Genetically modified mammalian cell having susceptibility to human sapovirus infection, genetically modified mammal having said cell, and method for producing human sapovirus, method for imparting infection susceptibility, and screening method using said cell or mammal

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

Genetically modified mammalian cells susceptible to infection with human sapovirus, genetically modified mammals harboring said cells, and methods for producing, imparting susceptibility to, and screening for human sapovirus using these cells

[0001] The present invention relates to a means for propagating a virus, and more specifically to a means for propagating a human sapovirus.

[0002] Sapoviruses (SaVs) are small, non-enveloped, spherical viruses with a positive-stranded single-stranded RNA genome. They belong to the genus Sapovirus and the family Caliciviridae. They have been detected in many animals, including humans, pigs, dogs, sea lions, and chimpanzees. They are classified into at least 19 genetic groups (GI-GXIX) and 52 genotypes. Human sapoviruses (HuSaVs) were discovered in 1977 and, like human noroviruses (HuNoVs) in the genus Norovirus, are one of the major enteric viruses detected in nonbacterial acute gastroenteritis worldwide. Outbreaks have also been reported in Japan, including a mass food poisoning incident (65 cases) during a school trip in Yokohama in 2007, a food poisoning incident (109 cases) at a wedding hall in Matsuyama in 2007, and a large-scale food poisoning incident (680 cases) caused by school lunches in Aichi Prefecture in 2010. Human sapoviruses are classified as GI, GII, GIV, and GV, with GI being the most commonly detected, followed by GII. From 2019 to 2023, the number of virus detections reported was 6,269 for human norovirus, 1,071 for human sapovirus, 885 for rotavirus, and 353 for astrovirus. Human sapovirus is second only to human norovirus in the number of reported detections. The suspected infection routes and symptoms of human norovirus and human sapovirus are difficult to distinguish, and human sapovirus is difficult to diagnose based on clinical symptoms. A major difference between human norovirus and human sapovirus is the structure of their genomic RNA (Figure 1). In human norovirus, the nonstructural proteins and structural proteins (capsid protein, VP1) are encoded by separate ORFs, whereas in human sapovirus, the nonstructural proteins and VP1 are encoded by the same ORF. Therefore, it is thought that human sapovirus VP1 expression occurs either through translation of ORF1 as a polyprotein containing VP1 and nonstructural proteins, which is then cleaved by the human sapovirus's own protease, or through direct translation of VP1 from the subgenome.

[0003] Although human norovirus and human sapovirus were discovered in the 1970s and over 40 years ago, research into both viruses has stalled. This is due to the lack of culture systems or animal models capable of infecting and propagating them. Regarding human norovirus, only an in vitro culture system using human intestinal stem cell-derived intestinal organoids (hIEOs) was reported in 2016.

[0004] In 2020, a culture system using a limited cell line, in which the human duodenal cancer cell line HuTu80 was passaged for a long period under a special environment, was finally reported for human sapovirus (Non-Patent Document 1). In 2023, infection and proliferation in human intestinal stem cell-derived hIEOs (Non-Patent Document 2) and iPS cell-derived hIEOs (Non-Patent Document 3) were reported.

[0005] Takagi H. et al., 2020. PNAS 117:32708-32085Euller-Nicolas G. et al., 2023. J Virol 97: e00383-23Matsumoto N. et al., 2023. Viruses 15: 1929

[0006] However, the human sapovirus propagation systems available to date do not use normal mammals (including humans), limiting their scope of application. To solve this problem, it is essential to identify the infection receptor that human sapovirus uses when it infects humans, and to provide genetically modified mammalian cells into which the infection receptor gene has been introduced, as well as the technology based on this.

[0007] The present inventors recently succeeded in establishing a cell line derived from intestinal cancer cells capable of serially amplifying human Sapovirus (PCT / JP2025 / 012990). They then detected gene level variations in the cell line and identified one of the candidate receptors, the human CD36 gene, in a human Sapovirus infection test using a cell line whose wild-type cell line is not susceptible to human Sapovirus infection. As a result, they determined that the human Sapovirus infection receptor is indeed human CD36 (platelet glycoprotein 4).

[0008] That is, the contents of the present invention are as follows.

[0009] First, we provide genetically modified mammalian cultured cells (also referred to as 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 with human Sapovirus.

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

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

[0012] Furthermore, the mammalian cultured cells include HEK293T cells (human origin), Caco2 cells (human origin), HCT116 cells (human origin), Intestine407 cells (human origin), macrophage cultured cells 15310-LN cells (human origin), NALM-6 cells (human origin), HuTu80 cells (human origin), HT29 cells (human origin), MRC5 cells (human origin), RAW264.7 cells (mouse origin), and NIH3T3 cells (mouse origin). Examples of such cells include, but are not limited to, M1 cells (derived from mice), BHK cells (derived from hamsters), CHO cells (derived from hamsters), CRFK cells (derived from cats), MDCK cells (derived from dogs), PK-15 cells (derived from pigs), Vero cells (derived from monkeys), MA104 cells (derived from monkeys: there are MA104 cells derived from rhesus monkeys and MA104 cells derived from African green monkeys), GL37 cells (derived from monkeys), and COS7 cells (derived from monkeys).

