Humanized Cell Lines

Abstract

Mammalian or avian cell lines that express human influenza virus receptors at high levels are provided. In one embodiment, the cell lines support the isolation and propagation of human influenza viruses, e.g., human A / H3 influenza viruses, much more efficiently than in corresponding conventional (unmodified) cells or corresponding human virus receptor-overexpressing cells, and the propagated viruses are able to maintain greater genetic stability than in the corresponding cells.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the filing date of U.S. Application No. 62 / 803,266, filed February 8, 2019, the disclosure of which is incorporated herein by reference.

[0002] Declaration of Government Rights This invention was made with Government support funded by the National Institutes of Health under HHSN272201400008C. The Government has certain rights in this invention. [Background technology]

[0003] Influenza A and B viruses have two major surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). HA recognizes sialic acid-containing receptors on the cell surface, whereas NA cleaves sialic acid from the cell surface receptor, facilitating the release of progeny virions from the surface of infected cells (Gamblin and Skehel, 2010). HA is also the major antigen that stimulates host immunity, especially the production of neutralizing antibodies.

[0004] Virus isolation from clinical specimens is an important tool for the identification and characterization of circulating viruses. Currently, two subtypes of influenza A virus (A / H1N1 and A / H3N2) and two lineages of influenza B virus (B / Yamagata and B / Victoria lineages) are circulating in human populations and causing seasonal influenza epidemics. Madin-Darby canine kidney (MDCK) cells are the most widely used cell line for the isolation and propagation of human influenza viruses. Although this cell line shows great susceptibility to influenza viruses, it only poorly supports the growth of recent A / H3N2 viruses. Moreover, passaging of influenza viruses in MDCK cells often leads to the selection of variants with mutations in their HA and / or NA genes (Chambers et al., 2014; Lee et al., 2013; Tamura et al., 2013; Lin et al., 2017; Li et al., 2009; Oh et al., 2008). The emergence of such variants with mutations important for influenza virus adaptation to cell culture may distort the evaluation of antigenic, genetic, and antiviral properties of circulating influenza viruses. For example, the emergence of mutations that confer receptor-binding activity to the NA of A / H3N2 viruses (e.g., aspartic acid to glycine substitution at position 151 (D151G) (Moh et al., 2015; Lin et al., 2010; Zhu et al., 2012)) is problematic for characterization of HA antigenicity by hemagglutination inhibition assays, virus neutralization assays, and infected cell foci reduction assays, because the receptor-binding activity of NA contributes to the results of these assays. Nevertheless, many laboratories use MDCK cells to isolate A / H3N2 viruses. An analysis of the GISAID EpiFlu database by Lee et al. (2013) found that approximately 30% of A / H3N2 isolates cultured in MDCK had an amino acid change at position 151. Therefore, currently circulating A / H3N2 strains should be isolated and propagated in cell lines that can faithfully maintain their characteristics.

[0005] Human influenza virus HAs preferentially bind to glycans that terminate with sialic acid linked to galactose via an α2,6-linkage, whereas avian virus HAs preferentially bind to glycans that terminate with sialic acid linked to galactose via an α2,3-linkage (Connor et al., 1994; Rogers and Paulson, 1983; Stevens et al., 2006). Correspondingly, epithelial cells in the human upper respiratory tract predominantly express α2,6-sialoglycan (van Riel et al., 2006; Shinya et al., 2006). Although MDCK cells expressing both α2,6-sialoglycan and α2,3-sialoglycan are suitable for isolating influenza viruses from many animal species, this cell line has been shown to express relatively low levels of α2,6-sialoglycan (Lin et al., 2017; Hatakeyama et al., 2005; Matrosovich et al., 2003). Previously, our group and others have engineered MDCK cells to overexpress α2,6-sialoglycan (Hatakeyama et al., 2005; Matrosovich et al., 2003). These engineered MDCK cells (named AX4 or MDCK-SIAT1) showed greater susceptibility to human influenza virus isolates than the conventional MDCK cell line (Oh et al., 2008; Hatakeyama et al., 2005), but still expressed α2,3-sialoglycan. Importantly, similar to conventional MDCK cells, variants with mutations in the HA or NA have been detected when seasonal influenza viruses were passaged through MDCK-SIAT1 cells (Tamura et al., 2013; Li et al., 2009). Therefore, alternative cell systems that support efficient isolation and growth of human influenza viruses without any cell culture adaptation mutations are needed for accurate characterization of circulating viruses and, potentially, for effective vaccine production in cells. Summary of the Invention

[0006] The present disclosure relates to genetically modified mammalian or avian cell lines to support, for example, more efficient isolation and / or amplification (propagation) of human influenza viruses, particularly human H3 influenza viruses. The disclosed cell lines can be genetically modified to reduce cell surface expression of alpha-2,3-linked sialic acid and / or increase expression of alpha-2,6-linked sialic acid compared to a parent cell line that has not been modified to alter expression of alpha-2,3-linked sialic acid, alpha-2,6-linked sialic acid, or both. In one embodiment, the modified mammalian or avian cell line is modified to express high levels of human influenza virus receptors and low levels of influenza virus receptors. In one embodiment, the cell line is a mammalian cell line (e.g., a non-human cell line, such as a primate cell line or a canine cell line). In one embodiment, the modified cell line is a modified MDCK cell line that has reduced expression of alpha-2,3-linked sialic acid compared to AX-4 or increased expression of alpha-2,6-linked sialic acid compared to an unmodified MDCK cell line. In one embodiment, the modified cell line is hCK, which supports more efficient isolation and amplification of human influenza virus compared to MDCK and AX-4 cells. In one embodiment, the reduced expression of alpha-2,3-linked sialic acid is due to a genetic modification that reduces or eliminates expression of one or more sialyltransferases that produce alpha-2,3-linked sialic acid, non-limiting examples of genetic modifications include an insertion of one or more nucleotides in one or more sialyltransferase genes, a deletion of one or more nucleotides, a substitution of one or more nucleotides, or any combination thereof. In one embodiment, the genetic modification comprises an insertion of one or more nucleotides in one or more sialyltransferase genes. In one embodiment, the genetic modification comprises a deletion of one or more nucleotides in one or more sialyltransferase genes. In one embodiment, the genetic modification comprises a substitution of one or more nucleotides in one or more sialyltransferase genes.In one embodiment, the genetic modification comprises an insertion of one or more nucleotides in at least one sialyltransferase gene. In one embodiment, the genetic modification comprises a deletion of one or more nucleotides in at least one sialyltransferase gene. In one embodiment, the genetic modification comprises a substitution of one or more nucleotides in at least one sialyltransferase gene. The genetic modification to reduce expression of alpha-2,3-linked sialic acid can be the result of any method to "knock down" or "knock out" expression, including the use of recombinase systems (such as CRISPR / Cas, TALEN, or zinc finger binding proteins). In one embodiment, the increased expression of alpha-2,6-linked sialic acid results from a genetic modification that increases the expression of one or more sialyltransferase genes that produce alpha-2,6-linked sialic acid, non-limiting examples of which include an expression cassette comprising a nucleotide sequence encoding a sialyltransferase that produces alpha-2,6-linked sialic acid (e.g., the human β-galactoside α2,6-sialyltransferase I (ST6Gal I) gene).

[0007] In one embodiment, an isolated recombinant mammalian or avian cell is provided that comprises a reduced amount of cell surface β-galactoside α2,3-sialyl residues and an increased amount of human β-galactoside α2,6-sialyl residues compared to a corresponding non-recombinant mammalian or avian cell. In one embodiment, the isolated recombinant cell is a non-human mammalian cell. In one embodiment, the isolated recombinant cell is a canine cell or a non-human primate cell. In one embodiment, the decrease in the amount of surface β-galactoside α2,3-sialyl residues is the result of a decrease in the amount or activity of one or more α2,3 sialyltransferases (e.g., the amount or activity of one or more α2,3 sialyltransferases may be decreased by at least 5%, 10%, 20%, 50%, 70%, 80%, 90%, 95% or more in the recombinant cell, which may result in at least 5%, 10%, 20%, 50%, 70%, 80%, 90%, 95% or more decrease in 2,3-sialyl residues). In one embodiment, the amount or activity of α2,3 sialyltransferase or the amount of α2,3 sialyl residues in the recombinant cell is not detectable. In one embodiment, the modified α2,3 sialyltransferase gene encodes an α2,3 sialyltransferase that is at least 80%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical in amino acid sequence to any one of SEQ ID NOs: 6, 8, 10, 12, 14, 16, or 18, or encodes a nucleotide sequence that is at least 80%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical in nucleotide acid sequence to any one of SEQ ID NOs: 5, 7, 9, 11, 13, 15, or 17. In one embodiment, the isolated recombinant cell comprises an expression cassette encoding human β-galactoside α2,6-sialyltransferase I (ST6Gal I) or ST6Gal II. In one embodiment, ST6Gal I or ST6Gal II comprises a protein having at least 80% amino acid sequence identity to any one of SEQ ID NOs: 1-4, 101 or 150.In one embodiment, the α2,6-sialyltransferase gene encodes an α2,6-sialyltransferase that has at least 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to any one of SEQ ID NOs: 1-4, 101, or 150, or encodes a nucleotide sequence that has at least 80%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% nucleotide acid sequence identity to SEQ ID NO: 101 or 151. In one embodiment, the amount or activity of human β-galactoside α2,6-sialyltransferase in the recombinant cell is increased by at least 1%, 5%, 10%, 20%, 50%, 70%, 80%, 90%, 95%, or more. In one embodiment, one or more β-galactoside α2,3-sialyltransferase genes are mutated to reduce the amount of cell surface β-galactoside α2,3-sialyl residues. In one embodiment, two or more of the ST3Gal-I gene, ST3Gal-II gene, ST3Gal-III gene, ST3Gal-IV gene, ST3Gal-V gene, ST3Gal-VI gene, or ST3Gal-II-like gene are mutated. In one embodiment, three, four, five, six, or seven of the ST3Gal-I gene, ST3Gal-II gene, ST3Gal-III gene, ST3Gal-IV gene, ST3Gal-V gene, ST3Gal-VI gene, or ST3Gal-II-like gene are mutated. In one embodiment, the ST3 gene has a nucleic acid sequence that is at least 80% identical to any one of SEQ ID NOs: 5, 7, 9, 11, 13, 15, or 17. In one embodiment, the reduction in cell surface β-galactoside α2,3-sialyl residues is the result of reduced expression of one or more ST3 sialyltransferases, in one embodiment, the one or more ST3 sialyltransferases have an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 6, 8, 10, 12, 14, 16, or 18. In one embodiment, the influenza H3 virus replicates more efficiently in recombinant cells than in non-recombinant cells.

[0008] Also provided is an isolated recombinant mammalian or avian cell comprising a reduced amount of cell surface β-galactoside α2,3-sialyl residues compared to a corresponding non-recombinant mammalian or avian cell. In one embodiment, one or more of the ST3Gal-I, ST3Gal-II, ST3Gal-III, ST3Gal-IV, ST3Gal-V, ST3Gal-VI, or ST3Gal-II-like genes in the recombinant cell are mutated. In one embodiment, a combination of the ST3Gal-I, ST3Gal-II, ST3Gal-III, ST3Gal-IV, ST3Gal-V, ST3Gal-VI, or ST3Gal-II-like genes in the recombinant cell are mutated. In one embodiment, the ST3Gal-I gene, the ST3Gal-II gene, the ST3Gal-III gene, the ST3Gal-IV gene, the ST3Gal-V gene, the ST3Gal-VI gene, and the ST3Gal-II-like gene in the recombinant cell are mutated.

[0009] The recombinant cell described herein is useful, for example, for virus isolation, vaccine production, and diagnosis. For example, the recombinant cell allows progeny virus isolation and / or amplification. Furthermore, HA assay is generally not used to detect recent human H3N2 viruses. The recombinant cell may be advantageous in this respect (e.g., virus amplification).

[0010] Furthermore, recombinant cells with increased β-galactoside α2,6-sialyl residues can be used as a source of isolated β-galactoside α2,6-sialyl residues, which can in turn be used to coat surfaces (such as beads) to inhibit galectins, and to isolate or detect elderberry agglutinin (SNA), elderberry (SSA), or trichosanthes agglutinin I (TJA-I).

[0011] In one embodiment, a method is provided for altering the amount of cell surface β-galactoside α2,3-sialyl residues and human β-galactoside α2,6-sialyl residues in a mammalian or avian cell. In one embodiment, the method comprises mutating one or more β-galactoside α2,3 sialyltransferase (ST3Gal) genes and overexpressing a human β-galactoside α2,6 sialyltransferase (ST6Gal) gene in a parent mammalian or avian cell, resulting in a modified mammalian or avian cell having a reduced amount of cell surface β-galactoside α2,3 sialyl residues and an increased amount of human β-galactoside α2,6 sialyl residues on its surface compared to the corresponding parent cell. In one embodiment, the one or more ST6Gal genes are mutated using a genome editing system, such as a CRISPR / Cas9 system, a zinc finger nuclease (ZFN) system, or a transcription activator-like effector nuclease (TALEN) system. In one embodiment, the mutation comprises an insertion of one or more nucleotides, or a deletion of one or more nucleotides, or both, in one or more ST3 genes. In one embodiment, the modified cell comprises an expression cassette comprising an ST6Gal open reading frame. In one embodiment, the modified cell is a kidney cell. In one embodiment, the modified cell is a canine cell.

[0012] Methods utilizing recombinant cells include methods for propagating influenza viruses (e.g., human influenza viruses) for vaccine production. In one embodiment, the influenza virus is an influenza A virus. In one embodiment, the influenza virus is an influenza B virus. In one embodiment, the influenza virus is an H3 virus. In one embodiment, the virus is an A / H1N1, A / H3N2, B / Yamagata lineage influenza B virus, or B / Victoria lineage influenza B virus.

[0013] Yet another method utilizing recombinant cells is a method for isolating influenza virus. The method includes providing a sample from a bird or mammal suspected of being infected with influenza virus; and contacting the sample with recombinant cells. In one embodiment, the method further includes determining whether the sample is infected with influenza virus. In one embodiment, the method further includes identifying the HA and / or NA subtype of the virus.

[0014] In one embodiment, the cell line is a modified MDCK cell line ("hCK" means "humanized MDCK" cells) prepared using CRISPR / Cas-mediated gene knockout techniques to downregulate multiple sialyltransferases that catalyze the synthesis of alpha-2,3-linked sialic acid and overexpress one sialyltransferase that catalyzes the synthesis of alpha-2,6-linked sialic acid. hCK cells express low levels of alpha-2,3-linked sialic acid (similar to human epithelial cells of the upper respiratory tract) and high levels of alpha-2,6-linked sialic acid. As disclosed herein, hCK cells allow for the isolation of H3N2 human influenza viruses 10-100 times better than the AX-4 cell line. Efficient isolation and amplification of influenza viruses, including human influenza viruses, is advantageous (presumably better supporting replication) for vaccine production (e.g., vaccine production of seasonal influenza viruses). This is because seasonal human influenza viruses often replicate inefficiently in unmodified MDCK cells, and even in MDCK(AX-4) cells, which overexpress alpha-2,6-linked sialic acid on their surface and to which human influenza viruses bind efficiently. In one embodiment, the titers of human influenza viruses on the surface of the modified cell lines disclosed herein are at least one order of magnitude, at least two orders of magnitude, at least three orders of magnitude, or more, greater than in unmodified MDCK, MDCK(AX-4), or MDCK-SIAT1 cells (Li et al., 2009). [Brief description of the drawings]

[0015] [Figure 1A-1B] Figure 1A-P. Sensitivity of hCK cells to human influenza virus growth and isolation. A-L) Growth kinetics of seasonal influenza viruses in MDCK, AX-4, and hCK cells. MDCK, AX-4, and hCK cells were infected with viruses at a multiplicity of infection (MOI) of 0.002. Supernatants from infected cells were harvested at the indicated time points, and virus titers were determined by plaque assay in hCK cells. Error bars indicate standard deviations from three independent experiments. P values ​​were calculated using the linear mixed model described in Methods (*P<0.05; **P<0.01). Red and blue asterisks indicate comparisons between hCK and AX4 cells and MDCK cells. Grey asterisks indicate comparisons between the cell lines shown in red and blue. M-P) Comparative sensitivity of hCK and AX4 cells to seasonal influenza viruses. Serial two-fold dilutions (21 to 220) of clinical samples were prepared and inoculated into AX4 and hCK cells. Cells were observed for the development of cytopathic effect (CPE) for 7 days. Triplicate wells were inoculated with each virus dilution. The horizontal bar indicates the highest dilution of CPE among all three wells. The numbers at the end of each horizontal bar indicate the ratio of the highest dilution of hCK to the highest dilution of AX4. [Fig. 1C-1D] Same as above. [Fig. 1E-1F] Same as above. [Fig. 1G-1H] Same as above. [Fig. 1I-1J] Same as above. [Fig. 1K-1L] Same as above. [Fig. 1M-1N] Same as above. [Fig. 1O-1P] Same as above.

[0016] [Figure 2A] FIG. 2A-B. Overall schematic of the generation of MDCK cells expressing significantly low levels of α2,3-linked sialic acid and high levels of α2,6-linked sialic acid. [Figure 2B] Same as above.

[0017] [Figure 3A-3B] Figure 3A-C. Flow cytometry analysis of cell surface expression of α2,6-linked sialic acid and α2,3-linked sialic acid. Clones 6-11 (orange outline) and parental cells (black outline) were incubated with biotinylated Malus domestica II agglutinin (MAL II) lectin (specific for α2,3-linked sialic acid) or Sambucus nigra agglutinin (SNA) lectin (specific for α2,6-linked sialic acid) followed by incubation with Alexa 488-labeled streptavidin and then analyzed by flow cytometry. Unstained cells served as negative controls (no lectin). [Figure 3C] Same as above.

[0018] [Figure 4A-4B]Figure 4A-J. Characterization of MDCK cells expressing significantly lower levels of α2,3-linked sialic acid and higher levels of α2,6-linked sialic acid. MDCK cells carrying mutations in seven different β-galactoside α2,3 sialyltransferase (ST3Gal) genes were generated by utilizing the CRISPR / Cas9 genome editing system as described in Methods. Edited MDCK cells were further modified to overexpress human β-galactoside α2,6 sialyltransferase I (ST6Gal-I) by transfection of a plasmid containing the ST6Gal-I gene. Modified cell clones were selected by puromycin and blasticidin and characterized. A-D) Flow cytometric analysis of cell surface expression of α2,6-linked sialic acid and α2,3-linked sialic acid. Engineered MDCK cells (green and red outlines) and parental MDCK cells (black outlines) were incubated with biotinylated Malus domestica II agglutinin (MAL II) lectin (specific for α2,3-linked sialic acid) or Sambucus nigra agglutinin (SNA) lectin (specific for α2,6-linked sialic acid) followed by incubation with Alexa 488-labeled streptavidin and then analyzed by flow cytometry. E-G) Immunofluorescence analysis of the expression of α2,3-linked sialic acid. Engineered and parental MDCK cells were fixed and stained with a monoclonal antibody recognizing Siaα2,3Galβ1,4GlcNAc (green). Nuclei were stained with Hoechst dye (blue). H and J) Flow cytometric analysis of cell surface expression of α2,6-linked sialic acid. Engineered MDCK cells (red outline), parental MDCK cells (black outline), and AX4 cells (blue outline) were incubated with SNA lectin or elderberry (SSA) lectin (specific for α2,6-linked sialic acid) followed by incubation with streptavidin labeled with Alexa 488 and then analyzed by flow cytometry. I) Flow cytometric analysis of cell surface expression of α2,3-linked sialic acid.Modified MDCK cells (red outline), parental MDCK cells (black outline), and AX4 cells (blue outline) were incubated with MAL II lectin followed by incubation with Alexa 488-labeled streptavidin and then analyzed by flow cytometry. [Fig. 4C-4D] Same as above. [Fig. 4E-4G] Same as above. [Fig. 4H-4I] Same as above. [Figure 4J] Same as above.

