[0001] This application claims the priority benefit of U.S. Provisional Application

No. 62/723,233 filed August 27, 2018, which application is incorporated herein by reference in its entirety.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on August 27, 2019, is named 24978-05l6_SL.txt and is 2,156 bytes in size.

TECHNICAL FIELD

[0003] The present invention relates to methods to control viral infection of mammalian cells.

BACKGROUND [0004] Chinese hamster ovary (CHO) cells are extensively used to produce biopharmaceuticals (Walsh 2014) for numerous reasons. Though one advantage is their reduced susceptibility to many human vims families (Berting et al. 2010; Poiley et al. 1991; Weiebe et al. 1989), there have been episodes of animal viral contamination of biopharmaceutical production runs, mostly from trace levels of viruses in raw materials. These infections have led to expensive decontamination efforts and threatened the supply of critical drugs (Dinowitz et al. 1992; Garnick 1998; Nims 2006). Viruses that have halted production of valuable therapeutics include RNA viruses such as Cache Valley vims (Nims 2006), Epizootic hemorrhagic disease vims (Rabenau et al. 1993), Reovirus (Nims 2006) and Vesivirus 2117 (Bethencourt 2009). Recently, a strategy was proposed to inhibit infection of CHO cells by a limited number of rodent viruses by engineering glycosylation (Mascarenhas et al. 2017), there is a need to understand the mechanisms by which CHO cells are infected and how the cells can be universally engineered to enhance their viral resistance (Merten 2002).

[0005] Many studies have investigated the cellular response to a diverse range of viruses in mammalian cells, and detailed the innate immune responses that are activated upon infection. For example, type I interferon (IFN) responses play an essential role in regulating the innate immune response and inhibiting viral infection (Perry et al. 2005; Sadler and Williams 2008; Schoggins and Rice 2011; Taniguchi and Takaoka 2002) and can be induced by treatment of cells with poly I:C (Green and Montagnani 2013; Pantelic et al. 2005; Plant et al. 2005). However, the detailed mechanisms of vims infection and the antiviral response in CHO cells remain largely unknown. Understanding the role of type I IFN-mediated innate immune responses in CHO cells could be invaluable for developing effective virus-resistant CHO bioprocesses. Fortunately, the application of recent genome sequencing (Chen et al. 2017; Lewis et al. 2013; Rupp et al. 2018; van Wijk et al. 2017; Vishwanathan et al. 2016; Xu et al. 2011; Yusufi et al. 2018) and RNA-Seq tools can now allow the analysis of complicated cellular processes in CHO cells (Fomina- Yadlin et al. 2015; Hsu et al. 2017; Vishwanathan et al. 2015; Wang et al. 2009; Yuk et al. 2014), such as vims infection.

SUMMARY OF THE INVENTION

[0006] The present invention provides, in embodiments, a method of inhibiting viral infection in a biological sample comprising administering to the sample an effective amount of: a) a type I interferon or poly I:C; b) a compound activating an innate immune response in the sample; c) a compound suppressing expression of Gfil, Trim24 and/or Cbl in the sample; and/or d) a compound activating expression of IRF7, IRF3, STAT1, STAT3, NFATC2, IRF5, STAT4, IRF9, IRF8, NFKB1, TP53, JUN and/or EBF1 in the sample. Activation or suppression of additional genes provided herein are also contemplated in all methods of the present invention.

[0007] In embodiments, the biological sample is a cell culture. In embodiments, biological sample comprises mammalian cells. In embodiments, the biological sample comprises CHO cells. In embodiments, the method is conducted in a biopharmaceutical manufacturing process. [0008] The present invention provides, in embodiments, a non-naturally occurring mammalian cell culture comprising cells genetically modified for suppressed expression of Gfil, Trim24 and/or Cbl, or activated expression of IRF7, IRF3, STAT1, STAT3, NFATC2, IRF5, STAT4, IRF9, IRF8, NFKB1, TP53, JUN and/or EBF1, as compared to wild- type cells of the same mammalian species.

[0009] The present invention provides, in embodiments, a method of producing a biopharmaceutical protein from a mammalian cell culture, comprising culturing mammalian cells having non-naturally occurring genetically suppressed expression of Gfil, Trim24 and/or Cbl, or genetically activated expression of IRF7, IRF3, STAT1, STAT3, NFATC2, IRF5, STAT4, IRF9, IRF8, NFKB1, TP53, JUN and/or EBF1, or both, as compared to wild-type cells of the same mammalian species; and isolating a protein of interest from the cultured cells.

[0010] The present invention provides, in embodiments, a method of treating or preventing viral infection in a mammalian cell comprising administering to the cell an effective amount of: a) a type I interferon or poly I:C; b) a compound activating an innate immune response in the sample; c) a compound suppressing expression of Gfil, Trim24 and/or Cbl in the sample; and/or d) a compound activating expression of IRF7, IRF3, STAT1, STAT3, NFATC2, IRF5, STAT4, IRF9, IRF8, NFKB1, TP53, JUN and/or EBF1 in the sample.

[0011] The present invention provides, in embodiments, a method for increasing vims infectivity in a mammalian cell comprising increasing expression of Gfil, Trim24 and/or Cbl, or decreasing expression of IRF7, IRF3, STAT1, STAT3, NFATC2, IRF5, STAT4, IRF9, IRF8, NFKB1, TP53, JUN and/or EBF1 in the cell. In embodiments, the method further comprises isolating virus or viral particles from the cell. In embodiments, genetic material is delivered to the sample by viral transduction to increase or decrease expression of said gene.

