SYSTEM FOR PROPAGATION OF HUMAN SAPOVIRUS USING Caco-2 CELLS TRANSFORMED WITH Cas9 GENE

Abstract

The present invention addresses the problem of providing a cultured cell line that has a constant susceptibility to infection by human sapovirus as well as providing measures for establishing said cultured cell line, and of contributing to research on human sapovirus infections and to the development of therapeutic and preventive measures; the inventors of the present invention achieved the present invention after finding that, by performing transformation using a Cas9 gene on Caco-2, which is an easily obtainable commercial cell line derived from human colon cancer, it is possible to acquire, as clones, cells having constant susceptibility to infection by human sapovirus, that a dramatically improved propagation efficiency of human sapovirus is observed in this human sapovirus infection-susceptible cell line, and that propagation by using a subculture is also possible.

Description

Human Sapovirus propagation system using cells transformed with the Cas9 gene of Caco-2

[0001] The present invention relates to transformed cells that can be used to culture human sapovirus in large quantities, and to the propagation of human sapovirus using such cells.

[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. Sapovirus genogroups GI, GII, GIV, and GV are human sapoviruses that infect humans, with GI being the most commonly detected, followed by GII. From 2019 to 2023, virus detections included 6,269 human noroviruses, 1,071 human sapoviruses, 885 rotaviruses, and 353 astroviruses, with human sapoviruses being the second most frequently detected virus after human noroviruses. The suspected infection routes and symptoms are difficult to distinguish between human noroviruses and human sapoviruses, and human sapoviruses are difficult to diagnose based on clinical symptoms. A major difference between human noroviruses and human sapoviruses is the structure of their genomic RNA (Figure 1). In human noroviruses, the nonstructural proteins and structural proteins (capsid protein, VP1) are encoded by separate ORFs, whereas in human sapoviruses, the nonstructural proteins and VP1 are encoded by the same ORF. Therefore, it is known that there are two routes for the expression of VP1 in human sapovirus: one in which VP1 is directly translated from the subgenome and expressed, and one in which VP1 is translated from ORF1 as a polyprotein containing VP1 and nonstructural proteins, and then cleaved by HuSaV's own protease or a protease derived from the host cell, resulting in expression.

[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] A culture system for human sapovirus using the human duodenal cancer cell line HuTu80 was finally reported in 2020 (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, these existing methods for propagating Sapovirus still have the problem that the amount of replication increase is small, making it difficult to passage human Sapovirus.

[0007] That is, the most recent human intestinal stem cell-derived organoid hIEOs (Non-Patent Document 2) and iPS cell-derived hIEOs (Non-Patent Document 3) have a smaller increase in human sapovirus replication compared to the reported HuTu80 value in Non-Patent Document 1, and passage of sapovirus is difficult even when using these cells. In particular, organoids derived from human intestinal stem cells are a system that reproduces intestinal epithelial cells in the human body, and are thought to be able to reproduce human sapovirus infection in the human body and subsequent intracellular replication, and are like a textbook on the life cycle from human sapovirus infection to intracellular replication and release of infectious particles. However, in the cell proliferation mode that maintains the stem cell state in the above organoids, they grow in cell clusters (spheroids), and if differentiation is induced as is, the inside becomes intestinal epithelium, making it infectible to human sapovirus. Therefore, measures have been taken to stop the proliferation of cells that have differentiated into intestinal epithelial cells by dispersing the spheroids into single cells using trypsin or the like, forming a monolayer, and then inducing differentiation. It is known that by doing so, only less than 10% of the monolayer becomes cells susceptible to human sapovirus infection. With this level of infection-susceptible cell ratio, human sapovirus in the culture supernatant or cell extract (lysate) cannot be passaged, making it virtually impossible to grow and culture sapovirus using organoids.

[0008] Next, a reproduction test of human sapovirus growth was carried out again using HuTu80, which is disclosed in Non-Patent Document 1 as being "capable of propagating human sapovirus at a level allowing passage." However, the growth efficiency of human sapovirus in HuTu80 was not very high, and at best only first-generation growth was achieved, with no passage growth possible. Thus, it cannot be said that a means for propagating human sapovirus at a level allowing passage has yet been established.

[0009] Starting from the aforementioned reproducibility test of human sapovirus growth using HuTu80, the present inventors conducted extensive research aimed at establishing a means for truly propagating human sapovirus at the serial culture level. As a result, they discovered that by transforming established human colon cancer-derived cells, "Caco-2," with the Cas9 gene, it is possible to obtain "transformed cells consistently susceptible to human sapovirus infection" with a certain probability. Furthermore, they discovered that by adding bile acids to such transformed cells susceptible to human sapovirus and culturing them with human sapovirus, human sapovirus can be propagated and serially propagated. This stable susceptibility to infection makes it possible to serially propagate human sapovirus. Furthermore, they discovered that, in order to improve the probability of obtaining cells consistently susceptible to human sapovirus infection, a first screening method can be used to select cells susceptible to or susceptible to infection with human astrovirus from a cell population of "Caco-2 transformed cells with the Cas9 gene."

[0010] This application is an international patent application claiming priority from a Japanese patent application (Patent Application No. 2024-58745) filed on April 1, 2024, and incorporates the entire contents of the priority application. In this application, the results of tests to confirm the invention disclosed in the priority application have been added to the priority application.

[0011] The present invention includes the following inventions.

[0012] First, the present invention provides Caco-2 cells transformed with the Cas9 gene (hereinafter also referred to as the Cas-transformed cells of the present invention) that have constant susceptibility to infection with human sapovirus. The term "constant susceptibility to infection with human sapovirus" means that even when the target Cas-transformed cells (clones) are repeatedly propagated, the propagated cells also have susceptibility to infection with human sapovirus; in other words, the proliferative ability of human sapovirus is maintained within the propagated cells. The Cas-transformed cells of the present invention preferably have susceptibility to infection or infection lethality (hereinafter abbreviated as infection susceptibility or lethality) with human astrovirus as a property.

[0013] Here, "infection susceptibility" refers to the property of target cells to accept a virus into the cell, regardless of whether or not this results in cell death. In some cases, cell death occurs as a result, and in other cases, the cells may survive despite being infected. Test methods for confirming infection susceptibility include flow cytometry, fluorescent immunostaining, and quantitative RT-PCR.

[0014] In contrast, "infectious lethality" refers to the property of a target cell dying as a result of the virus being introduced into the cell. Test methods for confirming infectious lethality include the cytopathic effect (CPE) observation method, the MTT test, the MTS test, etc.

[0015] In the present invention, the "cells" in "Caco-2 cells" and "Cas-transformed cells" are used with emphasis on "predetermined properties or contents," and may refer to one cell or multiple cells depending on the context. To emphasize the number, the term "one or more cells" refers to "one or more cells."

[0016] Furthermore, in the Cas-transformed cells of the present invention, it is preferable that cells consistently susceptible to infection with human sapovirus account for 50% or more of the total cell population in the directly amplified cell population, more preferably 70% or more, and most preferably 90% or more. In this preferred embodiment, the "cell" in the "amplified cell population" is a cell as a "unit of cell count," as described above, and is one cell. Furthermore, while a "cell population" is technically composed of "two or more cells," in reality it is a "cell constituting a cell culture" and is composed of so many cells that accurate counting is meaningless; if one were to specify a lower limit, it would be "approximately 100 cells," with no practical upper limit. Even if a cell population is a culture of a single clone, not all cells constituting the culture have the same properties. In particular, since Caco-2 cells are derived from a tumor (colon cancer), the Cas gene-transformed cells also tend to have the same properties.

[0017] Second, there is provided a method for propagating human sapovirus in Cas-transformed cells of the present invention (hereinafter also referred to as the propagation method of the present invention), which comprises contacting the cells with human sapovirus and then adding a bile acid to the cells, followed by culturing. It is preferable that all or part of the bile acid is glycocholic acid (GCA). It is also preferable that the cells are susceptible to or lethal to human astrovirus infection. The propagation method of the present invention can also be carried out as a serial propagation method.