[0013] Second, we provide a genetically modified mammal (hereinafter also referred to as the recombinant animal of the present invention) that possesses the recombinant cell of the present invention as its own cell. The recombinant animal of the present invention is a so-called transgenic animal or knock-in animal. The genetically modified animal of the present invention excludes humans.

[0014] The method for introducing the human CD36 gene into cultured mammalian cells is not particularly limited and can be performed according to conventional methods. Typical examples include vectors for gene transfer into mammals, such as murine leukemia virus vectors incorporating all or part of the human CD36 gene, retroviral vectors such as lentiviral vectors, adenoviral vectors, adeno-associated virus vectors, herpes simplex type I vectors, modified viral vectors such as HVJ-liposomes, and plasmid vectors having a CMV promoter, GAC promoter, EF-1α promoter, or SV40 promoter that function in mammalian cells. In addition to recombinant viruses, calcium phosphate methods, lipofection methods, commercially available transfection reagents, microinjection methods, stamporation methods, particle gun methods, and the like can also be used for gene transfer. Furthermore, knock-in methods using gene editing techniques such as the CRISPR / Cas9 system can also be used.

[0015] The human CD36 gene can be obtained by conventional methods. That is, all or a portion of the desired human CD36 gene can be easily obtained by amplifying the gene region by gene amplification methods such as PCR using cDNA from one or more species of animal from which the human CD36 gene is derived as a template. This can be done in-house, outsourced, or commercially available. As described below, the human CD36 gene may be the entire gene encoding CD36, or a portion of the nucleotide sequence excluding non-essential regions. Specifically, a gene encoding the extracellular domain, transmembrane domain, and intracellular domain as essential regions is preferred. Furthermore, in non-human mammalian cells, such as mouse-derived cells, the native CD36 gene of the mammalian cells (e.g., mouse CD36 gene) can be preserved intact and transformed with the human CD36 gene. Alternatively, the native CD36 gene can be inactivated by expression suppression treatment such as knockout. For human-derived cells that originally contain the human CD36 gene, such gene suppression treatment is not necessary.

[0016] The recombinant animal of the present invention can be produced by conventional methods. For example, the human CD36 gene can be introduced into a fertilized egg of a target mammal by conventional methods such as the microinjection method described above, and then the resulting embryo can be cultivated to produce the recombinant animal of the present invention. Alternatively, the recombinant animal of the present invention can be produced by knocking in the human CD36 gene using the CRISPR / Cas9 system or the like. Mammals include, but are not limited to, mice, rats, hamsters, pigs, monkeys, dogs, cats, horses, and the like, excluding humans. As with the mammalian cells described above, the native CD36 gene of a mammal other than human can be preserved as is to produce the recombinant animal of the present invention. Alternatively, the native CD36 gene can be inactivated by knockout or other expression suppression treatment, and a chimeric gene of the human CD36 gene and the host's CD36 gene can be introduced. Furthermore, by selecting a promoter for expressing the human CD36 gene, such as the villin promoter, which is highly active in intestinal epithelial cells, it is possible to express the CD36 gene in large amounts in the target organ of viral infection (e.g., intestinal epithelium, etc.).

[0017] Thirdly, we provide a method for producing human Sapovirus (hereinafter also referred to as the method for producing the Sapovirus of the present invention), which involves infecting a recombinant cell of the present invention or a recombinant animal of the present invention with human Sapovirus and allowing the human Sapovirus to grow in the mammalian cell or mammal (excluding humans).

[0018] Fourth, we provide a method for conferring infection susceptibility to mammalian cells or mammals (excluding humans) by introducing the human CD36 gene into said mammalian cells or mammals (excluding humans), thereby conferring susceptibility to infection with human Sapovirus (hereinafter also referred to as the method for conferring infection susceptibility of the present invention).

[0019] Fifth, we provide a screening method (hereinafter also referred to as the first screening method of the present invention) characterized in that a substance to be screened for its effect on human Sapovirus is contacted with a recombinant cell of the present invention or a recombinant animal of the present invention that has been infected with human Sapovirus, and the proliferation of human Sapovirus in the cultured mammalian cells or mammals (excluding humans) is detected, thereby obtaining information on the effect of the substance to be screened (hereinafter also referred to as the first screening method of the present invention).

[0020] Examples of the action to be screened in the first screening method of the present invention include, but are not limited to, the action of inactivating human sapovirus or the action of inhibiting its proliferation.

[0021] Sixth, we provide a screening method (hereinafter also referred to as the second screening method of the present invention) characterized by contacting a screening target substance and human Sapovirus with a recombinant cell of the present invention or a recombinant animal of the present invention, and detecting the invasion or proliferation of human Sapovirus into the mammalian cultured cell or mammal (excluding humans), thereby obtaining information on the action of the screening target substance.

[0022] Examples of the action to be screened in the second screening method of the present invention include, but are not limited to, the inhibitory action on cell invasion by human sapovirus or the inhibitory action on intracellular proliferation.

