Genetically modified mammalian cells susceptible to human sapovirus infection, genetically modified mammals possessing such cells, and methods for producing human sapovirus using these, methods for conferring infection susceptibility, and screening methods.

Abstract

The present invention addresses the problem of providing: a genetically modified cultured cell or a genetically modified animal each allowing a human sapovirus to proliferate regardless of host characteristics as barriers to proliferation in mammalian cells; and an application of the cell or animal to a screening method. The present inventors have solved the problem by finding that a genetically modified cultured mammalian cell having a human CD36-encoding gene incorporated therein and a mammal having such a cell as a self-cell show acquired susceptibility to human sapovirus infection, and providing, for example, a screening method for a drug relevant to a human sapovirus on the basis of the finding.

Description

[Technical Field]

[0001] This invention relates to a means for the proliferation of viruses, and more specifically, to a means for the proliferation of human sapoviruses. [Background technology]

[0002] Sapoviruses (SaV) are small, non-enveloped, spherical viruses with a positive-sense single-stranded RNA genome. Belonging to the genus Sapovirus in the family Caliciviridae, Sapoviruses have been detected in many animals, including humans, pigs, dogs, sea lions, and chimpanzees, and are classified into at least 19 gene groups (GI-GXIX) and 52 genotypes. Human sapovirus (HuSaV), discovered in 1977, is one of the major enteric viruses detected in nonbacterial acute gastroenteritis worldwide, similar to human norovirus (HuNoV), which belongs to the genus Norovirus in the same family. Outbreaks have also been reported, including a mass food poisoning incident during a school trip in Yokohama in 2007 (65 cases), a food poisoning incident at a wedding venue in Matsuyama in 2007 (109 cases), and a large-scale food poisoning incident caused by school lunch boxes in Aichi Prefecture in 2010 (680 cases). Human sapoviruses are classified as GI, GII, GIV, and GV, with GI being the most frequently detected, followed by GII. From 2019 to 2023, there were 6,269 detections of human norovirus, 1,071 of human sapovirus, 885 of rotavirus, and 353 of astrovirus, with human sapovirus being the second most frequently detected virus after human norovirus. The suspected routes of infection and symptoms are difficult to distinguish between human norovirus and human sapovirus, making human sapovirus difficult to diagnose based on clinical symptoms alone. A major difference between human norovirus and human sapovirus is the structure of their genomic RNA (Figure 1). Specifically, in human norovirus, non-structural proteins and structural proteins (capsid protein, VP1) are encoded in separate ORFs, while in human sapovirus, non-structural proteins and VP1 are encoded in the same ORF. Therefore, it is thought that VP1 expression in human sapoviruses can occur either by being expressed after being translated from ORF1 into a polyprotein containing VP1 and non-structural proteins, and then cleaved by the human sapovirus's own protease, or by being directly translated and expressed from the subgenome.

[0003] Although more than 40 years have passed since the discovery of human norovirus and human sapovirus in the 1970s, research on both has stagnated. One reason for this is the lack of culture systems or animal models that can enable infection and proliferation. In the case of human norovirus, the only report so far is the in vitro culture system using human intestinal stem cell-derived intestinal organoids (hIEOs) reported in 2016.

[0004] In 2020, a culture system using a limited cell line, specifically the human duodenal cancer cell line HuTu80, which had been passaged for a long period in a special environment, was finally reported for human sapovirus (Non-Patent Literature 1). Furthermore, in 2023, infection and proliferation in hIEOs derived from human intestinal stem cells (Non-Patent Literature 2) and hIEOs derived from iPS cells (Non-Patent Literature 3) were reported. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Takagi H. et al., 2020. PNAS 117:32708-32085 [Non-Patent Document 2] Euller-Nicolas G. et al., 2023. J Virol 97: e00383-23 [Non-Patent Document 3] Matsumoto N. et al., 2023. Viruses 15: 1929 [Disclosure of the Invention] [Problems that the invention aims to solve]

[0006] However, the human sapovirus replication systems currently available do not use typical mammals (including humans), thus limiting their range of applications. To overcome this challenge, it is essential to identify the infection receptor used by human sapoviruses when infecting humans and to provide genetically modified mammalian cells into which this infection receptor gene has been introduced, as well as technologies based on these cells. [Means for solving the problem]

[0007] The inventors recently succeeded in establishing a cell line derived from intestinal cancer cells that can be passage-amplified with human sapovirus (PCT / JP2025 / 012990). They then detected gene-level variations in this cell line and performed infection tests with human sapovirus on a cell line in which the wild-type cell line did not show susceptibility to human sapovirus infection, targeting the human CD36 gene, one of the candidate receptors identified by this detection. As a result, they confirmed that the infection receptor for human sapovirus is indeed human CD36 (platelet glycoprotein 4).

[0008] In other words, the contents of this invention are as follows:

[0009] Firstly, the present invention provides genetically modified mammalian cultured cells (also referred to as the recombinant cells of the present invention) that are transformed with a gene encoding human CD36 (hereinafter, the gene encoding CD36 is referred to as the CD36 gene) and are susceptible to infection by human sapovirus.

[0010] The recombinant cells of the present invention are preferably, but not limited to, established cell lines, organoids derived from biopsy samples, immortalization-inducing cells, or iPS cells.

[0011] Furthermore, the recombinant cells of the present invention are preferably mammalian cultured cells derived from humans, mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, pigs, or monkeys (for example, rhesus macaques or African green macaques), but are not limited to these.