[0018] Thirdly, the present invention provides a method for preparing a cell population in which the probability of the presence of cells that are consistently susceptible to infection by human Sapovirus is increased by obtaining cells that are susceptible to or lethal to infection by human Astrovirus from a cell population of Caco-2 cells transformed with the Cas9 gene; or a method for preparing a cell population in which the probability of the presence of cells that are consistently susceptible to infection by human Sapovirus is increased by obtaining cells that are susceptible to infection by human Astrovirus by transforming Caco-2 cells with the Cas9 gene (also referred to as a first screening method).

[0019] The present invention can be summarized as "the Cas-transformed cells of the present invention, and related means for propagating human sapovirus and their applications." The present invention relates to the yield of cells consistently susceptible to human sapovirus infection obtained from a cell population of Caco-2 cells transformed with the Cas9 gene. It is believed that the Cas-transformed cells of the present invention are cells consistently susceptible to human sapovirus infection due to random insertion of the Cas9 gene into the genetic sequence of Caco-2 cells. Furthermore, Caco-2 cells are originally a cell line derived from human colon cancer, possessing a certain degree of diversity. Therefore, a cell population of Caco-2 cells transformed with the Cas9 gene is not composed of a single clone, but is provided as a cell population containing cells with various characteristics. Therefore, the first screening method, an invention for increasing the proportion of cells consistently susceptible to the desired human sapovirus infection from a provided cell population of Caco-2 cells transformed with the Cas9 gene and using the resulting cells for propagating human sapovirus, is important.

[0020] Fourth, there is provided a method for obtaining cells that are consistently susceptible to infection with human sapovirus (hereinafter also referred to as the second screening method), which comprises contacting a population of Caco-2 cells transformed with the Cas9 gene with human sapovirus in the presence of bile acid, detecting susceptibility of each cell to human sapovirus infection, and obtaining cells for which susceptibility has been detected. It is efficient and preferable to perform the first screening method as a prerequisite for performing the second screening method.

[0021] While the first screening method is an initial selective screening method, the second screening method is an essential or final step, in which Caco-2 cells transformed with the Cas9 gene and consistently susceptible to human sapovirus infection are directly obtained as clones. It is also possible to select and obtain clones with high human sapovirus proliferation ability by focusing on the proliferation ability of individual cell units (clones), or to select and obtain clones suitable for the proliferation of a particular type of human sapovirus by focusing on the genotype, etc., of the human sapovirus. As originally disclosed in the Industrial Applicability section of this specification, repeated use of the second screening method can also increase the content of clones susceptible to human sapovirus infection when the obtained clones are propagated as a cell population. In Example 2, described below, it was confirmed that the first screening yielded a content of approximately 80%, and the second screening yielded a content of more than 90%. Conversely, it was also shown that by intentionally selecting clones with a low frequency of susceptibility to human sapovirus infection, it is possible to achieve a content ratio of clones susceptible to human sapovirus infection of only 0.05%, confirming that the acquisition of clones susceptible to human sapovirus infection (Cas-transformed cells of the present invention) using the second screening method is a process that involves non-accidental certainty.

[0022] As mentioned above, Caco-2 is a cell line derived from human colon cancer and is readily available commercially. When cultured on a porous membrane filter, Caco-2 cells exhibit morphological characteristics similar to those of small intestinal circular epithelial cells, such as brush borders and tight junctions, and form a monolayer that also expresses metabolic enzymes such as P-glycoprotein and peptidases.

[0023] The present invention first provides Caco-2 cells transformed with the Cas9 gene (Cas-transformed cells of the present invention) that have constant susceptibility to infection by human sapovirus, and further provides a cell population in which the Cas-transformed cell trait of the present invention has been propagated.

[0024] Secondly, a method for propagating human sapovirus is provided, which also allows for serial propagation of human sapovirus in the Cas-transformed cells of the present invention.

[0025] Thirdly, a method for preparing a cell population containing the Cas-transformed cells of the present invention is provided, which increases the probability of the presence of cells that have constant susceptibility to infection by human Sapovirus.

[0026] 1A and 1B are diagrams showing the genomic RNA structures of human sapovirus and human norovirus. Human sapovirus infection susceptibility was examined in various cells, including the Cas-transformed cells of the present invention, using the quantitative results of qRT-PCR as an indicator ((a)(b)). (c) shows the results of examining the relationship between the type of bile acid and the acquisition of infection susceptibility in the Cas-transformed cells of the present invention. (a)(b) shows the results of examining the possibility of passage in the human sapovirus propagation process in various cells, including the Cas-transformed cells of the present invention, using the quantitative results of qRT-PCR as an indicator. (a)(b) shows electron microscope images examining the presence and morphology of human sapovirus particles in the culture supernatant and lysate fractions of the Cas-transformed cells of the present invention infected with human sapovirus, and (b) shows electrophoretic images examining the detection of VP1 by Western blotting. (a) Confirmation of the absence of human sapovirus particles in the culture supernatant of HEK293T, where human sapovirus passage is difficult, and (b) confirmation of the absence of the original VP1 band by Western blotting. (b) Shows the results of flow cytometry showing the proportion of clones susceptible to human sapovirus infection in a cell population obtained by performing the second screening method on astrocyte-infected lethal Cas-transformed cells. (A) shows the results of a single run of the second screening method, while (B-1) and (B-2) show the results of flow cytometry of two of the clones obtained by performing the second screening method twice. Furthermore, (C) shows the results of two runs of selection of clones with a low frequency of human sapovirus infection-susceptible clones. (A) Time course of changes in human sapovirus RNA copy number and (B) TCID in cell lysates and culture supernatants of clone MC, a highly susceptible Caco-2 / Cas9 strain, infected with human sapovirus. ５０ (C) Phase-contrast microscopy images of clonal MC cultures (cell groups) from 0 to 7 dpi (upper panel: infected group, lower panel: uninfected group). The possibility of subculture using clonal MC of human sapovirus of each genotype (7 genotypes, 12 samples) was investigated based on the change in RNA copy number for each of the 12 samples.

[0027] 1. Cas-transformed cells of the present invention The Cas-transformed cells of the present invention are "Caco-2 transformed cells with the Cas9 gene, which have constant susceptibility to infection with human sapovirus." Transformed cells obtained by introducing the Cas9 gene into Caco-2 cells increase the probability of the presence of cells (clones) that are constantly susceptible to infection with human sapovirus in a population of cells grown using the transformed cells, and also significantly improve the growth efficiency of human sapovirus in such infection-susceptible cells.

[0028] The method for preparing Cas-transformed cells of the present invention involves, simply put, transformation of Caco-2 cells with the Cas9 gene to achieve "steady Cas9 expression." This transformation can be performed using currently commercially available "CRISPR / Cas9 construct delivery means." Examples include lentiviral vector plasmid DNA, adenoviral vector plasmid DNA, lentiviral transduction, and AAV transduction, and specific methods can be performed according to their respective protocols. For example, when using lentiviral vector plasmid DNA, this can be performed by infecting specific animal cells with the plasmid DNA to prepare Cas9 recombinant lentiviral particles, and then infecting Caco-2 cells with the recombinant lentiviral particles.

[0029] By using a drug resistance marker or the like incorporated into the above-mentioned plasmid DNA to select Caco-2 cells that grow even in the presence of the drug, "Caco-2 cells transformed with the Cas9 gene" can be obtained.