[0012] The present invention provides, in embodiments, a non-naturally occurring mammalian cell culture comprising mammalian cells having genetically activated expression of Gfil, Trim24 and/or Cbl, or genetically suppressed expression of IRF7, IRF3, STAT1, STAT3, NFATC2, IRF5, STAT4, IRF9, IRF8, NFKB1, TP53, JUN and/or EBF1, as compared to wild-type cells of the same mammalian species.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figures 1A-1E show RNA viruses induce cytopathic effects on CHO-K1 cells. (Figure 1A) Cytopathic effect of the three RNA viruses on CHO cells upon 30h (VSV), 54h (EMCV) or 78h (Reo) of infection. Fold change in (Figure 1B) IENb and (Figure 1C) Mxl gene expressions in CHO cells infected with the three RNA viruses compared to uninfected cells at the same time points. Figure 1D shows several pathways and processes were enriched for differentially expressed genes following viral infection (m vs. Vm). Figure 1E shows activated (red) or repressed (blue) upstream regulators following virus infection.

[0014] Figures 2A-2E show innate immunity genes in CHO cells are activated by poly I:C. (Figure 2 A) IFN- stimulated transcription was increased in cells treated with poly I:C /LyoVec for 24h, but not with other TLR ligands engaging TLR9, TLR4 or TLR7/8. (Figure 2B) Poly I:C triggered STAT1 phosphorylation in a dose dependent manner, and (Figure 2C) the levels of STAT2 phosphorylation and Mxl protein expression were comparable to those triggered by IFNa2c. (Figure 2D) Several pathways and processes were enriched for differentially expressed genes following poly I:C treatment (m vs. p). (Figure 2E) Upstream regulators that are activated (red greyscales) or repressed (blue greyscales) following poly I:C treatment.

[0015] Figures 3A-3E show Poly I:C pre-treatment prevents virus infection of

VCV, EMCV, and Reo. (Figures 3A-3C) Cell morphology (left panels) and cytopathic effect measured by crystal violet staining (right panels) of virus -infected CHO cells (Note that panels a, b, c and d corresponds to‘m’‘p’,‘Vm’ and‘Vp’, respectively); (Figure 3D) the enriched down-stream pathways under condition of Vm vs. Vp using RNA-Seq data. (Figure 3E) The top 35 upstream regulators that are activated or repressed.

[0016] Figures 4A-4B show a STATl-dependent regulatory network controls viral resistance (VSV and EMCV) in CHO cells. A STATl-dependent regulatory network induced by the pretreatment of poly I:C leads to the inhibition of (Figure 4A) VSV and (Figure 4B) EMCV replication in CHO cells, based on the comparison of Vm and Vp RNA-Seq. The greyscales denote the states inferred from the RNA-Seq data. For example, the blue greyscales of TRIM24 means that TRIM24 activity is suppressed, based on the differential expression of genes that are regulated by TRIM24.

[0017] Figure 5 shows enhanced vims resistance through genetic engineering on the repressors of STAT1. Schematic view of the genetic engineering approach in improving vims resistibility in CHO cells by knocking out repressors of STAT1.

[0018] Figures 6A-6C show RNA-Seq results of the Gfil and/or Trim24 KO engineered CHO cells. Gfil and Trim24 were knocked out compared to the control (susceptible) cells. Transcriptional regulatory networks were identified using IPA upstream regulatory analysis (Figure 6A), in which the innate immunity regulatory network (JAK-STAT network) is indicated by the arrow. Transcriptional factors of the identified JAK-STAT regulatory network in the knocked down cells (Figure 6B) and the activation of immune functions following Gfil and/or Trim24 genetic engineering were illustrated (Figure 6C).

[0019] Figures 7A-7C show viral resistance of the Gfil and/or Trim24 engineered

CHO cells. Gfil and Trim24 were knocked out and tested for resistance to EMCV and Reo-3 virus infection compared to the control (susceptible) cells (see details in Materials and methods). Cell density and viability was followed up for one week post infection (p.i.) for Gfil single knockout cells (Figure 7A), Trim24 single knockout cells (Figure 7B) and Gfil and Trim24 double knockout cells (Figure 1C). To assess robustness of the observed viral resistances in EMCV and Reo-3 virus infection, the reproducibility analysis was conducted for EMCV (three replicates) and Reo-3 (two replicates) virus. Susceptible CHO cell lines were used as positive controls for EMCV and Reo-3 virus infections during the first seven days (Figures 17A-17B). Resistant cultures were passaged and followed up for an additional week (Figures 18A-18C).

[0020] Figure 8 shows pretreatment of the cell culture with type I IFN protein limits VSV infection. Cells were cultured with the indicated concentration of human or murine IFN protein for 24h prior to infect with VSV, which was serially diluted (1: 10). Last row includes cells infected with VSV but not pretreated with IFN (6 wells) or non- infected cells (5 wells). The plate shows results from one experiment representative of 2, in which Hu-IFNa standard was used at 1000 IU/ml and gave comparable results.

[0021] Figures 9A-9B show enrichment strength of the interferon-alpha response.

(Figure 9A) Interferon-alpha response in the comparisons of m vs. Vm and Vm vs. Vp. (Figure 9B) Time effects on the Interferon-alpha response induced by poly I:C on non- infected cultures. The‘interferon-alpha response’ is a hallmark gene set of the gene set enrichment analysis (GSEA). The enrichment strength describes the leading-edge subset of a gene set (i.e., the interferon- alpha response in this study) (Subramanian et al. 2005). If the gene set is entirely within the first N positions in the ranked differentially expressed gene list, then the signal strength is maximal or 100%. If the gene set is spread throughout the list, then the signal strength decreases towards 0%.

