The stained slides were observed under a microscope. The primary antibodies used were anti-ionized calcium-binding adapter molecule 1 (Iba1) rabbit polyclonal antibody (Fujifilm Wako), anti-CD172a mouse monoclonal antibody (clone DH59B) (VMRD), anti-CD203a mouse monoclonal antibody (clone PM18-7) (Bio-Rad), and major histocompatibility complex class II (MHC-II) (clone MSA3) mouse monoclonal antibody (Kingfisher Biotech).

[0079] (Cell Culture) Cells were cultured in a growth medium (DMEM High glucose: containing 10% fetal bovine serum, 100 U / ml penicillin, 100 μg / mL streptomycin, 10 μg / mL insulin, 25 μM monothioglycerol, and 5 μg / mL fungin). ６ ) were seeded onto 90 mm suspension culture dishes (Sumitomo Bakelite) and subcultured every 4 to 5 days. At each subculture, cells were detached using TrypLE Express solution (Thermo Fisher Scientific) and the cell number was measured using a Bio-Rad TC20 automated cell counter.

[0080] (Confirmation of insertion of the SV40 large T antigen (SV40LT) gene and porcine telomerase reverse transcriptase (pTERT) gene) Genomic DNA was extracted from cells using a NucleoSpin kit (Takara Bio). A forward primer was designed within the lentiviral vector sequence, and a reverse primer sequence was designed within the SV40LT or pTERT gene. Gene insertion into the genome was confirmed by PCR. The lengths of the PCR products derived from the SV40LT and pTERT genes were 128 bp and 143 bp, respectively. PCR amplification was performed using KOD FX DNA polymerase (Toyobo). PCR products were separated by polyacrylamide gel electrophoresis and detected with GelGreen™ staining (Biotium, Inc.).

[0081] (African Swine Fever Virus (ASFV)) Three field isolates of ASFV, namely, the European / China epidemic strain Armenia 07 (genotype: II), the strain Kenya 05 / Tk-1 (genotype: I) isolated from soft ticks, and the ASFV standard strain Espana 75 (genotype: I), were introduced from the Complutense University of Madrid, Spain, which is the ASF reference laboratory of the World Organization for Animal Health (OIE). Information such as the year and place of outbreak of each virus strain is provided in Fernandez-Pinero J, et al. See, "Molecular diagnosis of African Swine Fever by a new real-time PCR using a universal probe library," Transbound Emerg Dis. 2013 Feb;60(1):48-58. The Vero cell-adapted strain Lisbon 60 / V was introduced from the Plum Island Animal Disease Center (PIADC), America. The Armenia 07, Kenya 05 / Tk-1, and Espana 75 strains were grown and cultured in PAM cells, and the Lisbon 60 / V strain was grown and cultured in Vero cells, and these were used as inoculated virus samples. The inoculated virus samples were aliquoted and stored at -80°C.

[0082] (ASFV Susceptibility Test) The susceptibility of cells to ASFV was determined by a cytopathic effect (CPE) test using CPE as an indicator, or a hemadsorption (HAD) test using the hemadsorption (HAD) reaction, which is specifically observed in ASFV-infected cells, as an indicator. The cells used in these tests were IPKM or RZJ / IBM cells. IPKM is an immortalized porcine kidney macrophage cell line previously established by the present inventors and described in Non-Patent Document 1. RZJ / IBM cells are an immortalized red river boar blood macrophage cell line established in this Example, as described below. Specifically, in these tests, IPKM or RZJ / IBM cells (3 x 10 ４ 10 pieces) were seeded and pre-cultured in each medium for 1-2 days, and then diluted 10 times (10 －１ ) to 100 million times (10 －８ 100 μL of the virus solution, which had been serially diluted 10-fold up to 100 μL per well, was inoculated into each of eight wells. Next, 20 μL of a 0.75% suspension of porcine red blood cells suspended in phosphate-buffered saline (PBS) was added to the HAD detection wells, and these wells were incubated together with the CPE detection wells at 37°C and 5% CO ２ The cells were cultured under the same conditions for 7 days and observed.