[0030] One of the characteristics of these "Caco-2 cells transformed with the Cas9 gene" is that the Cas9 gene is inserted at a random position on the gene. In contrast, the Cas-transformed cells of the present invention can be obtained by contacting the cells with human sapovirus in the presence of bile acid, detecting the susceptibility of each cell to human sapovirus infection, and obtaining cells in which the susceptibility to infection is consistently detected (second screening method: specifically, similar to the proliferation method of the present invention (described below)). However, when no measures are taken on a group of "Caco-2 cells transformed with the Cas9 gene," the proportion of "cells consistently susceptible to infection by human sapovirus" is low, and the yield of the screening is poor. As a means to solve this problem, cells susceptible to or lethal to human astrovirus infection are obtained from both or either of the pre-transformation Caco-2 cell population and the post-transformation "Caco-2 cell transformed with the Cas9 gene" cell population. By using these cells, the yield of "cells susceptible to human sapovirus infection" obtained in the second screening method can be significantly improved (first screening method). Caco-2 cells are known to be susceptible to or lethal to human astrovirus infection at a certain frequency. It has been found that these Caco-2 cells susceptible to or lethal to human astrovirus infection contain cells susceptible to human sapovirus infection at a relatively high frequency. Therefore, it is preferable to perform this first screening method as a prerequisite for the second screening method. Furthermore, when performing the first screening method, it is most preferable to perform it both before and after transformation with the Cas9 gene. If one of the methods is selected, it is preferable to perform it "after."In the first screening method, Caco-2 cells or the like are contacted with human astrovirus to obtain cells that are susceptible to or lethal to human astrovirus infection, and from these susceptible or lethal cells, when subjected to clonal proliferation, many clones, specifically clones in which 50% or more, preferably 80% or more of the cells are susceptible to or lethal to human astrovirus infection, are selected, and these are then preferably subjected to the above-mentioned second screening method to select cells in which susceptibility to human Sapovirus infection is consistently detected.

[0031] In this way, it is possible to prepare the Cas-transformed cells of the present invention, which serve as the basis for a population of Caco-2 cells transformed with the Cas9 gene, preferably in which cells in which susceptibility to human sapovirus infection is consistently detected account for 50% or more of the total cell population.

[0032] The ratio of "cells in which human Sapovirus infection susceptibility is consistently detected" in the Cas-transformed cell population of the present invention, i.e., the performance of the Cas-transformed cells of the present invention, can be improved by repeated cloning and screening (second screening method).

[0033] As shown in the Examples section, the present inventors have observed that, in Caco-2 cells (which have not been transformed), there are cell individuals that exhibit susceptibility to human sapovirus infection, albeit at a low probability, within the initial culture (cell population) of Caco-2 cells. The present inventors therefore suspected that stable susceptibility to human sapovirus might be observed in Caco-2 cells themselves, and conducted further investigations. However, even if the above-mentioned Caco-2 cell individuals that are susceptible to human sapovirus infection are susceptible to human sapovirus infection as one cell in a cell population composed of different clones, once the single cell is grown and converted into individual cell units (cloning), the susceptibility to human sapovirus observed in the cell population composed of different clones is lost, and it has been confirmed that "it is not possible to obtain from Caco-2 cells a cell clone in which constant susceptibility to human sapovirus infection is detectable."

[0034] 2. Propagation Method of the Present Invention The propagation method of the present invention is a method for propagating human sapovirus, which comprises contacting human sapovirus with the Cas-transformed cells of the present invention, which are "Caco-2 cells transformed with the Cas9 gene, which have cells in which infection susceptibility to human sapovirus is consistently detected," and then culturing the cells with the addition of bile acid. The Cas-transformed cells of the present invention are as described above.

[0035] The culture method for the Cas-transformed cells of the present invention, which is the basis for the proliferation method of the present invention, can be performed in accordance with the known culture method for Caco-2 cells. An example of this method is disclosed in the Examples section. Typically, the cells are cultured at 37°C and 5% CO. ２ Plate incubation is performed under the conditions below, but other conditions can also be selected.

[0036] In the growth method of the present invention, contacting the Cas-transformed cells of the present invention with human sapovirus and subsequent culturing are carried out in a manner similar to that for Caco-2 cells described above, typically by contacting the Cas-transformed cells of the present invention grown to confluence with human sapovirus, and then switching the culture medium to one containing bile acids immediately before or after the contact, while maintaining other standard culture conditions. By culturing for a predetermined period of time, the human sapovirus that has infected the Cas-transformed cells of the present invention grows within the Cas-transformed cells of the present invention and is released, or Sapovirus infection induces cell death, resulting in the culture supernatant and infected cell lysate containing the grown human sapovirus. The growth method of the present invention can be carried out by obtaining this culture supernatant and / or infected cell lysate containing human sapovirus. The obtained culture supernatant and / or lysate of infected cells is then contacted with "Cas-transformed cells of the present invention that are not infected with human sapovirus" and cultured in the presence of the above-mentioned bile acid. By repeating this subculture, human sapovirus can be grown extremely efficiently.

[0037] The type of bile acid is not particularly limited. Examples include glycocholic acid (GCA), glycochenodeoxycholic acid (GCDCA), cholic acid (CA), taurocholic acid (TCA), hyocholic acid (HCA), deoxycholic acid (DCA), lithocholic acid (LCA), hyodeoxycholic acid (HDCA), and ursodeoxycholic acid (UDCA). The bile acids (including the bile acids listed above) used in the proliferation method of the present invention can be selected individually or in mixtures of two or more. Bile extract (BEP) can also be used. Among these, glycocholic acid is a preferred bile acid, and it is preferable that all or part of the bile acids used be glycocholic acid. The amount of the bile acid added to the medium is not limited as long as it is an effective amount that promotes the proliferation of human sapovirus in the Cas-transformed cells of the present invention, but approximately 100 μM to 2000 μM is preferred. When a bile extract is used, it is preferably present in an amount of 0.5% by mass or more relative to the medium.

[0038] Furthermore, the culture time per passage unit is preferably a time period during which a sufficient amount of human sapovirus is contained in the culture supernatant and the infected cell lysate, and the amount of human sapovirus in the culture supernatant and the infected cell lysate does not increase proportionately with the increase in culture time even if the culture is continued for a longer period of time. Specifically, a period of about 3 to 10 days is preferable.

[0039] 3. Screening Methods of the Present Invention (1) First Screening Method As described above, the first screening method is a method for preparing a cell population in which the probability of the presence of cells susceptible to infection with human Sapovirus is increased by obtaining cells susceptible to or lethal to human astrovirus infection from a "cell population of cells transformed with the Cas9 gene of Caco-2 cells" (hereinafter referred to as the initial Cas-transformed cell population) (first screening method (1)), or a method for preparing a cell population in which the probability of the presence of cells susceptible to infection with human Sapovirus is increased by obtaining cells transformed with the Cas9 gene from Caco-2 cells susceptible to or lethal to human astrovirus (hereinafter referred to as astro-sensitive or lethal Caco-2 cells) (first screening method (2)).

[0040] <First screening method (1)> The initial Cas-transformed cell population is expected to contain Cas-transformed cells of the present invention that are susceptible to infection with human Sapovirus. The first screening method is a means for increasing the probability that the initial Cas-transformed cell population as a cell population will contain cells that are susceptible to infection with human Sapovirus.

[0041] Specifically, cells susceptible to or lethal to human astrovirus infection are obtained from the initial Cas-transformed cell population, and these are then prepared as a cell population containing the Cas-transformed cells of the present invention with a high probability. The initial Cas-transformed cells are cultured in Caco-2 cells in a conventional manner, preferably until they reach a confluent state. Then, human astrovirus is contacted with the cells, and cells that are infected with human astrovirus or that die upon infection are selected from the individual cells. These are then enriched as a cell population (hereinafter referred to as an astrovirus infection-susceptible or lethal Cas-transformed cell population). The cell population obtained by this first screening method has a significantly higher probability of containing cells consistently susceptible to human sapovirus infection than the initial Cas-transformed cell population.

[0042] That is, the first screening method can be performed by diluting the cell culture density so that individual initial Cas-transformed cells form independent cell groups in the culture medium as single clones, testing the cells that make up these mutually independent Cas-transformed clone cell groups (clonal populations) for susceptibility to or lethality against human astrovirus for each cell group, and screening cell groups to which clone populations that are found to be susceptible to or lethal against human astrovirus at a predetermined frequency (50% or more, preferably 80% or more) belong as "clonal cell groups of astrovirus-infected susceptible or lethal Cas-transformed cells."

[0043] <First screening method (2)> Astrovirus infection-susceptible or lethal Caco-2 cells are expected to have a high probability of originally having factors that make them susceptible to human sapovirus infection. Conversely, astrovirus infection-susceptible or lethal cell populations within a cell population of cells transformed with the Cas9 gene are expected to have a high probability of containing the Cas-transformed cells of the present invention.