[0022] Figure 9 A shows that the enrichment strength (65%, see EXAMPLE 2) of

‘interferon alpha response’ from the comparison of untreated media and Reo infected CHO cells (m vs. Vm) is smaller than those from the comparison of both virus presenting and poly I:C pretreated media (Vm vs. Vp; 77% and 77% for VSV and EMCV, respectively), which suggests that Reo-induced interferon alpha response might be insufficient for CHO cells limiting Reo infection. Indeed, Reo has been known to inhibit the type I IFN response using different strategies (Sherry 2009), such as modulation of cell RNA sensors (RIG-I and MDA5) and transcription factors (IRF3 and NF-kB) involved in induction of IFN. In consistent with our results, the IRF3 (z score = 4.96 and p-value < 0.05; Figure 1E) and NFkB pathways (p-value = 1.12c10 ² and NES = 2.22) have been observed to be activated in the comparison of m vs. Vm. While the underling mechanism of how these RNA viruses evade the (innate) immune system is still unclear, these data substantiate the inability of CHO cells to elicit protective anti-viral mechanisms by not mounting an effective protective (type I IFN) response. However, these data suggest that viral infection could likely be limited by further inducing IFN pathways.

[0023] Figure 9B further demonstrates that temporal difference might be another factor accounting for the variations of type I interferon response. Indeed, we observed the enrichment strength of‘interferon alpha response’ in the comparison of untreated cells and poly I:C pretreated cells (m vs. p) are different (73%, 70% and 78% for 30, 54 and 78 h, respectively). These differences might also result in the different magnitudes of downstream pathway/hallmark responses (Figure 2D) and upstream regulator expression variations (Figure 2E) across the different batches of samples that were collected from different time points.

[0024] Figures 10A-10B. PTNίb and Mxl gene expression kinetics by poly I:C.

Changes in RNA transcript levels of anti-viral genes IENb (Figure 10A) and Mxl (Figure 10B) in CHO cells treated with poly I:C (black squares) compared to untreated cultures (open circles) over time.

[0025] Figures 11A-11B. Poly I:C pre-treatment of CHO cells protect against viral infection through the IENb-mediated pathway. (Figure 11 A) Poly I:C can induce effective anti- viral mechanisms in CHO cells. (Figure 11B) PTNίb plays a protective role in the VSV infection, as treatment with hhΐί-IRNb neutralizing Ab abolishes the protective effect of poly I:C treatment.

[0026] Figure 12. Differential induction of antiviral genes (Mxl and IITMP3) by poly I:C and VSV or EMCV as opposed to Reo. Expression levels of Mxl and IITMP3 were measured by Taqman real-time PCR (qPCR) and RNA-Seq. The x-axis represents the comparisons of the two indicated culture conditions. The y-axis denotes the log 2 values of fold change (log2(FC)). The black bars represent the values of the differential fold change were calculated from the qPCR data, and the white bars denote the values of the differential fold change were calculated from the RNA-Seq data using the R package of DESeq2.

[0027] Figures 13A-13B. Up-regulated DEGs present in m vs. Vp and m vs. p but not in m vs. Vm. (Figure 13 A) Venn diagram of up regulated genes across different comparisons and the enriched KEGG pathways for the 30 DEGs that present with poly I:C treatment but not in Reo infection. (Figure 13B) Example of the most enriched pathway: “antigene processing and presentation” for the 30 DEGs. Note that, the criteria for identifying up regulated DEGs are: adjust p-value < 0.05 and fold change > 1.5 in the differential expressed genes test using DESeq2.

[0028] Figure 14. Poly I:C pretreatment activates STATl-dependent network in

CHO cells. A STATl-dependent regulatory network induced by the pretreatment of poly- I:C leads to several immune related responses activated in CHO cells, based on the comparison of m and p RNA-Seq. The greyscales denote the states inferred from the RNA-Seq data. For example, the blue greyscale of TRIM24 means that TRIM24 activity is suppressed, based on the differential expression of genes that are regulated by TRIM24.

[0029] Figures 15A-15B. NFATC2-dependent network in inducing STAT1 for inhibiting infection of mammalian cells. (Figure 15A) m vs. Vm. (Figure 15B) m vs. p. Note that, the six genes (IL15, NFKB1Z, IRF1, IL18, PML and REL) that are different in these two networks are highlighted in the green greyscales dashed circles.

[0030] Figures 16A-16B. IRF3-dependent network inducing STAT1 for the inhibition of viral infection. (Figure 16A) m vs. Vm. (Figure 16B) m vs. p. Note that, the three genes (DHX58, IL15 and IFIH1) that are different in these two networks are highlighted in the green dashed circles.

[0031] Figures 17A-17B. Positive controls of susceptible CHO cell lines in the

EMCV and Reo-3 virus infections. Susceptible CHO cell lines were used as positive controls for EMCV (Figure 17A) and Reo-3 (Figure 17B) virus infections (see Figure 5) during the first seven days.

[0032] Figures 18A-18C. Long term culture of vims infection assay. Resistant cultures were passaged and followed up for an additional week for Gfil and Trim24 double knockout cells (Figure 18A), Gfil single knockout cells (Figure 18B) and Trim24 single knockout cells (Figure 18C).