[0012] In addition, the above mammalian cultured cells include HEK293T cells (human-derived), Caco2 cells (human-derived), HCT116 cells (human-derived), Intestine407 cells (human-derived), macrophage-derived cultured cells 15310-LN cells (human-derived), NALM-6 cells (human-derived), HuTu80 cells (human-derived), HT29 cells (human-derived), MRC5 cells (human-derived), RAW264.7 cells (mouse-derived), and NIH3T3 cells (mouse-derived). Examples of such cells include, but are not limited to, M1 cells (derived from mouse), BHK cells (derived from hamster), CHO cells (derived from hamster), CRFK cells (derived from cat), MDCK cells (derived from dog), PK-15 cells (derived from pig), Vero cells (derived from monkey), MA104 cells (derived from monkey: there are MA104 derived from rhesus monkeys and MA104 derived from African green monkeys), GL37 cells (derived from monkey), or COS7 cells (derived from monkey).

[0013] Secondly, the present invention provides a genetically modified mammal (hereinafter also referred to as the recombinant animal of the present invention) that possesses the recombinant cells of the present invention as its own cells. The recombinant animal of the present invention is a so-called transgenic animal or knock-in animal. Humans are excluded from the genetically modified animals of the present invention.

[0014] The method for introducing the human CD36 gene into mammalian cultured cells is not particularly limited and is carried out according to standard procedures. Typically, methods include using vectors for gene transfer into mammals, such as retroviral vectors like mouse leukemia virus vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, herpes simplex virus type I vectors, modified viral vectors like HVJ-liposomes, and plasmid vectors containing CMV promoters, GAC promoters, EF-1α promoters, or SV40 promoters that function in mammalian cells, all or part of the human CD36 gene. In addition to recombinant viruses, methods such as calcium phosphate, lipofection, commercially available transfection reagents, microinjection, stamping, and particle gun can also be used for gene transfer. Furthermore, knock-in methods using gene editing technologies such as the CRISPR / Cas9 system can also be used.

[0015] Furthermore, the human CD36 gene can be obtained by conventional methods. That is, by using the cDNA of one or more animal species from which the human CD36 gene is derived, either all or part of it, as a template, and amplifying the gene region using gene amplification methods such as PCR, all or part of the desired human CD36 gene can be easily obtained. This can be done in-house, outsourced, or commercially available if available. As will be described later, the human CD36 gene may be the entire gene encoding CD36, or it may be a part of the base sequence from which non-essential regions have been removed. Specifically, it is preferable that the gene encodes the extracellular domain, transmembrane domain, and intracellular domain as essential regions. In addition, in mammalian cells other than human-derived cells, such as mouse-derived cells, it is possible to preserve the native CD36 gene of the mammalian cell (e.g., mouse CD36 gene) as is and transform it with the human CD36 gene, or it is possible to inactivate the native CD36 gene by performing expression suppression treatment such as knockout. For human-derived cells that naturally contain the human CD36 gene, such gene suppression treatment is not necessary.

[0016] The recombinant animals of the present invention can be produced by conventional methods. For example, the human CD36 gene described above can be introduced into a fertilized egg of a target mammal by a conventional method such as the above microinjection method, and the gene recombinant animal of the present invention can be produced by culturing this. In addition, gene recombinant animals of the present invention can be produced by performing knock-in of the human CD36 gene using the CRISPR / Cas9 system or the like. Mammals are not limited except for humans, and examples include mice, rats, hamsters, pigs, monkeys, dogs, cats, horses, etc. Similar to the case of the above mammalian cells, it is also possible to produce the gene recombinant animals of the present invention while preserving the native CD36 gene of mammals other than humans as it is. However, it is possible to perform an expression suppression treatment such as knocking out the native CD36 gene to inactivate it and introduce a chimeric gene of the human CD36 gene and the host CD36 gene. Also, by selecting a promoter for expressing the human CD36 gene such as the villin promoter that is highly active in intestinal epithelial cells, it is also possible to express a large amount of the CD36 gene in the virus infection target organs (for example, intestinal epithelium, etc.).

[0017] Thirdly, there is provided a method for producing human sapovirus (hereinafter, also referred to as the method for producing sapovirus of the present invention) in which the recombinant cells of the present invention or the recombinant animals of the present invention are infected with human sapovirus and the human sapovirus is propagated in the mammalian cells or the mammal (excluding humans).

[0018] Fourthly, there is provided a method for conferring susceptibility to infection with human sapovirus (hereinafter, also referred to as the method for conferring susceptibility to infection of the present invention) in mammalian cells or mammals by introducing the human CD36 gene into mammalian cells or mammals (excluding humans).

[0019] Fifth, a screening target substance for the action against human sapovirus is brought into contact with the recombinant cell of the present invention or the recombinant animal of the present invention infected with human sapovirus, and the growth of human sapovirus in the mammalian cultured cell or the mammalian (excluding human) is detected, thereby obtaining information on the action of the screening target substance. A screening method (hereinafter, also referred to as the first screening method of the present invention) is provided.

[0020] Examples of the screening target action in the first screening method of the present invention include, but are not limited to, the inactivating action of human sapovirus or the inhibitory action of growth.

[0021] Sixth, a screening target substance and human sapovirus are brought into contact with the recombinant cell of the present invention or the recombinant animal of the present invention, and the invasion or growth of human sapovirus in the mammalian cultured cell or the mammalian (excluding human) is detected, thereby obtaining information on the action of the screening target substance. A screening method (hereinafter, also referred to as the second screening method of the present invention) is provided.

[0022] Examples of the screening target action in the second screening method of the present invention include, but are not limited to, the inhibitory action of cell invasion of human sapovirus or the inhibitory action of growth in cells.

[0023] Seventh, the recombinant cell of the present invention or the recombinant animal of the present invention is immunized with a screening target vaccine, and the protective action against human sapovirus in the mammalian cultured cell or the mammalian (excluding human) is detected, thereby obtaining information on the action of the screening target vaccine. A screening method (hereinafter, also referred to as the third screening method of the present invention) is provided.