[0044] Astrovirus infection-susceptible or lethal Caco-2 cells can be obtained by performing a procedure on Caco-2 cells similar to the method for obtaining cells that are susceptible to or lethal to human astrovirus infection in the initial Cas-transformed cell group in the first screening method (1) above.

[0045] The method for transforming astrocyte-sensitive or lethal Caco-2 cells with the Cas9 gene is as described above in the section on Cas-transformed cells of the present invention.

[0046] (2) Second Screening Method As described above, the second screening method is a method for obtaining cells that have constant susceptibility to infection with human sapovirus, by contacting a group of cells transformed with the Cas9 gene of Caco-2 cells (an initial Cas-transformed cell group, or an astroinfection-susceptible or lethal Cas-transformed cell group) with human sapovirus in the presence of bile acid, detecting the susceptibility of each cell to human sapovirus infection, and obtaining cells for which the susceptibility to infection has been detected.

[0047] The "cell population of cells transformed with the Cas9 gene of Caco-2 cells" used in the second screening method is preferably an astroinfection-susceptible or lethal Cas-transformed cell population in which the probability of the presence of cells consistently susceptible to infection with the desired human sapovirus is increased, rather than using an initial Cas-transformed cell population.

[0048] The culture method (using bile acids) and human sapovirus infection method for the "Caco-2 cell population transformed with the Cas9 gene" used in the second screening method are the same as those used in the propagation method of the present invention. The second screening method can be performed by obtaining cells (clones) susceptible to human sapovirus infection from the propagated "Caco-2 cell population transformed with the Cas9 gene." In the second screening method, a clone cell population independently propagated as a clone is selected as a population. If at least 50%, preferably 70%, and more preferably 90% of the clone cell population is found to be susceptible to human sapovirus infection, the clone cell population in the population is a Cas-transformed cell population of the present invention. Because Caco-2 cells are originally tumor cells and exhibit diverse properties, it is difficult to efficiently confer susceptibility to human sapovirus infection in the propagated cell population. Furthermore, even if approximately 20% of the cells happen to exhibit susceptibility to human Sapovirus infection, this occurs because the cells interact with one another as a cell population exhibiting diverse properties, and their properties as cells consistently exhibiting Sapovirus susceptibility are not constant. Furthermore, even if cells that exhibit susceptibility to Sapovirus infection are cloned from that population, they will not be able to maintain consistent susceptibility to Sapovirus infection because their properties are initialized upon cell isolation. The cells used in the second screening are a population of cells (clones) that are susceptible to human Sapovirus infection from a "cell population of cells transformed with the Cas9 gene" so as to consistently exhibit Sapovirus susceptibility, and only cells that consistently exhibit Sapovirus susceptibility without losing susceptibility to human Sapovirus infection can be cloned.

[0049] Examples of the present invention will be disclosed below. In the examples, the percentages indicating the contents are by mass unless otherwise specified.

[0050] 1. Cells and human sapovirus strains used in the examples (1) Known cells The human duodenal cancer cell line HuTu80 was purchased from ATCC. HuTu80 was cultured in Iscove's Modified Dulbecco's Medium (IMDM: SIGMA, 13390-500ML) containing 1% GlutaMAX-I (Gibco, 35050-061), 1% penicillin-streptomycin (Nacalai Tesque, 26253-84), and 5% heat-inactivated fetal bovine serum (FBS: CAPRICORN, FBS-12A) at 37°C and 5% CO ２ The cells were cultured under the conditions.

[0051] The human embryonic kidney cell line HEK293T was purchased from ATCC and was cultured in Dulbecco's Modified Eagle Medium (DMEM: Nacalai Tesque, 08459-64) containing 1% penicillin-streptomycin and 10% FBS at 37°C and 5% CO. ２ The cells were cultured under the conditions.

[0052] Intestinal stem cell-derived organoids (hIEOs) were provided by Professor Toshiro Sato of the Keio University School of Medicine. hIEOs were cultured in Matrigel Matrix (CORNING, 356231) using IntestiCult OGM Human Basal Medium (VERITAS, ST-06010) containing 1% penicillin-streptomycin to form spheroids. The cells were then maintained in a dedifferentiated state during expansion. To induce differentiation of hIEOs, cells were seeded on a 96-well plate coated with collagen from human placenta (SIGMA, C5533-5MG) prepared at 1 mg / mL with 0.1 M acetic acid (Nacalai Tesque, 37307-15) and diluted 30-fold with distilled water (DW). The cells were then cultured in ENRA medium (Advanced DMEM / F12 (Gibco, 12634-010) containing 1% GlutaMAX-I, 10 mM HEPES (Gibco, 15630-080), and 1% penicillin-streptomycin, supplemented with 50 ng / mL EGF (Invitrogen, The cells were cultured in a medium containing 1% B27 (Invitrogen, #17504-044), 10 nM Gastrin (Sigma-Aldrich, G9145-1MG), 10% Noggin (Peprotech, #250-38), 10% R-spondine 1 (R&D, #4645-RS), 1 mM nAc (Sigma-Aldrich, A9165-5G), and 0.5 μM A83 (Tocris, #2939) at 37°C and 5% CO ２ The cells were cultured under these conditions to acquire susceptibility to human sapovirus infection and used in infection experiments.

[0053] Human colon carcinoma-derived Caco-2 cells were purchased from ATCC and cultured in Minimum Essential Media (MEM: Nacalai Tesque, 21442-25) containing 1 mM sodium pyruvate (Nacalai Tesque, 06977-34), 1% MEM Non-Essential Amino Acids (Nacalai Tesque, 06344-56), 1% Antibiotic-Antimycotic (Gibco, 15240-062), and 10% FBS at 37°C and 5% CO. ２ The human colon cancer-derived cells HCT15, HCT116, and C2BBe1 were also purchased from ATCC and cultured in the same manner as Caco-2 and used in the study.

[0054] 2. Methods Used in the Examples (1) qRT-PCR qRT-PCR was performed to quantify the human sapovirus genome copy number in the culture supernatant using the Norovirus Detection Kit GI / GII - High-Speed ​​Probe Detection Quick Step - (TOYOBO, FIK-273), substituting primers and probes for those used to detect human sapovirus RNA. Table 1 shows the primers and probes used. An 8 μM primer solution was prepared by mixing the forward primer and reverse primer and diluting them with DW, and a 3 μM probe solution was prepared by diluting the probe with DW. Next, 2.5 μL each of the primer solution, probe solution, and enzyme were mixed with 15 μL of the reaction solution to prepare a reaction buffer. 2 μL of the sample and 6 μL of the pretreatment solution were mixed, and 2 μL of this mixture was added to the reaction buffer. qRT-PCR was performed under the conditions in Table 2 using a LightCycler 96 (Roche).

[0055]

[0056]

[0057] (2) Western Blotting: The sample was mixed with an equal volume of SDS sample buffer and subjected to protein denaturation at 100°C for 10 minutes. After polyacrylamide gel electrophoresis using a 5-20% e-PAGEL (ATTO, E-R520L), the protein was transferred to a PVDF membrane (Bio Rad, 1704156) using a Trans-Blot Turbo (Bio Rad). The PVDF membrane was blocked at room temperature for 60 minutes using PVDF Blocking Reagent (TOYOBO, NYPBR01). The PVDF membrane was washed three times with PBS-T (D-PBS(-) containing 0.05% Tween 20 (Nacalai Tesque, 35624-15)), and Gp2 anti-HuSaV VLP was diluted 1000-fold with Can Get Signal Immunoreaction Enhancer Solution 1 (TOYOBO, NKB-101) as a primary antibody solution, which was then added to the PVDF membrane and allowed to react at room temperature for 60 minutes. After removing the primary antibody solution and washing three times with PBS-T, Goat pAb to Gpig IgG (HRP) (abcam, ab6908) was diluted 10,000-fold with Can Get Signal Immunoreaction Enhancer Solution 2 to serve as the secondary antibody solution, which was added to the PVDF membrane and allowed to react for 60 minutes at room temperature. After washing the PVDF membrane three times with PBS-T, the membrane was illuminated with Chemi-Lumi One L Solution A / B (nacalai tesque, 07880-54) and photographed with a ChemiDoc touch (Bio Rad).