[0019] [Figure 5A-5C] Figure 5A-F. Location of amino acid changes in the HA and NA proteins. A-C) The three-dimensional structures of A / California / 04 / 2009 (H1N1pdm) HA (PDB ID: 3UBN), A / Wyoming / 3 / 2003 (H3N2) HA (PDB ID: 6BKR), and B / Hong Kong / 8 / 1973 HA (PDB ID: 2RFU) in complex with the human receptor analog are shown. Mutations identified in this study are shown in red. Mutations in influenza A HA are shown with H3 numbering. D-F) The three-dimensional structures of A / California / 04 / 2009 (H1N1pdm) NA (PDB ID: 3TI6), A / Tanzania / 205 / 2010 (H3N2) NA (PDB ID: 4GZP), and B / Brisbane / 60 / 2008 NA (PDB ID: 4CPM) in complex with oseltamivir carboxylate are shown. Mutations identified in this study are shown in red. All mutations are shown with N2 numbering. Figures were created using DS Visualizer v17.2. [Fig. 5D-5F] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] definition

[0021] A "vector" or "construct" (sometimes referred to as a "vehicle" for gene delivery or transduction) refers to a macromolecule or molecular complex that contains a polynucleotide that is delivered to a host cell in vitro or in vivo. The polynucleotide to be delivered can contain a coding sequence of interest or a sequence for introducing a mutation into the genome of the host cell, or both. Vectors include, for example, plasmids, viral vectors (such as adenoviruses, adeno-associated viruses (AAV), lentiviruses, herpes viruses, and retroviruses), liposomes and other lipid-containing complexes, and other macromolecular complexes that can mediate delivery of a polynucleotide to a host cell. Vectors can also contain other components or functions that further alter gene delivery and / or gene expression or that otherwise provide beneficial properties to the targeted cells. Such other components include, for example, components that affect binding or targeting to cells, including components that mediate cell type or tissue specific binding; components that affect uptake of the vector's nucleic acid by cells; components that affect the location of the polynucleotide within the cell after uptake, such as agents that mediate nuclear localization; and components that affect expression of the polynucleotide. Such components may also include markers, such as detectable and / or selectable markers, that can be used to detect or select cells that have taken up and are expressing the nucleic acid delivered by the vector. Such components can be provided as natural features of the vector, such as the use of some viral vectors that have components or functions that mediate binding and uptake, or the vector can be modified to provide such functions. A wide variety of such vectors are known in the art and are generally available. When the vector is maintained in a host cell, it can be stably replicated by the cell during mitosis, integrated into the genome of the host cell, or maintained in the nucleus or cytoplasm of the host cell.

[0022] "Recombinant viral vector" refers to a viral vector that contains one or more heterologous genes or sequences. Because many viral vectors exhibit size constraints associated with packaging, the heterologous genes or sequences are typically introduced by replacing one or more portions of the viral genome. Such viruses can become replication-deficient (biologically inhibited), and therefore the deleted functions must be provided in trans during viral replication and encapsidation (e.g., by using a helper virus or packaging cell line carrying genes required for replication and / or encapsidation). Modified viral vectors in which the polynucleotide to be delivered is outside the viral particle have also been reported.

[0023] "Gene delivery", "gene transfer" and the like are terms used herein to refer to the introduction of an exogenous polynucleotide (sometimes referred to as a "transgene") into a host cell, regardless of the method used for transfer. Such methods include a variety of well-known techniques, such as vector-mediated transfer (e.g., by viral infection / transfection or various other protein- or lipid-based gene delivery complexes), as well as techniques that facilitate the delivery of "naked" polynucleotides (e.g., electroporation, "gene gun" delivery, and various other techniques used to transfer polynucleotides). The transferred polynucleotide can be stably or transiently maintained in the host cell. Stable maintenance typically requires that the transferred polynucleotide contains an origin of replication compatible with the host cell, or is integrated into a replicon (such as an extrachromosomal replicon (e.g., a plasmid)) or integrated into a nuclear or mitochondrial chromosome of the host cell. As known in the art, numerous vectors are known to be capable of mediating the transfer of genes into mammalian cells.

[0024] "Transgene" means any piece of nucleic acid molecule (e.g., DNA) that is inserted, either transiently or permanently, into a cell by any device and becomes part of that organism (if integrated into the genome) or remains extrachromosomally. Such a transgene can contain at least a portion of an open reading frame of a gene that is in part or in whole heterologous (i.e., foreign) to the transgenic organism, or can represent at least a portion of an open reading frame of a gene that is homologous to an endogenous gene of the organism, the portion encoding a polypeptide that, as the case may be, has substantially the same activity as, or at least one activity of, the corresponding full-length polypeptide.

[0025] "Transgenic cell" refers to a cell that contains a transgene. For example, a cell that has been stably or transiently transformed with a vector containing an expression cassette is a transgenic cell that can be used to generate a population of cells with altered phenotypic characteristics. "Recombinant cell" is a cell that has been genetically altered, for example, by the insertion, deletion, or replacement of sequences in a non-recombinant cell by genetic engineering.

[0026] The term "wild-type" or "naturally occurring" refers to a gene or gene product that has the characteristics of that gene or gene product when isolated from a natural source. A wild-type gene is that gene that is most frequently observed in a population and is therefore arbitrarily designated the "normal" or "wild-type" form of that gene. In contrast, the term "altered" or "mutant" refers to a gene or gene product that exhibits altered sequence or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. It is noted that naturally occurring mutants may be isolated. Naturally occurring mutants are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product.

[0027] The term "gene transfer" refers to the delivery of a polynucleotide to a recipient cell in vivo or in vitro via a viral vector (such as a replication-defective viral vector).

[0028] The term "heterologous" when referring to nucleic acid sequences (gene sequences encoding proteins, control sequences, etc.) refers to sequences that are not normally associated with a particular cell, and / or sequences that are not normally associated with a particular cell, e.g., sequences from a different source (e.g., sequences from a virus are heterologous to sequences in the genome of an uninfected cell). Thus, a "heterologous" region of a nucleic acid construct or vector is a segment of nucleic acid that is within or attached to another nucleic acid molecule that is not found associated with the other molecule in nature. For example, a heterologous region of a nucleic acid construct could include, as a coding sequence, a coding sequence flanked by sequences that are not found associated with that coding sequence in nature (i.e., a heterologous promoter). Another example of a heterologous coding sequence is a construct in which the coding sequence contained therein (e.g., a synthetic sequence with different codons than the native gene) is itself not found in nature. Similarly, a cell transformed with a construct that is not normally found in the cell would be considered heterologous.

[0029] "DNA" refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in either double- or single-stranded form found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. When discussing the structure of a particular DNA molecule, the sequence may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having the sequence complementary to the mRNA). The term encompasses molecules containing the four bases adenine, guanine, thymine, or cytosine, as well as base analogues known in the art.

[0030] As used herein, the terms "complementary" or "complementarity" are used in reference to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, the sequence "AGT" is complementary to the sequence "TCA." Complementarity may be "partial," in which case only some of the bases of the nucleic acids match according to the base-pairing rules. Alternatively, there may be "complete" or "total" complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between the nucleic acid strands. This is particularly important in amplification reactions and in detection methods that depend on binding between nucleic acids.

[0031] A DNA molecule is said to have a "5' end" and a "3' end" because mononucleotides react together to unidirectionally attach the 5' phosphate of one mononucleotide pentose ring to the 3' oxygen of an adjacent mononucleotide through a phosphodiester bond to form an oligonucleotide or polynucleotide. Thus, an end of an oligonucleotide or polynucleotide is referred to as the "5' end" if its 5' phosphate is not attached to the 3' oxygen of a mononucleotide pentose ring and as the "3' end" if its 3' oxygen is not attached to the 5' phosphate of a subsequent mononucleotide pentose ring. As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide or polynucleotide, can also be said to have a 5' end and a 3' end. In linear or circular DNA molecules, discrete elements are referred to as being "upstream" or 5' of the "downstream" or 3' elements. This terminology reflects the fact that transcription proceeds from 5' to 3' along the DNA strand. Promoter and enhancer elements that direct transcription of linked genes are generally located 5' or upstream of the coding region. However, enhancer elements can exert their effect even when they are located 3' of the promoter element and the coding region. Transcription termination and polyadenylation signals are located 3' or downstream of the coding region.

[0032] A "gene," "polynucleotide," "coding region," "sequence," "segment," "fragment," or "transgene" that in one embodiment "encodes" a particular protein is a nucleic acid molecule that, when placed under the control of appropriate regulatory sequences, is transcribed, and optionally translated, in vitro or in vivo into a gene product (e.g., a polypeptide). The coding region can exist in the form of cDNA, genomic DNA, or RNA. When present in the form of DNA, the nucleic acid molecule can be single-stranded (i.e., the sense strand) or double-stranded. The boundaries of a coding region are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. Non-limiting examples of genes can include cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic DNA sequences. A transcription termination sequence will usually be located 3' to the gene sequence.

[0033] The term "control elements" collectively refers to promoter regions, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, splice junctions, and the like, which collectively provide for the replication, transcription, post-transcriptional processing, and translation of a coding sequence in a recipient cell. Not all of these control elements need always be present so long as the replication, transcription, and translation of a selected coding sequence is possible in an appropriate host cell.

[0034] The term "promoter" is used herein in its ordinary sense to refer to a nucleotide region that contains a DNA regulatory sequence derived from a gene capable of binding RNA polymerase and initiating transcription of a downstream (3' direction) coding sequence.

[0035] By "enhancer" is meant a nucleic acid sequence that, when located near a promoter, confers increased transcriptional activity relative to the transcriptional activity resulting from the promoter in the absence of that enhancer domain.

[0036] "Operably linked" with respect to nucleic acid molecules means that two or more nucleic acid molecules (e.g., a nucleic acid molecule to be transcribed, a promoter element, and an enhancer element) are connected in a manner that allows for transcription of the nucleic acid molecules. "Operably linked" with respect to peptide and / or polypeptide molecules means that two or more peptide and / or polypeptide molecules are connected to produce a single polypeptide chain (i.e., a fusion polypeptide) that has at least one property of each of the peptide and / or polypeptide components of the fusion. Fusion polypeptides can be chimeric, i.e., composed of heterologous molecules.

[0037] "Homology" refers to the percentage of identity between two polynucleotides or two polypeptides. The correspondence between one sequence and another can be determined by techniques known in the art. For example, homology can be determined by directly comparing the sequence information between two polypeptide molecules by aligning the sequence information and using readily available computer programs. Alternatively, homology can be determined by hybridizing the polynucleotides under conditions that form stable duplexes between the homologous regions, followed by digestion with a single-strand specific nuclease and determining the size of the digested fragments. As determined using the above methods, two DNA sequences or two polypeptide sequences are "substantially homologous" to each other when at least about 80%, at least about 90%, or at least about 95% of the nucleotides or amino acids, respectively, match over a defined length of the molecule.

[0038] "Mammal" means any member of the class Mammalia, non-limiting examples of which include humans and non-human primates (chimpanzees and other apes and monkey species); farm animals (such as cows, sheep, pigs, goats, and horses); domesticated mammals (such as dogs and cats); and laboratory animals (including rodents such as mice, rats, rabbits, and guinea pigs).

[0039] "Derived from" means that a nucleic acid molecule is created or designed from a parent nucleic acid molecule, where the derivative retains substantially the same functional characteristics of the parent nucleic acid molecule from which it was created or designed (e.g., encodes a gene product that has substantially the same activity as the gene product encoded by the parent nucleic acid molecule).

[0040] "Expression construct" or "expression cassette" refers to a nucleic acid molecule capable of directing transcription. An expression construct contains at least a promoter. Additional elements, such as enhancers and / or transcription termination signals, may also be included.

[0041] The term "exogenous" when used with reference to a protein, gene, nucleic acid, or polynucleotide in a cell or organism means a protein, gene, nucleic acid, or polynucleotide that has been introduced into the cell or organism by artificial or natural means. An exogenous nucleic acid can be derived from a different organism or cell, or it can be one or more additional copies of a nucleic acid that occurs naturally in the organism or cell. As a non-limiting example, an exogenous nucleic acid is in a different chromosomal location than in the native cell, or is otherwise flanked by different nucleic acids than are found in nature.

[0042] The term "isolated" when used in reference to a nucleic acid, peptide, polypeptide, virus, or cell means a nucleic acid sequence, peptide, polypeptide, virus, or cell that has been identified and separated from at least one contaminating nucleic acid, polypeptide, or other biological component that is normally associated with the natural source. For example, because it is not associated with an in vivo substance or has been substantially purified from an in vitro substance. An isolated nucleic acid, peptide, polypeptide, or virus exists in a form or setting that is different from that found in nature. For example, a given DNA sequence (e.g., a gene) is found in close proximity to neighboring genes on the chromosome of a host cell. An RNA sequence (such as a particular mRNA that encodes a particular protein) is found in a cell as a mixture with many other mRNAs that encode a variety of proteins. An isolated nucleic acid molecule can exist in single-stranded or double-stranded form. When an isolated nucleic acid molecule is used to express a protein, the molecule will contain at least the sense or coding strand, but may contain both the sense and antisense strands (i.e., the molecule can be double-stranded).

[0043] As used herein, the term "recombinant nucleic acid" or "recombinant DNA sequence, molecule, or segment" refers to a nucleic acid (e.g., DNA) that is derived from or isolated from a source and may then be chemically modified in vitro, including, but not limited to, sequences that are naturally occurring, sequences that are not naturally occurring, or sequences that correspond to naturally occurring sequences that are not in the position in the genome where they would be located in nature. An example of DNA "derived" from a source would be a DNA sequence that is identified as a useful fragment and then chemically synthesized in substantially pure form. An example of such DNA "isolated" from a source would be a useful DNA sequence that has been excised or removed from the source by chemical means (e.g., using a restriction endonuclease). This DNA sequence can then be further manipulated (e.g., amplified) by genetic engineering methods for use.

[0044] The term "recombinant protein" or "recombinant polypeptide" as used herein refers to a protein molecule that is expressed from a recombinant DNA molecule.

[0045] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein, unless otherwise specified.

[0046] The term "sequence homology" refers to the percentage of base matches between two nucleic acid sequences, or the percentage of amino acid matches between two amino acid sequences. When sequence homology is expressed as a percentage (e.g., 50%), the percentage indicates the percentage of matches over the entire length of the selected sequence compared to some other sequence. Gaps (in either of the two sequences) are allowed to maximize matching. Gap lengths of 15 bases or less are usually used, with 6 bases or less, such as 2 bases or less. When using oligonucleotides as probes or treatments, the sequence homology between the target nucleic acid and the oligonucleotide sequence is generally less than 17 target base matches (85%) out of 20 possible oligonucleotide base pair matches, less than 9 matches (90%) out of 10 possible oligonucleotide base pair matches, or less than 19 matches (95%) out of 20 possible oligonucleotide base pair matches.

[0047] Generally, the nucleic acid sequence homology between the polynucleotides, oligonucleotides, and fragments and the nucleic acid sequence of interest is at least 65%, and more typically at least about 70%, about 90%, about 95%, about 98%, 100%, and increasing homology.

[0048] Two amino acid sequences are homologous if there is a partial or complete match between these sequences. For example, 85% homology means that 85% of the amino acids are the same when the two sequences are aligned for maximum matching. Gaps (in either of the two sequences being matched) are allowed to maximize matching. The gap length is 5 or less, for example 2 or less. Alternatively, two protein sequences (or polypeptide sequences derived therefrom and at least 30 amino acids in length) are homologous as this term is used herein if their alignment score (in standard deviation units) is greater than 5 using the program ALIGN with a mutation data matrix and a gap penalty of 6 or greater. Two sequences or parts thereof can be considered to be homologous if their amino acids are 50% or more the same when optimally aligned using the ALIGN program.

[0049] The term "corresponding to," as used herein, means that a polynucleotide sequence is homologous (e.g., identical, although not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence encoding a polypeptide, or its complementary strand, or that a polypeptide sequence is identical in sequence or function to a reference polypeptide sequence. Specifically, the nucleotide sequence "TATAC" corresponds to the reference sequence "TATAC" and is complementary to the reference sequence "GTATA."

[0050] The following terms are used to describe sequence relationships between two or more polynucleotides: "reference sequence", "comparison window", "sequence identity", "percentage of sequence identity", and "substantial identity". A "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence can be a portion of a larger sequence (e.g., a segment of a full-length cDNA or gene sequence provided in a sequence listing) or can include a complete cDNA or gene sequence. Generally, a reference sequence is at least 20 nucleotides in length, frequently at least 25 nucleotides in length, and often at least 50 nucleotides in length. Because two polynucleotides each may (1) contain sequences that are similar between the two polynucleotides (i.e., a portion of the complete polynucleotide sequence) and (2) further contain sequences that differ between the two polynucleotides, sequence comparison between two (or more) polynucleotides is typically performed by comparing the sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity.

[0051] A "comparison window" herein refers to a conceptual segment of at least 20 contiguous nucleotides, that portion of a polynucleotide sequence within the comparison window may contain no more than 20% additions or deletions (i.e., gaps) compared to a reference sequence (not including additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for alignment in the comparison window can be performed by using local homology algorithms or by similarity search methods, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA Genetics Software Packages), or by eye, and the best alignment (i.e., the one that results in the highest percentage of homology over the comparison window) generated by the various methods is selected.

[0052] The term "sequence identity" means that two polynucleotide sequences are identical over a window of comparison (i.e., on a nucleotide-by-nucleotide basis). The term "percentage of sequence identity" means that two polynucleotide sequences are identical over a window of comparison (i.e., on a nucleotide-by-nucleotide basis). The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over a window of comparison, determining the number of positions where the same nucleobase (e.g., A, T, C, G, U, or I) occurs in both sequences to generate the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the size of the window), and multiplying the result by 100 to obtain the percentage of sequence identity. The term "substantial identity" as used herein refers to a characteristic of a polynucleotide sequence, which comprises a sequence that has at least 85% sequence identity, at least 90-95% sequence identity, and more usually at least 99% sequence identity, relative to a reference sequence over a comparison window of at least 20 nucleotide positions, and frequently over a comparison window of at least 20-50 nucleotides, where the percent sequence identity is calculated by comparing the reference sequence to a polynucleotide sequence that may contain deletions or additions that total no more than 20% of the reference sequence over the comparison window.

[0053] The term "substantial identity" when applied to polypeptides means that two peptide sequences, when optimally aligned using, for example, the programs GAP or BESTFIT with default gap weights, share at least about 80% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity, and / or at least about 99% sequence identity.

[0054] As used herein, "substantially pure" means that the species of interest is the predominant species present (i.e., on a molar basis, more abundant than any other individual species in the composition), and a substantially purified fraction is one in which the species of interest comprises at least about 50% (on a molar basis) of the total macromolecular species present. Generally, a substantially pure composition will comprise greater than about 80%, greater than about 85%, greater than about 90%, greater than about 95%, and greater than about 99% of the total macromolecular species present in the composition. The species of interest can be purified to essential homogeneity (contaminating species cannot be detected in the composition by conventional detection methods), and the composition consists essentially of a single macromolecular species.