[0023] Seventh, we provide a screening method (hereinafter also referred to as the third screening method of the present invention), which is characterized by immunizing a recombinant cell of the present invention or a recombinant animal of the present invention with a vaccine to be screened, and detecting the protective effect against human sapovirus in the mammalian cultured cells or mammals (excluding humans), thereby obtaining information on the effect of the vaccine to be screened.

[0024] CD36, the human sapovirus infection receptor that forms the basis of the present invention, is a known glycoprotein present in humans and is typically known as platelet glycoprotein 4. Throughout this application, the term "human CD36" or simply "CD36" will be used throughout. Unless otherwise specified, CD36 is referred to as "human CD36." A typical example of the human CD36 gene sequence is shown as the cDNA sequence shown in SEQ ID NO: 1 (accession number KU177961). Human CD36 is generally considered to be a multifunctional glycoprotein that acts as a receptor for a wide range of ligands, including proteinaceous ligands such as thrombospondin, fibronectin, collagen, and amyloid beta, as well as lipid ligands such as oxidized low-density lipoprotein, anionic phospholipids, long-chain fatty acids, and bacterial diacylated lipopeptides. Because they are generally multivalent, multiple receptors may be simultaneously involved, resulting in the formation of CD36 clusters, which initiate specific signal transduction and internalization of receptor-ligand complexes. Cellular responses to these ligands are believed to be involved in angiogenesis, inflammatory responses, lipid metabolism, taste, and intestinal processing of dietary fat.

[0025] In the present invention, the CD36 gene capable of transforming mammalian cells and conferring susceptibility to human Sapovirus infection to the transformed cells may be the entire gene encoding CD36, or a portion of the nucleotide sequence excluding non-essential regions. Specifically, a gene encoding the extracellular domain, transmembrane domain, and intracellular domain as essential regions can be defined as the CD36 gene.

[0026] The present invention provides a means for artificially propagating human sapovirus, enabling the production of pharmaceuticals such as various vaccines including live vaccines. Furthermore, it provides a method for screening drugs and vaccines against human sapovirus using the human sapovirus propagation system.

[0027]

[0023] Figure 1 shows the structure of genomic RNA of human sapovirus and human norovirus. Figure 2 shows the results of a comprehensive analysis of mRNA expression for each gene in human sapovirus infection-susceptible clones. Figure 3 shows the results of a human sapovirus infection susceptibility test in CD36 gene-introduced cells (clones produced by the present inventors). Figure 4 shows the results of a human sapovirus infection susceptibility test in CD36 gene-introduced cells (HEK293T cells). Figure 5 shows the results of a human sapovirus infection susceptibility test in CD36 gene-introduced cells (HCT116 cells). Figure 6 shows the results of a human sapovirus infection susceptibility test in CD36 gene-introduced cells (HuTu80 cells). Figure 7 shows the results of a human sapovirus infection susceptibility test in CD36 gene-introduced cells (CHO cells). Figure 8 shows the results of a human sapovirus infection susceptibility test in CD36 gene-introduced cells (Vero cells). 1 shows the results of measuring cytotoxicity (CPE) caused by human sapovirus using Vero cells transfected with the CD36 gene. ５０ TCID values ​​of human sapoviruses of each genotype grown in Vero cells transfected with the CD36 gene were measured. ５０ 1 is a diagram showing the results of a test of susceptibility to infection with human sapovirus in intestinal organoid-associated cells. ５０ 1 is a diagram showing the results of measurements of values.

[0028] 1. Creation of cells susceptible to human sapovirus infection The cell line derived from intestinal cancer cells capable of serially amplifying human sapovirus, which the present inventors have succeeded in, was established from a "Cas-transformed cell group" in which human colon cancer-derived Caco2 cells were transformed with the Cas9 gene.

[0029] Caco2 cells, derived from human colon cancer, obtained from ATCC, were cultured in EMEM medium (Wako Pure Chemical Industries, Ltd., 051-07615) supplemented with 10% fetal bovine serum (Biosera, FB-1365 / 500, Lot. 10259), 1% 100 mmol / L 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) at 37°C and 5% CO in an incubator. ２ The culture was carried out under the conditions of . The dishes used for the above culture were treated with Collagen I-C (Nitta Gelatin Co., Ltd.) for approximately 10 minutes to form a collagen coating, and then washed twice with PBS(-). The collagen coating solution used was adjusted to a collagen concentration of 0.15 mg / mL using filter-sterilized 0.01 N HCl / PBS as the solvent. The cultured Caco2 cells were detached from the dishes using 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).

[0030] Caco2 cells maintained under the above conditions were seeded onto a 10 cm dish. Approximately 24 hours later, 1 mL of lentiviral vector stock prepared using pLentiCas9-Blast was used to infect the Caco2 cells with lentivirus at an moi of 1 to introduce the Cas9 expression cassette. Four days after transfection, 3 μg / mL of blasticidin was added to select Cas-transformed cells. The cells after selection were maintained as initial Cas-transformed Caco2 cells.

[0031] The lentiviral vector stock is a stock solution of Cas9-mediated recombinant lentivirus 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 expanded cells were susceptible to human astrovirus infection). These clones were then infected with human astrovirus, and lethal clones infected with astrovirus were further selected.