[0024] CD36, the human sapovirus infection receptor that forms the basis of this invention, is a known glycoprotein that also exists in humans and is typically known as platelet glycoprotein 4. In this application, the name human CD36 or simply CD36 will be used consistently. Unless otherwise specified, CD36 refers to "human CD36". A typical sequence of the human CD36 gene is shown as the cDNA sequence in Sequence ID No. 1 (accession number KU177961). General knowledge about human CD36 is that it is a multifunctional glycoprotein that acts as a receptor for a wide range of ligands. These ligands can be proteinaceous, such as thrompospondin, fibronectin, collagen, and amyloid-beta, as well as lipidaceous, such as oxidized low-density lipoproteins, anionic phospholipids, long-chain fatty acids, and bacterial diacylated lipopeptides. Since these ligands are generally polyvalent, multiple receptors may be involved simultaneously, and as a result, when a CD36 cluster is formed, a predetermined signal transduction and internalization of the receptor-ligand complex are initiated. Cellular responses to these ligands are thought to be involved in angiogenesis, inflammatory responses, lipid metabolism, taste, and the processing of dietary fats in the gut.

[0025] In the present invention, the CD36 gene capable of conferring susceptibility to human sapovirus infection to transformed mammalian cells by transformation may be the entire gene encoding CD36, or it may be a part of such a base sequence excluding non-essential regions. Specifically, a gene encoding an extracellular domain, a transmembrane domain, and an intracellular domain as essential regions can be defined as the CD36 gene. [Effects of the Invention]

[0026] The present invention provides a means for artificially propagating human sapoviruses, enabling the manufacture of various pharmaceuticals, including live vaccines and other vaccines. Furthermore, the invention provides a screening method for drugs and vaccines against human sapoviruses using the human sapovirus propagation system. [Brief explanation of the drawing]

[0027] [Figure 1] This diagram shows the structures of the genomic RNA of human sapovirus and human norovirus. [Figure 2] This diagram shows the results of a comprehensive analysis of mRNA expression for each gene in human sapovirus infection-susceptible clones. [Figure 3] This diagram shows the results of a human sapovirus infection susceptibility test in cells transduced with the CD36 gene (clones created by the inventors). [Figure 4] This diagram shows the results of a human sapovirus infection susceptibility test in cells transduced with the CD36 gene (HEK293T cells). [Figure 5] This diagram shows the results of a human sapovirus infection susceptibility test in cells transduced with the CD36 gene (HCT116 cells). [Figure 6] This diagram shows the results of a human sapovirus infection susceptibility test in cells transduced with the CD36 gene (HuTu80 cells). [Figure 7] This diagram shows the results of a human sapovirus infection susceptibility test in cells transduced with the CD36 gene (CHO cells). [Figure 8] This diagram shows the results of a human sapovirus infection susceptibility test in cells (Vero cells) that have been transduced with the CD36 gene. [Figure 9] This figure shows the results of measuring cytotoxicity (CPE) caused by human sapovirus using Vero cells into which the CD36 gene has been introduced. [Figure 10] This diagram shows the results of measuring the TCID50 value of human sapovirus using Vero cells into which the CD36 gene has been introduced. [Figure 11] This diagram shows the results of measuring the TCID50 values ​​of each genotype of human sapovirus grown in Vero cells into which the CD36 gene was introduced. [Figure 12] This diagram shows the results of infection susceptibility testing for human sapovirus in intestinal organoid-associated cells. [Figure 13] This diagram shows the results of measuring the TCID50 value of human sapovirus derived from intestinal organoid-associated cells. [Modes for carrying out the invention]

[0028] 1. Creation of human sapovirus infection-susceptible cells The cell line derived from intestinal cancer cells that the inventors have successfully established, which is capable of serially amplifying human sapovirus, was created from a "Cas-converted cell group" obtained by transforming human colon cancer-derived cells Caco2 with the Cas9 gene.

[0029] Caco2 cells, derived from human colon cancer and obtained from ATCC, were cultured in an incubator at 37°C under 5% CO2 conditions using EMEM medium (Wako Pure Chemical Industries, Ltd., 051-07615) containing 10% fetal bovine serum (Biosera, FB-1365 / 500, Lot. 10259), 1% 100 mmol / 1L sodium pyruvate solution (Wako Pure Chemical Industries, Ltd., 190-14881), 1% MEM non-essential amino acid solution (Wako Pure Chemical Industries, Ltd., 139-15651), and 1% penicillin-streptomycin-amphotericin B suspension (Wako Pure Chemical Industries, Ltd., 161-23181). The dishes used for the culture were treated with Collagen IC (Nitta Gelatin Co., Ltd.) for approximately 10 minutes to coat with collagen, and then washed twice with PBS(-) before use. The collagen coating solution used was prepared by adjusting the collagen concentration to 0.15 mg / mL using filter-sterilized 0.01N HCl / PBS as the solvent. 250-300 μL of 0.25 w / v% trypsin-1 mmol / L EDTA·4Na solution (containing phenol red) (Wako Pure Chemical Industries, Ltd., 209-16941) was used to detach the cultured Caco2 cell population from the dish.

[0030] Caco2 cell populations maintained under the above conditions were seeded in 10 cm dishes. Approximately 24 hours later, lentiviral infection was performed using 1 mL of lentiviral vector stock prepared with pLentiCas9-Blast at moi=1, and the Cas9 expression cassette was introduced into the Caco2 cells. Four days after introduction, 3 μg / mL of blastosidine was added, and Cas-converted cells were selected. Cells selected thereafter were maintained as initial Cas-converted Caco2 cells.

