JP7477914B2 - Immortalized fetal porcine small intestinal macrophages - Google Patents

Description

The present invention relates to immortalized porcine small intestinal macrophages and a method for producing the macrophages. The present invention also relates to a method for producing a porcine infectious virus or a vaccine using the macrophages. Furthermore, the present invention relates to a method for detecting a porcine infectious virus or a method for detecting a neutralizing antibody against a porcine infectious virus using the macrophages.

In pig farming, diarrhea is one of the causes of death and growth retardation in livestock, and is a serious problem in stable and efficient pork production. Diarrhea occurs due to various factors, such as pathogen infection, nutrition, and host stress, either alone or in combination with each other. Small intestinal macrophages, which are innate immune cells, play a major role in the activation and termination of host immunity, and their response has a significant impact on the severity of diarrhea and the amount of pathogen excretion. In addition, enteritis accompanied by diarrhea can also occur due to changes in nutrition and the associated changes in the intestinal flora and their metabolic products, and it is becoming clear that the response of small intestinal macrophages plays a part in the pathology.

Based on these findings, an in vitro immunological response analysis system of small intestinal macrophages is considered necessary as a fundamental tool for elucidating the pathology of diarrhea in pigs and developing preventive methods. However, to date, no cell line of porcine small intestinal macrophages that can be passaged has been established, and its creation was also required in order to build the above-mentioned analysis system.

Takenouchi T. et al., Front Vet Sci., August 2017, 21;4:132.

The present invention has been made in consideration of the problems associated with the above-mentioned conventional technology, and aims to provide a cell line of porcine small intestinal macrophages that can be passaged.

The present inventors have previously succeeded in isolating and immortalizing macrophages from the kidneys of pigs (6 days old) (Non-Patent Document 1). Therefore, they attempted to isolate and immortalize macrophages from the small intestines of pigs (approximately 1 month old). However, even when a medium containing antibiotics and antifungals was used, there was severe microbial contamination, and it was not possible to isolate macrophages from the small intestines of pigs.

Therefore, we first carried out intensive research and studies to enable the primary culture of macrophages from pig small intestine. As a result, we found that the small intestine of a fetus does not cause the above-mentioned contamination and it is possible to isolate and culture small intestinal macrophages.

Then, the SV40 large T antigen gene and the pig-derived telomerase reverse transcriptase gene were introduced into the porcine small intestinal macrophages thus obtained and transformed, and it was found that the obtained cells were capable of dividing at least 60 times. It was also found that the immortalized cells maintained the expression of marker molecules and the functions of macrophages (activation of intracellular signaling molecules and induction of inflammatory cytokine production in response to bacterial components).

Furthermore, the thus obtained immortalized fetal pig small intestine macrophages were subjected to a susceptibility test against African swine fever virus (ASFV). As a result, it was revealed that the cells were able to detect ASFV in both cytopathic effect (CPE) and hemadsorption (HAD) tests. Furthermore, it was revealed that the cells were susceptible to all tested ASFV strains and were also able to grow these virus strains. Moreover, the susceptibility was surprisingly orders of magnitude higher than that of primary cultured porcine alveolar macrophages (PAM) (100 to 10,000 times higher than PAM in the CPE test and 10 to 100 times higher than PAM in the HAD test). Furthermore, susceptibility tests were also performed against porcine reproductive and respiratory syndrome virus and porcine circovirus type 2. As a result, it was confirmed that the immortalized fetal pig small intestine macrophages were highly susceptible to these viruses, as well as ASFV.

Thus, the present invention is based on the first successful production of immortalized porcine small intestinal macrophages and the discovery that the produced immortalized porcine fetal small intestinal macrophages have high susceptibility to viral infection, and is specifically as follows.
<1> Immortalized macrophages from the small intestine of fetal pigs.
<2> The cell according to <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 small intestinal macrophage of a pig fetus.
<3> The cell according to <2>, wherein the expression is from a lentivirus encoding the protein.
<4> The cell according to <2> or <3>, wherein the telomerase reverse transcriptase is porcine-derived telomerase reverse transcriptase.
<5> The cell according to any one of <1> to <4>, wherein the pig fetus is a pig fetus of 30 to 114 days of fetal age.
<6> A method for producing an immortalized porcine small intestinal macrophage, comprising a step of expressing at least one protein selected from the group consisting of SV40 large T antigen and telomerase reverse transcriptase in a porcine fetal small intestinal macrophage.
<7> The method according to <6>, wherein the step is a step of introducing a lentivirus encoding the protein into small intestinal macrophages of the pig fetus to express the protein.
<8> The method according to <6> or <7>, wherein the telomerase reverse transcriptase is porcine-derived telomerase reverse transcriptase.
<9> The method according to any one of <6> to <8>, wherein the pig fetus is a pig fetus of 30 to 114 days of gestation.
<10> A method for producing a porcine-infecting virus, comprising the steps of contacting the cell according to any one of <1> to <5> with a porcine-infecting virus and propagating the virus in the cell.
<11> A step of contacting the cell according to any one of <1> to <5> with a porcine infectious virus and propagating the virus in the cell;
A method for producing a vaccine containing a porcine infectious virus, comprising the steps of: isolating the propagated porcine infectious virus; and mixing the isolated porcine infectious virus with a pharmacologically acceptable carrier or medium.
<12> The production method according to <10> or <11>, wherein the porcine-infecting virus is at least one virus selected from the group consisting of African swine fever virus, porcine epidemic diarrhea virus, porcine rotavirus, porcine circovirus, and porcine reproductive and respiratory syndrome virus.
<13> The cell according to any one of <1> to <5>, which has DNA in which a reporter gene is operably linked downstream of a promoter region of a gene derived from a porcine infectious virus.
<14> The cell according to <13>, wherein the porcine-infecting virus is at least one virus selected from the group consisting of African swine fever virus, porcine epidemic diarrhea virus, porcine rotavirus, porcine circovirus, and porcine reproductive and respiratory syndrome virus.
<15> A method for detecting a porcine infectious virus, comprising the steps of: culturing the cell according to <13> or <14> in the presence of a test sample;
detecting expression of the reporter gene in the cells; and determining that the test sample contains the porcine infectious virus when expression of the reporter gene is detected.
<16> A method for detecting a neutralizing antibody against a porcine-infecting virus, comprising the steps of: contacting the cell according to any one of <1> to <5> with a porcine-infecting virus in the presence of a biological sample isolated from a test pig, and allowing the virus to grow in the cell;
detecting the number of the proliferated viruses; and determining that the biological sample contains a neutralizing antibody against the virus when the number of the viruses detected in the step is smaller than the number of the viruses proliferated in the cell according to any one of <1> to <5> in the absence of the biological sample.
<17> The method according to <15> or <16>, wherein the porcine-infecting virus is at least one virus selected from the group consisting of African swine fever virus, porcine epidemic diarrhea virus, porcine rotavirus, porcine circovirus, and porcine reproductive and respiratory syndrome virus.