[0083] (ASFV Growth Test) Cells were seeded in T25 flasks (Sumitomo Bakelite) and inoculated with an ASFV strain at a multiplicity of infection (MOI) of 0.01. After culturing at 37°C for 1 hour, the inoculated virus was removed by washing with DPBS, and growth medium was added. The culture supernatant was collected 1, 2, 3, 4, and 5 days after inoculation, and the virus titer (TCID50 / ml) was measured using CPE activity against IPKM cells as an indicator.

[0084] (Classical swine fever virus (CSFV)) The JPN / 1 / 2018 strain is a virus strain isolated from pigs infected with CSF that occurred in Gifu Prefecture, Japan in September 2018. The strain was established at the National Institute of Animal Health (NIH), National Agriculture and Food Research Organization (NARO), Ibaraki Prefecture, Japan, and subcultured for four generations in pig kidney epithelial cells (CPK cells) to be used as an inoculation virus sample. The ALD strain is a highly pathogenic and virulent strain isolated in the United States and commonly used as a standard strain. It was provided to the then Livestock Hygiene Research Institute (now NIH) by Dr. Kesteven K. V. L., a veterinary officer at the Food and Agriculture Organization of the United Nations (FAO) in 1950, and subcultured for one generation in CPK cells to be used as an inoculation virus sample. GPE － The strain was a vaccine strain established at the National Institute of Animal Health (NIAH) by passage and adaptation in cultured cells such as guinea pigs (GP), and was subcultured for one generation in CPK cells for use as the inoculation virus sample. The C strain is a live vaccine strain (Chinese strain) widely used worldwide, which was attenuated by subculture of a highly virulent wild-type strain in rabbits for 420 generations. The C strain was isolated from an oral CSF vaccine (product name: Pestiporc Oral, manufactured by IDT Biologika GmbH, Germany), and was subcultured for two generations in CPK cells for use as the inoculation virus sample.

[0085] (Fluorescent Immunostaining) RZJ / IBM cells were plated in a 96-well plate at 3 x 10 ４ Cells were seeded at 1 / well and cultured. The next day, the medium was removed, and each CSFV-containing sample was inoculated and cultured at 37°C for 1 hour. After removing the virus solution and culturing for a certain period, the medium was removed, and the cells were fixed with ethanol (4°C, 1 hour). After removing the ethanol, the plate was air-dried, and the cells were incubated with a primary antibody (anti-swine fever virus mouse monoclonal antibody, clone WH303, APHA Scientific) (37°C, 30 minutes). After washing, the cells were fluorescently labeled with a secondary antibody (Alexa Fluor 488 Goat anti-mouse IgG (H+L), Invitrogen) (37°C, 30 minutes) and observed under a fluorescence microscope.

[0086] (Virus growth test) RZJ / IBM cells (4 x 10 ６ ) to 75cm ２ The cells were seeded into flasks (SUMILON suspension culture) and culture was initiated. The next day, the medium was removed, and each CSFV-containing sample was inoculated into growth medium at a multiplicity of infection (MOI) of 0.01, followed by culture at 37°C for 1 hour. After removing the virus solution, 20 ml of growth medium was added, and the culture supernatant was collected 0, 1, 2, 3, 7, 10, and 14 days later. The culture supernatant from each day after inoculation was serially diluted 10-fold, and the virus titer was measured using fluorescent immunostaining.

[0087] The results obtained using the above materials and methods are shown below.

[0088] <Results> (Mixed culture of red river boar blood and pig kidney cells) First, pig kidney cells for feeder use were the same as those previously reported (Non-Patent Document 1), i.e., a primary culture system derived from pig kidneys from which macrophages had been removed and mainly containing epithelial cells, fibroblasts, and myofibroblasts. These cells were pre-cultured in a growth medium in a T150 flask (Sumitomo Bakelite). Two milliliters of red river boar blood (provided by Zoorasia, Yokohama Zoological Gardens) was then added to initiate the mixed culture.