[0033] Figures 19A-19B. Negative regulatory scores of STAT1 upstream regulators. (Figure 19A) Negative regulatory score of STAT1 upstream regulators in the comparison of m (Media) vs. p (poly I:C treated media). (Figure 19B) Negative regulatory score of STAT1 upstream regulators in the comparison of Vm (Virus+Media) vs. Vp (Virus-i- poly I:C treated media).

DETAILED DESCRIPTION

[0034] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0035] Unless defined otherwise, all technical and scientific terms and any acronyms used herein have the same meanings as commonly understood by one of ordinary skill in the art in the field of the invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the exemplary methods, devices, and materials are described herein.

[0036] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, 2 ^nd ed. (Sambrook et ak, 1989); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Animal Cell Culture (R. I. Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Current Protocols in Molecular Biology (F. M. Ausubel et ak, eds., 1987, and periodic updates); PCR: The Polymerase Chain Reaction (Mullis et ak, eds., 1994); Remington, The Science and Practice of Pharmacy, 20 ^th ed., (Lippincott, Williams & Wilkins 2003), and Remington, The Science and Practice of Pharmacy, 22 ^th ed., (Pharmaceutical Press and Philadelphia College of Pharmacy at University of the Sciences 2012).

DEFINITIONS

[0037] To facilitate understanding of the invention, a number of terms and abbreviations as used herein are defined below as follows:

[0038] When introducing elements of the present invention or the preferred embodiment(s) thereof, the articles“a”,“an”,“the” and“said” are intended to mean that there are one or more of the elements. The terms“comprising”,“including” and“having” are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0039] The term“and/or” when used in a list of two or more items, means that any one of the listed items can be employed by itself or in combination with any one or more of the listed items. For example, the expression“A and/or B” is intended to mean either or both of A and B, i.e. A alone, B alone or A and B in combination. The expression“A, B and/or C” is intended to mean A alone, B alone, C alone, A and B in combination, A and C in combination, B and C in combination or A, B, and C in combination.

[0040] It is understood that aspects and embodiments of the invention described herein include“consisting” and/or“consisting essentially of’ aspects and embodiments.

[0041] It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. Values or ranges may be also be expressed herein as“about,” from“about” one particular value, and/or to“about” another particular value. When such values or ranges are expressed, other embodiments disclosed include the specific value recited, from the one particular value, and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent“about,” it will be understood that the particular value forms another embodiment. It will be further understood that there are a number of values disclosed therein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. In embodiments,“about” can be used to mean, for example, within 10% of the recited value, within 5% of the recited value, or within 2% of the recited value.

[0042] As used herein, “patient” or“subject” means a human or mammalian animal subject to be treated.

[0043] As used herein the term “pharmaceutical composition” refers to a pharmaceutical acceptable compositions, wherein the composition comprises a pharmaceutically active agent, and in some embodiments further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition may be a combination of pharmaceutically active agents and carriers.

[0044] The term“combination” refers to either a fixed combination in one dosage unit form, or a kit of parts for the combined administration where one or more active compounds and a combination partner (e.g., another drug as explained below, also referred to as“therapeutic agent” or“co-agent”) may be administered independently at the same time or separately within time intervals. In some circumstances, the combination partners show a cooperative, e.g., synergistic effect. The terms“co-administration” or“combined administration” or the like as utilized herein are meant to encompass administration of the selected combination partner to a single subject in need thereof (e.g., a patient), and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time. The term “pharmaceutical combination” as used herein means a product that results from the mixing or combining of more than one active ingredient and includes both fixed and non-fixed combinations of the active ingredients. The term“fixed combination” means that the active ingredients, e.g., a compound and a combination partner, are both administered to a patient simultaneously in the form of a single entity or dosage. The term“non-fixed combination” means that the active ingredients, e.g., a compound and a combination partner, are both administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the two compounds in the body of the patient. The latter also applies to cocktail therapy, e.g., the administration of three or more active ingredients.

[0045] As used herein the term“pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopoeia, other generally recognized pharmacopoeia in addition to other formulations that are safe for use in animals, and more particularly in humans and/or non human mammals.

[0046] As used herein the term“pharmaceutically acceptable carrier” refers to an excipient, diluent, preservative, solubilizer, emulsifier, adjuvant, and/or vehicle with which demethylation compound(s), is administered. Such carriers may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents. Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for the adjustment of tonicity such as sodium chloride or dextrose may also be a carrier. Methods for producing compositions in combination with carriers are known to those of skill in the art. In some embodiments, the language“pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. See, e.g., Remington, The Science and Practice of Pharmacy, 20th ed., (Lippincott, Williams & Wilkins 2003). Except insofar as any conventional media or agent is incompatible with the active compound, such use in the compositions is contemplated.

[0047] As used herein, “therapeutically effective” refers to an amount of a pharmaceutically active compound(s) that is sufficient to treat or ameliorate, or in some manner reduce the symptoms associated with diseases and medical conditions. When used with reference to a method, the method is sufficiently effective to treat or ameliorate, or in some manner reduce the symptoms associated with diseases or conditions. For example, an effective amount in reference to age-related eye diseases is that amount which is sufficient to block or prevent onset; or if disease pathology has begun, to palliate, ameliorate, stabilize, reverse or slow progression of the disease, or otherwise reduce pathological consequences of the disease. In any case, an effective amount may be given in single or divided doses.