[0055] "Transfected," "transformed," or "transgenic," as used herein, includes any host cell or cell line that has been altered or augmented by the presence of at least one recombinant DNA sequence. The host cells of the invention are typically produced by transfecting a DNA sequence in a plasmid expression vector as an isolated linear DNA sequence, or by infection with a recombinant viral vector.

[0056] Representative cells and their modifications

[0057] Most influenza vaccines are produced in embryonated chicken eggs, but influenza vaccines are increasingly being produced in other systems. MDCK (Madin-Darby canine kidney) cells are one of two mammalian cell lines licensed for influenza vaccine production. Vaccine production in cells can be enhanced by altering the host cell or the virus. However, virus modification during passaging is not advantageous for vaccine production.

[0058] As disclosed herein, the genome of a cell (e.g., an avian or mammalian cell, non-limiting examples of which include canine, feline, equine, bovine, caprine, porcine, human, or non-human primate cells) can be modified to enhance influenza virus isolation, growth, or both. For example, some HA subtypes (HA subtypes H1-H18) can bind well to certain species or types of cells due to the number or composition of cell surface receptors for HA. These cells can be modified by increasing the number of cell surface receptors, or by changing the types of molecules found on the cell surface receptors, or both. For example, in mammals, there are about 20 sialyltransferases that transfer sialic acid residues to the oligosaccharide side chains of glycoconjugates. Genes encoding one or more of these enzymes can be modified to reduce (e.g., reduce by 1%, 5%, 10%, 50%, 70%, 80%, 90%, or more) or eliminate expression of the encoded enzyme. Alternatively, one or more open reading frames of these enzymes can be expressed intracellularly from an exogenously introduced expression cassette (e.g., a plasmid carrying the expression cassette). For example, α2,6-sialyltransferase transfers α2,6-linked sialic acid to terminal Gal (ST6Gal I and II) or GalNAc (ST6GalNAcI-VI); α2,8-sialyltransferase transfers α2,8-linked sialic acid (STSiaI-IV); and α2,3-sialyltransferase transfers α2,3-linked sialic acid to terminal Gal residues (ST3Gal). ST3GalI-II and IV transfer to Gal residues located at terminal Galβ1-3GlcNAc, ST3GalIV and VI transfer to Gal residues located at terminal Galβ1-4GlcNAc, ST3GalV transfers to Gal residues located at terminal Galβ1-4Glc-Cer, and ST3GalIII transfers to Gal residues located at terminal Galβ1-3GlcNAc or Galβ1-3GlcNAc. Thus, each of these sialyltransferase genes can be used to prepare the cells disclosed herein.In one embodiment, one or more α2,3-sialyltransferase genes are modified in the genome of the host cell to reduce (e.g., eliminate) expression of the encoded enzyme, and one or more α2,6-sialyltransferase genes are expressed from a recombinant expression vector introduced into the host cell. To reduce expression of sialyltransferase, one or more vectors or a combination of vectors and isolated proteins can be introduced into the cell. The vector and / or protein can be part of a recombinase system that can target specific genes in the cell, including systems such as CRISPR / Cas, TALEN, and zinc finger nucleases.

[0059] To prepare an expression cassette for transformation herein (to express RNA such as gRNA, or a protein including a recombinase or sialyltransferase), the recombinant DNA sequence or segment can be circular or linear, double-stranded or single-stranded. The DNA sequence encoding the RNA sequence substantially complementary to the mRNA sequence encoding the gene product of interest is typically the "sense" DNA sequence that is cloned into the cassette in the opposite orientation (i.e., 3' to 5' instead of 5' to 3'). Generally, the DNA sequence or segment is in the form of chimeric DNA (such as plasmid DNA), which can also contain a coding region flanked by regulatory sequences that facilitate expression of that DNA in the cell. As used herein, "chimeric" means that the vector contains DNA from at least two species, or contains DNA from the same species, linked or associated in a manner that does not occur "naturally" or wild-type in that species.

[0060] Apart from the DNA sequence or parts thereof that function as transcription units, parts of the DNA can be non-transcribed and serve regulatory or structural functions. For example, the DNA can itself contain a promoter active in eukaryotic cells (e.g., mammalian cells) or in a type of cell, or the DNA can utilize a promoter already present in the genome of the lymphotropic virus that is the transformation target. Such promoters include the CMV promoter, as well as the SV40 late promoter and retroviral LTRs (long terminal repeat elements), such as MMTV, RSV, MLV, or HIV LTR, although many other promoter elements well known in the art can be used.

[0061] Other elements that function in the host cell can also be part of the recombinant DNA, such as introns, enhancers, polyadenylation sequences, etc. Such elements may or may not be necessary for the function of the DNA, but can improve expression of the DNA by affecting transcription, mRNA stability, etc. Such elements can be included in the DNA when desired to obtain optimal performance of transforming the DNA in cells.

[0062] The recombinant DNA introduced into the cell can contain a selectable marker gene, or a reporter gene, or both, to facilitate identification and selection of transformed cells from a population of cells to be transformed. Alternatively, the selectable marker can be carried on a separate piece of DNA and used in a co-transformation procedure. Both the selectable marker and the reporter gene can be flanked by appropriate regulatory sequences to allow expression in the host cell. Useful selectable markers are well known in the art and include, for example, antibiotic and herbicide resistance genes (neo, hpt, dhfr, bar, aroA, puro, hyg, dapA, etc.). See also the genes listed in Table 1 of Lundquist et al. (U.S. Patent No. 5,848,956).

[0063] Reporter genes can be used to identify potentially transformed cells and to assess the function of regulatory sequences. Reporter genes that encode easily assayed proteins are well known in the art. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue, and which encodes a protein whose expression is indicated by some easily detectable property (e.g., enzymatic activity). Representative reporter genes include the chloramphenicol acetyltransferase gene (cat) from Tn9 of E. coli, the beta-glucuronidase gene (gus) at the uidA locus of E. coli, green, red, or blue fluorescent protein genes, and the luciferase gene. Expression of the reporter gene is examined at an appropriate time after the DNA is introduced into the recipient cells.

[0064] General methods for constructing recombinant DNA capable of transforming a target cell are well known to those skilled in the art. The same compositions and methods for construction can be used to generate DNA useful herein. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2002) provides suitable methods for construction.

[0065] Recombinant DNA can be readily introduced into a host cell (e.g., a mammalian, yeast, or insect cell) by transfecting an expression vector containing the recombinant DNA by any procedure (e.g., physical or biological methods) useful for introducing the recombinant DNA into a particular cell, resulting in a transformed (transgenic) cell carrying the recombinant DNA, such that the recombinant DNA of interest is expressed by the host cell. In one embodiment, at least one of the recombinant DNAs introduced into the cell is maintained extrachromosomally. In one embodiment, at least one of the recombinant DNAs is stably integrated into the genome of the host cell.

[0066] Physical methods for introducing recombinant DNA into host cells include calcium-mediated methods, particle bombardment, microinjection, electroporation, etc. Biological methods for introducing DNA of interest into host cells include the use of DNA or RNA vectors. Viral vectors (e.g., retroviral or lentiviral vectors) have become a widely used method for inserting genes into eukaryotic cells, such as mammalian (e.g., human) cells. Other viral vectors useful for introducing genes into cells can be derived from poxviruses, such as vaccinia virus, herpes virus, adenovirus, adeno-associated virus, baculovirus, etc.

[0067] A variety of assays can be performed to confirm the presence of the recombinant DNA sequence in the host cell, including, for example, molecular biological assays well known to those of skill in the art, such as Southern blotting, Northern blotting, RT-PCR, and PCR, biochemical assays, such as detecting the presence or absence of specific gene products by immunological means (ELISA and Western blot) or other molecular assays.

[0068] RT-PCR can be used to detect and quantify the RNA produced from the introduced recombinant DNA segment. This application of PCR requires first reverse transcribing the RNA into DNA using an enzyme (such as reverse transcriptase) and then amplifying the DNA through the use of conventional PCR techniques. In most cases, PCR techniques are useful but do not prove the integrity of the RNA product. Further information about the RNA product can be obtained by Northern blotting. This technique proves the presence of an RNA species and gives information about the integrity of that RNA. The presence or absence of an RNA species can also be revealed using dot or slot blot Northern hybridization. These techniques are modifications of Northern blotting and prove only the presence or absence of an RNA species.

[0069] Southern blotting and PCR can be used to detect recombinant DNA segments of interest, but they do not provide information regarding whether the recombinant DNA segment is expressed. Expression can be assessed by specifically identifying the peptide product of the introduced DNA sequence or by assessing the phenotypic changes resulting from expression of the introduced DNA segment in a host cell.

[0070] In vectors used to knock down or knock out the expression of one or more sialyltransferases, the vector has a sequence that results in one or more mutations in the genome of the cell. The mutations are effective to suppress or prevent the production of at least one functional sialyltransferase. In one embodiment, the mutations are deletions of 1, 10, 20, 50, 100, 500, or even several thousand nucleotides, such as deletions of 1%, 10%, 50%, 90%, or more of the sequence corresponding to the sialyltransferase gene (e.g., deletions in the region encoding a sialyltransferase, e.g., 2,3-sialyltransferase (ST3)). In one embodiment, the deleted sequence corresponds to a substantially identical sequence (e.g., at least 80% or more (e.g., 85%, 90%, or 95%) and up to 100%, or any integer in between, nucleic acid sequence identity with SEQ ID NO:5, 7, 9, 11, 13, 15, or 17, or any combination thereof). In one embodiment, the mutation is an insertion of 1, 2, 3, 5, 10, 20, 50, 100, 500, or even more than several thousand nucleotides into a sequence corresponding to a sialyltransferase gene, such as an insertion into a region encoding a sialyltransferase (e.g., 2,3-sialyltransferase (ST3)). In one embodiment, the insertion is into a sequence corresponding to a sequence that is substantially identical (e.g., at least 80% or more (e.g., 85%, 90%, or 95%) and up to 100% nucleic acid sequence identity, or any integer in between), to SEQ ID NO:5, 7, 9, 11, 13, 15, or 17, or any combination thereof. In one embodiment, the mutation comprises one or more nucleotide substitutions, such as 1, 2, 3, 4, 5, 6, 10, or up to several hundred nucleotide substitutions (e.g., substitutions within SEQ ID NOs: 5, 7, 9, 11, 13, 15, or 17, or any combination thereof) within a sequence corresponding to a region encoding a sialyltransferase, such as 2,3-sialyltransferase (ST3).In one embodiment, there is a combination of insertions, nucleotide substitutions, and / or deletions in a sequence that is substantially identical (e.g., at least 80% or more (e.g., 85%, 90%, or 95%) and up to 100%, or any integer nucleic acid sequence identity, to SEQ ID NO:5, 7, 9, 11, 13, 15, or 17, or any combination thereof) in the host cell. In one embodiment, the mutations result in a host cell with reduced expression of one or more ST3 genes that encode proteins that have an amino acid sequence identity of at least 80% or more (e.g., 85%, 90%, or 95%) and up to 100%, or any integer identity, to, for example, any of SEQ ID NOs:6, 8, 10, 12, 14, 16, or 18.

[0071] In one embodiment, the host cell expresses one or more ST6 genes encoding proteins having an amino acid sequence that is at least 80% identical (e.g., 85%, 90%, or 95% and up to 100%, or any integer in between) to any of SEQ ID NOs:1-4.

[0072] CRISPR / Cas system

[0073] Type II CRISPR is a well-characterized system that executes targeted DNA double-strand breaks in four sequential steps. First, two non-coding RNAs, the pre-crRNA array and the tracrRNA, are transcribed from the CRISPR locus. Second, the tracrRNA hybridizes to the repeat region of the pre-crRNA and mediates the processing of the pre-crRNA into mature crRNA containing a distinct spacer sequence. Third, the mature crRNA:tracrRNA complex directs Cas9 to the target DNA through Watson-Crick base pairing between the spacer on the crRNA and the protospacer on the target DNA next to the protospacer adjacent motif (PAM) (an additional requirement for label recognition). Finally, Cas9 mediates cleavage of the target DNA to generate a double-strand break in the protospacer. The activity of the CRISPR / Cas system involves three steps. (i) the insertion of foreign DNA sequences into the CRISPR array to thwart future attacks in a process called "adaptation", (ii) expression and processing of the array as well as expression of the relevant proteins, followed by (iii) RNA-mediated interference with the foreign nucleic acid. Thus, in bacterial cells, several so-called "Cas" proteins are involved in the natural function of the CRISPR / Cas system. The primary products of the CRISPR locus appear to be short RNAs containing sequences that target the invader, termed guide RNAs.

[0074] "Cas1" polypeptide refers to CRISPR-associated (Cas) protein 1. Cas1 (COG1518 in the Clusters of Orthologous Group of Protein Classification System) is the best marker of the CRISPR-associated system (CASS). Based on phylogenetic comparisons, seven different versions of the CRISPR-associated immune system have been identified so far (CASS1-7). The Cas1 polypeptide used in the methods described herein can be any Cas1 polypeptide present in any prokaryotic organism. In some embodiments, the Cas1 polypeptide is a Cas1 polypeptide of an archaeal microorganism. In some embodiments, the Cas1 polypeptide is a Cas1 polypeptide of a euryarchaeal microorganism. In some embodiments, the Cas1 polypeptide is a Cas1 polypeptide of a crenarchaeal microorganism. In some embodiments, the Cas1 polypeptide is a bacterial Cas1 polypeptide. In some embodiments, the Cas1 polypeptide is a Cas1 polypeptide of a gram-negative or gram-positive bacterium. In some embodiments, the Cas1 polypeptide is a Cas1 polypeptide of Pseudomonas aeruginosa. In some embodiments, the Cas1 polypeptide is a Cas1 polypeptide of Aquifex aeolicus. In some embodiments, the Cas1 polypeptide is a Cas1 polypeptide that is a member of CASs1-7. In some embodiments, the Cas1 polypeptide is a Cas1 polypeptide that is a member of CASS3. In some embodiments, the Cas1 polypeptide is a Cas1 polypeptide that is a member of CASS7. In some embodiments, the Cas1 polypeptide is a Cas1 polypeptide that is a member of CASS3 or CASS7.

[0075] In some embodiments, the Cas1 polypeptide can be found in GenBank, e.g., under Gene ID Nos. 2781520, 1006874, 9001811, 947228, 3169280, 2650014, 1175302, 3993120, 4380485, 906625, 3165126, 905808, 1454460, 1445886, 1485099, 4274010, 888506, 3169526, 99 7745, 897836, or 1193018, and / or an amino acid sequence exhibiting homology (e.g., greater than 80%, 90-99% (including 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) to the amino acid sequences encoded by these polynucleotides, and these polypeptides function as Cas1 polypeptides.

[0076] There are three types of CRISPR / Cas systems, all of which incorporate RNA and Cas proteins: type I and type III both have a Cas endonuclease that processes the pre-crRNA, which, when fully processed into the crRNA, assembles into a multi-Cas protein complex that can cleave nucleic acids complementary to the crRNA.

[0077] In type II CRISPR / Cas systems, crRNA is generated using a different mechanism, where a transactivating RNA (tracrRNA) complementary to the repeats in the pre-crRNA initiates double-strand-specific RNase III processing in the presence of Cas9 protein. Cas9 can then cleave the target DNA complementary to the mature crRNA, but cleavage by Cas9 depends on both base pairing between the crRNA and the target DNA and the presence of a short motif in the crRNA, called the PAM (protospacer adjacent motif) sequence. In addition, tracrRNA must be present because tracrRNA base pairs with the crRNA at its 3' end, and this association initiates Cas9 activity.

[0078] The Cas9 protein has at least two nuclease domains: one similar to the HNH endonuclease, while the other resembles the Ruv endonuclease domain. The HNH-type domain appears to be responsible for cleaving the DNA strand complementary to the crRNA, whereas the Ruv domain cleaves the non-complementary strand.

[0079] The need for a crRNA-tracrRNA complex can be circumvented by the use of engineered "single guide RNAs" (sgRNAs) that contain a hairpin that is normally formed by annealing of the crRNA and tracrRNA (see Jinek et al. (2012) Science 337:816; Cong et al. (2013) Sciencexpress / 10.1126 / science.1231143). In S. pyogenes, the engineered tracrRNA:crRNA fusion or sgRNA guides Cas9 to cleave the target DNA when a double-stranded RNA:DNA heterodimer forms between the Cas-associated RNA and the target DNA. This system includes the Cas9 protein and an engineered sgRNA.

[0080] "Cas polypeptide" includes full-length Cas polypeptides, enzymatically active fragments of Cas polypeptides, and enzymatically active derivatives of Cas polypeptides or fragments thereof. Non-limiting examples of suitable derivatives of Cas polypeptides or fragments thereof include mutants, fusions, covalent modifications, or fragments of Cas proteins.

[0081] The RNA component of CRISPR / Cas

[0082] The CRISPR / Cas system, related to Cas9, contains two RNA non-coding components: the tracrRNA and a pre-crRNA array containing nuclease guide sequences (spacers) separated by identical direct repeats (DRs). Both of these RNAs must be functional to achieve genome engineering using the CRISPR / Cas system (see Cong et al. (2013) Sciencexpress 1 / 10.1126 / science 1231143). In some embodiments, the tracrRNA and pre-crRNA are provided through separate expression constructs or as separate RNAs. In other embodiments, chimeric RNAs are constructed in which an engineered mature crRNA (which provides target specificity) is fused to a tracrRNA (which provides interaction with Cas9) to generate a chimeric crRNA-tracrRNA hybrid (also called a single guide RNA). (See Jinek, supra and Cong, supra).

[0083] Chimeric or sgRNAs can be engineered to contain sequences complementary to any desired target. The RNA contains 22 bases complementary to the target, in the form G[n19], followed by a protospacer adjacent motif (PAM) in the form NGG. Thus, in one method, sgRNAs can be designed using known ZFN targets in a gene of interest by (i) aligning the recognition sequence of the ZFN heterodimer with a reference sequence in the relevant genome (human, mouse, or a particular plant species); (ii) identifying the spacer region between the ZFN half-sites; (iii) identifying the location of the motif G[n20]GG closest to the spacer region (when two or more such motifs overlap the spacer, select a motif that is more central than the spacer); and (iv) using that motif as the core of the sgRNA. This method advantageously relies on a proven nuclease target. Alternatively, sgRNAs can be designed to target any region of interest by identifying an appropriate target sequence that matches the G[n20]GG format.

[0084] donor

[0085] As pointed out above, insertion of a foreign sequence (also called "donor sequence" or "donor" or "transgene" or "gene of interest"), for example, to correct a mutant gene or increase expression of a wild-type gene. It will be readily apparent that the donor sequence is typically not the same as the genomic sequence in which it is placed. The donor sequence can contain a non-homologous sequence flanked by two regions of homology to allow efficient HDR at the location of interest. Alternatively, the donor may not have a region of homology to the targeted location in the DNA and can integrate by NHEJ-dependent end joining after cleavage at the target site. In addition, the donor sequence can include a vector molecule that contains a sequence that is not homologous to the region of interest in cellular chromatin. The donor molecule can contain several discontinuous regions of homology to cellular chromatin. For example, a sequence that is not normally present in the region of interest can be present in the donor nucleic acid molecule flanked by regions of homology to sequences in the region of interest for targeted insertion.