[0089] (Isolation of Red River Boar Blood Macrophages) After approximately two weeks, spherical cells appeared, weakly adhering and growing on the pig kidney cell sheet (Figure 1A, arrow). Cells were collected from the culture supernatant, and cells adhering to a suspension culture dish (Sumitomo Bakelite) were isolated (Figure 1B). The isolated cells expressed macrophage marker molecules (Iba1, CD172a, CD203a, MHC-II) (Figure 1C).

[0090] (Generation of an immortalized red river boar blood macrophage cell line) Isolated red river boar blood macrophages were seeded onto 60 mm suspension culture dishes (Sumitomo Bakelite) and the following day exposed for 2 hours to a solution containing recombinant lentiviral particles individually transfected with two immortalization genes (SV40LT, pTERT). Approximately one week later, the cells were again exposed to the same lentiviral particles and cultured. Approximately six months later, we successfully obtained a subculture-ready immortalized cell line (RZJ / IBM cells: Red River Hog, Zoorasia, Japan / Immortalized Blood-derived Macrophages) and cryopreserved it (Figure 2A). RZJ / IBM cells expressed macrophage markers (Iba1, CD172a, and CD203a) (Fig. 2B) and were confirmed to have the ability to divide at least 50 times (Fig. 2C). Furthermore, PCR confirmed that the SV40LT and pTERT genes had been inserted into the RZJ / IBM genome (Fig. 2D).

[0091] (Biological species identification analysis) DNA was extracted from RZJ / IBM cells, and an analysis to identify the biological species was requested from Biotechnology Research Institute. As a result, as shown in Table 1 below, the cells were determined to be those of river boar (Potamochoerus porcus) using the mitochondrial DNA sequence as an indicator. In this table, "Merge 1" to "Merge 3" are the three most frequently occurring merged sequences obtained by merging reads obtained by MiSeq sequencing, and the three with the most merged reads (Read_count) are shown. These merged sequences are also shown as the DNA sequences set forth in SEQ ID NOs: 1 to 3, respectively.

[0092]

[0093] (Inflammatory Response Analysis) Total RNA was extracted from RZJ / IBM cells treated with 1 μg / mL LPS for 3 hours and untreated cells using the NucleoSpin RNA Kit (Takara Bio), and gene expression changes were comprehensively analyzed by RNA-seq (analysis requested by Biotechnology Research Institute). The results showed a significant increase in gene expression of inflammatory cytokines (IL1A, IL1B, IL18, TNF) and interferon INFB1 (Figures 3A-3F). Although not shown in the figures, the uptake of killed E. coli bacteria by RZJ / IBM cells was also confirmed. This revealed that the cells possess phagocytic ability.

[0094] (ASFV susceptibility test) When the susceptibility of each cell line to four different ASFV strains was compared using CPE as an indicator, CPE was confirmed for all virus strains inoculated into IPKM cells (Fig. 4, Table 2). On the other hand, in RZJ / IBM cells, CPE was not observed for the three field strains, and CPE was observed only for the cell-adapted strain Lisbon 60 / V (Fig. 5, Table 3).

[0095] When the susceptibility of each cell line to four different ASFV strains was compared using HAD as an indicator, HAD was detected in both IPKM and RZJ / IBM cells with all inoculated virus strains (Fig. 4, Table 2, Fig. 5, Table 3). However, the dilution limit at which HAD could be detected was 10- to 1000-fold higher in RZJ / IBM cells than in IPKM cells (Tables 2 and 3), indicating that RZJ / IBM cells were less susceptible to the virus.