[0048] As used herein, the terms“treat,”“treatment,” or“treating” embraces at least an amelioration of the symptoms associated with diseases in the patient, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g. a symptom associated with the disease or condition being treated. As such, “treatment” also includes situations where the disease, disorder, or pathological condition, or at least symptoms associated therewith, are completely inhibited (e.g. prevented from happening) or stopped (e.g. terminated) such that the patient no longer suffers from the condition, or at least the symptoms that characterize the condition.

[0049] As used herein, and unless otherwise specified, the terms "prevent,"

"preventing" and "prevention" refer to the prevention of the onset, recurrence or spread of a disease or disorder, or of one or more symptoms thereof. In certain embodiments, the terms refer to the treatment with or administration of a compound or dosage form provided herein, with or without one or more other additional active agent(s), prior to the onset of symptoms, particularly to subjects at risk of disease or disorders provided herein. The terms encompass the inhibition or reduction of a symptom of the particular disease. In certain embodiments, subjects with familial history of a disease are potential candidates for preventive regimens. In certain embodiments, subjects who have a history of recurring symptoms are also potential candidates for prevention. In this regard, the term "prevention" may be interchangeably used with the term "prophylactic treatment."

[0050] As used herein, and unless otherwise specified, a "prophylactically effective amount" of a compound is an amount sufficient to prevent a disease or disorder, or prevent its recurrence. A prophylactically effective amount of a compound means an amount of therapeutic agent, alone or in combination with one or more other agent(s), which provides a prophylactic benefit in the prevention of the disease. The term "prophylactically effective amount" can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent. As used herein, and unless otherwise specified, the term "subject" is defined herein to include animals such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, and the like. In specific embodiments, the subject is a human. The terms "subject" and "patient" are used interchangeably herein in reference, for example, to a mammalian subject, such as a human.

[0051] The term“antibody” as used herein encompasses monoclonal antibodies

(including full length monoclonal antibodies), polyclonal antibodies, multi-specific antibodies (e.g., bi-specific antibodies), and antibody fragments so long as they exhibit the desired biological activity of binding to a target antigenic site and its isoforms of interest. The term“antibody fragments” comprise a portion of a full length antibody, generally the antigen binding or variable region thereof. The term “antibody” as used herein encompasses any antibodies derived from any species and resources, including but not limited to, human antibody, rat antibody, mouse antibody, rabbit antibody, and so on, and can be synthetically made or naturally-occurring.

[0052] The term“monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The“monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques known in the art.

[0053] The invention may also refer to any oligonucleotides (antisense oligonucleotide agents), polynucleotides (e.g. therapeutic DNA), ribozymes, DNA aptamers, dsRNAs, siRNA, RNAi, and/or gene therapy vectors. The term“antisense oligonucleotide agent” refers to short synthetic segments of DNA or RNA, usually referred to as oligonucleotides, which are designed to be complementary to a sequence of a specific mRNA to inhibit the translation of the targeted mRNA by binding to a unique sequence segment on the mRNA. Antisense oligonucleotides are often developed and used in the antisense technology. The term“antisense technology” refers to a drug-discovery and development technique that involves design and use of synthetic oligonucleotides complementary to a target mRNA to inhibit production of specific disease-causing proteins. Antisense technology permits design of drugs, called antisense oligonucleotides, which intervene at the genetic level and inhibit the production of disease- associated proteins. Antisense oligonucleotide agents are developed based on genetic information.

[0054] As an alternative to antisense oligonucleotide agents, ribozymes or double stranded RNA (dsRNA), RNA interference (RNAi), and/or small interfering RNA (siRNA), can also be used as therapeutic agents for regulation of gene expression in cells. As used herein, the term“ribozyme” refers to a catalytic RNA-based enzyme with ribonuclease activity that is capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which it has a complementary region. Ribozymes can be used to catalytically 45- cleave target mRNA transcripts to thereby inhibit translation of target mRNA. The term “dsRNA,” as used herein, refers to RNA hybrids comprising two strands of RNA. The dsRNAs can be linear or circular in structure. The dsRNA may comprise ribonucleotides, ribonucleotide analogs, such as 2’-0-methyl ribosyl residues, or combinations thereof. The term “RNAi” refers to RNA interference or post-transcriptional gene silencing (PTGS). The term“siRNA” refers to small dsRNA molecules (e.g., 21-23 nucleotides) that are the mediators of the RNAi effects. RNAi is induced by the introduction of long dsRNA (up to 1-2 kb) produced by in vitro transcription, and has been successfully used to reduce gene expression in variety of organisms. In mammalian cells, RNAi uses siRNA (e.g. 22 nucleotides long) to bind to the RNA-induced silencing complex (RISC), which then binds to any matching mRNA sequence to degrade target mRNA, thus, silences the gene.

[0055] The present invention provides, in embodiments, a method of inhibiting viral infection in a biological sample comprising administering to the sample an effective amount of: a) a type I interferon or poly I:C; b) a compound activating an innate immune response in the sample; c) a compound suppressing expression of Gfil, Trim24 and/or Cbl in the sample; and/or d) a compound activating expression of IRF7, IRF3, STAT1, STAT3, NFATC2, IRF5, STAT4, IRF9, IRF8, NFKB1, TP53, JUN and/or EBF1 in the sample. Activation or suppression of additional genes provided herein are also contemplated in all methods of the present invention.