[0031] The lentiviral vector stock mentioned above is a stock solution of recombinant lentiviruses mediated by Cas9, prepared by introducing pLentiCas9-Blast, a lentiviral vector DNA incorporating the Cas9 gene, into HEK293T cells.

[0032] Two months later, cell cloning was performed to select cell clones highly susceptible to human astrovirus infection (clones in which 80% of the proliferated cells were susceptible to human astrovirus infection). Subsequently, these clones were infected with human astrovirus, and astrovirus-lethal clones were further selected.

[0033] In other words, the cell culture density is diluted so that individual Cas-converted cells form independent cell populations in the culture medium as single clones. Cells constituting these independent Cas-converted clone cell populations (clonal populations) are then picked out for each cell population and tested for lethality against human astrovirus. Cell populations to which susceptibility to human astrovirus infection is observed at a predetermined frequency (50% or more, preferably 80% or more) can be screened as "clonal populations of astrovirus-infected lethal Cas-converted cells." From the clones selected in this way, astrovirus-infected lethal clones that die when infected with human astrovirus are selected.

[0034] As a result, six clones were selected as astro-susceptible Cas-converting cells. These clones were mixed and used as a population of astro-infected lethal Cas-converting cells (parent stock). Verification revealed that some of these astro-infected lethal Cas-converting cells constitutively possessed susceptibility to human sapovirus infection (susceptible clones). At the same time, cell clones that did not possess susceptibility to human sapovirus infection were also obtained (unsusceptible clones). Two types each of these susceptible and unsusceptible clones were selected and used in the following tests.

[0035] 2. Identification of human sapovirus infection receptors (1) Cell line used mRNA was extracted by conventional methods from the human sapovirus infection-susceptible clones "MC" and "ME" and the non-susceptible clones "PG" and "PE" (these cells can be cultured in standard plastic flasks for cell culture, and no special coating such as collagen is required on the cell adhesion surface) established from the above-mentioned astro-infected lethal Cas-converted cells.

[0036] (2) Comprehensive analysis of mRNA The mRNA profiles of the four clones described above (two susceptible clones; two non-susceptible clones) were comprehensively analyzed and compared using "RNA-seq". Figure 2 shows the analysis results for the two susceptible clones using a "Volcano plot". Of the genes whose expression levels were increased or decreased, 775 genes decreased in susceptible cells, 508 genes increased, and 60,523 genes remained unchanged. In the figure, the left side of each plot represents a "decreased gene," and the right side represents a "increased gene." The target infection receptor gene was conditioned to "show a significant increase in expression and is normally expressed in intestinal cells." "CD36" was identified as a gene that met these conditions. The CD36 plot in Figure 2 is shown using a leader line. The expression level of CD36 in the two susceptible clones was 147 times the steady state, and its p-value was "1.23 × 10⁻⁶". -8 This demonstrates, with statistical significance, that "CD36 is overexpressed in susceptible clones."

[0037] (3) Human sapovirus infection susceptibility test in cells transduced with the CD36 gene 1 Culturing was performed on "susceptible clone MC," "unsusceptible clone PG," and "clone PG CD36," which was created by introducing the CD36 gene (the gene containing the nucleotide sequence of SEQ ID NO: 1, hereafter the same) into the unsusceptible clone PG using a recombinant lentivirus for forced expression. Figure 3 shows the results of infecting each with human sapovirus (GI.1 AH20 strain, DDBJ accession number: LC671561; accession number omitted hereafter), washing the culture supernatant with a culture medium, and immediately measuring the amount of human sapovirus gene (RNA) in the supernatant as copy number. The measurement results from zero dpi to 3 days later are "RNA copy number at 3 dpi," and the measurement results at 7 days later are "RNA copy number at 7 dpi." The susceptibility of each clone to human sapovirus infection was evaluated by comparing the RNA copy numbers at 3 dpi and 7 dpi. The RNA copy number was measured by qPCR on the culture supernatant of each clone. As a result, it was found that the RNA copy number increased in MC and PG+CD36. In contrast, no increase was observed in PG. This confirmed that clone MC is inherently susceptible to human sapovirus infection, and it also confirmed that clone PG is inherently not susceptible to human sapovirus infection. In contrast, it was found that the PG+CD36 clone acquired susceptibility to human sapovirus infection by introducing and expressing the CD36 gene into clone PG, which was originally not susceptible to human sapovirus infection. From this, it was revealed that human CD36 is an infection receptor for human sapovirus.

[0038] (4) Human sapovirus infection susceptibility test in cells transduced with the CD36 gene 2 Figure 4 shows the results of a study in which transformed cells—HEK293T cells derived from human fetal kidney (cells originally incapable of sapovirus replication: indicated as 293T in the figure) (ATCC)—were forcibly expressed using a recombinant lentivirus incorporating the CD36 gene, and then infected with human sapovirus (GI.1 AH20 strain). The increase in sapovirus gene (RNA) in the culture supernatant and the increase in sapovirus gene (RNA) in the cells in the residue were examined using qRT-PCR (quantitative RT-PCR). Since sapoviruses consist of newly generated viruses released extracellularly and newly generated viruses remaining inside the cells, the RNA copy number was examined in both the culture supernatant and inside the cells. The susceptibility of the test cells to human sapovirus infection was evaluated using the increase in sapovirus RNA copy number from zero dpi to 3 days later (3 dpi) as an indicator (the horizontal axis shows pairs of "0 and 3").

[0039] From left to right are the first group (supernatant of HEK293T cells), the second group (supernatant of HEK293T cells + CD36), the third group (intracellular HEK293T cells), and the fourth group (intracellular HEK293T cells + CD36). In the comparison of the first group and the second group (sapovirus RNA copy number in the culture supernatant), and the third group and the fourth group (intracellular sapovirus RNA copy number), it can be seen that the RNA copy number in the groups into which the CD36 gene was introduced (the second and fourth groups) is orders of magnitude higher than the RNA copy number in the groups without the introduction (the first and third groups).