[0058] (3) TCID ５０ Assay In Example 2, the infectious titer of human Sapovirus in clone MC (described below) was measured in TCID ５０ Clonal MC cultures (cell populations) were cultured in 96-well plates at 2 × 10 ４ Cells were seeded at 1000 x g / well and inoculated with human sapovirus in 5-fold serial dilutions. ２ After 5 days of incubation under 5°C, the virus infectivity was assessed by immunofluorescence assay and the TCID was determined by the Reed & Muench method. ５０values ​​were calculated.

[0059] The immunofluorescence assay was performed as follows: Human Sapovirus-infected cells were washed twice with D-PBS(-) and fixed with 4% paraformaldehyde for 15 minutes. After fixation, the cells were washed twice more and permeabilized with D-PBS(-) containing 0.1% Triton X-100 for 15 minutes at room temperature. After washing, the cells were blocked with D-PBS(-) containing 1% bovine serum albumin (BSA) for 60 minutes at room temperature. Next, the cells were incubated with the primary antibody (guinea pig anti-SaV VP1 hyperimmune serum (1:1000 dilution) raised against human Sapovirus GI.1 VLP) for 60 minutes at 37°C. After washing, the cells were incubated with Alexa Fluor® 488-conjugated goat anti-guinea pig secondary antibody (1:5000 dilution) (Invitrogen, MA, USA) for 30 minutes at room temperature in the dark. Hoechst dye was used for nuclear staining (1:10000 dilution). Cells were visualized using a BZ-X810 imaging system (Keyence, Osaka, Japan).

[0060] (4) Flow Cytometry For flow cytometry analysis in Example 2, cells infected with human sapovirus GI.1 at 4 dpi were used. After removing the culture supernatant, the cells were washed with D-PBS(-) and detached for 10 minutes using 0.5 g / L trypsin / 0.53 mmol / L EDTA (Nacalai Tesque). The cells were then pelleted by centrifugation, fixed with 4% formaldehyde for 15 minutes, and permeabilized with methanol for 10 minutes at 4°C using an Intra Cellular Flow Cytometry kit (Cell Signaling Technology, Massachusetts, USA). After blocking with 1% bovine serum albumin (BSA) in D-PBS(-) for 60 minutes, the cells were incubated overnight at 4°C with guinea pig anti-SaV VP1 hyperimmune serum (1:1,000 dilution). After three washes, the cells were incubated with Alexa Fluor® 488-conjugated goat anti-guinea pig secondary antibody (1:1,000 dilution) for 60 minutes at room temperature. After washing, the cells were filtered through a Falcon 5 mL polystyrene round-bottom tube with a 35 μm cell strainer cap (Corning, New York, USA) and analyzed using a BD FACSMelody cell sorter (BD Biosciences, California, USA). Data analysis was performed using FlowJo software (BD Biosciences).

[0061] Example 1: Creation of Caco-2 / Cas9 and investigation of its properties 1. Creation of astrocyte-sensitive Cas-transformed cells Caco-2 cells, which are human colon cancer-derived cells obtained from ATCC, were cultured in EMEM medium (Wako Pure Chemical Industries, Ltd., 051-07615) supplemented with 10% fetal bovine serum (Biosera, Inc., 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 Caco-2 cells were detached from the dishes using 250-300 μL of 0.25% trypsin-1 mmol / L EDTA 4Na solution (containing phenol red) (Wako Pure Chemical Industries, Ltd., 209-16941).

[0062] Caco-2 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 Caco-2 cells with lentivirus at an MOI of 1, thereby introducing the Cas9 expression cassette into the cells. 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 Caco-2 cells.

[0063] 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.

[0064] Two months later, cell cloning was performed to select cell clones with high lethality in human astrovirus infection (clones in which 80% of the expanded cells were lethal to human astrovirus infection). These clones were then infected with human astrovirus, and lethal clones in human astrovirus infection were further selected (astrovirus-infected lethal Cas-transformed cells: first screening method (1)).

[0065] As a result, six clones were selected as astrocyte lethal 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 lethal Cas-transformed cells consistently possessed susceptibility to infection with human sapovirus. Hereinafter, in the Examples section, unless otherwise specified, this will be abbreviated as "Caco-2 / Cas9."

[0066] 2. Purification of virus particles Human sapovirus particles were purified from the culture supernatant and cell lysate of Caco-2 / Cas9 infected with human sapovirus. ８The lysate of hpi168 cells was used. hpi168 cells were scraped with a cell scraper (BMBio, 99003) and centrifuged at 7,000 × g for 10 minutes to separate the culture supernatant and pellet. To remove lipid components, the pellet was vigorously suspended in FBS-free MEM (ZW(+)MEM(-)) containing 0.5% ZWITTERGENT (Merck Millipore, 693017-5GM, adjusted to 5% with DW) to disrupt the cell membrane and elute the virus particles. The lysate was then centrifuged at 15,000 × g for 10 minutes, and the supernatant, from which cell debris had been removed, was used for sucrose density gradient centrifugation. The culture supernatant and lysate were transferred to Ultra-Clear Tubes (25 x 89 mm) (Beckman Coulter, 344058) and diluted to 30 mL. 5 mL of MEM(-) containing 30% sucrose (Nacalai Tesque, 30404-45) was placed at the bottom of the tube. The tubes were then ultracentrifuged at 174,900 x g for 2 hours using an SW32Ti rotor and an ultracentrifuge (Beckman Coulter, A94471). MEM(-) was added to the culture supernatant pellet, and ZW(+)MEM(-) was added to the lysate pellet, and the mixture was allowed to soak overnight at 4°C. The softened sample was suspended and centrifuged at 15,000 × g for 10 minutes. The supernatant, after removing cellular debris, was used for cesium chloride (CsCl) density gradient centrifugation. For CsCl density gradient centrifugation, the sample was first mixed with 2.2 g of CsCl (Nacalai Tesque, 07807-11) and diluted to 5 mL with MEM(-) or ZW(+)MEM(-). The sample was transferred to an Ultra-Clear Tube (13 × 51 mm) (Beckman Coulter, 344057) and subjected to ultracentrifugation at 148,900 × g for 24 hours using an SW55Ti rotor and ultracentrifuge to purify virus particles. The gradient was fractionated into 12 samples of 417 μL each, and each fraction was transferred to an Ultra-Clear Tube (13 × 51 mm). The fraction was made up to 5 mL with MEM(-) and ultracentrifuged in an SW55Ti rotor at 367,600 × g for 2 hours to remove CsCl and ZWITTERGENT components and concentrate the virus particles.The pellet was soaked in MEM(-), and the sample was used for Western blotting and electron microscopic observation.

[0067] In addition to Caco-2 / Cas9, human sapovirus particles were purified from the culture supernatant of HEK293T. 60 mL of culture supernatant was used for purification. The purification process was the same as for Caco-2 / Cas9 up to sucrose density gradient centrifugation, followed by OptiPrep density gradient centrifugation. Specifically, OptiPrep (Cosmo Bio, AXS1114542) was first diluted 1.2-fold, 1.5-fold, 3-fold, and 6-fold with DW to prepare solutions, and 1 mL of each solution was layered into an Ultra-Clear Tube (13 x 51 mm). The sample was placed on top of the 6-fold diluted solution, and the virus particles were purified by ultracentrifugation at 367,600 x g for 2 hours using an SW55Ti rotor and ultracentrifuge. The purified human sapovirus particles between the 1.5-fold and 3-fold diluted solutions were collected in a Microfuge Tube Polypropylene (Beckman Coulter, 357448) and concentrated by ultracentrifugation at 186,000 × g for 2 hours using a TLA55 rotor and ultracentrifuge (Beckman Coulter, A95761). The pellet was soaked in MEM(-), and the sample was used for Western blotting and electron microscopy.