[0086] The donor polynucleotide can be DNA or RNA, single-stranded and / or double-stranded, and can be introduced into cells in linear or circular form. If introduced in linear form, the ends of the donor sequence can be protected (e.g., from exonuclease degradation) by methods known to those skilled in the art. For example, one or more dideoxynucleotide residues can be added to the 3' end of the linear molecule and / or self-complementary oligonucleotides can be ligated to one or both ends. See, for example, Chang et al. (1987) Proc. Natl. Acad. Sci. USA 84:4959-4963; Nehls et al. (1996) Science 272:886-889. Non-limiting examples of additional methods for protecting exogenous polynucleotides from degradation include the addition of terminal amino groups and the use of modified internucleotide linkages (e.g., phosphorothioates, phosphoramidates, and O-methyl ribose or O-methyl deoxyribose residues).

[0087] Polynucleotides can be introduced into cells as part of a vector molecule that has additional sequences, such as an origin of replication, a promoter, and genes encoding antibiotic resistance. Additionally, donor polynucleotides can be introduced as naked nucleic acid, as nucleic acid complexed with an agent (such as a liposome or poloxamer), or delivered by a virus (such as adenovirus, AAV, herpes virus, retrovirus, lentivirus, and integrase-deficient lentivirus (IDLV)).

[0088] The donor is generally inserted such that its expression is driven by an endogenous promoter (i.e., a promoter that drives expression of the endogenous gene into which the donor is inserted) at the integration site (e.g., highly expressed albumin, AAVS1, HPRT, etc.). However, it will be apparent that the donor can include a promoter and / or enhancer (e.g., a constitutive promoter, or an inducible promoter, or a tissue-specific promoter).

[0089] The donor molecule can be inserted into an endogenous gene to express all or part of the endogenous gene, or to not express the endogenous gene at all. For example, a transgene as described herein can be inserted into an albumin or other locus to express some of the endogenous albumin sequence (e.g., the N-terminus and / or C-terminus of the transgene encoding a lysosomal enzyme), e.g., as a fusion with a transgene encoding a lysosomal enzyme, or to not express the endogenous albumin sequence at all. In another embodiment, the transgene (with or without additional coding sequences, e.g., albumin) is integrated into any endogenous locus (e.g., a safe harbor locus). See, e.g., U.S. Patent Application Publication Nos. 2008 / 0299580; 2008 / 0159996; and 2010 / 0218264.

[0090] When an endogenous sequence (endogenous or part of a transgene) is expressed along with a transgene, the endogenous sequence (such as, for example, albumin) can be a full-length sequence (wild-type or mutant) or a partial sequence. In one embodiment, the endogenous sequence is functional. Non-limiting examples of functions of these full-length or partial sequences (such as, for example, albumin) include increasing the serum half-life of and / or acting as a carrier of a polypeptide expressed by a transgene (such as a therapeutic gene).

[0091] In addition, although not required for expression, exogenous sequences can also include sequences that regulate transcription or translation (e.g., promoters, enhancers, insulators, internal ribosome entry sites, sequences encoding 2A peptides, and / or polyadenylation sites).

[0092] Other nucleases (e.g., zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or others specific to the targeted gene and site) can be utilized to allow the transgene construct to be inserted by homology directed repair (HDR) or by end capture during a process driven by non-homologous end joining (NHEJ).

[0093] Representative Embodiments

[0094] In one embodiment, an isolated recombinant mammalian or avian cell is provided that comprises a reduced amount of cell surface β-galactoside α2,3 sialyl residues and an increased amount of human β-galactoside α2,6 sialyl residues compared to a corresponding non-recombinant mammalian or avian cell. In one embodiment, the isolated recombinant cell is a non-human cell. In one embodiment, the isolated recombinant cell is a canine or primate cell. In one embodiment, the isolated recombinant cell comprises an expression cassette encoding human β-galactoside α2,6 sialyltransferase I (ST6Gal-I) or ST6Gal-II. In one embodiment, ST6Gal-I or ST6Gal-II comprises a protein having at least 80% amino acid sequence identity to any one of SEQ ID NOs: 1-4 or 101. In one embodiment, one or more β-galactoside α2,3 sialyltransferase genes are mutated in the recombinant cell to reduce the amount of cell surface β-galactoside α2,3 sialyl residues. In one embodiment, two or more of the ST3Gal-I gene, the ST3Gal-II gene, the ST3Gal-III gene, the ST3Gal-IV gene, the ST3Gal-V gene, the ST3Gal-VI gene, or the ST3Gal-II-like gene are mutated in the recombinant cell. In one embodiment, the ST3 gene has a nucleic acid sequence that is at least 80% identical to any one of SEQ ID NOs: 5, 7, 9, 11, 13, 15, or 17. In one embodiment, the reduction in cell surface β-galactoside α2,3 sialyl residues is the result of reduced expression of one or more ST3 sialyltransferases. In one embodiment, the one or more ST3 sialyltransferases have an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 6, 8, 10, 12, 14, 16, or 18. In one embodiment, the influenza H3 virus replicates more efficiently in the recombinant cell compared to a non-recombinant cell.

[0095] In one embodiment, an isolated recombinant mammalian or avian cell is provided that comprises a reduced amount of cell surface β-galactoside α2,3 sialyl residues compared to the corresponding non-recombinant mammalian or avian cell. In one embodiment, one or more of ST3Gal-I, ST3Gal-II, ST3Gal-III, ST3Gal-IV, ST3Gal-V, ST3Gal-VI, or ST3Gal-II-like genes are mutated in the recombinant cell. In one embodiment, a combination of ST3Gal-I, ST3Gal-II, ST3Gal-III, ST3Gal-IV, ST3Gal-V, ST3Gal-VI, or ST3Gal-II-like genes are mutated in the recombinant cell. In one embodiment, ST3Gal-I, ST3Gal-II, ST3Gal-III, ST3Gal-IV, ST3Gal-V, ST3Gal-VI, and ST3Gal-II-like genes are mutated in the recombinant cell.

[0096] In one embodiment, a method is provided for altering the amount of cell surface β-galactoside α2,3 sialyl residues and human β-galactoside α2,6 sialyl residues in a mammalian or avian cell. In one embodiment, the method comprises mutating one or more β-galactoside α2,3 sialyltransferase (ST3Gal) genes and overexpressing a human β-galactoside α2,6 sialyltransferase I (ST6Gal) gene in a parent mammalian or avian cell, resulting in a modified mammalian or avian cell having a reduced amount of cell surface β-galactoside α2,3 sialyl residues and an increased amount of human β-galactoside α2,6 sialyl residues on its surface compared to the corresponding parent cell. In one embodiment, the one or more ST3Gal genes are mutated using a genome editing system. In one embodiment, the genome editing system comprises CRISPR / Cas9, zinc finger nucleases (ZFNs), or transcription activator-like effector nucleases (TALENs). In one embodiment, the mutation comprises an insertion of one or more nucleotides, or a deletion of one or more nucleotides, or both, in one or more ST3 genes. In one embodiment, the modified cell comprises an expression cassette comprising an ST6Gal open reading frame. In one embodiment, the modified cell is a kidney cell. In one embodiment, the modified cell is a canine cell. In one embodiment, the modified cell is a Madin-Darby canine kidney (MDCK) cell.

[0097] In one embodiment, a method of propagating influenza virus is provided. The method includes infecting a recombinant cell with the influenza virus; and recovering the progeny virus. In one embodiment, the influenza virus is a human influenza virus. In one embodiment, the influenza virus is an influenza A virus. In one embodiment, the influenza virus is an influenza B virus. In one embodiment, the influenza virus is an H3 virus. In one embodiment, the virus is an A / H1N1, A / H3N2, B Yamagata lineage type B influenza virus, or B / Victoria lineage type B influenza virus.

[0098] In one embodiment, a method of isolating influenza virus is provided, the method comprising: providing a sample from an avian or mammalian animal suspected of being infected with influenza virus; and contacting the sample with a recombinant cell. In one embodiment, the method comprises determining whether the sample is infected with influenza virus. In one embodiment, the method comprises identifying the HA and / or NA subtype of the virus.

[0099] In one embodiment, a method for diagnosing influenza virus infection is provided. The method includes infecting a recombinant cell with a sample from an avian or mammalian organism suspected of being infected with influenza virus; and determining whether the cell is infected with the virus. In one embodiment, a plaque assay is used to determine the presence of a quantity of virus. In one embodiment, a plaque assay is used to reveal the presence of a quantity of virus, for example, in the supernatant of infected cells.

[0100] Representative sialyltransferase sequences

[0101] Sialyltransferases in higher vertebrates are glycosyltransferases that mediate the transfer of sialic acid residues from activated sugar donors (CMP-β-Neu5Ac, CMP-β-Neu5Gc, and CMP-β-KDN) to terminal non-reducing positions of oligosaccharide chains of glycoproteins and glycolipids. The vertebrate sialyltransferase superfamily is divided into four families, ST6Gal, ST3Gal, ST6GalNAc, and ST8Sia, depending on the glycosidic bond formed and the monosaccharide acceptor used. Members of the mammalian and avian ST6Gal family catalyze the transfer of sialic acid residues to the terminal galactose residues of type 2 disaccharides (Gal(NAc) β1,4GlcNAc), forming an α2-6 glycosidic bond. Unlike other sialyltransferase families, this family contains only two paralogs in the human genome, designated ST6GAL1 and ST6GAL2, respectively. The human ST6GAL1 gene is ubiquitously expressed in a variety of tissues, whereas the ST6GAL2 gene is expressed in a tissue-specific (adult brain) and stage-specific (embryonic) manner. Mammalian st6gal1 gene expression is regulated by multiple promoters that direct the expression of several transcripts encoding the same polypeptide enzyme.

[0102] In one embodiment, one or more ST3 genes in an avian or mammalian cell (e.g., a canine or non-human primate cell) are modified resulting in reduced cell surface expression of α2,3-linked sialic acid. In one embodiment, one or more human ST6 genes are introduced into the canine or non-human primate cell. In one embodiment, one or more ST3 genes are modified before one or more ST6 genes are introduced into the cell. In one embodiment, one or more ST6 genes are introduced before the ST3 genes are modified in the cell. In one embodiment, the ST3 genes are modified and the ST6 genes are introduced into the cell, either simultaneously or sequentially.

[0103] In one embodiment, the expressed ST6Gal comprises: MIHTNLKKKFSCCVLVFLLFAVICWKEKKKGSYYDSFKLQTKEFQVLKSLGKLAMGSDSQSVSSSSTQDPHRGRQTLGSLRGLAKAKPEASFQVWNKDSSSKN LIPRLQKIWKNYLSMNKYKVSYKGPGPGIKFSAEALRCHLRDHVNVSMVEVTDFPFNTSEWEGYLPKESIRTKAGPWGRCAVVSSAGSLKSSQLGREIDDHDAV LRFNGAPTANFQQDVGTKTTIRLMNSQLVTTEKRFLKDSLYNEGILIVWDPSVYHSDIPKWYQNPDYNFFNNYKTYRKLHPNQPFYILKPQMPWELWDILQEISPEEIQPNPPSSGMLGIIIMMTLCDQVDIYEFLPSKRKTDVCYYYQKFFDSACTMGAYHPLLYEKNLVKHLNQGTDEDIYLLGKATLPGFRTIHC (SEQ ID NO: 1) or MIHTNLKKKFSCCVLVFLLFAVICWKEKKKGSYYDSFKLQTKEFQVLKSLGKLAMGSDSQSVSSSSTQDPHRGRQTLGSLRGLAKAKPEASFQVWNKDSSSKNL IPRLQKIWKNYLSMNKYKVSYKGPGPGIKFSAEALRCHLRDHVNVSMVEVTDFPFNTSEWEGYLPKESIRTKAGPWGRCAVVSSAGSLKSSQLGREIDDHDAVLRF NGAPTANFQQDVGTKTTIRLMNSQLVTTEKRFLKDSLYNEGILIVWDPSVYHPDIPKWYQNPDYNFFNNYKTYRKLHPNQPFYILKPQMPWELWDILQEISPEEIQPNPPSSGMLGIIIMMTLCDQVDIYEFLPSKRRTDVCYYYQKFFDSACTMGAYHPLLFEKNLVKHLNQGTDEDIYLLGKATLPGFRTIHC (SEQ ID NO: 150), mkphlkqwrq rmlfgifawg llfllifiyf tdsnpaepvp sslsfletrr llpvqgkqra imgaahepsp pggldarqal prahpagsfh agpgdlqkwa qsqdgfehke ffssqvgrks qsafypeddd yffaagqpgw hshtqgtlgf pspgepgpre gafpaaqvqr rrvkkrhrrq rrshvleegd dgdrlyssms raflyrlwkg nvsskmlnpr lqkamkdylt ankhgvrfrg kreaglsraq llcqlrsrar vrtldgteap fsalgwrrlv pavplsqlhp rglrscavvm sagailnssl geeidshdav lrfnsaptrg human ST6 (accession number BAC24793) containing yekdvgnktt iriinsqilt npshhfidss lykdvilvaw dpapysanln lwykkpdynl ftpyiqhrqr npnqpfyilh pkfiwqlwdi iqentkekiq pnppssgfig ilimmsmcre vhvyeyipsv rqtelchyhe lyydaactlg ayhpllyekl lvqrlnmgtq gdlhrkgkvv lpgfqavhcp apspviphs (SEQ ID NO:2); aamgsdsqsv sssstqdphr grqtlgslrg lakakpeasf qvwnkdsssk nliprlqkiw knylsmnkyk vsykgpgpgi kfsaealrch lrdhvnvsmv evtdfpfnts ewegylpkes irtkagpwgr cavvssagsl kssqlgreid dhdavlrfng aptanfqqdv gtkttirlmn sqlvttekrf lkdslynegi livwdpsvyh sdipkwyqnp dynffnnykt yrklhpnqpf yilkpqmpwe lwdilqeisp eeiqpnppss gmlgiiimmt lcdqvdiyef lpskrktdvc yyyqkffdsa ctmgayhpll Human ST6 (Accession No. SJL87798) containing yeknlvkhln qgtdediyll gkatlpgfrt ihc (SEQ ID NO: 3); or Human ST6 Gal-II (SEQ ID NO:4); MIHTNLKKKFSCCVLVFLLFAVICWKEKKKGSYYDSFKLQTKEFQVLKSLGKLAMGSDSQSVSSSSTQDPHRGRQTLGSLRGLAKAKPEASFQVWNKDSSSKNL IPRLQKIWKNYLSMNKYKVSYKGPGPGIKFSAEALRCHLRDHVNVSMVEVTDFPFNTSEWEGYLPKESIRTKAGPWGRCAVVSSAGSLKSSQLGREIDDHDAVLRF human ST6 Gal1 comprising NGAPTANFQQDVGTKTTIRLMNSQLVTTEKRFLKDSLYNEGILIVWDPSVYHPDIPKWYQNPDYNFFNNYKTYRKLHPNQPFYILKPQMPWELWDILQEISPEEIQPNPPSSGMLGIIIMMTLCDQVDIYEFLPSKRRTDVCYYYQKFFDSACTMGAYHPLLFEKNLVKHLNQGTDEDIYLLGKATLPGFRTIHC (SEQ ID NO: 101); or a protein having an amino acid sequence at least 80%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1-4, 101, or 150, or a nucleotide sequence having at least 80%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 100 or 151.