[0033] That is, the cell culture density is diluted so that individual Cas-transformed cells form independent cell populations in the medium as single clones, and the cells (clonal populations) that make up these independent Cas-transformed clone cell populations are selected for each cell population and tested for lethality against human astrovirus infection. Cell populations to which clonal populations that are susceptible to human astrovirus infection at a predetermined frequency (50% or more, preferably 80% or more) belong can be screened as "clonal cell populations of astrovirus-infectious lethal Cas-transformed cells." Among the clones selected in this way, astrovirus-infectious lethal clones that die when infected with human astrovirus were selected.

[0034] As a result, six clones were selected as astrocyte-sensitive Cas-transformed cells. These clones were mixed and used as a group of astrocyte-infected lethal Cas-transformed cells (parent strain stock). Verification revealed that some of these astrocyte-infected lethal Cas-transformed cells consistently possessed susceptibility to human sapovirus infection (susceptible clones). At the same time, cell clones that were not susceptible to human sapovirus infection were also obtained (insensitive clones). Two of these sensitive and insensitive clones were selected and used in the following tests.

[0035] 2. Identification of the infection receptor of human sapovirus (1) Cell lines used: Human sapovirus infection-susceptible clones "MC" and "ME" established from the above-mentioned astrocyte-infected lethal Cas-transformed cells; and human sapovirus infection-insensitive clones "PG" and "PE" (these cells can be cultured in ordinary plastic flasks for cell culture, and do not require special coatings such as collagen on the cell adhesion surface). mRNA was extracted from each cell line by standard methods.

[0036] (2) Comprehensive Analysis of mRNA The mRNA profiles of each of the four clones (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 in a Volcano plot. Of the genes whose expression was identified as increased or decreased, 775 genes decreased in susceptible cells, 508 genes increased, and 60,523 genes remained unchanged. Each plot in the figure indicates a "decreased gene" on the left and an "increased gene" on the right. The target infection receptor gene was conditioned to be a gene that "has 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 indicated by a lead line. The expression level of CD36 in the two susceptible clones was 147-fold higher than the normal level, and the p-value was 1.23 × 10 －８ " and "CD36 is overexpressed in sensitive clones" with a statistically significant difference.

[0037] (3) Human Sapovirus Infection Susceptibility Test 1 in CD36 Gene-Transduced Cells: "Sensitive Clone MC," "Non-Sensitive Clone PG," and "Clone PG CD36," in which the CD36 gene (a gene having the nucleotide sequence of SEQ ID NO: 1, hereinafter the same) was incorporated into the non-sensitive clone PG and the CD36 gene was introduced using a recombinant lentivirus to forcibly express the CD36 gene. Figure 3 shows the results of infecting each clone with human sapovirus (GI.1 AH20 strain, DDBJ Accession No. LC671561; hereinafter, the accession number is omitted). The culture supernatant was washed with medium, and the amount of human sapovirus gene (RNA) in the supernatant was measured immediately thereafter as a copy number. The measurement result of sapovirus RNA three days after zero dpi is the "RNA copy number at 3 dpi," and the measurement result seven days later is the "RNA copy number at 7 dpi." The susceptibility of each clone to human sapovirus infection was assessed by comparing RNA copy numbers at 3 and 7 dpi. RNA copy numbers were measured by qPCR analysis of the culture supernatants of each clone. The results showed 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, while clone PG is inherently not susceptible to human sapovirus infection. In contrast, the PG+CD36 clone was newly acquired susceptibility to human sapovirus infection by introducing and expressing the CD36 gene into clone PG, which is not inherently susceptible to human sapovirus infection. This demonstrates that human CD36 is a receptor for human sapovirus infection.

[0038] (4) Human Sapovirus Infection Susceptibility Test 2 in CD36 Gene-Transfected Cells. Figure 4 shows the results of human fetal kidney-derived HEK293T cells (cells incapable of Sapovirus replication; designated 293T in the figure) (ATCC) transformed with a recombinant lentivirus incorporating the CD36 gene, allowing forced expression. The cells were then infected with human Sapovirus (GI.1 AH20 strain). The increase in Sapovirus RNA in the culture supernatant and the increase in Sapovirus RNA in the residual cells were analyzed by qRT-PCR (quantitative RT-PCR). Because Sapovirus exists as both a nascent virus released outside the cells and a nascent virus that remains within the cells, the RNA copy numbers in both the culture supernatant and the cells were examined. The increase in Sapovirus RNA copy number from zero dpi to three days later (3 dpi) was used as an index to evaluate the susceptibility of the test cells to human Sapovirus infection (the "0 and 3" group on the horizontal axis).

[0039] From the left, the first group (supernatant of HEK293T cells), the second group (supernatant of HEK293T cells + CD36), the third group (inside HEK293T cells), and the fourth group (inside HEK293T cells + CD36 cells). Comparing the first group with the second group (Sapovirus RNA copy number in the culture supernatant) and the third group with 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 (groups 2 and 4) was increased by an order of magnitude compared to the RNA copy number in the groups into which the introduction was not performed (groups 1 and 3).