According to the present invention, it is possible to provide a cell line of fetal pig small intestine macrophages that can be passaged. Furthermore, since the immortalized fetal pig small intestine macrophages have high susceptibility to infection with viruses, it is possible to grow various viruses, which makes it possible to manufacture and develop vaccines against those viruses. Furthermore, due to the high susceptibility to infection, it is also possible to detect (test, diagnose) the viruses.

This is a micrograph showing the results of observing adherent primary cultured cells (primary cultured fetal pig small intestine cells) that had spread on the 22nd day after enzymatically treating and seeding pieces of fetal pig small intestine tissue and culturing them. The scale bar in the figure represents 200 μm. This is a micrograph showing spherical cells (macrophages, PIM) that are weakly attached and proliferating on a cell sheet formed by detaching and reseeding primary cultured cells from fetal pig small intestine. The scale bar in the figure represents 100 μm. This is a photograph showing the results of immunostaining of primary cultured cells of fetal pig small intestine after reseeding and culturing. In the figure, the top left shows the results of staining alpha smooth muscle actin (αSMA), a myofibroblast marker, and the top right shows the results of staining Vimentin, a mesenchymal cell marker. The center shows the results of staining cytokeratin, an epithelial marker (the left shows the results of staining cytokeratin 18 (CK18), and the right shows the results of staining cytokeratin 19 (CK19)). The bottom left shows the results of staining CD172a, a macrophage marker, and the bottom right shows the results of staining CD204, a macrophage marker. The scale bar represents 400 μm. In addition, under the color display, brown represents antibody staining and blue-purple represents nuclear staining. This is a photograph showing the results of immunostaining of PIM. The top left shows the results of staining Iba1, a macrophage marker, the top right shows the results of staining CD172a, a macrophage marker, and the middle left shows the results of staining CD204, a macrophage marker. The middle right shows the results of staining Vimentin, a mesenchymal cell marker. The bottom shows the results of staining cytokeratin, an epithelial marker (the left shows the results of staining CK18, and the right shows the results of staining CK19). The scale bar represents 400 μm. In addition, under the color display, brown represents antibody staining and blue-purple represents nuclear staining. 1 is a micrograph showing the results of observing immortalized macrophages (IPIM) isolated from the small intestine of a fetal pig. The scale bar in the figure represents 100 μm. 1 is a graph showing the results of testing the cell proliferation properties of IPIM. This is a photograph showing the results of immunostaining analysis of IPIM. The upper part shows, from the left, the results of staining the macrophage marker Iba1, the macrophage marker CD172a, the macrophage marker CD204, and the antigen-presenting cell marker MHC-II, which includes macrophages. The lower part shows, from the left, the results of staining the macrophage marker CD16, the M2 macrophage marker CD163, the M1 macrophage marker CD169, and the porcine macrophage marker CD203a. The scale bar in the figure represents 200 μm. In addition, under the color display, brown represents antibody staining and blue-purple represents nuclear staining. This is a photograph showing that IPIM activates intracellular signaling molecules (phosphorylates p38 MAP kinase) in response to bacterial-derived components (muramyl dipeptide: MDP, lipopolysaccharide: LPS). These are photographs showing that IPIM induces the production of inflammatory cytokines (IL-1β) in response to bacterial-derived components (MDP, LPS). 1 is a micrograph showing the results of IPIM inoculated with African swine fever virus (Armenia 07 strain) and observing cytopathic effect (CPE) two days later. In the figure, the left panel "non-infected" shows the results of the negative control not inoculated with the ASFV strain. 1 is a micrograph showing the results of detecting hemadsorption (HAD) in IPIM two days after inoculation with the Armenia 07 strain in the presence of red blood cells. In the figure, the left panel "non-infected" shows the results of the negative control not inoculated with the ASFV strain. These are micrographs showing the results of inoculating IPIM with porcine reproductive and respiratory syndrome virus (PRRSV), detecting viral antigens by indirect fluorescent antibody technique on day 3, and detecting CPE on day 7. In the figure, the left panel "non-inoculated" shows the results of the negative control not inoculated with a PRRSV strain. 1 is a graph showing the results of an analysis of the time course of PRRSV tissue culture infective dose 50 (TCID ₅₀ ) in IPIM. 1 is a graph showing the results of an analysis of the change in porcine circovirus type 2 (PCV2) gene quantity over time in IPIM.

The present invention relates to immortalized fetal pig small intestinal macrophages (immortalized fetal pig small intestinal macrophages).