[0040] Furthermore, when nuclear staining (blue) and CD36 staining (red) were performed at zero dpi in both HEK293T cells and HEK293T cells + CD36, and the images were merged, only the blue of nuclear staining was observed in the HEK293T cell images, and the red of CD36 was not observed, confirming that CD36 expression was not detected in HEK293T cells. In contrast, in the HEK293T cells + CD36 images, both the blue of nuclear staining and the red of CD36 were clearly observed in most cells, confirming that most cells were expressing the introduced CD36 gene in large quantities.

[0041] Therefore, it was confirmed that HEK293T cells, which are originally cells that do not have susceptibility to human sapovirus infection, acquired susceptibility to human sapovirus infection by transforming them with the human CD36 gene.

[0042] This also revealed that human CD36 is an infection receptor for human sapovirus.

[0043] (5) Human sapovirus infection susceptibility test in cells transduced with the CD36 gene 3 Following the HEK293T cells described above, we introduced the CD36 gene into other animal cell lines that are originally unable to replicate human sapoviruses, and confirmed the acquisition of susceptibility to human sapovirus infection. Specifically, we introduced the CD36 gene using recombinant lentiviruses incorporating the same CD36 gene as described above into HCT116 cells (human-derived), HuTu80 cells (human-derived), CHO cells (hamster-derived), and Vero cells (monkey-derived), and confirmed the acquisition of susceptibility to human sapovirus infection in these cells.

[0044] In this specification and in the drawings, when "+CD36" or " / CD36" is used, both refer to "cells transformed with the CD36 gene."

[0045] (a) HCT116 cells Figure 5 shows the results of quantitative RT-PCR analysis of the increase in sapovirus (RNA) in the culture supernatant and the increase in sapovirus (RNA) in the residue, after inducing forced expression of the CD36 gene in human colon adenocarcinoma-derived HCT116 cells (ATCC: HCT116 in the figure) using recombinant lentivirus. The cells were then infected with human sapovirus (GI.1 AH20 strain), and centrifugation was performed in each culture system. The susceptibility of the test cells to human sapovirus infection was evaluated using the copy number of sapovirus (RNA) from zero dpi to 5 days later (5 dpi) as an indicator (horizontal axis: "0 and 5 pairs").

[0046] From left to right are the first group (supernatant of HCT116 cells), the second group (supernatant of HCT116 cells + CD36), the third group (intracellular HCT116 cells), and the fourth group (intracellular HCT116 cells + CD36). In the comparison of the first group and the second group (sapovirus RNA copy number in the culture supernatant), and the third group and the fourth group (sapovirus RNA copy number in the cells), it can be seen that the RNA copy number in the groups into which the CD36 gene was introduced (the second and fourth groups) is orders of magnitude higher than the RNA copy number in the groups without the introduction (the first and third groups).

[0047] Furthermore, immunohistochemical staining targeting CD36 was performed in each system (0 dpi), and the effect of permeabilization using TritonX was investigated. As a result, no staining was observed in HCT116 cells, regardless of whether or not TritonX treatment was performed. In contrast, clear cell staining was observed in HCT116 cells + CD36, regardless of whether or not TritonX treatment was performed. In particular, since staining was observed even in the TritonX-untreated group, where the antibody for immunohistochemical staining did not penetrate into the cells and only the cell surface was stained, it was confirmed that in HCT116 + CD36, CD36 is distributed on the cell surface and exhibits its external form as a receptor.

[0048] Therefore, it can be concluded that transforming HCT116 cells, which are originally cells that do not have susceptibility to human sapovirus infection, with the human CD36 gene newly acquired susceptibility to human sapovirus infection.

[0049] This further confirms that human CD36 is an infection receptor for human sapovirus.

[0050] (b) HuTu80 cells Figure 6 shows the results of quantitative RT-PCR detection of the changes in human sapovirus (RNA) gene levels after infection with human sapovirus (GI.1 AH20 strain) in transformed cells (HuTu80 cells derived from human duodenal cancer, provided by ATCC; shown as HuTu80 in the figure) that were introduced using a recombinant lentivirus incorporating the CD36 gene to force expression of the CD36 gene. The susceptibility of the test cells to human sapovirus infection was evaluated using the copy number of sapovirus gene (RNA) from day zero to 3 days later as an indicator (horizontal axis: "Day 0 and Day 3 pair").

[0051] From left to right are the first group (RNA copy number extracted from HuTu80 cells) and the second group (RNA copy number extracted from HuTu80 cells + CD36). Comparing the first and second groups (sapovirus RNA copy numbers in cells), it can be seen that the RNA copy number of the group into which the CD36 gene was introduced (second group) is orders of magnitude higher than that of the group without the introduction (first group).

[0052] Furthermore, when nuclear staining (blue) and CD36 staining (red) were performed on day zero in both HuTu80 cells and HuTu80 cells + CD36, and the images were superimposed (marged), only the blue color of nuclear staining was observed in the HuTu80 cell images, and the red color of CD36 was not observed, confirming that CD36 expression was not observed in HuTu80 cells. In contrast, in the HuTu80 cells + CD36 images, both the blue color of nuclear staining and the red color of CD36 were clearly observed in most cells, confirming that most cells were expressing the introduced CD36 gene in large quantities.

[0053] Therefore, it can be concluded that transforming HuTu80 cells, which are originally cells that do not have susceptibility to human sapovirus infection, with the human CD36 gene newly acquired susceptibility to human sapovirus infection.

[0054] This further confirms that human CD36 is an infection receptor for human sapovirus.