[0040] In addition, nuclear staining (blue) and CD36 staining (red) were performed on both HEK293T cells and HEK293T cells + CD36 at zero dpi, and the images were merged. Only the blue nuclear staining was observed in the HEK293T cell image, and the red CD36 was not observed, confirming that CD36 expression was not observed in HEK293T cells. In contrast, in the HEK293T cell + CD36 image, both the blue nuclear staining and the red CD36 were clearly observed in most cells, confirming that most cells expressed large amounts of the introduced CD36 gene.

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

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

[0043] (5) Human Sapovirus Infection Susceptibility Test 3 in CD36 Gene-Transduced Cells Following the HEK293T cells described above, the CD36 gene was introduced into other animal cell lines in which human Sapovirus cannot originally grow, and the acquisition of susceptibility to human Sapovirus infection was confirmed. Specifically, the CD36 gene was introduced into HCT116 cells (human-derived), HuTu80 cells (human-derived), CHO cells (hamster-derived), and Vero cells (monkey-derived) using a recombinant lentivirus incorporating the CD36 gene as described above, and the acquisition of susceptibility to human Sapovirus infection in these cells was confirmed.

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

[0045] (a) HCT116 cells. Figure 5 shows the results of transforming human colon adenocarcinoma-derived HCT116 cells (ATCC: HCT116 in the figure) with a CD36 gene transfected using a recombinant lentivirus, forcing expression of the gene, and infecting them with human sapovirus (GI.1 AH20 strain). Each culture was centrifuged, and the increase in sapovirus RNA in the culture supernatant and the increase in RNA in the cellular residue were analyzed by quantitative RT-PCR. The susceptibility of the test cells to human sapovirus infection was assessed using the number of copies of sapovirus RNA five days after 0 dpi (the "0 and 5" group on the horizontal axis).

[0046] From the left, the first group (supernatant of HCT116 cells), the second group (supernatant of HCT116 cells + CD36), the third group (inside HCT116 cells), and the fourth group (inside HCT116 cells + CD36 cells). A comparison of the first group with the second group (Sapovirus RNA copy number in the culture supernatant) and the third group with the fourth group (Sapovirus RNA copy number in the cells) shows that the RNA copy number in the groups into which the CD36 gene was introduced (groups 2 and 4) was increased by an order of magnitude compared to the RNA copy number in the groups into which the gene was not introduced (groups 1 and 3).

[0047] Immunostaining of CD36 was performed in each system (0 dpi), and the effect of permeabilization using TritonX was examined. As a result, no staining was observed in HCT116 cells, regardless of whether or not they were treated with TritonX. In contrast, clear cell staining was observed in HCT116 cells + CD36, regardless of whether or not they were treated with TritonX. In particular, staining was observed in the TritonX-untreated group, in which the immunostaining antibody did not infiltrate into the cells and only the cell surface was stained. This confirmed that in HCT116 + CD36, CD36 is distributed on the cell surface and exhibits the appearance of a receptor.

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

[0049] This also confirmed that human CD36 is an infection receptor for human sapovirus.

[0050] (b) HuTu80 cells. Figure 6 shows the results of quantitative RT-PCR analysis of the changes in the amount of human sapovirus RNA when transformed cells (HuTu80 cells, provided by ATCC; HuTu80 in the figure) derived from human duodenal cancer were transformed with a recombinant lentivirus incorporating the CD36 gene, forcing expression of the CD36 gene. The cells were then infected with human sapovirus (GI.1 AH20 strain). The susceptibility of the test cells to human sapovirus infection was assessed using the copy number of the sapovirus RNA from day 0 to three days later (the "Day 0 and Day 3" combination on the horizontal axis).

[0051] From the left, the first group (RNA copy number extracted from HuTu80 cells) and the second group (RNA copy number extracted from HuTu80 cells + CD36) are shown. Comparing the first group with the second group (Sapovirus RNA copy number of cells), it can be seen that the RNA copy number in the group into which the CD36 gene was introduced (second group) was increased by an order of magnitude compared to the RNA copy number in the group without the introduction (first group).

[0052] In addition, nuclear staining (blue) and CD36 staining (red) were performed on both HuTu80 cells and HuTu80 cells + CD36 on day 0, and the images were merged. Only the blue nuclear staining was observed in the image of HuTu80 cells, and the red CD36 was not observed, confirming that CD36 expression was not observed in HuTu80 cells. In contrast, in the image of HuTu80 cells + CD36, both the blue nuclear staining and the red CD36 were clearly observed in most cells, confirming that most cells expressed large amounts of the introduced CD36 gene.

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

[0054] This also confirmed that human CD36 is an infection receptor for human sapovirus.

[0055] (c) CHO cells. Figure 7 shows the results of quantitative RT-PCR analysis of the changes in the amount of human sapovirus gene (RNA) when transformed cells were infected with human sapovirus (GI.1 AH20 strain) after Chinese hamster CHO cells (ECACC: CHO in the figure) were forcibly expressed by introducing a recombinant lentivirus incorporating the CD36 gene into the transformed cells. The susceptibility of the test cells to human sapovirus infection was assessed using the number of copies of sapovirus RNA from day 0 to 3 days later as an index (the "Day 0 and Day 3 combination" on the horizontal axis).