[0056] In embodiments, the biological sample is a cell culture. In embodiments, biological sample comprises mammalian cells. In embodiments, the biological sample comprises CHO cells. In embodiments, the method is conducted in a biopharmaceutical manufacturing process. In embodiments, the compound suppresses expression of Gfil, Trim24 and/or Cbl in the sample. In embodiments, the compound activates expression of IRF7, IRF3, STAT1, STAT3, NFATC2, IRF5, STAT4, IRF9, IRF8, NFKB1, TP53, JUN and/or EBF1 in the sample. In embodiments, the vims is VSV, EMCV, REO, or a RNA vims. The virus can also be a DNA vims.

[0057] The present invention provides, in embodiments, that the compound for activating/increasing genetic expression or suppressing/decreasing genetic expression can be a nucleic acid. The nucleic acid can be transduced into the cell by methods well-known to those of skill in the art. In embodiments, the compound for activating/increasing genetic expression or suppressing/decreasing genetic expression can be a small molecule, transcription factor, microRNA (miRNA), small interfering RNA (siRNA), RNAi, Zinc Finger Nucleases/Peptides, TALENS, antibody, aptamer, or other functional agent. Non coding nucleic acids can also be used as a compound for modulating the expression of genes: antisense oligonucleotides, antisense DNA or RNA, triplex- forming oligonucleotides, catalytic nucleic acids (e.g. ribozymes), nucleic acids used in co suppression or gene silencing, or similar systems to activate/increase or suppress/decrease the genetic expression. Well-known genetic engineering techniques such as site-directed knock-out (KO), knock-in (KI), knock-down (KD), gene mutation, gene transfection, CRISPR activation, CRISPR inhibition, CRISPR/Cas9, and other gene editing systems can also be used as compounds to modify genes and expressional levels as described herein. Compounds to modify expression can include poly I:C or drugs that activate/increase or suppress/decrease the innate immune response.

[0058] The present invention provides, in embodiments, a non-naturally occurring mammalian cell culture comprising cells genetically modified for suppressed expression of Gfil, Trim24 and/or Cbl, and/or activated expression of IRF7, IRF3, STAT1, STAT3, NFATC2, IRF5, STAT4, IRF9, IRF8, NFKB1, TP53, JUN and/or EBF1, as compared to wild- type cells of the same mammalian species.

[0059] The present invention provides, in embodiments, a method of producing a biopharmaceutical protein from a mammalian cell culture, comprising culturing mammalian cells having non-naturally occurring genetically suppressed expression of Gfil, Trim24 and/or Cbl, and/or genetically activated expression of IRF7, IRF3, STAT1, STAT3, NFATC2, IRF5, STAT4, IRF9, IRF8, NFKB1, TP53, JUN or EBF1 or TP53 or JUN and/or EBF1, as compared to wild-type cells of the same mammalian species; and isolating a protein of interest from the cultured cells.

[0060] The present invention provides, in embodiments, that the biological sample comprises CHO cells. In embodiments, the cells have suppressed expression of Gfil, Trim24 and/or Cbl. In embodiments, the cells have activated expression of IRF7, IRF3, STAT1, STAT3, NFATC2, IRF5, STAT4, IRF9, IRF8, NFKB1, TP53, JUN and/or EBF1. [0061] The present invention provides, in embodiments, a method of treating or preventing viral infection in a mammalian cell comprising administering to the cell an effective amount of: a) a type I interferon or poly I:C; b) a compound activating an innate immune response in the sample; c) a compound suppressing expression of Gfil, Trim24 and/or Cbl in the sample; and/or d) a compound activating expression of IRF7, IRF3, STAT1, STAT3, NFATC2, IRF5, STAT4, IRF9, IRF8, NFKB1, TP53, JUN and/or EBF1 in the sample.

[0062] The present invention provides, in embodiments, a method to block viral infection in mammalian cells in vivo having genetically or chemically decreased activity of Gfil, Trim24 and/or Cbl, and/or genetically or chemically increased expression and/or activity of IRF7, IRF3, STAT1, STAT3, NFATC2, IRF5, STAT4, IRF9, IRF8, NFKB1, TP53, JUN and/or EBF1, as compared to wild-type cells of the same mammalian species.

[0063] The present invention provides, in embodiments, a method for increasing vims infectivity in a mammalian cell comprising increasing expression of Gfil, Trim24 and/or Cbl, and/or decreasing expression of IRF7, IRF3, STAT1, STAT3, NFATC2, IRF5, STAT4, IRF9, IRF8, NFKB1, TP53, JUN and/or EBFlin the cell. In embodiments, the method further comprises isolating vims or viral particles from the cell. In embodiments, genetic material is delivered to the sample by viral transduction to increase or decrease expression of said gene.

[0064] The present invention provides, in embodiments, a non-naturally occurring mammalian cell culture comprising mammalian cells having genetically activated expression of Gfil, Trim24 or Cbl, and/or genetically suppressed expression of IRF7, IRF3, STAT1, STAT3, NFATC2, IRF5, STAT4, IRF9, IRF8, NFKB1, TP53, JUN and/or EBF1, as compared to wild-type cells of the same mammalian species.

[0065] These and other embodiments of the invention will be apparent to one of skill in the art upon a review of the present Specification. 48-

EXAMPLE 1

Materials and Methods

CHO-K1 cells and RNA viruses

[0066] The susceptibility of CHO-K1 cells to viral infection has been previously reported (Berting et al. 2010). Since infectivity was demonstrated for viruses of a variety of families (harboring distinct genomic structures), the following RNA viruses were selected from three different families to be used as prototypes: Vesicular stomatitis vims (VSV, ATCC VR-1238), Encephalomyocarditis virus (EMCV, ATCC VR-129B), and Reovirus-3 vims (Reo, ATCC VR-824). Viral stocks were generated in susceptible Vero cells as per standard practices using DMEM (Dulbecco's Modified Eagle's medium) supplemented with 10% FBS, 2m M L-glutamine, 100 U/ml penicillin and 100 pg/ml streptomycin (DMEM- 10). Viral stocks were tittered by tissue culture infectious dose 50 (TCID50) on CHO-K1 cells and used to calculate the multiplicity of infection in the experiments (Table 1).