In the present invention, "immortalized fetal pig small intestine macrophages" refers to immortalized macrophages present in the small intestine of a fetal pig (an animal of the order Cetacea, family Suidae, class Mammalia). "Fetal pig" refers to an individual within a mother pig before birth, preferably a pig with a gestational age of 30 to 114 days, and more preferably a pig with a gestational age of 90 to 114 days from the viewpoint of maturation in small intestinal tissue formation.

There are no particular limitations on the "small intestine" from which macrophages are collected, and examples include the ileum, jejunum, and duodenum, but from the viewpoint of the development of lymphatic tissue in which immune cells are concentrated, the ileum is preferred.

The "porcine small intestinal macrophages" to be immortalized are macrophages that are present in the pig small intestine and are differentiated from macrophages or monocytes derived from the egg yolk sac or fetal liver, and may be activated macrophages (inflammatory M1 type macrophages, anti-inflammatory M2 type macrophages) or resting macrophages. For example, as shown in the Examples below, such macrophages are prepared by removing the membrane from the small intestine taken from a pig fetus. The isolated small intestinal tissue is then cut into thin strips, washed with a buffer solution, subjected to an enzyme treatment, and then cultured. Macrophage-like cells that are loosely attached to a monolayer cell sheet that is generated during the culture process can be isolated to prepare the macrophage-like cells.

There are no particular limitations on the buffer solution used to wash the shredded small intestinal tissue, and examples include Dulbecco's phosphate buffered saline (DPBS), phosphate buffered saline (PBS), Tris-HCl buffer (TBS), and HEPES buffer.

There are no particular limitations on the enzymes used in the enzyme treatment, so long as they can separate and disperse cells from the tissue. Examples include collagenase, dispase, DNase (DNase I, etc.), trypsin, hyaluronidase, elastase, or pronase, with a preferred combination being collagenase, dispase, and DNase I. There are also no limitations on the temperature and time of treatment, and these can be adjusted as appropriate depending on the type of enzyme used and the degree of cell separation and dispersion.

The medium used for culturing the cells dispersed by enzyme treatment is not particularly limited as long as it can maintain small intestinal macrophages, but it can be prepared by appropriately adding well-known and commonly used medium additives to a known basal medium. Examples of "basal medium" 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's 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 of these. 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'-isopropyl ether, etc.), and the like. Examples of suitable antioxidants include, but are not limited to, phosphate, hypoxanthine, thymidine, etc.), metal salts (iron (III) nitrate, ferrous sulfate (II), copper sulfate, zinc sulfate, etc.), inorganic salts (sodium, potassium, calcium, magnesium, phosphorus, chlorine, etc.), carbon sources (glucose, galactose, fructose, sucrose, etc.), vitamins, inorganic compounds (selenium acid), organic compounds (para-aminobenzoic acid, ethanolamine, corticosterone, progesterone, lipoic acid, putrescine, pyruvic acid, lactic acid, triiodothyronine, etc.), buffer compounds (HEPES, sodium bicarbonate, etc.), and pH indicators (phenol red, etc.).

There are no particular limitations on the culture conditions when using such a medium, but the culture temperature is usually 30 to 40° C., preferably 37° C. The concentration of carbon dioxide in the gas in contact with the medium is usually 1 to 10% by volume, preferably 2 to 5% by volume.

Then, under such culture conditions, the cells derived from the small intestine tissue dispersed by the enzyme treatment are cultured to obtain primary cultured small intestine cells. There is no particular limit to the culture time for obtaining the cells, but it is usually one week to one month, preferably two to three weeks.

The resulting primary cultured small intestinal cells are then subjected to an enzyme treatment, detached from the culture vessel, dispersed, and reseeded. The enzyme treatment here can also be carried out using the enzymes described above, but preferably a combination of protease (an enzyme that has at least the activity of degrading non-collagenous proteins), collagenase, and DNase (for example, product name: Accumax (registered trademark), manufactured by Sigma-Aldrich).

Then, in the culture after reseeding, a mixed culture system is established, which consists of a cell sheet composed of myofibroblast-like cells and spherical cells (porcine small intestinal macrophages) that weakly adhere and grow on the cell sheet. There is no particular limit to the culture time for establishing the culture system, but it is usually between a few days (2-4 days) and a few weeks (2-3 weeks), preferably 1-2 weeks.

There is no particular limitation on the method for immortalizing the small intestinal macrophages of the pig fetus obtained in this manner, but it can be carried out by introducing at least one immortalization gene. Examples of immortalization genes include SV40 large T antigen (SV40T antigen), telomerase reverse transcriptase (TERT), Myc, and Ras. It is preferable to introduce SV40T antigen and TERT, and from the viewpoint of increasing the immortalization efficiency of pig macrophages, it is more preferable to introduce SV40T antigen and pig-derived TERT.

The introduction of an immortalizing gene can be carried out by using a vector that encodes the gene. The vector may be linear or circular, and examples of the vector include a viral vector, a plasmid vector, an episomal vector, an artificial chromosome vector, and a transposon vector.

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 porcine macrophages.

In addition to the immortalization gene, the vector of the present invention may contain expression control sequences such as a promoter, enhancer, poly A addition signal, and terminator, a replication origin and 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 3' untranslated region including a polyadenylation signal, an AU-rich element, a GU-rich element, etc., nucleotides encoding other proteins, etc.

By operably positioning the immortalization gene downstream of a promoter, it becomes possible 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, heat shock promoter, etc.

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

When introducing multiple types of immortalization genes, 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, 2A peptide sequence, etc. into the vector, it becomes possible to express multiple types of immortalization genes in a polycistronic manner.

Methods for introducing the vector into cells include lipofection, microinjection, calcium phosphate, DEAE-dextran, electroporation, and particle gun methods. In addition, when the vector of the present invention is a retrovirus vector, appropriate packaging cells may be selected based on the LTR sequence and packaging signal sequence possessed by the vector, and used to prepare retrovirus particles. 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. In addition, the virus particles prepared in this manner can be introduced into cells by the Polybrene method, the Protamine method, the RetroNectin method, and the like.