[0056] From the left, the first group (RNA copy number extracted from CHO cells) and the second group (RNA copy number extracted from CHO cells + CD36) are shown. Comparing the first group with the second group (Sapovirus RNA copy number of XX), it can be seen that the RNA copy number in the group into which the CD36 gene was introduced (second group) is increased by an order of magnitude compared to the RNA copy number in the group into which the introduction was not performed (first group).

[0057] In addition, nuclear staining (blue) and CD36 staining (red) were performed on both CHO cells and CHO cells + CD36 on day 0, and the images were merged. Only the blue nuclear staining was observed in the CHO cell image, and the red CD36 was not, confirming that CD36 expression was not observed in the CHO cells. In contrast, in the CHO cell + CD36 image, both the blue nuclear staining and the red CD36 were clearly observed in most cells, confirming that most of the cells expressed large amounts of the introduced CD36 gene.

[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 have newly acquired susceptibility to human Sapovirus infection.

[0059] This also confirmed that human CD36 is an infection receptor for human sapovirus.

[0060] (d) Vero cells. Figure 8 shows the results of transfection of Vero cells (provided by ATCC: Vero in the figure) derived from African green monkey kidney cells with a recombinant lentivirus incorporating the CD36 gene, forcing the CD36 gene expression. The cells were then infected with human sapovirus (GI.1 AH20 strain), and the viral load was measured in TCID 20. ５０ The results are shown as values ​​(described below). The susceptibility of the test cells to human sapovirus infection was evaluated using the viral load based on the RNA copy number from day 0 to 3 days later as an index (the "combination of Day 0 and Day 3" on the horizontal axis).

[0061] From the left, the first group (Sapovirus amount in Vero cells) and the second group (Sapovirus amount in Vero cells + CD36) are shown. Comparing the first and second groups, it can be seen that the Sapovirus amount in the group into which the CD36 gene was introduced (group 2) was increased by an order of magnitude compared to the Sapovirus amount in the group without the introduction (group 1).

[0062] In addition, both Vero cells and Vero cells + CD36 were stained for nuclei (blue) and CD36 (red) on day 0, and the images were merged. Only the blue nuclear staining was observed in the Vero cell image, and the red CD36 was not, confirming that no CD36 expression was observed in the Vero cells. In contrast, in the Vero cell + CD36 image, both the blue nuclear staining and the red CD36 were clearly observed in most cells, confirming that most of the cells expressed large amounts of the introduced CD36 gene.

[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 have newly acquired susceptibility to human Sapovirus infection.

[0064] This also confirmed that human CD36 is an infection receptor for human sapovirus.

[0065] 3. Cytotoxicity test of human sapovirus derived from CD36 recombinant cells Vero cells transformed with the CD36 gene (Vero cells + CD36), which were found to have acquired susceptibility to infection with human sapovirus in 2(5)(d) above, were subjected to a cytotoxicity test to confirm their usefulness as a screening tool for drugs (therapeutic drugs, 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% CO ２ The cells were incubated at 4°C for 1 hour, and then diluted with human sapovirus (GI.1 AH20 strain) particles at moi of 1, moi of 0.1, or moi of 0.01 were added thereto. The cells were then incubated under the same conditions as above to infect the Vero cells + CD36 with the human sapovirus. CPE was measured 2 days (2 dpi), 4 days (4 dpi), and 7 days (7 dpi) after addition using CellTiter-Glo® 2.0 (Promega).

[0067] CellTiter-Glo (registered trademark) 2.0 is a CPE assay kit that detects intracellular ATP released into the culture supernatant when cells are destroyed by a virus by measuring the fluorescence generated by luciferase catalyzed by luciferin, and the stronger the fluorescence, the more cytotoxicity (CPE) is observed in the cell culture system. It is generally 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] The results of this cytotoxicity test are shown in Figure 9. The vertical axis represents fluorescence intensity, and the four groups on the horizontal axis are, from left to right, "moi 1," "moi 0.1," "moi 0.01," "negative control (no infection)," and the positive control (cell lysate obtained by lysing infected cells (moi 1) in a well with a detergent-containing solubilizing agent to release all intracellular ATP). In accordance with the general theory above, it can be seen that the fluorescence intensity peaks earlier in the ascending order of moi. Furthermore, at moi 0.01, it can be seen that the fluorescence intensity increases stepwise over time on days 2, 4, and 7.

[0069] In addition, human sapovirus infection was performed on a 364-well plate using a culture system of Vero cells and Vero cells + CD36 cells at an moi of 0.01. On day 7 of infection, both cultures were stained with methylene blue, and the viability of the cultured cells was visually observed under a microscope. This cytotoxicity test utilizes the fact that even a single particle of human sapovirus causes severe cytotoxicity, resulting in cell death on day 7 of infection, and the cells are no longer stainable with methylene blue. While this method cannot express the degree of CPE numerically like CellTiter-Glo® 2.0, it is an inexpensive and simple cytotoxicity test. As a result of this methylene blue staining test, in the Vero cell system 7 days after infection, methylene blue-stained cells were observed throughout the entire well, without any gaps. In contrast, in the Vero cell + CD36 system 7 days after infection, methylene blue-stained cells were observed to be significantly sparse within the well.