[0055] (c)CHO cells Figure 7 shows the results of quantitative RT-PCR detection of the amount of human sapovirus (RNA) after infection with human sapovirus (GI.1 AH20 strain) in transformed cells (ECACC: CHO in the figure) derived from Chinese hamsters, which were introduced using a recombinant lentivirus incorporating the CD36 gene to force expression of the CD36 gene. The susceptibility of the test cells to human sapovirus infection was evaluated using the copy number of sapovirus RNA from day zero to 3 days later as an indicator (horizontal axis: "Day 0 and Day 3 pair").

[0056] From left to right are the first group (RNA copy number extracted from CHO cells) and the second group (RNA copy number extracted from CHO cells + CD36). Comparing the first and second groups (sapovirus RNA copy numbers of XX), it can be seen that the RNA copy number of the group with the CD36 gene introduced (second group) is orders of magnitude larger than that of the group without the introduction (first group).

[0057] Furthermore, when nuclear staining (blue) and CD36 staining (red) were performed on day zero in both CHO cells and CHO cells + CD36, and the images were superimposed (merged), only the blue of nuclear staining was observed in the CHO cell images, and the red of CD36 was not observed, confirming that CD36 expression was not observed in CHO cells. In contrast, in the CHO cells + CD36 images, both the blue of nuclear staining and the red of CD36 were clearly observed in most cells, confirming that most cells were expressing the introduced CD36 gene in large quantities.

[0058] Therefore, it can be concluded that by transforming CHO cells (which are not susceptible to human sapovirus infection) derived from an animal species other than humans with the human CD36 gene, they acquired new susceptibility to human sapovirus infection.

[0059] This further confirms that human CD36 is an infection receptor for human sapovirus.

[0060] (d) Vero cells Figure 8 shows the viral load when transformed cells derived from African green monkey kidney cells (provided by ATCC: Vero in the figure) were infected with human sapovirus (GI.1 AH20 strain) and then infected with TCID. 50 The results obtained as values ​​(described later) are shown. The susceptibility of test cells to human sapovirus infection was evaluated using the viral load, based on the RNA copy number from day zero to three days later, as an indicator (horizontal axis: "Day 0 and Day 3 pair").

[0061] From left to right are the first group (sapovirus load in Vero cells) and the second group (sapovirus load in Vero cells + CD36). Comparing the first and second groups, it can be seen that the sapovirus load in the group with the CD36 gene introduced (second group) is orders of magnitude higher than that in the group without the introduction (first group).

[0062] Furthermore, when nuclear staining (blue) and CD36 staining (red) were performed on day zero in both Vero cells and Vero cells + CD36, and the images were superimposed (merged), only the blue color of nuclear staining was observed in the Vero cell images, and the red color of CD36 was not observed, confirming that CD36 expression was not detected in Vero cells. In contrast, in the Vero cells + CD36 images, both the blue color of nuclear staining and the red color of CD36 were clearly observed in most cells, confirming that most cells were expressing the introduced CD36 gene in large quantities.

[0063] Therefore, it can be concluded that by transforming Vero cells (which are not susceptible to human sapovirus infection) derived from an animal species other than humans with the human CD36 gene, they acquired new susceptibility to human sapovirus infection.

[0064] This further confirms that human CD36 is an infection receptor for human sapovirus.

[0065] 3. Cytotoxicity test using human sapovirus derived from CD36 recombinant cells In 2(5)(d) above, cytotoxicity tests were conducted on Vero cells transformed with the CD36 gene (Vero cells + CD36) that were found to have acquired susceptibility to human sapovirus infection, in order to confirm their usefulness as a screening method for drugs (therapeutic agents, etc.) against human sapovirus.

[0066] (1) Measurement of cytotoxicity (CPE) Vero cells + CD36 were evenly seeded on a 384-well plate and incubated at 37°C and 5% CO2. Human sapovirus (GI.1 AH20 strain) particles were then added in diluted solutions adjusted to moi1, moi0.1, and moi0.01, and incubated under the same conditions as above to infect the Vero cells + CD36 with human sapovirus. CPE was measured at 2 days (2 dpi), 4 days (4 dpi), and 7 days (7 dpi) after addition using CellTiter-Glo® 2.0 (Promega Co., Ltd.).

[0067] CellTiter-Glo® 2.0 is a CPE (Cell-Pervasive Exposure) measurement kit that detects intracellular ATP released into the culture supernatant when cells are destroyed by a virus, using fluorescence generated by luciferin catalyst via luciferase. The stronger the fluorescence, the greater the cytotoxicity (CPE) in the cell culture system. Generally, it is known that the higher the MOI (Multiplicity of Infection: number of virus particles / number of cells) at the time of infection, the earlier cytotoxicity is observed.

[0068] Figure 9 shows the results of this cytotoxicity test. The vertical axis represents fluorescence intensity, and the four groups on the horizontal axis, from left to right, are "moi1," "moi0.1," "moi0.01," "negative control (no infection)," and positive control "a cell lysate obtained by lysing infected cells (moi1) in the well with a solubilizer containing a detergent, thereby releasing all intracellular ATP." Following the general principle mentioned above, it can be seen that the fluorescence intensity reaches its peak earlier in the order of higher moi. Furthermore, in the case of moi0.01, it can be seen that the fluorescence intensity increases stepwise over time on days 2, 4, and 7.