The gene introduction and the subsequent maintenance culture can be performed using the medium for primary culture of fetal pig small intestine described above and the culture conditions using the medium. The immortalized fetal pig small intestine macrophages established by introducing the immortalization gene in this way exhibit a proliferation rate of at least one month, preferably two months or more, and more preferably three months or more. The doubling time of the immortalized pig kidney macrophages is at least four days, preferably two days, and more preferably one day. The immortalized fetal pig small intestine macrophages preferably maintain the characteristics of macrophages, and for example, at least one gene of macrophage-specific genes CD172a, CD16, Iba-1, CD204 (MSR-A), and CD203a is expressed, preferably two or more genes are expressed, more preferably three or more genes are expressed, even more preferably four or more genes are expressed, and particularly preferably all of these genes are expressed. Furthermore, the immortalized fetal porcine small intestine macrophages 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. The immortalized fetal porcine small intestine macrophages according to the present invention retain at least one function, and preferably two or more functions, selected from the group consisting of phagocytosis, production of inflammatory cytokines by LPS stimulation, and IL-1β maturation associated with inflammasome activity, as characteristics of macrophages.

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

(Porcine-infected virus)
In the present invention, the "pig-infecting virus (virus that infects pigs)" may be any virus that is at least capable of infecting pigs, and may be a DNA virus (double-stranded (ds) DNA virus, single-stranded (ss) DNA virus, or DNA virus containing both ss and dsDNA regions) or an RNA virus (single-stranded (ss) RNA virus (positive-strand RNA virus or negative-strand RNA virus), double-stranded (ds) RNA virus).

Examples of swine-infecting viruses include African swine fever virus (ASFV), porcine epidemic diarrhea (PED) virus, porcine rotavirus, porcine circovirus 2 (PCV2), and porcine reproductive and respiratory syndrome (PRRS) virus. In Japan, the name of "African swine fever" defined in the Livestock Infectious Disease Prevention Act was revised to "African swine fever" on February 5, 2020.

(Method for producing porcine infectious virus)
The method for producing (growing, amplifying) a porcine infectious virus of the present invention is a method comprising the steps of contacting an immortalized fetal pig small intestine macrophage with a porcine infectious virus and growing the virus in the immortalized fetal pig small intestine macrophage.

The porcine infectious virus to be brought into contact with the immortalized fetal pig small intestine 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 tissues, cells, cultures thereof, washings, or extracts derived from pigs, or samples taken from the pig breeding environment (breeding facilities, etc.), washings, or cultures thereof.

"Contact" is usually performed by adding the porcine infectious virus to a culture medium for culturing immortalized fetal pig small intestine macrophages. Such a "culture medium" is not particularly limited, but may be the medium for primary culture of fetal pig small intestine described above.

The "proliferation" of the porcine infectious virus can be achieved by contacting and culturing the infected immortalized fetal pig small intestine macrophages 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 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 after contact with the porcine infectious virus is not particularly limited, but is usually 1 to 10 days, preferably 2 to 7 days, more preferably 3 to 5 days.

Those skilled in the art can determine whether the porcine infectious virus has proliferated by known methods. Examples of such methods include a cytopathic effect (CPE) test using CPE as an indicator, as shown in the Examples below, and a method for detecting the degree of intracellular ATP depletion associated with CPE (for example, the Viral ToxGlo assay provided by Promega). A method for detecting genes derived from porcine infectious viruses or their expression can also be used. Here, gene expression may be at the transcription level (RNA level) or 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 blotting or Southern blotting, in situ hybridization, dot blot, RNase protection assay, and mass spectrometry. In addition, gene or RNA levels can be quantitatively detected by counting the number of reads in the so-called next-generation sequencing method. Methods for detecting proteins include, for example, methods for detection using antibodies (immunological methods) such as ELISA, antibody arrays, immunoblotting, imaging cytometry, flow cytometry, radioimmunoassay, immunoprecipitation, and immunohistochemical staining, as well as mass spectrometry.

(Vaccine manufacturing method)
The method for producing a vaccine containing a porcine infectious virus of the present invention includes the steps of contacting an immortalized fetal pig small intestine macrophage with a porcine infectious virus and proliferating the virus in the immortalized fetal pig small intestine macrophage;
isolating the propagated porcine infectious virus; and mixing the isolated porcine infectious virus with a pharmacologically acceptable carrier or medium.

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

The swine-infectious viruses isolated in this manner may be used as a vaccine as is (so-called live vaccine), or in a live attenuated form (so-called live attenuated virus), or in an inactivated form. Furthermore, parts of these isolated swine-infectious viruses (proteins, polypeptides, sugars, glycoproteins, lipids, nucleic acids, etc.) may be used as a vaccine, so long as they have immunogenicity.

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 a mutagen, acclimation to cultured cells by continuous (long-term) passage in vitro, or growth of a porcine-infecting virus under conditions that deviate from the natural growth environment (e.g., high temperature conditions). Live attenuated viruses can also be obtained by deleting or recombining specific genes of the virus using genome editing, gene modification techniques, etc.

Those skilled in the art can inactivate viruses using known methods. Such inactivation methods include formaldehyde treatment, UV irradiation, X-ray irradiation, electron beam irradiation, gamma ray irradiation, alkylation treatment, ethylene-imine treatment, thimerosal treatment, β-propiolactone treatment, and glutaraldehyde treatment.

Examples of the "pharmacologically acceptable carrier" to be mixed with the isolated porcine 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 relevant field depending on the vaccine formulation and method of use. There are no particular limitations on the form of the vaccine, and it may be, for example, in the form of a suspension or in the form of a freeze-dried product.