[0070] These results demonstrate that measuring CPE in animal cells transformed with the CD36 gene is a reliable detection system. Using a test drug in this system, it was found possible to screen the presence or absence and strength of the test drug's activity against human sapovirus using cytotoxicity (CPE) as an indicator. This method enables high-throughput screening of anti-human sapovirus drugs. In this first example (Figure 9), when a candidate anti-human sapovirus drug inhibits viral proliferation, cell death is suppressed and fluorescence is no longer observed. In this second example, when a candidate anti-human sapovirus drug inhibits viral proliferation, cell death is suppressed and the decrease in methylene blue-stained cells is suppressed.

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

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

[0073] In all sets (a)-(e), the infectious titer of human Sapovirus produced in cells transformed with the CD36 gene was several orders of magnitude higher than that of human Sapovirus in native cells. ５０ In (f), the native cells showed high TCID ５０However, by knocking out the CD36 gene (in this example, knocking out the CD36 gene using the CRISPR / Cas9 system), ５０ They found that the levels dropped by orders of magnitude (meaning the virus was no longer able to infect cells).

[0074] Figure 11 shows the TCID of human Sapovirus of each genotype grown in Vero cells + CD36 used in (d) above. ５０ The values ​​shown are the results obtained using Vero cells + CD36. Specifically, the TCID values ​​for the human sapovirus genotypes 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) are shown. ５０ The value was considered.

[0075] As a result, all genotypes of Sapovirus grown using Vero cells + CD36 showed high TCID ５０ It was shown to show value.

[0076] These results confirmed that the infectious titer of human sapovirus cultured in cells transformed with the CD36 gene is suitable for testing purposes, and that, for example, an infectious titer detection system such as that in this example is suitable for use in screening drugs (therapeutic drugs, etc.) related to human sapovirus.

[0077] 4. Study using intestinal organoids: Intestinal organoids naturally contain and function the CD36 gene, making them susceptible to infection by human sapovirus. We investigated whether knocking out the naturally occurring CD36 gene alters susceptibility to infection.

[0078] (1) Infection susceptibility test Figure 12 shows the results of intestinal organoids (provided by Keio University; Ileum-1 in the figure) infected with human sapovirus (GI.1 AH20 strain) and quantitative RT-PCR analysis of viral load over time. The susceptibility of test cells to human sapovirus infection was assessed using the number of copies of human sapovirus RNA from day 0 (0 dpi), 1 day later (1 dpi), and 4 days later (4 dpi) as an index (horizontal axis: "0 dpi, 1 dpi, 4 dpi").

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

[0080] In the first set, human sapovirus was confirmed to be produced over time. In the second set, no increase in human sapovirus production over time was observed. In the third set, human sapovirus production over time was observed to be one order of magnitude higher than in the first set. In the fourth set, human sapovirus production was observed at approximately the same level as in the third set.

[0081] These results confirmed that susceptibility of animal cells to human sapovirus infection is dependent on the CD36 gene, and that the CD36 gene conferred by transformation has a stronger effect on susceptibility to human sapovirus infection than the native CD36 gene.

[0082] (2) Infectious titer (TCID ５０ Measurement of Infectious Titer of Human Sapovirus (GI.1 AH20 strain) derived from the intestinal organoids of (1) above was determined using Vero cells + CD36 cells in the same manner as in 3(2) above.

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

[0084] TCID of human sapovirus derived from intestinal organoids in which the CD36 gene was knocked out ５０ The values ​​were extremely low, and the TCID of human sapovirus derived from native intestinal organoids ５０ Based on this value, the TCID of human sapovirus derived from intestinal organoids into which the CD36 gene was additionally introduced was ５０ The TCID values ​​of human sapovirus derived from intestinal organoids in which the CD36 gene was knocked out and transformed with the CD36 gene were very high. ５０ The values ​​were an order of magnitude greater than those of native intestinal organoids.

[0085] These results confirmed that even in animal cells that already have the CD36 gene present and functioning, making them susceptible to infection by human sapovirus, it is possible to obtain human sapovirus with extremely high infectious titers by further transforming the cells with the CD36 gene.

[0086] 5. Relationship to the Present Invention (1) Infection susceptibility test 1 using the susceptible clone in Example 2(3) above; infection susceptibility test 2 using HEK293 cells in Example 2(4); infection susceptibility tests using various cells in Infection Susceptibility Tests 3(a)-(d) in Example 2(5); and infection susceptibility test of intestinal organoids in Example 4(1); it was demonstrated that transforming mammalian cells with the CD36 gene confers susceptibility to human sapovirus infection. This result applies to the recombinant cells of the present invention. Furthermore, this result clearly demonstrates that it is possible to confer susceptibility to human sapovirus infection in living mammals (excluding humans) by introducing the CD36 gene into the cells of the mammal using standard methods, and this result also applies to the recombinant animals of the present invention.