Virus infection and innate immune modulator treatment.

[0067] Cells were seeded in cell culture plates (3xl0 ⁵ and l.2xl0 ⁶ cells/well in 96- well and 6- well plates, respectively) and grown overnight in RPMI-1040 supplemented with 10% FBS, 2mM L-glutamine, 100 U/ml penicillin and 100 pg/ml streptomycin, 10 mM Hepes, lx non-essential amino acids and 1 mM sodium pyruvate (RPMI-10). IENa/b and innate immune modulators (LPS (TLR4) (Calbiochem), CpG-oligodeoxynucleotide (ODN) D-ODN, (Puig et al. 2012) and ODN-1555, (TLR9) (custom-synthesized at the Center for Biologies Evaluation and Research facility, FDA), imidazoquinoline R837 (TLR7/8) (Sigma) and poly I:C-Low molecular weight/LyoVec (polyinosinic- polycytidylic acid) (poly I:C) (Invivogen) were added to the cultures 16-24 h prior to vims infection, at the concentrations indicated in the figures. Viral infection was performed by adding virus suspensions to the cell monolayers at the indicated MOI in serum-free media and incubate at 37 °C 5% CO2 for 2h. Cell cultures were washed twice to discard unbound virus and further incubated at 37 °C for 30 h (VSV), 54 h (EMCV) or 78 h (Reo) (unless otherwise indicated in the figures). The cell harvesting time was established based on appearance of cytopathic effect in approximately 50% of the cell monolayer. Cytopathic effect was visualized by crystal violet staining as per standard practices. Infection/poly I:C 49- experiments were repeated twice, independently. In each replicate CHO cells were cultured as poly I:C untreated - uninfected (media control, m), poly I:C treated - uninfected (p), poly I:C untreated - virus infected (Vin) and poly I:C treated - virus infected (Vp). The antibodies and cytokines used as innate immune modulators were anti- STAT1 and pSTAT2 antibodies (Becton Dickinson), neutralizing anti-IENb antibody (R&D), anti-Mxl antibodies (a gift from Dr. O. Haller, Germany). Human IFNa (Avonex) and IFN (Roferon) are clinical grade drugs.

[0068] Western blot procedures. Cell lysates were prepared using mammalian protein extraction reagent M-PER (Thermo Fisher Scientific, Waltham, MA) with Protease and Halt™ phosphatase inhibitor cocktails (Thermo Fisher Scientific) using an equal number of cells per sample. Samples were analyzed by SDS-PAGE using 10-20% Tris-Glycine gels (Thermo Fisher Scientific) under reducing conditions. As a molecular weight marker, protein ladder (cat# 7727S) from Cell Signaling Technology (Danvers, MA) was used. Nitrocellulose membranes and iBlot™ transfer system (Thermo Fisher Scientific) were used for Western Blot analysis. All other reagents for Western Blot analyses were purchased from Thermo Fisher Scientific. Membranes were blocked with nonfat dry milk (BIO-RAD, Hercules, CA) for lh followed by incubation with primary antibodies against STAT1, pSTATl (pY70l, BD Transduction Lab, San Jose, CA), or Mxl (gift from O. Haller, University of Freiburg, Freiburg, Germany) O/N at 4°C. Secondary goat anti-mouse and anti-rabbit antibodies were purchased from Santa Cruz Biotechnology. SuperSignal West Femto Maximum Sensitivity Kit (Thermo Fisher Scientific) was used to develop membranes, and images were taken using LAS-3000 Imaging system (GE Healthcare Bio-Sciences, Pittsburgh, PA).

RNA extraction, purification, and quality control procedure

[0069] Cell cultures were resuspended in RLT buffer (Qiagen) and kept at -80°C until RNA was extracted using the RNeasy kit (Qiagen) and on-column DNAse digestion. RNA was eluted in 25 mΐ of DEPC water (RNAse/DNAse free); concentration and purity were tested by bioanalyzer. Total RNA levels for type I IFN related genes and viral genome were also assessed by RT-PCR. Complementary DNA synthesis was obtained from 1 pg of RNA using the High capacity cDNA RT kit (Thermo Fisher Scientific) as per manufacturer’s instructions. Semi-quantitative PCR reactions (25 mΐ) consisted in 1/20 cDNA reaction volume, lx Power Sybr master mix (Thermo Fisher Scientific), 0.5 mM Chinese hamster- specific primers for IENb, Mxl, IRF7 and IITMP3 sequences (SAbiosciences) (these genes were selected to assess type I IFN response). Eukaryotic 18S was used as a housekeeping gene and assessed in IX Universal master mix, 18S expression assay (1:20) (Applied Biosystems) using a 1/50 cDNA reaction volume. Fold changes were calculated by the 2-AACt method. cDNA library construction and Next-generation sequencing (RNA-Seq)

[0070] Library preparation was performed with Illumina’s TruSeq Stranded mRNA Library Prep Kit High Throughput (Catalog ID: RS-122-2103), according to manufacturer’s protocol. Final RNA libraries were first quantified by Qubit HS and then QC on Fragment Analyzer (from Advanced Analytical). Final pool of libraries was run on the NextSeq platform with high output flow cell configuration (NextSeq 500/550 High Output Kit v2 (300 cycles) FC-404-2004).