[0104] In one embodiment, the mutated ST3 gene is gtcaggtctc agaaaagtct agaataaggc ttacaggcac tttgttcagt tgtggaacac atggaaactt catacaccgc ccccctcctt gcagaccgga gagctctctg ctctaatttg ggcacaggcg cccaaccttg ggccccagag gaagcctgtt ctactccaccgcgct caggcatgct gtaacttggc acccacactg ttccaggctc tggtctcagt ttcactctct gcaaaatgag aggctcagag ttccgctgac atggcctcgg ggccgttaaa acctttcttc atgaagacag cctgcaccct tcctgcttct gctccaggtc catcctcaga acctccaga aggcagg aggcaggta cccggcagtg atgccccacc ccccacccct tcccttactc ataggcctg ctccagactc tcagagccca cacccacttg gtgaagtcat ttgccagtaa ttcttcgcac tggacattga gaggtttcag accccagaag tctcaggcgc tgggtctgaa accggctcactcactc ggtgcgtata gccgccggga ccattttcag actcaacctt tctgacctgg aaatgccaat agaagtaatc atcatcgcca ggggctgtgg tgagcaaatg cttggagctc tgtggccagg gcagtttcat ttgaggacca gagatggaca atccctcagc ctactgagat gagaaactg aggccagac aggttagact ggttgcacca ctgaggaaga ttgacctgac ttcccaagac catacatctg gtaaaccgga acctgcacct gcgccatctc aagcctactc tggaggcccg aggctaattggcagagtttg agggctgaga tgacagacaa ccctcagtgc cttcatcggg cgggctgctt acctgcacat ccctggtgac agcatgggaa agaccgcttc taattaagcg tcatcacaca tcaccctttt ccggaggaag agaggcaaag acagctcccc tctatcctgc actgtgaacc ttcctccgag gtcctcccct cacccccgag agtccttgcc ctgtcaccaa gattaattac ccctcaaccc ctttggatgg caaaggcagt cattttatta agtttgatta aagcttcaag agacattgcc ggatgtttca ggactgctga caaagcagcc tgcttgtttc ctgaaaagac caattatatg caagaagcgt cagccccact ccccgagggg tccacttagc ctccgaccac cacagggtga tgtccagcgc accggtgtgg ccatcacctg gcgggagtga ggtcggccgt agcaggtctg gaggcccccc tgcaagtccc tgcctcaccc tgtgactgag cctctcgtct ctgggaattt tgttccaaat tccccgtcta ccaggtgtga tttcctccag ccccaccagc cctgggaggc gcccatccag agagcagaga tggtgaccat gagaaagagg actctcaaag tgctcaccct cctcgtcctc ttcatcttcc tcacctcctt cttcctgaat tactcccaca ccatggtgac caccacctgg tttcccaagc agatggttgt cgagctctca gagaacttta agaagttcat gaaatacact cacaggcctt gcacctgtgc ccgctgcatc gggcagcaga gggtctcggc ctggttcgat gagaggttca accggtccat gcagccgctg ctgaccgccc agaacgccctcttggaggag gacacctaca gctggtggct gaggctccag cgggagaagc aacccaacaa cttgaacgat accatcaggg agctgttcca ggtggtgccc gggaacgtgg accccctgct ggagaagagg tcggtgggct gccggcgctg cgcagtcgtg ggcaactctg gcaacctccg agagtcctgg tacgggcctc agatcgacag ccacgacttc gtgctcagga tgaacaaggc ccccacggcg ggcttcgaga tggatgtcgg gagcaagacc acccaccacc tggtgtaccc cgagagcttc agggagctgg cggagaatgt cagcatggtc ctggtgccct tcaagaccac cgacctggag tgggtggtca gtgccaccac cacaggcacc atctctcaca cctatgttcc tgttcctgca aagatcaaag tgaaaaagga taagatcctc atctaccacc cggccttcat caagtacgtc ttcgacagct ggctgcaggg ccacgggcgg tacccgtcca ccggcatcct ctccgtcatc ttctcgctgc acatctgcga cgaggtggac ttgtacggct tcggggcaga cagtaagggg aactggcatc actactggga gaacaatcca tcggcggggg ctttccgcaa gaccggggtg cacgacggag actttgagtc caacgtgacg gccaccttgg cgtccatcaa taagatccgg atatttaagg ggagatgacg ctgccgagga gcaccggagc ccgcctcttt ggccagcccc agcctctgct ggagccgatc tgtgctgggg gctttgaggg ccagcctcgg gggcgtgttc aggtgcccct cgtgccccct cgcaccccga catttggcag catcgactca gcaaggccccagagccccgc tgggtctgca gagcgagtgt cagaactggt cttggatggg gacacccccc ctccacctcc ctgcaccgcc actgctgtcc tggagcaggg ggcaggtggg gagacgcggg aggaggtccg gtctccaaac gctcaagtca tttcggcttt ggggggcaga gggggagata tcgaagactc tgtgttctaa agtgaaggac actggccctg ggcagagggg tcccgggctg actctctgac tctgatgctt gctgggtgag gacgacctcg ggacagagcc acatcgggcc acgtggacgc tgggggtgag aaggcacctt cctctgcacg atgcccggcc cctccctcac cgcctctgcg gtcttcccga agctcctccg tggcggccag gagaggcgcc ctgcgccgag ggtcctcaca gatgcttggc caagtgtttc agactccagc aggtgtcttc ttcgcaagct gaggctccct tgagtgatcg atctttgtgg ccataaatga tggctgagag cagatctgac tacttccacg tgccttttgtg tctggggggag agctgtgcgc attggctgaa ataaggcaaa agccttaatt cgggagtggg gagctcccccc ctctccccgc ccccagcaat gccaccccct tgctctggag ctgggtaaca tctttactag tttcctgagg cggtgccgga gctggaatga agctaggatg atgctcaacg gcgtccagag gtgcgtcagc cgcggcacgt cattcttagg ttcacagact gatggatttg gggcccgtga ccttccaagg ccacacacag aacctccacc agccagcact ttgccccagg agcccaccct ccaggccaag tccctccggg ccacctgcac agtgtcctggggtttgtggg gccatcccag gaacatgacc ctcggagcgg gagaagacat acctcatcct ctcacttctg ggctgctctg attcctcctc atgattattt attgattttt ttttaattca gctgacattg cgtgcacttt gtggctccgt ggctggtcct gatgttttaa ttaagcttgc tctcgcttca cctggcagcc ggggcatggg gggcttaagt caaggtttgc aggagtcctc agacttggga gggggatgca tatctagggc ttgggggctc gtcggtgggc aaatgccagg gtttcaggtt ggtaggttct ccgagtgctc tgtacctcct tcccctcagc tctgcctccc ttctccatca ttgcgggttg ggatcattct caggctaaga atctgcaaaa cccagtgaga cggccttggg gccaggtggg gagtggagtc acacaggcag ggcagcgatg gttggctccg gttgctgaca ctgaatcaga aaatccacgt ttcctattga gagcatttcc taacaggcca ctgctacttc gaggaggtgt gacagtgtcc tggctgtcac gggggcccgt ctcctgtgct ccttgcagca gcactccggc aagttctgct ctcctggctt cagtctctgc ctctgcaaac ggagagggag gcatttgggg agctcagggg gccacaggtg gtttcagggg gttcacaagc gtccccagag agagcacacc acgcccgtgt tttaccccaa gcctgtgtgt atgagcatat attcctgggg agcggtgccg tagcgttcat tcaattgtca gaggttcaga acccagacaa tggtttacaa aaacacgaaa cgacagcaag caaacaaaaa cccgttcgag ctttaaaatc ctccaacacttagactctct tgacccctaa aggctcataa atcactacga ggtcagcaag ctgatcataa atcccaaaga ctatatagca ggcatctgtg ggttggctga tgaatccgct gagagaagtg ggtgcggagc ggcctccgag ctcctccccg agtcctggaa gagccgagtc cagtcagagc tgatgggtcc agagcgctca gcctgcgtcg gggctgtgct gagtcaggac aggtccccag agtaaggggg ggggtctctc tccctcccca ccccactgag ccgtccaggc tgttgagagg acttccacc tgccttccca tgtaacactt gagaaaagc aggcccggag aagggaagtg gcttgcaaag tgaacccgtg gtgctggaca acgcaagtca gaccctttga gcaaagatag agcacgattg ctacatgtt aacacacctc gaggtgggct ttcccagccg gcttccatcc agcctcccca aggactgtga ccgggcacag gtgggctcct caggctgcac gtggtggccc ccgcaagctc cggtgtctgc cttttcccc catccctcca gtgagggaga cacccttgct cttccctcag ccccagcaaa cttgtctttg tgttcgttgg cttagcaaag gggtctgacc cgaatgcacc aacgggcttc agccccctgg catctgtacc tggggtaagg gtggctgaga atccaaccag agtatttccc ccagagctcg tggctgtggg caggttaacg agaaccacccc gcccccccca ctcggaggag gctcctgaat gcccacccca gtgttgggct cccagcctag ggccctgctt gctcctcaag cacagagctc ccttggaggc cagtcctgga cccctgggca gggagcagaggcatccctgc ctctctcagt gctgagcccg aagccagga gaccagggcc gaggagggag agagagattt taacaccctc cccggccctg gggagccagg agctggagtc acctgccgca aatgcaaggc ctggttccac gtgcccccct ccgcccctgt cctcacccc ttgttagggt gggtttgtag cgtctggctc ccagagccat caggcaaaaa agaaagttct taaagagatg cttttagggg cctgtgctta gtgggagag ccagattcaa gaaaatggga cttaaaacgg ggagccaga cgagaaggtg gggggggggt tgtccggggtg tgattcttac aagtgctgag gccagaagga tggaccctct ccaggtccag agtaggtgg agtcaacggg gaatatttag gactgtgatg tttgcagtaa tggtcctctt caaggcagcc tggacacaaa gcaaaatcgg aaacagaaaa gaactcacca tcccttgcgt gggtaggacc aaaacagctt tctccagggga ggcgcaagcc ctgagtggcc gccgtctggc caaagccggc ctcgtgccat cgggaaccgg ccaccgccca tgcccctccg gagagctcgg ggtcgtcatc gtgtccacca cgggcatctg gtcggaagca aacgttccca caggccactg gctggtcatc gtcccctgtg gtctgggctg accggttga gaccaagtcc gctgccttgc tccggcctgt cctgagaca ccagcgcccc ggcctgtggg gtgccccgtg atgcctccat gcagggaga gcaggggacc agggaggag agcagagaca ggtgaagcga cagtccccgc gtcccagcct cagcattcgc atcctcttgg cccctacttttcctctccgc ccagcagaca tctgccctgc ccttgccctt gaccccattg ctgcgcttcc ctcaaggacg ggcctggcct tggtggccac ctgcggacag ccctgcgccc gacgcccgct tcaccccggg gcccgggtct ggaggggccg cccccaggac gaacgcggct gccccacggg gccggcccct caccggcttc gcgtccaagc caaagtttct cgagcacttt tttgttcttt gcaatcatgt tgggttcatt gttggtgttt taaaattttg cttccctctc cctctggcct cgctcctgtg tgtgttttgt agccgagcgc taacctggat gcttttttga at least 80%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% nucleic acid sequence identity with canine ST3GalI, including atgacctttg caagagcctg ccttcctcgg cctctgctct gttttattta ttgttgaata tttccaatga tccaaatcaa agtgaattaa aacaaagcta ttttatcgtt (SEQ ID NO: 5) (Accession No. XM_022426722); mvtmrkrtlk vltllvlfif ltsfflnysh tmvtttwfpk qmvvelsenf kkfmkythrp ctcarcigqq rvsawfderf nrsmqpllta qnalleedty swwlrlqrek qpnnlndtir elfqvvpgnv dpllekrsvg crrcavvgns gnlreswygp qidshdfvlr mnkaptagfe mdvgsktthh lvypesfrel aenvsmvlvp fkttdlewvv satttgtish tyvpvpakik vkkdkiliyh pafikyvfds wlqghgryps tgilsvifsl hicdevdlyg fgadskgnwh hywennpsag afrktgvhdg dfesnvtatl asinkirifk an ST3 gene encoding a protein with an amino acid sequence that is at least 80%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to gr (SEQ ID NO: 6; accession no. XP_022282430); cggctcgctc cgggagggca gagccggcaa gggcgggact ggcctgctgc gggcggacgg gggagccgcg gagctaacgg gtccggacgt cgccagcggc ggagctttct gcacggggcgg aggcggcggc aggagcggca agcatggccc gcgcggcgcc cggcgctgac tgagggtcat gctgcggcag cctggcgcct gacgagtgag ggggaacctg ccaggggatt actgctagca cgagctaaca gcaggacagc aggcctcagg ggagtgggga tgggcatagc agcctgcctg caacgctggg ccagtggcac caaggaggcc caggccctgg cagggagctc ggcacacgct cagctgaccc agacagcatt caaagcaggt ccctgctgag cagcagctgt tcccaggccc tggaggaccg caaacgcctc agcaaccaca tttggggctg tgaccatcag gagaagggac caacacctcg aatgggaagc agaagggcag aacctgctgc caggaggatc ttggtctctg cagcaaagtc ttccgacacc cgtggagctt cttgggtttc tcctctcagg actggggcct tgtttcctcc tctggtggag ggggtaggcc aggagggcca gctgaggtgg ggacgactct cggatggaat ccaggccact tgaagcccat gggctctctg ctgtgattgg ggttcgaggg cttccccact gcaggggacg aggggccgcc tccgtgtctg ctcatgaacc acaaggaccc cgaatgctcc agactggacc atttcgagcc accaaagagt ggggccccca gagctggcag ccagcagctc caagggacta gagagctggg atggactgac ctccccctca ccagggcatt aggagagtcagagcctctgt ggcccagcta gtgacagaga gacccgatga ggccataagc tggcacccga gtgactcagg gacaggagag ccactcctgc ctactgtgct ttctctttac attgagggag ggtgtggtaa gggacccctg cctctgttcc ctcctcagca cgtgccccta tgccctttgc acgtggtgcc aggagacgca ggctacactg tggctggccc atcagtgggc tgggaaggga gtggccacgg tgacacccac ccgcccacca ggctggtggt ctagcccccc ggggcgccaa accatgaagt gctccctgcg ggtgtggttc ctctctgtgg ccttcctgct ggtgttcatc atgtcactgc tctttaccta ctcccaccac agcatggcca ccttgcccta cctggactca ggggccctgg gcggtaccca ccgggtgaag ctggtgcctg gctatgctgg tctgcagcgc ctcagcaagg aggggctcac cggtaagagc tgtgcctgcc gccgctgcat gggtgacact ggcgcctctg actggtttga cagccacttc aacagcaaca tttcccctgt gtggacccga gagaatatgg atctgcctcc agatgtccag aggtggtgga tgatgctgca gccccagttc aagtcacaca acaccaacga ggtactggag aagctgttcc agatagtacc aggcgagaac ccctaccgtt tccgggaccc ccaccagtgc cggcgctgtg cggtagtggg gaactcaggc aacctgcggg gctctggcta cggcccagat gtggatgggc ataacttcat catgaggatg aatcaggcgc caaccgtggg ctttgagcag gatgttggca gccgaactac ccaccatttc atgtaccccgagagtgccaa gaaccttcct gccaacgtca gctttgtgtt ggtgcccttc aaagctctgg acctactatg gattgccagc gctttgtcca cagggcaaat cagattcacc tatgcgccag tgaagtcctt ccttcgagtg gacaaaa aggttcagat ctacaacccg gcattcttca agtacatcca cgaccggtgg acagagcatc acgggcggta cccttccaca gggatgctgg tgctcttctt tgccctgcat gtttgtgatg aggtgaacgt gtacgggttc ggggccgaca gccggggcaa ctggcaccac tactgggaga ataaccggta tgcgggcgag ttccggaga cgggagtgca cgacgccgac ttcgaggccc acatcatcga catgctggcc aaggccagca agatcgaggt ctaccgaggc aactgagcc ggcctcgccg cgacccttcc ggcccagccg cgaggctgcg acgctcgctc cgcagccggg actcccggcc agcccgaggg cggcggcctt ggcgagggcg cggcgcccgg ttgggcgtct ccagccctgg gagcgacgcc aagtcgcggt ctggaccaat catgctgcaa gtccagcgag cgccggctgt cccccgccaa tcaggagact ttggggctg gcccaggcct ggcacccaat cagcgctgca gtgggagcgg aggctctttc tcccagccaa tcatgcgact caaggagaac ttccggcgct gggcccggtc tcctccaatc aatggccttc ggaggcgggc cggccgccgc tgaatcccca ctcccctatg ctttgggtag gattttatt tatgctttt aaggagatgt ggttggttcc ggcctcagtg gagtactttc ctcaggctctgcgggaggag tgttggtggc ctgtcggcgg tactcggcca ggggcaccga ggaggaagcg ggggggaagg tgcggggcag cagcggctgg gcctcccttg gccgggggcc cctcgcgacc tcggggcggg cgggggggcg ggggcgttgg cctcccgctt ctggaggtcc ggggggaatc aggtggtttc cggggagcgc gctttcatcc ccgggaagag ctagatccct ctcaaacctt ttcaggcctc agagcactct agaccgcgta tttcctttat ctgtcgggcc cagatgggtg agtgtaaacc caccaaagaa aggcagtgga gcgtgggtcc cctcgtcctc ccttgtcccc atccccacct ttggccaccc tatgggatgg ccttcctgac cagggcattg agcatcccac ctggaaacta gaactgtatt cacccgttgc tagggctcgg agttcgccga tggcctaact cgagcgcagg ggtaaggaac agggcagggg acccggaagc cccgtcactt cagatgtaag gtgcttctca ctctcgtgta ctctcgcggc cccttactgt tcgcccacaa cttttttagt gtcccttctt aagccctggg cctcctcacc agcctgctcg tcctgggatt gggggtgggg ggtggggcac tgctggcttc tccaaccccc tacccctcct cgctcgtctt tagccggctc tagggagagg aaggcaggct ggagatgggg agcccagctg cctggtgcat gcaccgtttt cctccgccca tcaccccaaa gaggagtagg aaacctcttg cttgggggtg gaatttgctt tggtctccta atttagttaa cttgaggtta ccagggatgg ctgaccaaca aagattcttt taaaattccaggctggccat gcaaattgct gggatcctag ctggggagga gtcgactgac ttgcccgcct tgcatgtctc ctctcctgcc cctgcgtccc ctccctctgc cacgctcact tcctgcctca tctctccaac ccattttcca tttcagctc tagaagggca gcatcacgc aagttacttc caagactgaa cccagcttaa gtcccttagag gaagctgctg atgatattct cacccttcaa ggttggggaa gtttcggagg gggaaagtgc ttctgtgaag cttccaaacc actaatagga tcccccttcc caacaatgag gaacacaaac accaccttg atccactg attaccat g ggccattggg gtaattcctg cagctggctg gggtaaccag caggggagta tattagagga ggattggggc agggcagtgg gcacccctaa agttaatata ttgagaactt agcttaagcc taagtcttag ttccttccca attccaaaag taggaggagc aacgagtggg ggtggattccta cgggctgctgctgctgct ggcttccccc accattttag agagtcaagg caccagccat tcatgccagt ctcctctcag tgcttcctga agaggctgtt tggagtgttc ggaaaatgaa aaaaacaatg caattatgcc aaacagtatt gagcagaata atttattttt tttttgttcc ttttctttct ttttgcttg tttaatctactctat agaggtaggt cccagcatcc agcccagatc tccttttctg caatagttat ttaaacaaat gttttgtttgt ttttttatt tcttcccttt ctctctttan ST3 gene having a nucleic acid sequence that is at least 80%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the canine ST3GalII gene, including ctgaattaaa aaaaaagaaa actccta (SEQ ID NO: 7) (Accession No. XM_014114023); mkcslrvwfl svafllvfim sllftyshhs matlpyldsg alggthrvkl vpgyaglqrl skegltgksc acrrcmgdtg asdwfdshfn snispvwtre nmdlppdvqr wwmmlqpqfk shntnevlek lfqivpgenp yrfrdphqcr rcavvgnsgn lrgsgygpdv dghnfimrmn qaptvgfeqd vgsrtthhfm ypesaknlpa nvsfvlvpfk aldllwiasa lstgqirfty apvksflrvd kekvqiynpa ffkyihdrwt ehhgrypstg mlvlffalhv cdevnvygfg adsrgnwhhy wennryagef rktgvhdadf eahiidmlak an ST3 gene encoding a protein having an amino acid sequence that is at least 80%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to askievyrgn (SEQ ID NO:8) (Accession No. XP_013969498); ttggtggtcg cgctccgccc gcgctgcgt ccccaccatg gcggcgccg tgcagcccc cgcgtcgtgg gcgcccgccg ggctccccg cggctgtggc ggcgcccgc gccgcggcgcgcgcgccgcgcgcgcgc ctgcctccgt ctccgctgcg gtcatgtagg aaatcgtaaa tcatgtgaag atgggactct tggtatttgt acgcaatctg ctgctagccc tctgccttt tctggtactg ggatttttgt attattctgc gtggaagctg catttactcc agtgggagga ctccactcttcttc cgctggacaa acactaggct cagagtatga tcggttgggt ttcctcctga agctggactc taaactgcct gctgagttag ccaccaagta tgcaaacttt tcagagggag cttgcaagcc tggctatgct tcggccttga tgactgccat ctttcccc gcaagtgggc taggattcgg gagtttgtgc cgccttttgg gatcaaaggt caagacaatc tgatcaaagc catcttgtca gtcaccaaag agtaccgcct gacccctgcc ttggacagcc tcagctgccg ccgctgcatc atcgtgggca acggaggtgt cctaggct agtcactgct tgactgaatc gacattgtgg tcagactgaa ctccgcacca gtgaaaggct ttgagaagga cgtgggcagc aaaactacac tgcgcatcac ctaccctgag ggcgccatgc agcggcctga gcaatatgaa cgcgattc tatttgtcct cgctggctc aagtggcaggcacttcaagtg gttgaagtac atcgtctac aggagagt gctctgggcc cgcagggata cctgccaatc tgtctggcc catccccctc tccctccac cagctgtcac cagccacccc agggagggg acctgcag tcaggccact atacgagtagccact tgacagacag cctctggcga caagtgcatc agatggctc tggaatccg tggcacacg agtgcccaag gagccctg agatcgc cttcacccg tactcatcc aggaggcgccttcaccctc atcggactcc ccttcaggcag cggcccggccac gtggcagtga ccatggcgct acacggctgt gatgaggtgg cagtcgcagg ctttggctac gatagagca caccaacgc gcccctgcac tactatga ccgtgcgcat ggcagccatc aaagaggtca ccagcgactc accagcaatggtt tgccagct tgccactc acctcccaga aatgctttt ctgttgacca ctccttcctc tttgaaactt ttcctgctca gactgtcctg gacacacaat atccagcgag agaagagtt tctgcgcaag ctggtgaagg cgcgcgtcat caccgaccta accagccaggca tcccaggg tccagggag ccgccaagag gaagccgcag ccactgccac ctgtccactt cattggcctc ggtctggctc tgctgaaag gcgcaggagt cttcagaccc agagaggac agtgccaaggan ST3 gene having a nucleic acid sequence that is at least 80%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the canine ST3GalIII gene, including gg (SEQ ID NO: 9) (Accession No. XM_025420404); mgllvfvrnl llalclflvl gflyysawkl hllqwedsns vvlsfdsagq tlgseydrlg fllkldsklp aelatkyanf segackpgya salmtaifpr fskpapmfld dsfrkwarir efvppfgikg qdnlikails vtkeyrltpa ldslscrrci ivgnggvlan kslgsriddy divvrlnsap vkgfekdvgs kttlritype gamqrpeqye rdslfvlagf kwqdfkwlky ivykervlwa rrdtcqsvwa hpplpstsch qppqgrgpae frpfffqyps llleenddrq platsasdgf wksvatrvpk eppeirilnp yfiqeaaftl iglpfnnglm grgniptlgs vavtmalhgc devavagfgy dmstpnaplh yyetvrmaai kevtsdsaqg cqiqwthgsl ifpdlpemlf llttpsslkl fllrlswthn iqrekeflrk lvkarvitdl tsgi (SEQ ID NO: 10) (Accession No. XP_025276189), an ST3 gene encoding a protein having an amino acid sequence that is at least 80%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to vavtmalhgc devavagfgy dmstpnaplh yyetvrmaai kevtsdsaqg cqiqwthgsl ifpdlpemlf llttpsslkl fllrlswthn iqrekeflrk lvkarvitdl tsgi (SEQ ID NO: 10) (Accession No. XP_025276189); gccctacagg cccgagctgc cggggtcggg cctccccggg ttcccgctcc cgggtcctcc tggacacacc ggcctggcct ggctcccggg gaactctcgt ctgctagcgg ggagcctccc tccgcctcgc ccacgggcac ccctcccacc cagtatcctt ggcctcttgc aggtggcccg aggcagccgg gatgacagct ctccccagga accctgctac cctctgagaa acatgatcag caagtcccgc tggaagctcc tggccatgtt ggctctggtc ctggtcgtca tggtgtggta ttccatctcc cgagaagaca ggtacattga gcttttttat tttcccatcc cagagaagaa ggaaccgtgc ttccagggtg aggcagagag aaaggcctct aagctctttg gcaactactc ccgagatcag cccatcttcc tgcagatgaa ggattatttc tgggtcaaga caccgtctgc ctacgagctg ccctatggga ccaaggggag cgaagacctg ctcctccggg ttctagccat caccagctac tccattccag agagcatcca gagtctcaag tgtcgccgct gcgtggtggt gggcaatggg catcggctgc gcaacagctc gctgggagat gccatcaaca agtacgacgt ggtcatcaga ctgaacaacg cccccgtggc tggctacgag ggtgacgtgg gctcgaagac caccatgcgt ctcttctacc cggagtcagc ccacttcaac cccaaagtgg agaacaaccc agacacactt ctcgtcctgg tggccttcaa ggcaatggac ttccactgga ttgagaccat cctgagtgat aagaagaggg tacgaaaggg cttctggaag cagcctcccc tcatctggga cgtcaaccccaggcaggttc ggattctcaa ccctttcttt atggagattg cagctgacaa actgctgaac ctgccaatga aacagccacg caagatttcc cagaagccca ccacgggcct gctggccatc acgctggctc tccacctctg cgacctggtg cacatcgccg gcttcggcta cccggacgcc cacaacagga agcagaccat tcactactat gaacagatca cgctcaagtc catggcgggg tcaggccaca acgtctccca ggaggccctg gccatcaagc ggatgctgga gatcggagca gtcaagaacc tcacgttctt ctgacgggga caggagctct agccgtcagt ctgcccgccc tgccgcctga gcgaccaacc acggctgtgg gggcgccggc gtgacctgct tggattcccc ctccccgtgt ggagaggggg cctggtacag gcggggcctg agatggggcc gcgcccctgg ctgctcttgg ggcggccgga tccagtcagg gtggaggccc cgggtggcgg gaggccttcc gaggcgcggg gtgtgtggct gaggcacccc ttctcaccag ccccgggagc ttatttaatg ggctatttaa ttaaaagggt aggaatgtgc ctcgggctgg tcccatggca tccggaaacg ggggcatagc acagtggtct gcccactgtg gataaaaaca cacaagtgct tggcccacta gagcctagag ccagagcagg cctcccagga gggcaggggc gtctggagcg ggtgggtgcc ctccagagag gggctgctac ctcccagcgg gcatgggaag agcattggga tgaggtccca cggagaatgg gacctcatgt agaaaagagg tttgaaacct aacattaaac tattttttccan ST3 gene having a nucleic acid sequence that is at least 80%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the canine ST3GalIV gene, including taaaacggaa (SEQ ID NO: 11) (Accession No. XM_014113293); misksrwkll amlalvlvvm vwysisredr yielfyfpip ekkepcfqge aerkasklfg nysrdqpifl qmkdyfwvkt psayelpygt kgsedlllrv laitsysipe siqslkcrrc vvvgnghrlr nsslgdaink ydvvirlnna pvagyegdvg skttmrlfyp esahfnpkve nnpdtllvlv afkamdfhwi etilsdkkrv rkgfwkqppl iwdvnprqvr ilnpffmeia adkllnlpmk qprkisqkpt tgllaitlal hlcdlvhiag fgypdahnrk qtihyyeqit lksmagsghn vsqealaikr mleigavknl an ST3 gene encoding a protein with an amino acid sequence that is at least 80%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to tff (SEQ ID NO: 12) (accession no. XP_013968768); cgctctggaa ccacttacag ccacctggtg catcctcctt tggggtgcgt ttggagggcc tggttcctgc tcagccacat cttctgccac tttcaccagc aatgcccagt gagtataact atgtaaaact gagaagcgat cgctcaagac cctctgca atggtacacct acagaaccac ttgttgttaa aagacatcct taagtgtaca ttgcttgtgt ttggagtgtg gatcctttat attctcaagt taaattatac tactgaagaa tgtgacatga aaaaaatgca ttatgtggac ccagaccgtg taaagagagc tcagaaatat gctcagcaag tcttgcaaaa ggaggttc gatggcgcag ttgttcgagc acaggtacag cacggacttg ccacctttcg tgaaggagac ccccaaaatg aatgaagccg agtacaagta tgatcctcct tttggattcc gaaagttctc cagtgaagtc cagaccctgt tggaaatact gcccgagcat gacatgcccctagagagagc tgtaggcgtt gtgtggtcat cggaagcggt ggcatactcc acggactagc actgggccag gccctcaacc agttcgatgt ggtgataagg ttaaacagtg caccagttga gggatattct gagcatgttg gtaataaaac tactataagg atgacttatc cagagggcgcgtc gccatc tgacttgttt gttgctgttt tattcaagag tgttgacttc aactggcttc aagcaatggt aaaaaatgaa accctgccat tttgggtgcg gctcttcttt tggaagcagg tggcggaaaaaatcccacta cagccaaaac atttcaggat tttgaatcca gttattatca aagagactgc ctttgacatc cttcaatact cagagcccca gtcaaggttc tggggccgag ataagaacgt gcccaccatt ggtgtcattg ccgttgtctt agccacacat ctgtgtgatg aagtcagctt ggcaggcttt ggatatgacc tcaatcaacc caaaacacct ttgcactact ttgacaatct ctgcatggct gccatgaact ttcaaaccat gcataatgtg acaacggaga ccaggttcct cctcaagctg gtcaaagagg gcgtggtgaa ggatctcagc ggaggcatcc attgtgaatt ttgaacacag ggaaacctca an ST3 gene having a nucleic acid sequence that is at least 80%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to a canine ST3GalV, including tgtgacaatg caactctgac tctgaaggct gtttttcgta gccttctcga tgcagcgcat cctgcaaaat acttagaggt gcagctgggg ttttt (SEQ ID NO: 13) (Accession No. XM_022404744); mpseynyvkl rsdrsrpslq wytraqnkmr rpnlllkdil kctllvfgvw ilyilklnyt teecdmkkmh yvdpdrvkra qkyaqqvlqk ecrpkfakks maqlfehrys tdlppfvket pkmneaeyky dppfgfrkfs sevqtlleil pehdmpehlr akscrrcvvi gsggilhgla lgqalnqfdv virlnsapve gysehvgnkt tirmtypega plsdleyysn dlfvavlfks vdfnwlqamv knetlpfwvr lffwkqvaek iplqpkhfri lnpviiketa fdilqysepq srfwgrdknv ptigviavvl athlcdevsl agfgydlnqp ktplhyfdnl cmaamnfqtm hnvttetrfl lklvkegvvk dlsggihcef (SEQ ID NO: 14) (Accession No. XP_022260452), an ST3 gene encoding a protein having an amino acid sequence that is at least 80%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to agfgydlnqp ktplhyfdnl cmaamnfqtm hnvttetrfl lklvkegvvk dlsggihcef (SEQ ID NO: 14) (Accession No. XP_022260452); ST3 gene ggtcgattgc cccttggctg ctgtggaggc tgtgatgacc tccagggccg cggccctccg ggcgatgctt ctccaggggc tgaggccaac gcagaactcc cgtggcaccc actcggactc gcggcgtgtt cacatgtggg gttttattaa atcctcccac caaccgtgtg agacaggaac agttagcccc ggtgtgtccg ccaagattgc cccgcacaag tggctccgga tggatcacac gaagcacttg caagtgaaga agcagcacag ccctttatct tgggctattt cctgtggaga gactccaaca atttagcagc caggctcctg ggcctctggg accctcacca catcacatcc ttcaccttca ggagcagagc gcctttggga aacagacttc taaaagtgca ggtgggccag ccatgagagg gtacctagtg gccatattcc tgagtgctgt ctttctctat tatgtgctgc attgtatatt gtggggaaca aacatctatt gggtgccacc tgtggaaatg aagcggagaa ataagatcca gccttgttta gcgaagccag cttttgcctc tctcctgagg tttcatcagt ttcacccttt tctgtgtgca gctgatttta aaaagattgc ttccttgtat ggtagcgata agtttgatct gccctatggg ataagaacat cagcggaata ttttcgactc gctctttcaa aactgcagag ttgtgatctc tttgatgagt ttgacaatgt gccgtgtaaa aagtgcgtgg tggttggtaa tggaggagtt ctgaagaata agacattagg agaaaaaatt gactcctatg atgtcataat aagaatgaat aatggtcctg ttttaggaca tgaagaggaa gttgggagaaggacaacctt ccgacttttt tatccagaat ctgttttttc agatcccaat cacaatgatc ctaatactac agcgattctc actgctttta agccgcttga cttaaagtgg ctgtgggaag tgttgacggg tggcaaaata aacactaatg gtttttggaa gaaaccagct ttaaacttga tctacaaacc ttatcaaatc agaatattag atcctttcat tatcagaatg gcagcttatg aactgcttca cttcccaaaa gtgtttccca aaaaccagaa acccaaacac ccaacaacag gaattattgc catcacgctg gcctttcaca tatgtcacga agttcacctt gctggtttta aatacaattt ttctgacctc aagagccctt tacactatta tgggaacgcg accatgtctt tgatgaataa gaatgcgtat cacaatgtga cagcggaaca gctctttttg aaggacattc tagaaaaaaa ctttgtaatc aacttgactg aagattgacc ctacagactc tgcagatgat gctaagagta ttagttttat ttttatactg caatttttag tttattttta aatatgttgg atgcacttgt caaaaaattg tgtatagtca gtctgttgct gcctggtgat tcataaccac cagcttaatt tctgtgaata tatttaattt ataaaaacca agaagatatg cttagatatc cgggaagttt tgattgcgtt ggttttaaaa caaccttagt tctctgaagt gtttttaaac atctttttta atagttactt catctttgac ttctgagggc atgtgacgtc caagtaaggg gctttagctt gaccaccaca aactctgaac agagttggtg gcggattcgg ctactgtaaa ttggtggggaatagccatgt gattgtgcaa actggaaccg gtttaggcaa gtacgagtt cctttact gaacccgagg aaacggattt gatcttaa gcaggccca ccatagcagt aggtacggtt atgaatcta agatcataat ggttcatta agcttttt cctgttag tagtagtagtagtagta aaccagatta tttaaggtgg aggaacagg ctacatgtga aattctggat gagtaaacaa cctaggaatg cattactaa agtctggtgg ctgcattatt ttaaagttca tacaagaag cagagctagg ccacctcaag gagacagttc ttaaacgtca tcttgcct ggattaattact ggattatatact aggaaagagatg atacggcac agtaggtaaa tccttcagac tcctcaggct gtttggat ttaatggtc ctttcgtgaa aaatctcact tgtccacggt gaaatcccat cttcaaggg aaggctacc cggctaccta gggtgcatca gagagacagccactc c tgtccaacaa acgtgcgccc gtctctctc tcaagaggg atggaatgaa cagctcag agaggtaag agttgaagga cttgttacc tctgagcgat aatcgtg gagacact gctggtagttc ctgaaccacca gcctgcaccgact gagagagact gagagact atcgtgaagc acggcataag gggggagaag cctcgtagtt gattgaaccc atgtctacgt ggctcagct gattcccctg taacggaggt ggaagttcc cgcacgtaca cagctgcacg ctgcagcctggcggctggga ttccatgggt ggactcattc agggtacaaa gacagtcctg gctgcaaagt gaaaaacccc aggtggcatt ttcaagtgtt tatggactga aataatggct gtacggtatc tggcggatgc tcaacttgag gaatcggcat ttttgtacag tgggagctga ggctataaac ctcagcgtgg cttcacataa gccagaagaa actctcagcc cgatacatat gtacaattta ttaaaaacac atgaacacgt taaaatctca ctatttatac aatctacatt ctagcaacat atacaaatac cgagtgacta cagtacatgc cgaggtaaga aaagtacatt cggggagact atcactgaca ctcaagccat ttttatttcc an ST3 gene having a nucleic acid sequence that is at least 80%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to canine ST3GalVI, including aatatgtttt gctttcacct ttcccagtgc caaaaaaaaa aaaaaaaaa (SEQ ID NO: 15) (Accession No. XM_005639375); mrgylvaifl savflyyvlh cilwgtniyw vppvemkrrn kiqpclakpa fasllrfhqf hpflcaadfk kiaslygsdk fdlpygirts aeyfrlalsk lqscdlfdef dnvpckkcvv vgnggvlknk tlgekidsyd viirmnngpv lgheeevgrr ttfrlfypes vfsdpnhndp nttailtafk pldlkwlwev ltggkintng fwkkpalnli ykpyqirild pfiirmaaye llhfpkvfpk nqkpkhpttg iiaitlafhi chevhlagfk ynfsdlkspl hyygnatmsl mnknayhnvt aeqlflkdil eknfvinlte d (SEQ ID NO: 16) (Accession No. XP_005639432)) encoding a protein having an amino acid sequence that is at least 80%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to (SEQ ID NO: 16) (Accession No. XP_005639432); aaagacttca ctgggtatca gtctcctttg ggagaccaca ggacacgtgt cacctctccc atcctctcag cctccagccc agaccttggc agagttcctt ttaggagtta gcaagtggct gaggaggcaa gaggtgccag agccaatcta ctatctgctg ggggatgatt gccagggcca gagatgaggg ctcaatactt gaagtggggt ctggtagctg cctgtatagt tacgttatgg ctgatgatga tgaacttcct ggaccaggag ttcaaacaga atgacttccc taaaaagaca agaatacaat tatgccactg ccccaggaac tctttcagaa agtgtaggtg ttcgtttgag atccgcaagt gctctgcctg cctccgcgta cgtggaacgt ctgtctggtt tgatgaacgc ttcgaaacgg ctattgagcc tgtgcagaga ccagaagatc ccatatcctc tgatgctctg atattgtggt tgggtgtcca atcaaagagg gagtttgaga ctcagaagcc aatagaagag cctcctgggc aaccactggg ctacgtggag tccagttgtc ggacctgtgc agtggttgga aactcaaggt gcctacgggg ctctggccat ggattcagga ttaaccaaaa tgacatggtc ctcaggatga accaggcccc cgtccaagga tttgagatgg atgtggggaa cacaaccacc atgcgcataa tgtaccccga tatggctagc acgcagaatc ctggcaccaa attgctgctg cttcctctga attcatctgg tctaaagtgg tttatggaag tactacagga acagagcttc agaaagccca taaaccctgg atttcagata gtccagtttc ctggtggaag taacacgagc aaagacgaggtcttggtgat cagcctcacc tttcttcagt acatccagga tcattggctg cgaaaacgtc atcgttttcc atccttgggg tttgtgggtc tgttatatgc cctgcacact tgtgaccagg tatccttatt tggttttggg acagatcagc tcatgaggtg gtcccattac tgggatgata aatatcggtt cgagagtaac atgcacagtt tcaaagaaga gcagaagctc atcctccagc tgcaatgtga ggggaagatt gttatctaca gctgacatgt ttctgtcctg ttcagcccac tggaggcccc aggaggctga caggtagtca aggggaccac agagtgtcag agaggactg gggcttcaag an ST3 gene having a nucleic acid sequence that is at least 80%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to a canine ST3GalII-like comprising tggaccctgg atatagatca gtctgctgct aaataaaact acagcttatt tctccca (SEQ ID NO: 17) (Accession No. XM_025469036); or mraqylkwgl vaacivtlwl mmmnfldqef kqndfpkktr iqlchcprns frkcrcsfei rkcsaclrvr gtsvwfderf etaiepvqrp edpissdali lwlgvqskre fetqkpieep pgqplgyves scrtcavvgn srclrgsghg frinqndmvl rmnqapvqgf emdvgntttm rimypdmast qnpgtkllll plnssglkwf mevlqeqsfr kpinpgfqiv qfpggsntsk devlvisltf lqyiqdhwlr krhrfpslgf vgllyalhtc dqvslfgfgt dqlmrwshyw ddkyrfesnm hsfkeeqkli It has an ST3 gene encoding a protein having an amino acid sequence that is at least 80%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to lqlqcegkiv iys (SEQ ID NO: 18) (Accession No. XP_025324821).