[0068] 3. Relationship between susceptibility to human sapovirus infection and bile acids Various cells seeded on plates were cultured until confluent and used in infection experiments. At the time of infection, the amount of FBS in the medium was changed from 5% to 3% for IMDM and from 10% to 2% for MEM. The above human sapovirus strain (HuSaV GI.1 AH20 strain) was inoculated for 10 min. ６The virus solution was adjusted to a concentration of 1 genome copy / μL. The prepared virus solution was filtered through a Millex-HV Syringe Filter Unit, 0.45 μm (Merck Millipore, SLHVR33RS), and then glycochenodeoxycholic acid (GCDCA: SIGMA, G0759-100MG prepared in D-PBS(-)), glycocholic acid (GCA: Nacalai Tesque, 17123-51 prepared in DW), and bile extract (BEP: SIGMA, B8631-100MG prepared in DW) were added to the solution at concentrations of 500 μM, 1000 μM, and 0.5%, respectively. After virus inoculation, the cells were cultured for 3 hours (Caco-2 / Cas9) or 24 hours (HuTu80, hIEOs, HEK293T). After washing three times with Dulbecco's phosphate-buffered saline (D-PBS(-): Nacalai Tesque, 14249-24), fresh medium supplemented with the bile acids was added and the cells were cultured for 7 days (168 h: hpi168). The HuSaV genome copy number in the culture supernatant up to 168 hours post infection (hpi) was quantified by qRT-PCR. A control experiment without bile acids was also performed.

[0069] The results are shown in Figure 2.

[0070] Figures 2(a) and (b) show the results of quantification of the human sapovirus copy number in the culture supernatant by qRT-PCR when various cells were inoculated with a human sapovirus strain (HuSaV GI.1 AH20) in the presence of 1000 μM glycocholate and cultured for 24 hours (a) or 3 hours (n=5). After removing the inoculum, the cells were washed three times, and fresh medium was added. The cells were cultured for 168 hours. The results are shown relative to the genome copy number in the initial culture [(a) is hpi24, (b) is hpi3].

[0071] qRT-PCR results showed that hIEOs, HuTu80, and HEK293T showed a 58-fold, 109-fold, and 243-fold increase in replication by hpi72, respectively, and maintained this increase until hpi168 (Figure 2(a)). Caco-2 / Cas9 and Caco-2 showed a 2191-fold and 2304-fold increase in replication by hpi48, respectively, and increased to 96260-fold and 3478-fold at hpi168 (Figure 2(b)). HCT15, HCT116, and C2BBe1 all showed a 5-fold or less increase in replication by hpi168 (Figure 2(b)).

[0072] From the above results, although human sapovirus (HuSaV GI.1 AH20 strain) showed a slight increase in replication in hIEOs, HuTu80, and HEK293T, the infection susceptibility was not sufficient to practically grow human sapovirus. In contrast, it was shown that replication increased highly efficiently in Caco-2 and Caco-2 / Cas9. However, subsequent studies revealed that Caco-2 cells lose the initially observed susceptibility to human sapovirus infection during the cell cloning process. Therefore, the cells that were found to be susceptible to practical infection by human sapovirus were narrowed down to Caco-2 / Cas9, which showed a highly efficient increase in human sapovirus replication.

[0073] Figure 2(c) shows the results of an investigation into the effect of bile acids on human sapovirus infection and proliferation in Caco-2 / Cas9. Cultures were performed up to hpi168 in a medium containing the same bile components as used at the time of inoculation, and the genome copy number of human sapovirus in the culture supernatant was quantified by qRT-PCR. qRT-PCR results showed that GCA, GCDCA, and BEP showed a 5640-fold, 2140-fold, and 646-fold increase in replication, respectively, by hpi72, and a 9913-fold, 4864-fold, and 1815-fold increase by hpi168. In the absence of bile acids, the increase in replication up to hpi168 was less than 10-fold.

[0074] The above results demonstrate that the increase in human sapovirus replication is promoted by bile acids, and that the increase is most efficient in the presence of GCA.

[0075] 4. Investigation into the Possibility of Passaging Human Sapoviruses Passage was investigated for Caco-2 / Cas9, Caco-2, HuTu80, and HEK293T.

[0076] HuTu80 and HEK293T were passaged in 6-well plates, while Caco-2 and Caco-2 / Cas9 were passaged in Tissue Culture Flask VTC-F75V (VIOLAMO, 2-8589-02). The time point at which the cells were infected with human sapovirus (AH20, GI.1) was designated as passage 0 (P0). Confluently seeded cells were inoculated with 1 mL (Caco-2, Caco-2 / Cas9) or 500 μL (HuTu80, HEK293T) of the P0 hpi168 culture supernatant in the presence of 1000 μM GCA, and cultured for 3 or 24 hours. After washing the cells, new medium containing bile acids was added and the cells were cultured until hpi168, up to passage 5 (P5). The number of human sapovirus genome copies in the culture supernatant was quantified by qRT-PCR to determine whether the passage was successful. The results are shown in Figure 3.

[0077] As a result of qRT-PCR, an increase in replication was detected in both Caco-2 / Cas9 and Caco-2 from P0 to P5 (Figures 3(a) and 3(b)). That is, the number of HuSaV genome copies in hpi168 from P0 to P5 was 10 in Caco-2 / Cas9. ６ -10 ７ genome copies / μL, and 10 in Caco-2 ４ -10 ６ The genome copies / μL was approximately 100 times higher than hpi3 at all time points, and a 100-fold or greater increase in replication was confirmed compared to hpi3. On the other hand, HuTu80 and HEK293T showed a 100-fold or greater increase in replication at P0, but no increase thereafter. However, as mentioned above, subsequent verification revealed that it was difficult to establish Caco-2 as a cell line (clone) capable of amplifying human sapovirus.

[0078] These results demonstrate that only Caco-2 / Cas9 can amplify human sapovirus by passage.

[0079] 5. Intracellular Virus Particle Production and Extracellular Release Based on the results above, we used Caco-2 / Cas9, which allows human sapovirus passage, and HEK293T, which does not allow passage, to perform electron microscopy of virus particles purified from the culture supernatant and lysate (only culture supernatant for HEK293T). Electron microscopy revealed virus-like structures with irregular surfaces in both the Caco-2 / Cas9 culture supernatant and lysate (Figure 4(a)). Specifically, in the culture supernatant, a clean particle structure was observed only in fraction 1 (Fraction 1); no particle structure was observed in fractions (F) 2, 9, and 10. A distorted structure was observed in F2. In contrast, in the lysate, more clean virus particles were observed in fractions (F) 9, 10, and 11 than in the culture supernatant. As a result of Western blotting, in addition to the fractions in which particulate structures were observed in Caco-2 / Cas9 (culture supernatant Fraction 1; lysate F9, 10, 11), a band of approximately 60 kDa was also detected in culture supernatant F2 (FIG. 4(b)).

[0080] In contrast, no virus-like structures were observed in the HEK293T culture supernatants in electron microscopy images, and many structures of different sizes and shapes were observed (Fig. 5(a)). Furthermore, Western blotting detected a VP1 band, but it was not present at the 60 kDa position ("feces" lane), which is the original size of VP1, but was detected at positions of 250 kDa and 130 kDa (Fig. 5(b)).

[0081] These results suggest that human sapovirus replication increased with Caco-2 / Cas9 is synthesized intracellularly as infectious particles with a structure similar to that of fecal virus particles, and then partially released outside the cells. In contrast, no virus particles with normal VP1 size were detected in the culture supernatant of HEK293T cells.

[0082] Example 2: Creation of Caco-2 / Cas9 highly susceptible to human sapovirus infection and examination of its properties 1. Confirmation of human sapovirus susceptibility in Caco-2 / Cas9 In order to confirm the proportion of clones that are susceptible to infection with human sapovirus [HuSaV AH20 strain (GI.1): stored in the Laboratory of Viral Infection Control, Omura Satoshi Memorial Institute] in the astrocyte-infected lethal Cas-transformed cells obtained in Example 1, an examination was performed using flow cytometry.