[0069] Furthermore, Vero cells and Vero cells + CD36 culture systems on 364-well plates were infected with human sapovirus at a dose of "moi0.01". On day 7 after infection, both systems were stained with methylene blue, and the viability of the cultured cells was observed visually under a microscope. This cytotoxicity test utilizes the fact that even a single particle of human sapovirus infecting a cell will cause severe cytotoxicity, resulting in the death of the cell by day 7, making it impossible to stain with methylene blue. This method cannot numerically indicate the degree of CPE like CellTiter-Glo(registered trademark) 2.0, but it is characterized by being inexpensive and simple. As a result of this methylene blue staining test, in the Vero cell system after 7 days of infection, cells stained with methylene blue were observed throughout the wells without any gaps. In contrast, in the Vero cell + CD36 system after 7 days of infection, methylene blue stained cells were observed to be significantly sparse within the wells.

[0070] These results demonstrate that measuring CPE in animal cells transformed with the CD36 gene is a reliable detection system. Using the test drug in this system, it is possible to screen for the presence and intensity of the drug's effect on human sapoviruses, using cytotoxicity (CPE) as an indicator. This method enables high-throughput screening of anti-human sapovirus drugs. In the previous example (Figure 9), when the anti-human sapovirus drug candidate inhibited viral replication, cell death was suppressed and fluorescence was no longer observed. In the subsequent example, when the anti-human sapovirus drug candidate inhibited viral replication, cell death was suppressed, and the decrease in methylene blue-stained cells was inhibited.

[0071] (2) Infectious titer (TCID) 50 ) measurement Cell lines of various cells transformed with the CD36 gene (control: native cells) on a 384-well plate were infected with human sapovirus (GI.1 AH20 strain), and the infectivity titer (TCID) of the resulting human sapovirus was measured. 50 ) was measured using Vero cells + CD36 (VERO / CD36).

[0072] Figure 10 shows the results. The vertical axis represents TCID. 50 The values ​​are per mL. The horizontal axis shows, from left to right: (a) pairs of native and CD36 transformed HEK293T cells, (b) pairs of HuTu80 cells, (c) pairs of HCT116 cells, (d) pairs of Vero cells, (e) pairs of CHO cells, and (f) pairs of Caco2MC and its CD36 gene knockout cells (Caco2MC / CD36KO).

[0073] In all pairs (a)-(e), the infectivity titer of human sapovirus produced in CD36 gene-transformed cells was orders of magnitude higher than that of human sapovirus in native cells. 50 It was found that the value was shown. In (f), native cells showed high TCID. 50showed a value, but by knocking out the CD36 gene (in this case, knocking out the CD36 gene using the CRISPR / Cas9 system), the TCID 50 value decreased significantly (that is, the virus could no longer infect the cells).

[0074] Figure 11 shows the TCID 50 values of human sapoviruses of each genotype grown in Vero cells + CD36 used in (d) above. Specifically, for the genotypes of human sapovirus GI.1 (AH20 strain), GI.2 (EH-32 strain; DDBJ accession number: LC504352), GI.3 (I21-042 strain; DDBJ accession number: LC842323), GII.1 (TKC19-5 strain; DDBJ accession number: LC842325), and GII.3 (EH-37 strain; DDBJ accession number: LC504413), the respective TCID 50 values were examined.

[0075] As a result, it was shown that sapoviruses of any genotype grown in Vero cells + CD36 showed high TCID 50 values in Vero cells + CD36.

[0076] From these results, it was confirmed that the infectivity titer of human sapovirus cultured in transformed cells by the CD36 gene is suitable for testing, and for example, it is suitable to use a detection system for infectivity titer such as in this example for screening drugs (therapeutic drugs, etc.) related to human sapovirus.

[0077] 4. Examination using intestinal organoids Intestinal organoids originally have the CD36 gene present and functioning and have infectivity to human sapovirus. Changes in infectivity, etc. when the originally present CD36 gene was knocked out were examined.

[0078] (1) Infectivity test Figure 12 shows the results of quantitative RT-PCR analysis of viral load over time in intestinal organoids (provided by Keio University: Ileum-1 in the figure) infected with human sapovirus (GI.1 AH20 strain). The susceptibility of the test cells to human sapovirus infection was evaluated using the copy number of the human sapovirus gene (RNA) from day zero (0 dpi) to day one (1 dpi) and day four (4 dpi) (horizontal axis: "0 dpi, 1 dpi, 4 dpi pairs").

[0079] From left to right are the first group (intestinal organoids), the second group (intestinal organoids with the CD36 gene knocked out), the third group (intestinal organoids transformed with the CD36 gene), and the fourth group (intestinal organoids transformed with the CD36 gene after the CD36 gene knockout). The CD36 gene knockout was performed using the CRISPR / Cas9 system.

[0080] In the first group, it was confirmed that human sapovirus was being produced over time. In the second group, no increase in human sapovirus production over time was observed. In the third group, the amount of human sapovirus produced over time was an order of magnitude higher than in the first group. In the fourth group, the amount of human sapovirus produced was almost the same as in the third group.

[0081] These results confirmed that susceptibility to human sapovirus infection in animal cells is dependent on the CD36 gene. Furthermore, it was found that the CD36 gene introduced through transformation had a stronger effect on susceptibility to human sapovirus infection than the native CD36 gene.

[0082] (2) Infectious titer (TCID) 50 ) measurement Similar to 3(2) above, the infectivity titer of the human sapovirus (GI.1 AH20 strain) derived from the intestinal organoids described in (1) above was determined using Vero cells + CD36.

[0083] The results are shown in Figure 13. The vertical axis represents TCID. 50 The values ​​are per mL. The horizontal axis represents the four types of intestinal organoid-associated cells shown on the horizontal axis of Figure 12.

[0084] TCID, a human sapovirus derived from intestinal organoids with the CD36 gene knocked out. 50 The values ​​were extremely low, and the TCID was derived from native intestinal organoids of human sapovirus. 50 Based on the value, TCID of human sapovirus derived from intestinal organoids in which the CD36 gene was additionally introduced. 50 The values ​​were very high. TCID was derived from human sapoviruses (TCID) from intestinal organoids that had been transformed with the CD36 gene after the CD36 gene was knocked out. 50 The values ​​were an order of magnitude larger than those of native intestinal organoids.