In order 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 (BCG, muramyl dipeptide, Bordetella pertussis, pertussis toxin, cholera toxin, etc.), surfactants (saponin, deoxycholic acid, etc.), emulsions of oily substances (mineral oil, vegetable oil, animal oil, etc.), alum, etc.

(Methods for detecting swine-infecting viruses)
The present invention provides an immortalized fetal pig small intestinal macrophage having DNA in which a reporter gene is functionally linked downstream of a promoter region of a gene derived from a porcine-infectious virus.

The present invention also provides a method for detecting a porcine infectious virus, the method comprising the steps of culturing immortalized fetal pig small intestine macrophages having the DNA in the presence of a test sample, detecting expression of the reporter gene in the immortalized fetal pig small intestine macrophages, and determining that the test sample contains the porcine infectious virus when expression of the reporter gene is detected.

The "immortalized fetal pig small intestine macrophages" into which the DNA is introduced are as described above. The DNA may be in the form of a vector as described above in the "immortalization gene". Furthermore, the DNA can be introduced into immortalized fetal pig small intestine macrophages by a person skilled in the art using the methods listed in the description of the "immortalization gene" above.

The "promoter region of a gene derived from a porcine-infectious virus" in the DNA is not particularly limited as long as it is derived from a porcine-infectious virus and is a region capable of activating the expression of a gene downstream in response to infection and proliferation of the virus, and may be any of the following genes: immediate-early, early, late, or very-late.

A person skilled in the art can select a gene derived from a porcine infectious virus to be used 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 pp. 155-157 of Virus Taxnomy, 9th Edition, ICTV (International Committee on Taxonomy of Viruses), 2012, and the promoter region of the p72, U104L, CD2v, DNA polymerase, or p30 gene is preferably used (see Portugal RS. et al., Virology, August 2017, Vol. 508, pp. 70-80).

There are no particular limitations on the "reporter gene" that is functionally (operably) linked downstream of the promoter region, and any known reporter gene may be used as appropriate. Examples include fluorescent protein genes, luminescent enzyme 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 luminescent protein/enzyme 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.

In the detection method of the present invention, the fluorescence, luminescence, color development, etc. that arise in response to the expression of these reporter genes can be used as indicators to detect whether or not immortalized fetal pig small intestinal macrophages are infected with a porcine infectious virus, and ultimately the presence of a porcine infectious virus in a test sample.

There are no particular limitations on the test sample, so long as it is a sample in which porcine infectious viruses may be present, and examples include tissues, cells, cultures thereof, washings, or extracts derived from pigs, or washings or cultures thereof from the pig breeding environment (breeding facilities, etc.).

The "culture medium" used for culture in the detection method of the present invention is not particularly limited, but may be the medium for primary culture of fetal pig small intestine described above. 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 the test sample until the expression of the reporter gene is detected is not particularly limited, but is usually 1 to 10 days, preferably 2 to 7 days, more preferably 2 to 5 days.

(Method for detecting neutralizing antibodies)
The method for detecting neutralizing antibodies against a virus that infects pigs according to the present invention comprises the steps of:
contacting immortalized fetal pig small intestine macrophages with a virus that infects pigs in the presence of a biological sample isolated from a test pig, and allowing the virus to grow in the immortalized fetal pig small intestine macrophages;
detecting the number of the proliferated viruses; and, if the number of viruses detected in the step is smaller than the number of viruses proliferated in immortalized fetal pig small intestinal macrophages in the absence of the biological sample, determining that the biological sample contains a neutralizing antibody against the virus.

In the present invention, a "neutralizing antibody" refers to an antibody that inhibits infection or proliferation of a porcine infectious virus. Such antibodies include all classes and subclasses of immunoglobulin. Furthermore, there are no particular limitations on the "subject pig" as long as it is a pig, regardless of whether it has been infected with a porcine infectious virus. "Biological samples" isolated from subject pigs include samples derived from pigs (for example, blood (serum, plasma, etc.), mucus (saliva, nasal secretions, milk, digestive tract secretions, etc.), and antibodies purified therefrom).

The term "contact" is as described above. Regarding the growth conditions, the culture temperature is not particularly limited, but is usually 30 to 40°C, preferably 37°C. The concentration of carbon dioxide 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 or absence of a biological sample is not particularly limited, but is usually 1 to 10 days, preferably 2 to 7 days, more preferably 3 to 5 days. In addition, the grown virus can be detected by the CPE test or the like, or by detecting a gene derived from a porcine infectious virus or its expression, as described above. Thus, in the detection method of the present invention, the presence or absence of a neutralizing antibody may be determined using not only the number of viruses themselves, but also the gene (such as the amount of genomic DNA) that reflects the number of viruses or its expression level as an indicator.

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.

<Isolation of porcine small intestinal macrophages from pig fetuses>
Approximately 5 cm of small intestine (ileum) was collected from a pig fetus (about 108 days old), and after removing the adherent membrane, it was cut vertically and opened in half. The small intestine tissue was thoroughly cut into small pieces with scissors, transferred to a conical tube, washed with Dulbecco's phosphate buffered saline (DPBS), and then immersed in DPBS containing collagenase/dispase/DNase I and enzyme treated at 37°C for 1 hour. The tissue was dispersed by pipetting, washed with DPBS, and then suspended in growth medium (DMEM High glucose: containing 10% fetal bovine serum, 10 μg/mL insulin, 25 μM monothioglycerol, 100 U/mL penicillin, 100 μg/mL streptomycin, and 5 μg/mL pimaricin (product name: Fungin, manufactured by InvivoGen)) and seeded in a T75 flask.

Initially, we attempted primary culture by treating small intestines taken from pigs (approximately 1 month old) in the same manner as above. However, even when using a medium containing antibiotics (penicillin, streptomycin) and an antifungal drug (pimaricin) as described above, severe microbial contamination occurred 2-3 days after the start of culture, making it impossible to perform primary culture.