[0083] Specifically, the parental strain stock (astrocytically infected lethal Cas-transformed cells) and Caco-2 cells were exposed to the HuSaV AH20 strain (GI.1) in a fecal suspension environment (containing bile acids) for 4 days, and flow cytometry was performed on these cells. Guinea pig anti-SaV GI.1VP1 hyperimmune serum was used as the primary antibody, and Alexa Fuor 488-conjugated anti-guinea pig goat IgG was used as the secondary antibody. Uninfected cells were represented by blue fluorescence, while infected cells were represented by red fluorescence, as indicated by flow cytometry. The percentage of infection-susceptible cells in Caco-2 cells (infected cells / infected cells + uninfected cells × 100) was 19.1%, compared with 38.8% for the parental strain stock.

[0084] 2. Creation of Caco-2 / Cas9 with High Susceptibility to Human Sapovirus Infection As described above, the proportion of cells susceptible to human sapovirus infection in the parent strain stock was only 38.8%, so an optimization step (second screening method) was further carried out with the aim of enhancing susceptibility to human sapovirus infection.

[0085] This involves the selection of HuSaV-susceptible clones obtained by contact with human sapovirus, as was done with the parent strain stock secured in Example 1. Specifically, the current Caco-2 / Cas9 cells are cultured at a diluted density so that individual cells form independent cell populations in the medium as single clones, and the cells constituting each of these independent Caco-2 / Cas9 cell populations (clonal populations) are tested for susceptibility to human sapovirus (the above-mentioned HuSaV AH20 strain (GI.1)) using flow cytometry. Cell populations containing clonal populations with a high rate of susceptibility to human sapovirus infection are screened and obtained as "human sapovirus infection-susceptible Caco-2 / Cas9 clonal cell populations." This process can be repeated until the infection susceptibility rate is observed at a predetermined frequency (50% or more, preferably 70% or more, and preferably 90% or more). In this example, the above optimization process was performed twice on the parent strain stock, thereby obtaining the desired Caco-2 / Cas9 strain with high susceptibility to infection with human sapovirus.

[0086] The first cycle was an optimization step for the HuSaV-susceptible clone group obtained at a frequency of 38.8% among the constituent clones of the parent strain stock secured in Example 1. Among the 16 cell groups designated A-P, clone M (Caco-2M) showed the highest fluorescence signal (79.9%) against VP1 by flow cytometry (Figure 6(A)).

[0087] In the second cycle, the same optimization process as in the first cycle was performed on the clone M (Caco-2M). Among the 16 cell groups designated MA-MP, clone MC (Caco-2MC) and clone ME (Caco-2ME) showed the highest fluorescence signal (91.5%) against VP1 by flow cytometry (Figures 6(B-1) and (B-2)). Of these, clone MC (Caco-2MC) was designated as "Caco-2 / Cas9 highly susceptible to human sapovirus infection" and used for the infection test in Example 2 below. Furthermore, this clone MC was internationally deposited with the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (NIET) under the identification label "Caco-2MC cells" (International Receipt Number: NITE ABP-04354). The susceptibility of this clone MC (Caco-2MC) to human sapovirus infection remains unchanged up to at least 79 passages. Furthermore, it has been determined that this clone MC (Caco-2MC) does not express the blasticidin resistance protein or Cas9 protein. Therefore, clone MC (Caco-2MC) is a cell that cannot be selectively cultured using blasticidin or subjected to genome-wide knockout using Cas9.

[0088] We also investigated whether clones with low sensitivity to human sapovirus could be intentionally obtained using the above process. Specifically, clone P (Caco-2P), which had the lowest VP1 fluorescence among the 16 cell groups designated AP in the first cycle, was further subjected to the optimization process in the second cycle. Among the 20 cell groups designated PA-PT, clone PG was found to be a low-sensitivity clone with a VP1 fluorescence signal of only 0.05% (Figure 6(C)).

[0089] Thus, it was confirmed that the second screening method is a screening method that can non-accidentally obtain clones (Cas-transformed cells of the present invention) that are susceptible to human sapovirus in Caco-2 / Cas9 and can be reproduced with an extremely high probability.

[0090] 3. Dynamics of RNA copy number and infectivity after human sapovirus infection in Caco-2 / Cas9, a strain highly susceptible to human sapovirus. Electron microscopy observations in Example 1 revealed that human sapovirus was abundantly present within cells rather than in the culture supernatant after infection with Caco-2 / Cas9. To confirm this in Caco-2 / Cas9, a strain highly susceptible to human sapovirus, we examined the dynamics of human sapovirus RNA copy number and infectivity in the culture supernatant and intracellular space of clone MC. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) and median tissue culture infectious dose (TCID50) assays were performed, and these parameters were compared over time. Clone MC was infected with HuSaV AH20 strain (GI.1) at a multiplicity of infection (MOI) of 0.01.

[0091] As a result, the number of human sapovirus RNA copies in the culture supernatant was 2.0 × 10 at 0 dpi. ２ From copies / μL, 4.2 × 10 at 7 dpi ５ In cell lysates, the number of human sapovirus RNA copies increased steadily to 6.9 × 10 at 0 dpi. ３ From copies / μL, 5.4 × 10 at 5 dpi ７ The number of copies / μL increased by 3.9 log10 fold and remained stable up to 7 dpi (6 dpi: 2.9 × 10 ７ Copies / μL, 7 dpi: 8.5 × 10 ７ copies / μL) (Figure 7(A)).

[0092] TCID ５０ The assay showed that the infectious titers of the culture supernatant and cell lysate gradually increased from 1 dpi, peaked at 5 dpi, and then slightly decreased at 6 and 7 dpi (Fig. 7(B)). At 5 dpi, the infectious titer of the cell lysate was 6.4 × 10 ７ TCID ５０ / mL, and the culture supernatant (1.0 × 10 ５ TCID ５０ / mL).

[0093] Clone MC infected with HuSaV AH20 strain (GI.1) began to show cytopathic effect (CPE) around 3 days after infection, and cell detachment was observed on days 6 and 7 after infection (Fig. 7(C)). In the control, uninfected clone MC, no significant CPE was observed even 7 days after infection.

[0094] These results confirmed that the tendency observed in Example 1 above, that "human sapovirus after infection is abundantly present within the cells rather than in the culture supernatant of Caco-2 / Cas9," was also observed in highly infection-susceptible clones.

[0095] 4. Serial passage of multiple genotypes of human Sapovirus in highly susceptible clones. Cell lysates were prepared from clone MC infected and propagated with multiple genotypes of human Sapovirus. These cell lysates were then used to infect clone MC again, according to their genotype, and serial passage of the human Sapovirus was attempted for each genotype.

[0096] Specifically, 50 μL of human sapovirus-positive stool suspension and 450 μL of medium were inoculated into the clonal MC cultures (cell populations) in the wells of two 12-well plates, and the cultures were incubated at 37°C and 5% CO ２ The mixture was incubated under reduced pressure for 3 hours.

[0097] The above-mentioned human sapovirus-positive stool suspension was prepared by dissolving an appropriate amount of feces (classified by the following genotype) collected from Japanese patients infected with human sapovirus in a solution of Mg ２＋ and Ca ２＋ The fecal suspension was thoroughly mixed and centrifuged at 5000 × g for 10 minutes, and the supernatant was frozen and stored at −80°C in individual tubes as a human sapovirus-positive fecal suspension.

[0098] The breakdown of genotyped strains of the human sapovirus-positive stool suspensions used is as follows (a)-(g). The numbers in parentheses indicate the date of fecal sample collection for each positive stool suspension. (a) GI. type 1: AH20 (February 2020), M13-18 (November 2013), Ni17-9 (March 2017), and E22-22 (May 2022) (b) GI. type 2: M13-6 (June 2013) (c) GI. type 3: I21-042 (June 2021) (d) GII. type 1: TKC19-5 (November 2019) and TKC19-10 (November 2019) (e) GII. Type 3: I22-124 (January 2023) (f) GIV. Type 1: KA-31 (2013) and IB-02 (2008) (g) GV. Type 1: EH-40 (2014) and IB-24 (2014)

[0099] After the incubation, the cells in the wells were washed three times with D-PBS(-), and 1 mL of medium containing 1000 μM GCA was added. Then, the cells were incubated at 37°C, 5% CO ２ At 5 dpi, the culture supernatant was removed and 1 mL of DW was added to extract the virus.