[0087] (2) Using the steps disclosed in Infection Susceptibility Test 1 using the susceptible clone in Example 2(3) above; Infection Susceptibility Test 2 using HEK293 cells in Example 2(4); Infection Susceptibility Tests using various cells in Infection Susceptibility Tests 3(a)-(d) in Example 2(5); and Infection Susceptibility Test of Intestinal Organoids in Example 4(1), it was demonstrated that by transforming various mammalian cells with the CD36 gene, these recombinant mammalian cells were conferred susceptibility to human sapovirus infection. This result applies to the method of conferring infection susceptibility of the present invention.

[0088] (3) In the infectious titer measurement in Example 3(2) above, the human Sapoviruses produced in various recombinant cells of the present invention showed high TCID in Vero cells + CD36. ５０ In the infectious titer measurement in Example 4(2), the human Sapovirus produced in the intestinal organoids transformed with the CD36 gene also showed a high TCID value in Vero cells + CD36. ５０ These results apply to the method for producing Sapovirus of the present invention.

[0089] (4) The cytotoxicity measurement in Example 3(1) above and the infectious titer measurement in Example 4(2) above were all successful. The success of these measurement systems provides a basis for screening drugs and vaccines using these measurement systems. In other words, in these measurement systems, the effect of a test drug or vaccine on human sapovirus can be detected by first exposing the recombinant cells or recombinant animals of the present invention to the test drug or vaccine and then measuring the cytotoxicity or infectious titer. This result applies to the first screening method of the present invention, which targets drugs, and to the third screening method of the present invention, which targets vaccines. Furthermore, in these measurement systems, the effect of a test drug on human sapovirus can be detected by using the test drug and human sapovirus together. This result applies to the second screening method of the present invention.

Claims

1. A genetically modified mammalian cultured cell line that has been transformed with a gene encoding human CD36 and is susceptible to infection by human sapovirus.

2. The genetically modified mammalian cultured cells according to claim 1, characterized in that the mammalian cultured cells are established cultured cells, organoids derived from biopsy samples, immortalized cells, or iPS cells.

3. The genetically modified mammalian cultured cells according to claim 1, characterized in that the mammalian cultured cells are mammalian cultured cells of human, mouse, rat, hamster, guinea pig, rabbit, cat, dog, pig, or monkey origin.

4. The genetically modified mammalian cultured cells according to claim 1, wherein the mammalian cultured cells are HEK293T cells (human origin), Caco2 cells (human origin), HCT116 cells (human origin), Intestine407 cells (human origin), macrophage cultured cells 15310-LN cells (human origin), NALM-6 cells (human origin), HuTu80 cells (human origin), HT29 cells (human origin), MRC5 cells (human origin), RAW264.7 cells (mouse origin), NIH3T3 cells (mouse origin), M1 cells (mouse origin), BHK cells (hamster origin), CHO cells (hamster origin), CRFK cells (cat origin), MDCK cells (dog origin), PK-15 cells (pig origin), Vero cells (monkey origin), MA104 cells (monkey origin), GL37 cells (monkey origin), or COS7 cells (monkey origin).

5. A genetically modified mammal (excluding humans) that possesses the genetically modified mammalian cultured cells according to claim 1 as its own cells.

6. A method for producing human sapovirus, comprising infecting the genetically modified mammalian cultured cells of claim 1 or the genetically modified mammal (excluding humans) of claim 5 with human sapovirus, and allowing the human sapovirus to grow in the mammalian cells or mammal (excluding humans).

7. A method for conferring susceptibility to infection in mammalian cells or mammals (excluding humans) by introducing a gene encoding human CD36 into said mammalian cells or mammals (excluding humans), thereby conferring susceptibility to infection with human Sapovirus in said mammalian cells or mammals (excluding humans).

8. A screening method characterized by contacting a substance to be screened for its effect on human sapovirus with a genetically modified mammalian cultured cell as described in claim 1 or a genetically modified mammal (excluding humans) as described in claim 5 that has been infected with human sapovirus, and detecting the proliferation of human sapovirus in the mammalian cultured cell or mammal (excluding humans), thereby obtaining information on the effect of the substance to be screened.

9. The screening method according to claim 8, wherein the function to be screened is the inactivation of human sapovirus or the inhibition of its proliferation.

10. A screening method characterized by contacting a substance to be screened and human sapovirus with a genetically modified mammalian cultured cell as described in claim 1 or a genetically modified mammal (excluding humans) as described in claim 5, and detecting the invasion or proliferation of human sapovirus into the mammalian cultured cell or mammal (excluding humans), thereby obtaining information on the action of the substance to be screened.

11. The screening method according to claim 10, wherein the activity to be screened is an inhibitory effect on cell invasion by human sapovirus or an inhibitory effect on intracellular proliferation.

12. A screening method characterized by obtaining information on the action of the vaccine to be screened by immunizing the genetically modified mammalian cultured cells described in claim 1 or the genetically modified mammal (excluding humans) described in claim 5 with the vaccine to be screened, and detecting the protective effect against human sapovirus in the mammalian cultured cells or mammal (excluding humans).

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