[0105] The present invention is further illustrated by the following non-limiting examples. EXAMPLES

[0106] method

[0107] Cells. MDCK and AX4 cells were maintained in Eagle's minimum essential medium (MEM) containing 5% newborn calf serum (NCS) or 10% fetal calf serum (FCS). All cells were incubated at 37°C with 5% CO2 and periodically tested for mycoplasma contamination by using PCR to confirm the absence of mycoplasma.

[0108] Clinical specimens. Respiratory specimens were obtained from patients with influenza-like symptoms who visited clinics in Yokohama City (Japan) during the 2017–2018 influenza season and submitted to the Yokohama City Institute of Health for virus isolation. These clinical specimens were collected under the National Epidemiological Surveillance of Infectious Diseases (NESID) program of Japan. Respiratory specimens were also obtained from patients with influenza-like symptoms who visited clinics in Tokyo (Japan) during the 2013–2014, 2015–2016, 2016–2017, and 2017–2018 seasons and submitted to the Department of Virology, Division of Infection and Immunology, Institute of Medical Science, University of Tokyo for virus isolation. These specimens were collected by the examining physicians after obtaining informed consent. Our study protocol was approved by the Research Ethics Committee of the Institute of Medical Science, University of Tokyo (approval number 26-42-0822). Samples that were positive by real-time RT-PCR (see below) or rapid diagnostic kits were used in this study.

[0109] Viruses. Human influenza viruses were propagated in hCK cells in MEM containing 1 μg L-1-tosylamido-2-phenylethylchloromethylketone (TPCK)-trypsin / ml.

[0110] Real-time RT-PCR. RNA was extracted from clinical specimens by using the Simply RNA Tissue Kit (Promega) or RNeasy Mini Kit (Qiagen). Real-time PCR amplification and detection were performed using the Applied Biosystems 7900HT Fast Real-Time PCR System (Applied Biosystems) or StepOnePlus Real-Time PCR System (Applied Biosystems). RT-PCR was performed using the QuantiTect multiplex RT-PCR kit (Qiagen) or QuantiTect Probe RT-PCR kit (Qiagen). The probes contained oligonucleotides with 6-carboxyfluorescein (FAM) reporter dye or hexachloro-6-carboxyfluorescein (HEX) reporter dye at the 5' end and Black Hole Quencher-1 (BHQ-1) quencher dye or 6-carboxytetramethylrhodamine (TAMRA) quencher dye at the 3' end. The list of primers and probes used is shown in Table 5.