On the other hand, such contamination could be suppressed for small intestines taken from pig fetuses, and primary culture could be performed. After about three weeks, the primary cultured cells (Figure 1A) that had adhered and spread were detached and collected using a cell separation/dispersion solution (product name: Accumax (registered trademark), manufactured by Sigma-Aldrich) and reseeded onto a 100 mm tissue culture dish.

A cell sheet was formed in about one week, and spherical cells appeared on the cell sheet, weakly adhering and proliferating (Figure 1B). Immunostaining confirmed that the cell sheet was composed of α-smooth muscle actin (αSMA)-positive myofibroblast-like cells, and that a mixed culture system had been established on top of the cell sheet, with macrophage marker (CD172a, CD204)-positive cells proliferating. Furthermore, the epithelial cell markers (cytokeratin 18 and 19 (CK18, CK19)) were negative (Figure 2A).

In addition, cells were collected from the culture supernatant, and cells attached to the Sumilon suspension culture dish were separated and immunostained. As a result, it was found that cells positive for macrophage markers (Iba1, CD172a, CD204) were isolated. Epithelial cell markers (CK18, CK19) were negative (Figure 2B). Furthermore, the formation of multinucleated giant cells, which is known to be a characteristic of primary cultured macrophages, was confirmed in the collected cells (Figure 2B). Thus, porcine intestinal macrophages (PIM) could be obtained by this method. The above markers were detected by immunostaining as described below.

(Immunostaining)
The cells were seeded on an 8-well chamber slide and cultured for a certain period of time, after which they were immersed in 4% paraformaldehyde phosphate buffer (Nacalai) and fixed. After washing with PBST, the cells were treated with 1% Triton X-100/PBS solution and blocked with a hydrogen peroxide solution (manufactured by DAKO) and a blocking agent (product name: Blocking One Histo, manufactured by Nacalai). Then, primary antibodies [anti-αSMA (manufactured by Progen), anti-Vimentin (manufactured by Progen), anti-CK18 (manufactured by Millipore), anti-CK19 (manufactured by Progen), anti-CD172a (manufactured by VMRD), anti-CD204 (manufactured by TransGenic, Inc.), Iba1 (manufactured by Wako), anti-MHC-II (manufactured by Kingfisher), and anti-αSMA (manufactured by Progen), anti-Vimentin (manufactured by Progen), anti-CK18 (manufactured by Millipore), anti-CK19 (manufactured by Progen), anti-CD172a (manufactured by VMRD), anti-CD204 (manufactured by TransGenic, Inc.), Iba1 (manufactured by Wako), anti-MHC-II (manufactured by Kingfisher), anti-αSMA (manufactured by Progen), anti-Vimentin (manufactured by Progen), anti-CK18 (manufactured by Millipore), anti-CK19 (manufactured by Progen), anti-CD172a (manufactured by VMRD), anti-CD204 (manufactured by TransGenic, Inc.), anti-MHC-II (manufactured by Kingfisher), anti-αSMA (manufactured by Progen), anti-VIMENTIN (manufactured by Progen), anti-CK18 (manufactured by Millipore), anti-CK19 (manufactured by Progen), anti-CD172a (man The sections were incubated with anti-CD163 (Biotech), anti-CD169 (Bio-Rad), and anti-CD203a (Bio-Rad) for 1 hour. After washing with PBST, the sections were stained with diaminobenzidine using an immunohistochemical staining detection system (product name: EnVision (registered trademark) system, DAKO). Furthermore, the nuclei were stained with Mayer's hematoxylin solution (Wako).

<Creation of immortalized fetal pig small intestinal macrophage cells>
Isolated PIM does not grow. Therefore, immortalization of PIM was attempted to impart proliferation ability. Specifically, first, PIM was seeded in a Sumilon suspension culture dish, and the next day, it was exposed for 2 hours to a solution containing recombinant lentivirus particles into which two types of immortalization genes (SV40 Large T antigen (SV40LT) gene and porcine telomerase reverse transcriptase (pTERT) gene) were individually introduced. Note that, as described in Non-Patent Document 1, the recombinant lentivirus particles were prepared by introducing pLVSIN-EF1α neo vectors encoding the SV40LT gene and the pTERT gene, respectively, together with a packaging vector into a lentivirus packaging cell line (product name: Lenti-X 293T cells, Takara Bio, Inc.). Then, about one week later, PIM was exposed to the same lentivirus particles again, and the culture was continued.

As a result, after about one month, we succeeded in obtaining immortalized PIM (IPIM) that could be subcultured, and cryopreserved them (Figure 3A). In addition, the proliferation ability of these IPIM cells was evaluated in the test shown below, and it was revealed that they could divide at least 60 times, as shown in Figure 3B.

(Cell proliferation test)
IPIM cells (1 x ¹⁰⁶ cells) were seeded on a 90 mm suspension culture dish (SUMILON), and subcultured every 3 to 5 days by detaching the cells with TrypLE Express reagent (ThermoFisher). When the cells were harvested, the cell number was counted using a TC10 fully automated cell counter (Bio-Rad).

Furthermore, similar to PIM, various macrophage markers in IPIM cells were positive when analyzed by the above-mentioned immunostaining (Figure 4). Specifically, Iba1, CD172a, CD204, CD16, and CD203a, which are macrophage markers common to M1 and M2 types, were positive in almost 100% of IPIM cells. In addition, a certain percentage of IPIM cells were positive for markers of specific macrophage subpopulations (M2 type macrophage marker: CD163, M1 type macrophage marker: CD169) and antigen-presenting cell markers (MHC-II), depending on the state of each individual cell.