[0100] For the genotypes GI.1, GI.2, and GI.3, the virus was diluted with medium to an MOI of 0.01, and 500 μL of the prepared virus suspension (supplemented with 1000 μM GCA) was inoculated to infect a fresh layer of confluent clonal MC cultures (cell populations). For the genotypes GII.1, GII.3, GIV.1, and GV.1, 500 μL of the virus suspension (supplemented with 1000 μM GCA) adjusted to the same RNA copy number as GI.1 (M13-18) was inoculated onto the cells, and infection was carried out in the same manner as above. The cells infected with human sapovirus in this manner were incubated at 37°C and 5% CO. ２ The cells were cultured under the same conditions until 5 dpi. At the end of the culture at 5 dpi, the culture supernatant was removed, and 1 mL of DW was added to extract the virus. This passage was repeated for two cycles (P+2), and the number of human sapovirus RNA copies was measured at 0 dpi and 5 dpi for each passage cycle.

[0101] The results are shown in Figure 8. The 12 graphs in Figure 8 represent the RNA copy number counts (logarithmic) for the genotyped samples. P+0 represents the RNA copy number at the time of initial infection; P+1 represents the RNA copy number at the first passage cycle; and P+2 represents the RNA copy number at the second passage cycle. For each cycle, the left bar represents the RNA copy number at 0 dpi, and the right bar represents the RNA copy number at 5 dpi. In all 10 samples, except for two GIV.1 samples, a significant increase in human sapovirus RNA copy number was observed from 0 dpi to 5 dpi by the second passage (P+2) (1.7-4.4 log 10-fold increase), indicating successful passage. In contrast, in the GIV.1 sample, the RNA copy number was maintained after inoculation with a fecal suspension, but further passages were not possible. This result deviates to a limited extent from reasonable expectations when compared with the results of other samples. The GIV.1 samples used here were It is possible that the virus in one sample was destroyed due to poor storage conditions and could not withstand the stress of passage.

[0102] The industrial-level advantages of the Cas-transformed cells of the present invention compared to the organoids derived from human intestinal stem cells (abbreviated as "organoids" in this section) described above are as follows: (1) While the use of organoids is subject to medical ethical restrictions, the use of the Cas-transformed cells of the present invention is free from such restrictions and can be used freely for research. (2) When organoid clones are grown as cell groups, the presence rate of cells susceptible to human sapovirus infection is at most about 10%, whereas in the Cas-transformed cells of the present invention, this is 50% or more. By repeatedly performing the second screening method described above, it is possible to obtain clones with this presence rate increased to 80% or more, even close to 100%. This is as shown in the clone MC of Example 2. (3) The culture medium used to culture organoids requires expensive supplements and reagents such as R-spondin, HGF, and Matrigel, and is very expensive, costing approximately 150,000 yen for 100 mL. However, the culture medium for the Cas-transformed cells of the present invention is inexpensive, costing approximately 2,000 yen for 500 mL. (4) Organoids must be maintained as spheroids and then monolayered to induce differentiation. Further monolayering stops cell proliferation, but the Cas-transformed cells of the present invention can be maintained by conventional subculture. (5) While organoids are limited to human sapovirus infectious titer testing using 96-well plates, the Cas-transformed cells of the present invention can be used to perform human sapovirus infectious titer testing using 384-well plates, making this extremely efficient. (6) While organoids cannot be used to generate human sapovirus using reverse genetics, this is easily achieved with the Cas-transformed cells of the present invention. (7) It is difficult to use human sapovirus propagated in organoids as an antigen for ELISA, etc., but it is easy to do so in the Cas-transformed cells of the present invention. (8) In organoids, the cells are fragile and difficult to maintain, making it difficult to propagate viruses with manipulated human sapovirus genomes. However, in the Cas-transformed cells of the present invention, it is easy to do so because ordinary maintenance methods and gene transfer methods can be used.(9) While only those contained in fecal samples can be used as the base for human sapovirus propagation in organoids, the Cas-transformed cells of the present invention allow for the passage of self-propagated human sapovirus. (10) Human sapovirus propagated in the Cas-transformed cells of the present invention can be easily identified at the genetic sequence level and can be used as a seed virus for research, drug screening, and other purposes. (11) Quantification of human sapovirus in the Cas-transformed cells of the present invention by qRT-PCR can be achieved as quickly as 24 hours after infection, whereas organoids require as little as three days. (12) Human sapovirus genome cDNA or RNA sequences can be introduced into the Cas-transformed cells of the present invention to produce infectious virus particles or virus-like hollow particles, which can then be used. In contrast, organoids are inherently prone to cellular alteration due to the introduction of cDNA or RNA, and specialized equipment is required to introduce these nucleic acids while maintaining normal organoid function. Even if infectious viruses are formed from small amounts of nucleic acid introduced into organoids using such special procedures, if the nascent viruses cannot be passaged, it is difficult to propagate and utilize the viruses. Therefore, as described above, in organoids where human sapovirus passage is virtually impossible, it is impossible to produce infectious virus particles by introducing cDNA or RNA from the human sapovirus genome. Because virus-like hollow particles cannot self-replicate, their production in organoids is inherently difficult. (13) By introducing guide RNA (gRNA) capable of randomly knocking out all human sapovirus genes into the Cas-transformed cells of the present invention, it is possible to search for host cell factors, including receptors, essential for human sapovirus replication and proliferation. However, this is difficult for the same reasons as in (12) above, because the virus propagation mode in organoids is virtually impossible to passage. (14) The Cas-transformed cells of the present invention can be used as an evaluation system for growth inhibitors and disinfectants against human sapovirus and viruses that use human sapovirus as a surrogate, such as human norovirus. However, this is difficult to achieve with organoids, which cannot be passaged or titered.

Claims

1. Caco-2 cells transformed with the Cas9 gene, which are constitutively susceptible to infection by human sapovirus.

2. The transformed cell according to claim 1, which exhibits susceptibility to or lethality upon infection with human astrovirus.

3. The transformed cell according to claim 1, wherein the number of cells consistently susceptible to infection with human Sapovirus accounts for 50% or more of the total number of cells in a directly amplified cell population of the transformed cell.

4. A method for growing human sapovirus in Caco-2 cells transformed with the Cas9 gene, which are constantly susceptible to infection by human sapovirus, by contacting the cells with human sapovirus and culturing them with the addition of bile acids.

5. A method for propagating a human sapovirus according to claim 4, wherein the bile acid is glycocholic acid (GCA) in whole or in part.

6. A method for propagating a human sapovirus according to claim 4 or 5, wherein the propagation method is a passage propagation method.

7. A method for preparing a cell population with an increased probability of containing cells that are consistently susceptible to infection with human sapovirus, by obtaining cells that are susceptible to or lethal to infection with human astrovirus from a cell population of Caco-2 cells transformed with the Cas9 gene.

8. A method for preparing a cell population with an increased probability of containing cells that consistently exhibit susceptibility to infection with human sapovirus, by obtaining cells by transforming Caco-2 cells, which are susceptible to or lethal to infection with human astrovirus, with the Cas9 gene.

9. A method for obtaining cells that are consistently susceptible to infection with human sapovirus, comprising contacting a group of Caco-2 cells transformed with the Cas9 gene with human sapovirus in the presence of bile acid, detecting the susceptibility of each cell to infection with human sapovirus, and obtaining cells for which susceptibility to infection has been detected.

10. The method for obtaining cells described in claim 9, wherein the cells constituting the cell population of Caco-2 cells transformed with the Cas9 gene are cells that are susceptible to or lethal to infection with human astrovirus.

11. Caco-2 cells transformed with the Cas9 gene, which are identified as Caco-2MC cells and have the international receipt number NITE ABP-04354 and are constantly susceptible to infection by human sapovirus.

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