[0111] Virus isolation. NDCK, AX4, and hCK cells grown in 12-well plates were inoculated with 0.2 ml of clinical sample per well and incubated at 34°C for at least 30 minutes. One microliter of MEM containing 2.5 μg / ml acetylated trypsin was then added to the cells. Cultures were then incubated for up to 7 days until CPE was evident. Cell culture supernatants were harvested and hemagglutination assays were performed using guinea pig red blood cells (see below).

[0112] Hemagglutination assay. Virus (50 μl) was serially diluted in 50 μl of PBS in microtiter plates. An equal volume (i.e., 50 μl) of 0.75% (vol / vol) guinea pig red blood cell suspension was added to each well. Plates were kept at 4°C and incubated for 90 min before hemagglutination was examined.

[0113] RT-PCR and sequencing of viral genes. Viral RNA was extracted from 140 μl of culture supernatant using the QIAamp Viral RNA Mini Kit (Qiagen). Samples were amplified using the SuperScript III One-step RT-PCR System with Platinum Taq High Fidelity DNA Polymerase (Invitrogen) and specific primers for the HA or NA genes. PCR products were then analyzed by 1.5% agarose gel electrophoresis in Tris buffer, and target bands were visualized by staining with GelRed (Biotium). PCR products were purified and directly sequenced. The level of mutation frequency was examined based on the wave height at each position on the sequencing chromatogram. The detection limit for small populations was 10% to 20%. The list of primers used is shown in Table 5.

[0114] Serial passage of human influenza virus. Serial 10-fold dilutions of the virus (10 1 From 10 6 ) was prepared in MEM. Each dilution was inoculated into monolayers of MDCK, AX4, and hCK cells in 24-well culture plates, one well per dilution. Plates were incubated at 33°C for 3 days. The endpoint was the highest dilution of the sample that showed CPE. Culture supernatants were harvested from wells inoculated at a dilution 10-fold greater than the endpoint dilution and used for the next round of infection. Sequence analysis was performed on viruses sampled in the supernatants from each cell line after the first and sixth passages.

[0115] Statistical analysis. Data are presented as mean ± SD. To analyze the growth curve data, we performed a linear mixed-effects analysis. As fixed effects, we used the different cell lines and time points of measurement (and the interaction term between these fixed effects). As random effect, we used an intercept for each individual animal. Viral titer values ​​were transformed to the log 10 scale and statistically analyzed using the R statistical package ( www.r-project.org ), lme4 ( Bates et al., 2015 ), and lsmeans packages ( Lenth, 2016 ). p values ​​were adjusted using Holm's method and considered significant when less than 0.05.

[0116] Generation of MDCK cells expressing significantly low levels of α2,3-linked sialic acid and high levels of α2,6-linked sialic acid

[0117] To mimic the expression pattern of sialic acid (Sia) molecules on the surface of human upper airway epithelial cells, we first attempted to knock out the β-galactoside α2,3 sialyltransferase (ST3Gal) gene (whose product catalyzes the transfer of α2,3-linked Sia to terminal galactose (Gal) residues) by utilizing the clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein 9 (Cas9) gene editing system ( Cong et al., 2013 ; Jinek et al., 2012 ; Han et al., 2018 ; Shalem et al., 2014 ). Dogs have seven different ST3Gal proteins (ST3Gal-I, -II, -III, -IV, -V, -VI, and ST3Gal-II-like proteins), each of which is encoded by a separate gene. ST3Gal-I, -II, -III, -IV, and -VI utilize oligosaccharides on the surface of glycoproteins or on the surface of glycoproteins and glycolipids as acceptors, whereas ST3Gal-V utilizes oligosaccharides on the surface of glycolipids only (Takashima and Tsuji, 2011). One previous study reported that N-linked glycoproteins are required for productive entry of influenza virus into host cells (Chu and Whittaker, 2004). Therefore, to inhibit the transfer of α2,3-linked Sia to glycoproteins, MDCK cells were transfected with a mixture of six plasmids, each containing a Cas9 gene expression cassette and an expression cassette for an individual guide RNA (gRNA) targeting ST3Gal-I, -II, -III, -IV, -VI, or ST3Gal-II-like protein gene (Figure 2). After transfection, puromycin was added to the cells, and 33 drug-resistant clones were randomly picked. Genomic DNA analysis revealed that only one clone (6-11) contained a mutation in the gRNA target region for the six ST3Gal genes (data not shown).Cell surface Sia was measured by flow cytometry using Malamute II agglutinin (MAL II) lectin, which is specific for α2,3-linked Sia, and Sambucus nigra agglutinin (SNA) lectin, which is specific for α2,6-linked Sia. Unexpectedly, the reactivity with MAL II was very similar between the parental MDCK cells and clone 6-11, indicating that the clone still expressed high levels of α2,3-linked Sia (Fig. 3). This was due to the compensatory activity of ST3Gal-V.

[0118] To more efficiently inhibit the transfer of α2,3-linked Sia and to express high levels of α2,6-linked Sia on the cell surface, clones 6-11 were cotransfected with a plasmid encoding human β-galactoside α2,6 sialyltransferase (ST6Gal-I), which catalyzes the addition of α2,6-linked Sia to Gal-containing glycans, and a plasmid containing an expression cassette for Cas9 and a gRNA targeting ST3Gal-V. Eighteen cell clones were selected with blasticidin, and genomic DNA was analyzed. Nine of the drug-resistant clones had mutations in the gRNA target region for the ST3Gal-V gene (data not shown). Flow cytometry analysis using MAL II and SNA lectins revealed that the expression of α2,3-linked Sia was significantly decreased compared to the parental MDCK cells, and the expression level of α2,6-linked Sia was higher than that of the parental cells in two of the nine clones (clones 6-11#2 and 6-11#10) (SyFigure ​(Figure4a; data only for clones 6-11#2 and 6-11#10 are shown). Terminal Sia is attached to several types of oligosaccharide structures on the surface of glycoproteins or glycolipids, including Galβ1,4GlcNAc (GlcNAc; N-acetylglucosamine), Galβ1,3GalNAc (GalNAc; N-acetylgalactosamine), and Galβ1,4Glc (Glc; glucose) (Takashima and Tsuji, 2011). MAL II lectin selectively recognizes Siaα2,3Galβ1,3GalNAc (Hidari et al., 2013). To assess whether the above two clones express different types of α2,3-linked oligosaccharide structures on the cell surface, indirect immunofluorescence assay (IFA) analysis was performed using a monoclonal antibody against Siaα2,3Galβ1,4GlcNAc7. IFA showed that the level of Siaα2,3Galβ1,4GlcNAc was undetectable or significantly lower in one of these two clones (6-11#10) (Figure 4b). This suggests that many types of oligosaccharides containing terminal α2,3-linked Sia are expressed at lower levels in 6-11#10 than in the parental cells.We next compared the cell surface expression levels of α2,6-linked Sia on the surface of AX4 cells and clone 6-11#10 using SNA and elderberry (SSA) lectins, both of which recognize Siaα2,6Gal or Siaα2,6GalNAc structures (Shibuya et al., 1989). Flow cytometry analysis showed that there was no difference in the expression levels of α2,6-linked Sia between AX4 cells and clone 6-11#10 (Figure 4c). However, the expression level of α2,3-linked Sia measured using MAL II lectin was significantly lower in clone 6-11#10 compared to AX4 cells (Figure 4d). Clone 6-11#10 was confirmed to contain the desired mutations within the gRNA target regions for the seven ST3Gal genes (Table 1). These results indicate that clone 6-11#10 expresses mainly human virus receptors and limited amounts of avian virus receptors. The resulting clone, 6-11#10, was designated hCK and was subsequently expanded and further analyzed.

[0119] [Table 1] a Influenza viruses isolated from clinical specimens were passaged 10 times in MDCK, AX4, or hCK cells, and the viral HA and NA genes were sequenced after a single passage (P1), six passages (P6), and ten passages (P10). b Mutations in influenza A viruses are shown with H3 numbering. c All mutations are shown with N2 numbering. d - No mutations were detected compared to the sequence from the original clinical specimen. e T / I, mixed threonine and isoleucine at position 167; f N / S, a mixture of asparagine and serine at position 296; g N / S, a mixture of asparagine and serine at position 446; h H / Y, a mixture of histidine and tyrosine at position 411c; i C / Y, mixed cysteine ​​and tyrosine at position 53; jNo influenza virus was isolated from clinical specimens in MDCK cells. k N / K, a mixture of asparagine and lysine at position 158; l D / N, a mixture of aspartic acid and asparagine at position 408; m S / N, a mixture of serine and asparagine at position 148; n D / N, a mixture of aspartic acid and asparagine at position 459; o G / R, a mixture of glycine and arginine at position 208; p N / S, a mixture of asparagine and serine at position 196; q L / F, a mixture of leucine and phenylalanine at position 72;

[0120] Establishment of stable cell lines carrying mutations in the ST3Gal gene and expressing the ST6Gal-I and HAT genes. Using the sgRNA Design Tool from Michael Boutros lab (http: / / www.e-crisp.org / E-CRISP / ), gRNA sequences were designed targeting the ST3Gal-I, -II, -III, -IV, V, -VI, and ST3Gal-II-like protein loci, respectively. Oligo DNA for gRNA was cloned into the Cas9 / gRNA dual expression vector pSpCas9(BB)-2APuro(PX459) (addgene), which encodes puromycin resistance. The resulting constructs were named PX459-ST3Gal-I, PX459-ST3Gal-II, PX459-ST3Gal-III, PX459-ST3Gal-IV, PX459-ST3Gal-V, PX459-ST3Gal-VI, and PX459-ST3Gal-II-like. They express gRNAs targeting ST3Gal-I, -II, -III, -IV, V, -VI, and ST3Gal-II-like protein genes, respectively. The human ST6Gal-I gene was amplified by PCR from the pCAGGS-FLAG-PUR-ST6Gal-I plasmid (Hatakeyama et al., 2005) and then digested with NotI and XhoI. The digested fragment was cloned between the NotI and XhoI sites of the eukaryotic expression vector pCAG-Bsd (Wako), which encodes blasticidin resistance. The resulting construct was named pCAG-Bsd-ST6Gal-I, which expresses ST3Gal-I. The sequences of all constructs were confirmed by Sanger sequencing. Cycle sequencing was performed using BigDye Terminator version 3.1 Cycle Sequencing Kits (Thermo Fisher Scientific), and sequences were analyzed on an ABI Prism 3130xl Genetic Analyzer (Thermo Fisher Scientific).

[0121] Electroporation was performed using an AMAXA Nucleofector II machine (Lonza) according to the manufacturer's instructions. Briefly, 5 × 10 5 MDCK cells were suspended in 100 μl of the desired electroporation buffer and mixed with 5 μg of Cas9 / gRNA dual expression vector (1 μg PX459-ST3Gal-I, 1 μg PX459-ST3Gal-II, 1 μg PX459-ST3Gal-III, 1 μg PX459-ST3Gal-IV, 1 μg PX459-ST3Gal-VI, and 1 μg PX459-ST3Gal-II-like) or 1.7 μg PX459-ST3Gal-V, and 1.7 μg pCAG-Bsd-ST6Gal-I. The resuspended cells were transferred to a cuvette and immediately electroporated using program A-024. Cells were cultured in MEM supplemented with 5% NCS in the presence of 2 μg / ml puromycin or 10 μg / ml blasticidin to select for transfected cells. Clones were isolated using cloning rings, dissociated with trypsin and EDTA, and expanded. Genomic DNA was isolated using a genomic isolation kit (Promega) according to the manufacturer's instructions. Target regions were amplified by PCR using primers surrounding each target site, and the amplified products were cloned using a Zero Blunt TOPO PCR Cloning Kit (Invitrogen). At least eight clones for each gene were randomly selected, and isolated plasmids were sequenced. A list of the primers used is shown in Table 5.

[0122] Flow cytometry analysis. Cells were detached by incubation in PBS containing 0.125% trypsin-20 mM EDTA (Dojindo) for 10 min. After washing with PBS, cells were blocked with Carbo-Free Blocking Solution (Vector) for 15 min at 4°C. Cells were incubated with biotinylated MAL II, SNA, or SSA for 30 min at 4°C. Cells were then rinsed with PBS and incubated with Alexa 488-labeled streptavidin (Invitrogen) for 30 min at 4°C (Invitrogen). Fluorescence was measured using a FACS Calibur or FACS Verse (Becton Dickinson) and analyzed using FlowJo software (Becton Dickinson).

[0123] To confirm the binding of sialic acid-specific lectins, cells were treated and then incubated with lectins and Clostridium perfringens (Roche) for 1 h at 37° C. Cell-bound lectins were detected as described above.

[0124] Immunofluorescence staining. Cells grown in 24-well plates were incubated at 4°C with a mouse monoclonal antibody recognizing Siaα2,3Galβ1,4GlcNAc (HYB4; Wako). After incubation, cells were fixed with 10% trichloroacetic acid for 10 min at -20°C. Cells were then washed with PBS and incubated with Alexa 488-labeled goat anti-mouse immunoglobulin G (IgG) (Invitrogen) for 30 min. Cell nuclei were counterstained with Hoechst 33342 trihydrochloride trihydrate (Molecular Probes). Samples were examined using a Zeiss fluorescence microscope (model Imager Z1; Carl Zeiss).

[0125] [Table 2] aThe PCR products of each gene were cloned into a blunt-end vector and subjected to sequencing analysis.

[0126] [Table 3] The sequences of the sgRNAs are shown in bold. The underlined sequences indicate the PAM sequences. The sequences shown in red indicate the insertions or deletions in hCK cells compared to MDCK cells (wild type). 1 This sequence matched a portion of the sequence of the PX459 vector.

[0127] [Table 4] a Clinical specimens that were shown to be influenza virus positive by real-time RT-PCR or rapid diagnostic kits were used for virus isolation. b Clinical specimens were inoculated into MDCK, AX4, and hCK cells. The cells were observed for cytopathic effect (CPE) for 7 days. After 7 days of inoculation, supernatants from CPE-negative cell culture samples were tested by using a rapid diagnostic kit and a hemagglutination assay with guinea pig red blood cells.

[0128] [Table 5-1] [Table 5-2] [Table 5-3] a Influenza viruses isolated from clinical specimens in MDCK, AX4, or hCK cells. Viral HA and NA gene sequences were determined after inoculation in MDCK, AX4, or hCK cells. b - No mutations were detected compared to the sequence from the original clinical specimen.c D / G, a mixture of aspartic acid and glycine at position 151; d K / R, a mixture of lysine and arginine at position 394; e D / N, a mixture of aspartic acid and asparagine at position 151; f S / P, a mixture of serine and proline at position 221; g D / N, a mixture of aspartic acid and asparagine at position 246; h K / I, a mixture of lysine and isoleucine at position 160; i K / T, mixed lysine and threonine at position 160; j K / T, mixed lysine and threonine at position 148; k No influenza virus was isolated from clinical specimens in MDCK and AX4 cells. l No influenza virus was isolated from clinical specimens in MDCK cells. m T / I, mixed threonine and isoleucine at position 148; n E / K, a mixture of glutamic acid and lysine at position 433; o S / P, a mixture of serine and proline at position 44;

[0129] [Table 6] a Clinical specimens that were shown to be influenza virus positive by real-time RT-PCR or rapid diagnostic kits were used for virus isolation. b Serial 2-fold dilutions of clinical samples (2 1 From 2 20 ) were prepared and inoculated into AX4 and hCK cells. The development of CPE in the cells was observed for 7 days. Triplicate wells were used to infect with the same dilution of virus, and the maximum dilution that showed CPE in all three wells is shown.

[0130] [Table 7-1] [Table 7-2] a FAM, 6-carboxyfluorescein; HEX, hexachloro-6-carboxyfluorescein; BHQ-1, black hole quencher; MGB, minor groove binder; TAMRA, 6-carboxytetramethylrhodamine.

[0131] result

[0132] To mimic the sialic acid expression pattern of human upper airway epithelial cells, we generated a new MDCK cell line (designated hCK) that overexpresses α2,6-sialoglycan and expresses extremely low levels of α2,3-sialoglycan (see Figures 2-4 and Table A).

[0133] To determine whether hCK cells can support efficient replication of human influenza viruses, the growth kinetics of viruses [three A / H1N1 2009 pandemic (A / H1N1pdm), three A / H3N2, three B / Yamagata lineages, and three B Victoria lineages] were examined in hCK cells. The three A / H1N1pdm isolates grew efficiently in MDCK, AX4, and hCK cells, with no substantial differences in titers observed (Figure 1A). The six influenza B isolates also replicated with similar efficiency in all three cell lines. In contrast, for A / H3N2 viruses, all three isolates grew much faster and to greater titers in hCK cells than in AX4 cells (2.03–2.91 log units greater at 48 h postinfection). As reported elsewhere (Chambers et al., 2014), these recent A / H3N2 isolates replicate poorly in MDCK cells. These findings demonstrate that hCK cells, which express very low levels of α2,3-sialoglycan and high levels of α2,6-sialoglycan, support recent A / H3N2 virus replication more efficiently than MDCK or AX4 cells.

[0134] To evaluate the susceptibility of hCK cells to human influenza virus isolates, aliquots of 90 respiratory specimens (30 A / H1N1pdm, 30 A / H3N2, and 30 B Yamagata lineages) were inoculated into MDCK, AX4, and hCK cells. The cells were observed for 7 days for the development of cytopathic effect (CPE). For MDCK, AX4, and hCK cells, A / H1N1pdm viruses were successfully recovered from RT-PCR-positive samples without the need for blind passaging (100% isolation efficiency) (Table 2). Similarly, these three cell lines showed 100% efficiency for the isolation of influenza B viruses. For A / H3N2-positive samples, five and two viruses were not recovered from MDCK and AX4 cells, respectively. These results are consistent with previous reports that conventional MDCK cells have a relatively low sensitivity for detecting recent A / H3N2 viruses ( Oh et al., 2008 ; Hatakeyama et al., 2005 ).

[0135] Agglutination of red blood cells by influenza viruses is thought to be due to viral binding to sialic acid on the cell surface. Since 2005, A / H3N2 isolates have lost the ability to agglutinate turkey red blood cells (Lin et al., 2013). In addition, current A / H3N2 isolates show reduced or no agglutination of guinea pig red blood cells (Lin et al., Influenza Other Respir Viruses, 2017). This indicates that the avidity of these isolates for sialic acid receptors has changed. Indeed, Lin et al. (2013) measured the avidity of recent A / H3N2 viruses for α2,6-linked sialic acid receptors and showed that it was dramatically reduced. Glycan array analysis revealed that recent A / H3N2 isolates have a preference for binding to branched sialylated N-linked glycans with extended poly-N-acetyllactosamine chains (Peng et al., Cell Host Microbe, 2017).

[0136] In contrast, virus isolation from hCK cells was successful in all samples without any subsequent blind passaging, suggesting that this cell line is more effective than AX4 or MDCK cells for isolating human A / H3N2 viruses from clinical specimens.

[0137] During replication of recent A / H3N2 human isolates in MDCK cells, the viruses rapidly acquired amino acid changes at positions 148 and 151 of the NA protein (e.g., T148I and D151G), which affect the biological properties of the NA. To examine whether the A / H3N2 viruses isolated from the three cell lines harbored mutations in their HA and NA proteins, the nucleotide sequences of the HA and NA segments were determined by Sanger sequencing (Table 3). Sequence analysis revealed that 7 of the 25 MDCK-grown isolates contained amino acid changes at position 151 of the NA compared to the sequence from the original specimen: NA-151N, NA-151D / G, and NA-151D / N (a mixed population of amino acids at position 151). Amino acid changes leading to loss of the glycosylation site at position 158 in HA were found in the viral population of several other MDCK-grown isolates: HA-158K, HA-160K, HA-160K / I, and HA-160K / T. These changes are known to alter the antigenic properties of HA (Lin et al., 2017; Chambers et al., 2015; Skowronski et al., 2016). Importantly, cell culture-adapted mutations were also found in the NA protein of several isolates grown in AX4 cells: NA-148K / T, NA-148T / I, NA-151D / N, and NA-151D / G. Surprisingly, no mutations were detected in the hCK-grown isolates, with the exception of one isolate carrying a S44P mutation in the NA stalk. These findings suggest that hCK cells support efficient growth of A / H3N2 viruses without cell culture adaptation mutations.