[0085] These results confirm that even animal cells that originally possess and function the CD36 gene, making them susceptible to human sapovirus infection, can be transformed with the CD36 gene to obtain human sapoviruses with extremely high infectivity titers.

[0086] 5. Relationship with the present invention (1) Infection susceptibility tests using susceptible clones in Example 2(3) above; infection susceptibility tests using HEK293 cells in Example 2(4); infection susceptibility tests using various cells in Infection Susceptibility Tests 3(a)-(d) of Example 2(5); and infection susceptibility tests of intestinal organoids in Example 4(1) demonstrated that mammalian cells can be transformed with the CD36 gene to confer susceptibility to human sapovirus infection. This result is applicable to the recombinant cells of the present invention. Furthermore, this result clearly shows that it is possible to confer susceptibility to human sapovirus infection to living mammalian cells (excluding humans) by introducing the CD36 gene into the cells of the mammal by conventional methods, and this result is also applicable to the recombinant animals of the present invention.

[0087] (2) It has been demonstrated that infection susceptibility to human sapovirus can be conferred to various mammalian cells by performing transformation with the CD36 gene using the steps disclosed in the infection susceptibility test 1 using the susceptible clone in Example 2(3) above; infection susceptibility test 2 using HEK293 cells in Example 2(4) above; infection susceptibility tests using various cells in Example 2(5) above; and infection susceptibility test of intestinal organoids in Example 4(1) above. This result is consistent with the method for conferring infection susceptibility of the present invention.

[0088] (3) When measuring the infectivity titer in Example 3(2) above, the human sapovirus produced in various recombinant cells of the present invention showed a high TCID in Vero cells + CD36. 50 The values ​​were shown; in the measurement of infectivity titer in Example 4(2), human sapovirus produced in intestinal organoids transformed with the CD36 gene showed high TCID in Vero cells + CD36 as well. 50 The values ​​shown are applicable to the sapovirus production method of the present invention.

[0089] (4) The measurement of cytotoxicity in Example 3(1) and the measurement of infectivity titer in (2) above; furthermore, the measurement of infectivity titer in Example 4(2) above were all successfully completed. The success in these measurement systems provides a basis for screening drugs and vaccines using these measurement systems. That is, in these measurement systems, it is possible to detect the effect of the test drug or test vaccine on human sapovirus by first treating recombinant cells or recombinant animals of the present invention with the test drug or test vaccine and then measuring the cytotoxicity or infectivity titer. This result applies to the first screening method of the present invention for drugs and to the third screening method of the present invention for vaccines. Furthermore, in these measurement systems, it is possible to detect the effect of the test drug on human sapovirus by using the test drug and human sapovirus together. This result applies to the second screening method of the present invention.

Claims

1. Recombinant mammalian cultured cells selected from (1)-(3) below, transformed with the human CD36 encoding gene and susceptible to human sapovirus infection: (1) HCT116 cells (human-derived), (2) HuTu80 cells (human-derived), and (3) Vero cells (derived from monkeys).

2. A method for producing human sapovirus, comprising infecting genetically modified mammalian cultured cells that are susceptible to infection with human sapovirus, transformed with a human CD36 gene, or a genetically modified mammal (excluding humans) that possesses genetically modified mammalian cultured cells that are susceptible to infection with human sapovirus, transformed with a human CD36 gene, as its own cells, with human sapovirus, and propagating the human sapovirus in the mammalian cultured cells or the mammal (excluding humans).

3. A method for conferring susceptibility to human sapovirus infection in mammalian cells or mammals (excluding humans) by introducing a gene encoding human CD36 into the mammalian cells or mammals (excluding humans).

4. A screening method characterized by obtaining information on the effects of a screening substance by contacting genetically modified mammalian cultured cells that have been infected with human sapovirus, transformed with a human CD36 encoding gene, and are susceptible to infection by human sapovirus, or a genetically modified mammal (excluding humans) that possesses genetically modified mammalian cultured cells transformed with a human CD36 encoding gene and susceptible to infection by human sapovirus as its own cells, with a screening substance for its effects on human sapovirus, and detecting the proliferation of human sapovirus in the mammalian cultured cells or the mammal (excluding humans).

5. The screening method according to claim 4, characterized in that the target effect for screening is the inactivation effect or the inhibition effect of proliferation of human sapoviruses.

6. A screening method characterized by obtaining information on the effects of the screening substance by contacting genetically modified mammalian cultured cells that have been transformed with a human CD36 gene and are susceptible to infection by human sapovirus, or genetically modified mammals (excluding humans) that possess genetically modified mammalian cultured cells that have been transformed with a human CD36 gene and are susceptible to infection by human sapovirus as their own cells, with a screening substance and human sapovirus, and detecting the invasion or proliferation of human sapovirus into the mammalian cultured cells or the mammal (excluding humans).

7. The screening method according to claim 6, characterized in that the target effect of the screening is the inhibitory effect on the entry of human sapovirus into cells or the inhibitory effect on its proliferation within cells.

8. A screening method characterized by obtaining information on the action of the screening target vaccine by immunizing genetically modified mammalian cultured cells that are transformed with a human CD36 gene and are susceptible to infection with human sapovirus, or genetically modified mammals (excluding humans) that possess genetically modified mammalian cultured cells that are transformed with a human CD36 gene and are susceptible to infection with human sapovirus as their own cells, with the screening target vaccine, and detecting the protective effect against human sapovirus in the mammalian cultured cells or the mammal (excluding humans).

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