[0096]

[0097]

[0098] (ASFV Growth Test) We compared the growth efficiency of four different ASFV strains in IPKM cells and RZJ / IBM cells. We found that the Armenia 07 strain grew very slowly in RZJ / IBM cells (Fig. 6A). Growth of other virus strains (Kenya 05 / Tk-1, Espana 75, and Lisbon 60 / V strains) was also observed in RZJ / IBM cells, but the growth efficiency was lower than that in IPKM cells (Figs. 6B-6D).

[0099] (CSFV susceptibility test) Fluorescent immunostaining was performed on RZJ / IBM cells 5 days after inoculation for the domestic epidemic strain JPN / 1 / 2018, and on 7 days after inoculation for the virulent strain ALD and vaccine strain (GPE-, C). The results showed that all CSFV strains infected RZJ / IBM cells (Figure 7).

[0100] (CSFV growth test) RZJ / IBM cells were inoculated with CSFV strains, and the virus titer in the culture supernatant was measured after each incubation period. As a result, growth of all CSFV strains was confirmed after one week ( FIG. 8 ).

[0101] As described above, according to the present invention, it is possible to provide a cell line of river boar macrophages that can be passaged. Furthermore, since the immortalized river boar macrophages are susceptible to boar-infecting viruses such as African swine fever virus, it is possible to grow various viruses, enabling the production and development of vaccines against these viruses. In particular, since the immortalized river boar macrophages can grow infected viruses while suppressing their cell death, it is possible to provide these viruses with high productivity. Furthermore, because they are susceptible to infection, it is also possible to detect (test, diagnose) these viruses.

Claims

1. Immortalized wild boar macrophage cells.

2. The cell according to claim 1, which is obtained by expressing at least one protein selected from the group consisting of SV40 large T antigen and telomerase reverse transcriptase in a macrophage of a river boar.

3. The cell of claim 2, wherein the expression is from a lentivirus encoding the protein.

4. The cell according to claim 2, wherein the telomerase reverse transcriptase is porcine-derived telomerase reverse transcriptase.

5. A method for producing immortalized macrophages, comprising the step of expressing at least one protein selected from the group consisting of SV40 large T antigen and telomerase reverse transcriptase in macrophages of river boars.

6. The method according to claim 5, wherein the step is a step of introducing a lentivirus encoding the protein into macrophages of the river boar and expressing the protein.

7. The method according to claim 5 or 6, wherein the telomerase reverse transcriptase is porcine-derived telomerase reverse transcriptase.

8. A method for producing a boar-infective virus, comprising the steps of contacting the cells of claim 1 with a boar-infective virus and propagating the virus in the cells.

9. A method for producing a vaccine containing a boar-infectious virus, comprising the steps of: contacting the cells of claim 1 with a boar-infectious virus and propagating the virus in the cells; isolating the propagated boar-infectious virus; and mixing the isolated boar-infectious virus with a pharmacologically acceptable carrier or medium.

10. The method of claim 8 or 9, wherein the boar-infecting virus is African swine fever virus.

11. The cell according to claim 1, which has DNA in which a reporter gene is functionally linked downstream of the promoter region of a gene derived from a boar-infectious virus.

12. The cell of claim 11, wherein the boar-infecting virus is African swine fever virus.

13. A method for detecting boar infectious virus, comprising the steps of: culturing the cells of claim 11 in the presence of a test sample; detecting expression of the reporter gene in the cells; and determining that the test sample contains boar infectious virus when expression of the reporter gene is detected.

14. A method for detecting neutralizing antibodies against boar-infecting viruses, comprising the steps of: contacting the cells described in claim 1 with the boar-infecting virus in the presence of a biological sample isolated from a test boar and allowing the virus to grow in the cells; detecting the number of the grown viruses; and determining that the biological sample contains neutralizing antibodies against the virus if the number of viruses detected in the step is lower than the number of viruses grown in the cells described in claim 1 in the absence of the biological sample.

15. The method of claim 13 or 14, wherein the boar-infecting virus is African swine fever virus.

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