[0138] Seasonal influenza viruses from clinical specimens grow better in AX4 cells than in NDCK cells (Hatakeyama et al., 2005). To determine whether hCK cells are superior to AX4 cells for virus isolation, we compared the sensitivity of hCK and AX4 cells by testing serial two-fold dilutions of specimens. Aliquots of 24 specimens (6 A / H1N1pdm, 6 A / H3N2, 6 B / Yamagata, and 6 B / Victoria strains) were inoculated in triplicate into AX4 and hCK cells. All culture wells were examined for CPE 7 days after inoculation, and the ratio of the maximum dilution observed in AX4 cells to the maximum dilution showing CPE was determined. In one of the six A / H1N1-positive samples (sample ID, HP79), hCK cells were slightly less sensitive than AX4 cells (Figure 1B and Table 4). However, in the remaining samples, the sensitivity of hCK cells was similar to or greater than that of AX4 cells. For B / Yamagata- and B / Victoria-lineage-positive samples, hCK cells showed similar or somewhat greater sensitivity than AX4 cells. For all A / H3N2-positive samples, hCK cells showed greater sensitivity than AX4 cells, and for some samples, hCK cells showed approximately 100- to 2,000-fold greater sensitivity than AX4 cells. Taken together, these results indicate that hCK cells are more suitable than AX4 or MDCK cells for the primary isolation of recent seasonal A / H3N2 viruses.

[0139] To evaluate the genetic stability of the HA and NA genes of viruses isolated in hCK cells, aliquots of 12 clinical specimens (three A / H1N1pdm, three A / H3N2, and three B Yamagata and three B Victoria lineages) were inoculated into MDCK, AX4, and hCK cells, and the isolates were serially passaged 10 times. After the first, fourth, and tenth passages, the viral HA and NA sequences were determined by Sanger sequencing and compared with those in the clinical specimens. In the A / H1N1pdm-positive specimens, after the first passage, a mixed population of viruses encoding either N or S at position 296 in the HA was detected in one of the three hCK-grown viruses (BB139) (Table 1). The hCK-grown virus also harbored an S153G substitution mutation in its NA. Another hCK-grown virus (HP79) encoded a D27N substitution in its HA after ten passages. A mixed population encoding either T or I at position 167 in HA was found in one MDCK-grown virus after 10 passages (BB139). Another MDCK-grown virus (BB131) had a mixed population encoding either HA-446N and HA-446S at 10 passages. This MDCK-grown virus also contained a mixed population encoding either H or Y at position 411c in NA. Mixed C53Y / C in NA was observed in one AX4-grown virus after 10 passages (BB131).

[0140] For A / H3N2-positive samples, viruses recovered from AX4 and hCK, but not from MDCK, were serially passaged. After six passages, a mixed population encoding HA-158N and HA-158K (leading to loss of the glycosylation site at position 158 in HA) was detected in one of the three AX4-propagated viruses (DA30). In addition, another AX4-propagated virus (DA29-1) encoded a T148K substitution in its NA after six passages. A mixed population encoding HA-408D and HA-408N was detected in one hCK-propagated virus after 10 passages (DA29-1).

[0141] For B / Yamagata lineage viruses, no changes were detected in any isolate after the first, sixth, or tenth passages, with the exception of a mixed population encoding HA-148S and HA-148N detected in one hCK-grown virus (HP70-2) at the sixth passage. For B Victoria lineage viruses, a mixed population encoding NA-208G and NA-208R was found in one of three AX4-grown viruses (BB139) after the first passage. After six passages, one hCK-grown virus encoded a mixture of L72L / F in its NA (WD28). At the tenth passage, one MDCK-grown virus (WD28) contained the N196S mutation known to lead to the loss of a glycosylation site at position 196 in HA (B / Victoria lineage). This can significantly alter the antigenicity of influenza B viruses. Another MDCK-grown virus (HP015) had a D459D / N mix within its NA.

[0142] Taken together, A / H1N1pdm and B viruses were slightly more prone to mutation when passaged in MDCK or hCK cells than in AX4 cells, whereas A / H3N2 viruses propagated in hCK cells were maintained with greater genetic stability than those in AX4 cells.

[0143] In conclusion, cell lines derived from MDCK cells, hCK, express large amounts of α2,6-sialoglycan and small amounts of α2,3-sialoglycan that will be useful for influenza virus research, especially research involving human A / H3N2 influenza virus, and vaccine production.

[0144] References Bates et al., J. Stat. Softw., 61:1 (2015). Chambers et al., Cell Rep., 12:1 (2015). Chambers et al., J. Viral, 88:10986 (2014). Chu and Whittaker , Proc. Natl. Acad. Sci. USA, 101:18153 (2004). Cong et al., Science, 339:819 (2013). Connor et al., Virology, 205:17 (1994). Gamblin and Skehel, J. Biol. Chem., 285:28403 (2010). He et al., Cell Rep., 23:596 (2018). Hatakeyama et al., J. Clin. Microbiol., 43:4139 (2005). Hegde, Hum. Vaccine Immunother., 11:1223 (2015). Hidari et al., Biochem. Biophys. Res. Commun., 436:394 (2013). Science, 337:816 (2012). Lee et al., PLoS One 8 (2013). Lenth , J. Stat. Softw., 69 (2016) Li et al., J. Clin. Microbiol., 47:466 (2009). Lin et al., Influenza Other Respiratory Viruses, 11:263 (2017). Lin et al., J. Viral., 84:6769 (2010). Lin et al., PLoS One, 12, 72299 (2017). Matrosovich et al., Journal of Virology, 77:8418 (2003). Mohr et al., Viral J., 12:67 (2015). Oh et al., J. Clin. Microbial., 46:2189 (2008). Rogers and Paulson, Virology, 127:361 (1983). Shalem et al., Science, 343:84 (2014). Shibuya et al., J Biochem., 106:1098 (1989). Shinya et al., Nature, 440:435 (2006). Skowronski et al., Euro. Surveill., 21:30112 (2016). Stevens et al., J. Mal. Biol., 355:1143 (2006). Takashima and Tsuji, Trends in Glycoscience and Glycotechnology, 23:178 (2011). Tamura et al., Antimicrob. Agents Chemother., 57:6141 (2013). van Riel et al., Science, 312:399 (2006). Zhu et al., J. Viral, 86:13371 (2012).

[0145] All publications, patents, and patent applications are incorporated herein by reference. Although the present invention has been described herein above with respect to certain preferred embodiments thereof, and numerous details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the present invention is capable of additional embodiments and that several of the details set forth herein may be significantly modified without departing from the underlying principles of the invention. Disclosure Summary [Section 1] An isolated recombinant mammalian or avian cell which comprises a reduced amount of cell surface β-galactoside α2,3 sialyl residues and an increased amount of human β-galactoside α2,6 sialyl residues compared to a corresponding non-recombinant mammalian or avian cell. [Section 2] 2. The isolated recombinant cell of paragraph 1, which is a non-human cell. [Section 3] 3. The isolated recombinant cell of paragraph 1 or 2, which is a canine cell or a primate cell. [Section 4] 4. The isolated recombinant cell of any one of items 1 to 3, comprising an expression cassette encoding human β-galactoside α2,6 sialyltransferase I (ST6Gal-I) or ST6Gal-II. [Section 5] Item 5. The isolated recombinant cell of Item 4, wherein the ST6Gal-I or ST6Gal-II comprises a protein having an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 1 to 4 or 101. [Section 6] 6. The isolated recombinant cell of any one of claims 1 to 5, wherein one or more β-galactoside α2,3 sialyltransferase genes are mutated to reduce the amount of cell surface β-galactoside α2,3 sialyl residues. [Section 7] 7. The isolated recombinant cell of paragraph 6, wherein two or more of the ST3Gal-I gene, the ST3Gal-II gene, the ST3Gal-III gene, the ST3Gal-IV gene, the ST3Gal-V gene, the ST3Gal-VI gene, or the ST3Gal-II-like gene are mutated. [Section 8] 8. The isolated recombinant cell of claim 7, wherein the ST3 gene has a nucleic acid sequence that is at least 80% identical to any one of SEQ ID NOs: 5, 7, 9, 11, 13, 15, or 17. [Section 9] 9. The isolated recombinant cell of any one of paragraphs 1 to 8, wherein said reduction in cell surface β-galactoside α2,3 sialyl residues is the result of reduced expression of one or more ST3 sialyltransferases. [Section 10] 10. The isolated recombinant cell of claim 9, wherein the one or more ST3 sialyltransferases have an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 6, 8, 10, 12, 14, 16, or 18. [Section 11] 11. The isolated recombinant cell of any one of paragraphs 1 to 10, wherein influenza H3 virus replicates more efficiently in the recombinant cell compared to the non-recombinant cell. [Section 12] An isolated recombinant mammalian or avian cell which contains a reduced amount of cell surface β-galactoside α2,3 sialyl residues compared to a corresponding non-recombinant mammalian or avian cell. [Section 13] 13. The recombinant cell of paragraph 12, wherein one or more of the ST3Gal-I gene, the ST3Gal-II gene, the ST3Gal-III gene, the ST3Gal-IV gene, the ST3Gal-V gene, the ST3Gal-VI gene, or the ST3Gal-II-like gene is mutated. [Section 14] 13. The recombinant cell of paragraph 12, wherein a combination of ST3Gal-I gene, ST3Gal-II gene, ST3Gal-III gene, ST3Gal-IV gene, ST3Gal-V gene, ST3Gal-VI gene, or ST3Gal-II-like gene is mutated. [Section 15] 15. The recombinant cell of paragraph 14, wherein the ST3Gal-I gene, the ST3Gal-II gene, the ST3Gal-III gene, the ST3Gal-IV gene, the ST3Gal-V gene, the ST3Gal-VI gene, and the ST3Gal-II-like gene are mutated. [Section 16] 1. A method for altering the amount of cell surface β-galactoside α2,3 sialyl residues and human β-galactoside α2,6 sialyl residues on a mammalian cell or an avian cell, comprising: The method comprises mutating one or more β-galactoside α2,3 sialyltransferase (ST3Gal) genes and overexpressing a human β-galactoside α2,6 sialyltransferase I (ST6Gal) gene in a parent mammalian or avian cell to produce a modified mammalian or avian cell having a reduced amount of cell surface β-galactoside α2,3 sialyl residues and an increased amount of human β-galactoside α2,6 sialyl residues on its surface compared to the corresponding parent cell. [Section 17] Item 17. The method of item 16, wherein one or more ST3Gal genes are mutated using a genome editing system. [Section 18] The method of claim 17, wherein the genome editing system comprises CRISPR / Cas9, zinc finger nuclease (ZFN), or transcription activator-like effector nuclease (TALEN). [Section 19] 19. The method of any one of paragraphs 16 to 18, wherein the mutation comprises an insertion of one or more nucleotides, or a deletion of one or more nucleotides, or both, in one or more ST3 genes. [Section 20] 20. The method of any one of items 16 to 19, wherein the modified cell comprises an expression cassette comprising an ST6Gal open reading frame. [Section 21] 21. The method of any one of items 16 to 20, wherein the modified cells are kidney cells. [Section 22] 22. The method of any one of items 16 to 21, wherein the modified cells are canine cells. [Section 23] 23. The method of any one of claims 16 to 22, wherein the modified cells are Madin-Darby canine kidney (MDCK) cells. [Section 24] 1. A method for propagating influenza viruses, comprising: Infecting the recombinant cell according to any one of items 1 to 11 with an influenza virus; and recovering the progeny virus. [Section 25] 25. The method of paragraph 24, wherein the influenza virus is a human influenza virus. [Section 26] 26. The method of paragraph 24 or 25, wherein the influenza virus is an influenza A virus. [Section 27] 26. The method of paragraph 24 or 25, wherein the influenza virus is an influenza B virus. [Section 28] 27. The method of paragraph 24, 25, or 26, wherein the influenza virus is an H3 virus. [Section 29] 27. The method of paragraph 24, 25, or 26, wherein the virus is an A / H1N1, A / H3N2, B / Yamagata lineage B influenza virus, or B / Victoria lineage B influenza virus. [Section 30] 1. A method for isolating influenza virus, comprising: Providing a sample from a bird or mammal suspected of being infected with an influenza virus; A method comprising contacting the sample with the recombinant cell according to any one of items 1 to 11. [Section 31] 31. The method of paragraph 30, further comprising determining whether the sample is infected with influenza virus. [Section 32] 32. The method of claim 30 or 31, further comprising identifying the HA and / or NA subtype of the virus. [Section 33] 1. A method for diagnosing influenza virus infection, comprising: Infecting the recombinant cell of any one of items 1 to 11 with a sample from a bird or mammal suspected of being infected with an influenza virus; determining whether the cell is infected with a virus.

Claims

1. An isolated recombinant canine or avian cell comprising a reduced amount of cell surface β-galactoside α2,3 sialyl residues and an increased amount of human β-galactoside α2,6 sialyl residues compared to a corresponding non-recombinant canine or avian cell, wherein a plurality of β-galactoside α2,3 sialyltransferase genes have been mutated to reduce the amount of cell surface β-galactoside α2,3 sialyl residues, wherein the plurality of β-galactoside α2,3 sialyltransferase genes are selected from the group consisting of ST3Gal-I gene, ... The isolated recombinant canine or avian cell comprises a ST3Gal-II gene, a ST3Gal-III gene, a ST3Gal-IV gene, a ST3Gal-V gene, a ST3Gal-VI gene and a ST3Gal-II-like gene, wherein the recombinant cell contains an expression cassette encoding human β-galactoside α2,6 sialyltransferase I (ST6Gal-I) or ST6Gal-II, and wherein a reduced amount of cell surface β-galactoside α2,3 sialyl residues is the result of a recombinase knockout of the ST3 gene in the cell.

2. The isolated recombinant cell of claim 1, which is a canine cell.

3. 3. The isolated recombinant cell of claim 1 or 2, which is a canine kidney cell.

4. The isolated recombinant cell of any one of claims 1 to 3, comprising an expression cassette encoding human β-galactoside α2,6 sialyltransferase I (ST6Gal-I).

5. The isolated recombinant cell of claim 1, wherein the ST6Gal-I or ST6Gal-II comprises a protein having at least 90% amino acid sequence identity with any one of SEQ ID NOs: 1-4 or 101 and maintains the biological activity of SEQ ID NO: 1-4 or 101, respectively.

6. The isolated recombinant cell of claim 1, wherein the ST3 gene has a nucleic acid sequence that is at least 90% identical to any one of SEQ ID NOs: 5, 7, 9, 11, 13, 15, or 17 and maintains the biological activity of any one of SEQ ID NOs: 5, 7, 9, 11, 13, 15, or 17, respectively.

7. The isolated recombinant cell of any one of claims 1 to 6, wherein the reduction in cell surface β-galactoside α2,3 sialyl residues is the result of reduced expression of the ST3 sialyltransferase.

8. The isolated recombinant cell of claim 7, wherein the ST3 sialyltransferase has an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 6, 8, 10, 12, 14, 16, or 18 and maintains the biological activity of SEQ ID NOs: 6, 8, 10, 12, 14, 16, or 18, respectively.

9. 9. The isolated recombinant cell of any one of claims 1 to 8, wherein influenza H3 virus replicates more efficiently in said recombinant cell compared to said non-recombinant cell.

10. 1. An isolated recombinant MDCK cell comprising a reduced amount of cell surface β-galactoside α2,3 sialyl residues compared to a corresponding non-recombinant MDCK cell, wherein a plurality of β-galactoside α2,3 sialyltransferase genes have been mutated to reduce the amount of cell surface β-galactoside α2,3 sialyl residues, the plurality of β-galactoside α2,3 sialyltransferase genes comprising an ST3Gal-I gene, an ST3Gal-II gene, an ST3Gal-III gene, an ST3Gal-IV gene, an ST3Gal-V gene, an ST3Gal-VI gene and an ST3Gal-II-like gene, wherein the recombinant cell comprises an expression cassette encoding human β-galactoside α2,6 sialyltransferase I (ST6Gal-I) or ST6Gal-II, and wherein the reduced amount of cell surface β-galactoside α2,3 sialyl residues is the result of a recombinase knockout of the ST3 gene in the cell.

11. A method for altering the amount of cell surface β-galactoside α2,3 sialyl residues and human β-galactoside α2,6 sialyl residues in a canine cell comprises mutating multiple β-galactoside α2,3 sialyltransferase (ST3Gal) genes and overexpressing the human β-galactoside α2,6 sialyltransferase I (ST6Gal) gene in a parent canine cell to produce a decreased amount of cell surface β-galactoside α2,3 sialyl residues and an increased amount of human β-galactoside α2,6 sialyl residues compared to the corresponding parent cell. wherein the plurality of β-galactoside α2,3 sialyltransferase (ST3Gal) genes include an ST3Gal-I gene, an ST3Gal-II gene, an ST3Gal-III gene, an ST3Gal-IV gene, an ST3Gal-V gene, an ST3Gal-VI gene and an ST3Gal-II-like gene, and wherein a reduced amount of cell surface β-galactoside α2,3 sialyl residues is the result of a recombinase knockout of the ST3 gene in the cell.

12. The method of claim 11, wherein multiple ST3Gal genes are mutated using a genome editing system.

13. The method of claim 12, wherein the genome editing system comprises CRISPR / Cas9, zinc finger nuclease (ZFN), or transcription activator-like effector nuclease (TALEN).

14. 14. The method of any one of claims 11 to 13, wherein the mutation comprises an insertion of one or more nucleotides, or a deletion of one or more nucleotides, or both, in multiple ST3 genes.

15. The method of any one of claims 11 to 14, wherein the modified cell comprises an expression cassette comprising an ST6Gal open reading frame.

16. The method of any one of claims 11 to 15, wherein the modified cells are kidney cells.

17. 17. The method of any one of claims 11 to 16, wherein the modified cells are Madin-Darby canine kidney (MDCK) cells.

18. 1. A method for propagating influenza viruses, comprising: Infecting the recombinant cell of any one of claims 1 to 9 with an influenza virus; and Recovering progeny viruses The method includes:

19. 20. The method of claim 18, wherein the influenza virus is a human influenza virus.

20. 20. The method of claim 18 or 19, wherein the influenza virus is an influenza A virus.

21. 20. The method of claim 18 or 19, wherein the influenza virus is an influenza B virus.

22. 21. The method of claim 18, 19, or 20, wherein the influenza virus is an H3 virus.

23. 21. The method of claim 18, 19, or 20, wherein the virus is A / H1N1, A / H3N2, B / Yamagata lineage B influenza virus, or B / Victoria lineage B influenza virus.

24. 1. A method for isolating influenza virus, comprising: Providing a sample from a bird or mammal suspected of being infected with an influenza virus; and contacting the sample with a recombinant cell according to any one of claims 1 to 9; The method includes:

25. 25. The method of claim 24, further comprising determining whether the sample is infected with influenza virus.

26. 26. The method of claim 24 or 25, further comprising identifying the HA and / or NA subtype of the virus.

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