In addition, when their macrophage function was evaluated using the method described below, IPIM cells were found to activate intracellular signaling molecules (phosphorylation of p38 MAP kinase) in response to bacterial-derived components (muramyl dipeptide: MDP, lipopolysaccharide: LPS) (Figure 5A), and were also able to induce the production of inflammatory cytokines (IL-1β) (Figure 5B).

(Macrophage function evaluation test)
IPIM cells were seeded at 3 x ¹⁰⁵ cells per well in a 24-well suspension culture plate (SUMILON). The next day, the medium was replaced with serum-free DMEM (400 μL) containing bacterial components (MDP or LPS) at the concentrations shown in Figures 5A and 5B to stimulate the cells. After another 24 hours, the culture supernatant was collected and a cell lysate was prepared using a lysis buffer (50 mM Tris-HCl buffer pH 7.5 containing 150 mM sodium chloride, 0.5% Triton X-100, 0.5% sodium deoxycholate, protease inhibitors, and phosphatase inhibitors). Protein components were concentrated from the culture supernatant (300 μL) by trichloroacetic acid/acetone precipitation. Proteins contained in the culture supernatant and cell lysate were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrically transferred to a polyvinylidene fluoride (PVDF) membrane (Merck). For the transferred PVDF membrane, the target protein was detected by Western blotting using primary antibodies [anti-phosphorylated p38 MAP kinase (Cell Signaling Technology), anti-p38 MAP kinase (Cell Signaling Technology), anti-IL-1β (R&D Systems)].

Thus, it was revealed that immortalized fetal pig intestinal macrophages (IPIM cells) introduced with the SV40 large T antigen gene and telomerase reverse transcriptase gene exhibited high proliferation while maintaining not only the expression of marker proteins but also their functions.

<African swine fever virus susceptibility test>
Next, the virus susceptibility of IPIM cells to African swine fever virus (ASFV) was evaluated by the method described below.

(ASFV strain used)
Three ASFV field isolates, the European/Chinese epidemic strain Armenia 07 (genotype: II type), the strain Kenya 05/Tk-1 (genotype: I type) isolated from soft ticks, and the ASFV standard strain Espana 75 (genotype: I type), were introduced from the Complutense University of Madrid, Spain, which is the ASF reference laboratory of the International Organization for Animal Health (OIE). For information on the year and place of outbreak of each virus strain, please refer to Fernandez-Pinero J, et al. (2013) (Transboundary and Emerging Diseases. 60 (2013) 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 using PAM cells, and the Lisbon 60/V strain was grown and cultured using Vero cells, and these were used as inoculated virus samples. The inoculated virus samples were dispensed and stored at -80°C.

(About the cells used)
Porcine alveolar macrophage (PAM) cells were collected from pig lungs as previously reported (Carrascosa AL. et al., Curr Protoc Cell Biol, December 2011, Chapter 26, UNIT 26.14, pp. 1-26). African green monkey kidney-derived immortalized cell lines Vero (CCL-81), COS-1 (CRL-1650), and porcine alveolar macrophage-derived immortalized cell line 3D4/21 (CRL-2843) were introduced from the American Type Culture Collection (ATCC). Wild boar lung-derived cell line WSL cells were obtained from the Friedrich-Loeffler Institute (FLI, Germany).

(Viral susceptibility testing)
The susceptibility of cells to viruses was determined by a cytopathic effect (CPE) test using CPE as an index or a hemadsorption (HAD) test using a hemadsorption (HAD) reaction specifically observed in ASFV-infected cells as an index. Specifically, PAM cells were seeded at 1×10 ⁵ cells per well in a 96-well plate, Vero cells and COS-1 cells were seeded at 1.5×10 ⁴ cells per well, WSL cells and 3D4/21 cells were seeded at 2×10 ⁴ cells per well, or IPIM cells were seeded at 3×10 ⁴ cells per well. After pre-cultivation in each medium for 1 to 2 days, 25 μL of virus solution serially diluted 10-fold from 10-fold (10 ⁻¹ ) to 10 billion-fold (10 ⁻¹⁰ ) in the same medium was inoculated into 8 wells each to prepare a CPE detection plate. Furthermore, 20 μL of a 0.75% suspension of pig red blood cells in PBS was added to the plate to prepare a plate for detecting HAD. Then, both the CPE detection plate and the HAD detection plate were cultured at 37° C. in a 5% CO ₂ environment for 7 days and observed.

As a result, as shown in Table 1, CPE was detected in PAM cells and IPIM cells for all virus strains inoculated. On the other hand, in Vero cells, COS-1 cells, WSL cells, and 3D4/21 cells, no CPE was observed in the three field strains, and only in the cell-adapted strain Lisbon 60/V (Table 1). Thus, it has become clear that IPIM cells, like PAM cells, can be used not only in CPE tests (Figure 6A), but also in HAD tests (Figure 6B).

Next, the dilution limit at which the virus could be isolated (virus detection limit) was evaluated for both PAM and IPIM cells. As a result, as shown in Table 2, in the CPE test, the detection limit concentration for IPIM cells was 100-10,000 times lower than that of PAM cells. In the HAD test, the detection limit concentration for IPIM cells was also 10-100 times lower than that of PAM cells (Table 3). In other words, it was revealed that IPIM cells are more susceptible to ASFV than PAM cells.

<Susceptibility testing for porcine reproductive and respiratory syndrome virus or porcine circovirus>
Next, the virus susceptibility of IPIM cells to porcine reproductive and respiratory syndrome virus (PRRSV) or porcine circovirus type 2 (PCV2) was evaluated by the method described below.

(About the virus strain used)
The EDRD-1 strain, a domestic isolate of porcine reproductive and respiratory syndrome virus (PRRSV), was used, and the Yamagata strain, a domestic isolate of porcine circovirus type 2 (PCV2), was used.

(PRRSV Susceptibility Testing)
IPIM cells were seeded in a 96-well suspension culture plate (manufactured by SUMILON) at 1 x ¹⁰⁵ cells/0.1 mL medium per well. After 3 days, the cell culture medium was aspirated and then 0.1 mL of virus liquid obtained by serially diluting the virus stock solution from 10-fold to 10,000-fold was inoculated per well to prepare a plate for detecting virus antigens by indirect fluorescent antibody method. After aspirating and removing the cell culture medium 3 days after virus inoculation, the cells were fixed with ice-cold 80% acetone, and PRRS virus antigens were detected by indirect fluorescent antibody method using anti-PRRS virus monoclonal antibody (clone: SR30-A). The results obtained are shown in the upper part of FIG. 7.

To observe virus proliferation, IPIM cells were seeded in a 6-well suspension culture plate (manufactured by SUMILON) at 3 x ¹⁰⁶ cells/3 mL of medium per well. After 3 days, the cell culture medium was aspirated and then 0.5 mL of virus solution, which was a 100-fold dilution of the stock solution, was inoculated per well and the virus was allowed to adsorb to the cells for 1 hour. After 1 hour, the medium containing the virus was removed, the cells were washed with 2 mL of medium per well, and then 5 mL of new medium was added and cultured at 37°C in the presence of 5% _CO2 . CPE was observed on the 1st, 2nd, 3rd, 5th, and 7th days after inoculation, and 0.5 mL of the culture supernatant was collected. The collected culture supernatant was serially diluted 10-fold from 10-fold to 10,000,000-fold, and then inoculated into IPIM cells, which had been seeded at 1 x ¹⁰⁵ cells/0.1 mL medium per well and cultured for 3 days, at 0.1 mL per well (4 wells per dilution), and cultured for 7 days at 37°C in a 5% _CO2 environment to observe CPE. The results of the observation on the 7th day after inoculation are shown in the lower part of Figure 7. Furthermore, the 50% tissue culture infectious dose ( _TCID50 ) was calculated based on the Behrens-Karber method. The results obtained are shown in Figure 8.

(PCV2 susceptibility test)
IPIM cells were suspended in 2 x ¹⁰⁵ cells/180 μL medium per well, mixed with 20 μL of virus stock solution, and 200 μL of the cell-virus mixture per well was seeded on a 48-well suspension culture plate (manufactured by SUMILON). Two hours after seeding, the medium containing the virus was removed, the cells were washed with 0.5 mL of medium per well, and then 200 μL of new medium was added and cultured at 37 ° C in the presence of 5% carbon dioxide. The culture supernatant and cells were collected together immediately after the addition of the medium, and used as a sample two hours after inoculation. Thereafter, the culture supernatant and cells were collected together on the second, fourth, and sixth days. Total DNA was extracted from the collected sample as a final 50 μL solution using DNeasy Blood & Tissue Kit (manufactured by QIAGEN), and quantitative PCR targeting the PCV2 capsid gene was performed using 2 μL of DNA from that. The results are shown in FIG. 9 as relative values, with the viral gene amount 2 hours after virus inoculation taken as 1.

Regarding the PRRSV sensitivity of IPIM cells, as shown in Figure 7, PRRSV was sensitive to IPIM cells, with viral antigens detectable in the cytoplasm 3 days after inoculation and clear CPE observed 7 days after inoculation. Furthermore, as shown in Figure 8, the viral infectivity titer in the culture supernatant of PRRSV-inoculated IPIM cells peaked 2 to 3 days after inoculation and increased by nearly 100,000-fold. The infectivity titer tended to be maintained without a significant decrease up to 7 days after inoculation.

Regarding the PCV2 sensitivity of IPIM cells, as shown in Figure 9, there was a tendency for the amount of PCV2 genes to double as the number of IPIM cells doubled after virus inoculation.

These findings confirmed that IPIM cells are also sensitive to field strains of PRRSV and PCV2.

As described above, according to the present invention, it is possible to provide immortalized porcine small intestinal macrophage cells. Furthermore, since the immortalized cells have high susceptibility to infection with porcine infectious viruses, it is possible to grow various strains of porcine infectious viruses, making it possible to manufacture and develop vaccines against those viruses. Furthermore, due to the high susceptibility to infection, it is also possible to detect (test, diagnose) porcine infectious viruses.

Claims (6)

A method for producing immortalized porcine small intestinal macrophages, comprising a step of expressing SV40 large T antigen and porcine-derived telomerase reverse transcriptase in small intestinal macrophages of a pig fetus. The method according to claim 1, wherein the step is a step of introducing a lentivirus encoding the SV40 large T antigen and porcine telomerase reverse transcriptase into small intestinal macrophages of the pig fetus and expressing the SV40 large T antigen and porcine telomerase reverse transcriptase . The method according to claim 1 or 2 , wherein the pig fetus is a pig fetus of gestational age 30 to 114 days. A step of producing an immortalized porcine small intestinal macrophage by the method according to any one of claims 1 to 3;
A method for producing a porcine infectious virus, comprising the steps of contacting the immortalized porcine small intestinal macrophages with a porcine infectious virus and allowing the virus to grow in the immortalized porcine small intestinal macrophages . A step of producing an immortalized porcine small intestinal macrophage by the method according to any one of claims 1 to 3;
contacting the immortalized porcine small intestinal macrophages with a porcine infectious virus and allowing the virus to grow in the immortalized porcine small intestinal macrophages ;
A method for producing a vaccine containing a porcine infectious virus, comprising the steps of: isolating the propagated porcine infectious virus; and mixing the isolated porcine infectious virus with a pharmacologically acceptable carrier or medium. 6. The method according to claim 4 , wherein the porcine-infecting virus is at least one virus selected from the group consisting of African swine fever virus, porcine epidemic diarrhea virus, porcine rotavirus, porcine circovirus, and porcine reproductive and respiratory syndrome virus.