RNA-Seq quantification and differential gene expression analysis

[0071] RNA-Seq quality was assessed using FastQC. Adapter sequences and low quality bases were trimmed using Trimmomatic (Bolger et al. 2014). Sequence alignment was accomplished using STAR (Dobin et al. 2013) against the CHO genome (GCF_0004l9365.l_C_griseus_vl.0) with default parameters. HTSeq (Anders et al. 2015) was used to quantify the expression of each gene. Differential gene expression analysis using DESeq2 (Anders and Huber 2010). After Benjamini-Hochberg FDR correction, genes with adjusted p-values less than 0.05 and fold change greater than 1.5 were considered as differentially expressed genes (DEGs). Table 3 shows the number of identified DEGs in the three different comparisons: 1) untreated - uninfected vs. untreated - vims infected (m vs. Vm); 2) untreated - uninfected vs. poly I:C treated - uninfected (m vs. p); and 3) untreated - virus infected vs. poly I:C treated - virus infected (Vm vs. Vp).

Genetic engineering (Gill, Trim24, Gfil/Trim24) of CHO-S cell lines

[0072] CHO-S cells (Thermo Fisher Scientific Cat. # Al 155701) and KO clones were cultured in CD CHO medium supplemented with 8 mM L-glutamine and 2 mL/L of anti-clumping agent (CHO medium) in an incubator at 37°C, 5% CO2, 95% humidity. Cells were transfected using FuGENE HD reagent (Promega Cat. # E2311). The day prior to transfection, viable cell density was adjusted to 8xl0 ⁵ cells/mL in an MD6 plate well containing 3 mL CD CHO medium supplemented with 8 mM L-glutamine. For each transfection, 1500 ng Cas9-2A-GFP plasmid and 1500 ng gRNA plasmid were diluted in 75 uL OptiPro SFM. Separately, 9 uL FuGene HD reagent was diluted in 66 uL OptiPro

SFM. The diluted plasmid was added to the diluted FuGENE HD and incubated at room temperature for 5 minutes and the resultant 150 pL DNA/lipid mixture was added dropwise to the cells. For viability experiments, CHO-S KO cell lines were seeded at 3xl0 ⁶ cells in 30 ml in CHO medium and incubated at 37 °C, 5% CO2, 125 rpm for up to 7 days. Infections were conducted with EMCV and Reo-3 at the same MOI calculated in CHO-K1 cells for 2h prior to wash cells twice to discard unbound particles. Control cell lines showing susceptibility to either vims were infected in parallel to those with Gfil and Trim24 gene KO.

[0073] The plasmids we used to generate Gfi, Trim24, and Gfi+Trim24 knock-out cell lines are: Plasmids 2632 (GFP_2A_Cas9), Plasmids 6016 (Gfil-665755) and 6018 (Trim24- 1009774). The Plasmids 2632 (GFP_2A_Cas9) is described in (Grav et ak, 2015). The Plasmids 6016 (Gfil-665755) and 6018 (Trim24- 1009774) were constructed as described in (Ronda et ak, 2014) with the following modification: sgRNA plasmid sgRNAl_C described in (Ronda et ak, 2014) was used as template in the PCR reaction to generate the backbone of gRNA plasmids.

[0074] Oligos used in the cloning reaction were:

[0075] PRIMERS USED FOR MISEQ ANALYSIS WERE:

Single cell sorting, clone genotyping and expansion

[0076] Transfected cells were single cell sorted 48 hours post transfection, using a

FACSJazz, based on green fluorescence with gating determined by comparison to non- transfected cells. Sorting was done into MD384 well plates (Coming Cat. # 3542) containing 30 pL CD CHO medium supplemented with 8 mM L-glutamine, 1% antibiotic- antimycotic agent (Thermo Fisher Scientific Cat. # 15240-062) and 1.5% HEPES buffer (Thermo Fisher Scientific Cat. # 15630-056). After 15 days, colonies were transferred to an MD96F well plate (Falcon Cat. # 351172) containing 200 pL CD CHO medium supplemented with 8 mM L-glutamine, and 1% antibiotic-antimycotic. After additional two days, 50 pL cell suspension from each well was transferred to a MicroAmp Fast 96 well reaction plate (Thermo Fisher Scientific Cat. # 4346907), along with 5xl0 ⁵ wildtype control cells. The plate was centrifuged at 1000 x g for 10 minutes and then the supernatant was removed by rapid inversion. Twenty pL of 65 °C QuickExtract DNA Extraction Solution (Epicentre Cat. # QE09050) was added to each well and mixed. The plate was then placed in a thermocycler at 65 °C for 15 minutes followed by 95 °C for 5 minutes. Amplicons were generated for each gene of interest per well using Phusion Hot Start II DNA Polymerase and verified to be present visually on a 2% agarose gel. Amplicons from each well had unique barcodes, allowing them to be pooled and purified using AMPure XP beads (Beckman Coulter Cat. # A63881) according to manufacturer’s protocol, except using 80% ethanol for washing steps and 40 pL beads for 50 pL sample. Samples were indexed using the Nextera XT Index kit attached using 2 x KAPA HiFi Hot Start Ready mix (Fisher Scientific Cat. # KK2602). AMPure XP beads were used to purify the resulting PCR products. DNA concentrations were determined with the Qubit 2.0 Fluorometer and used to pool all indices to an equimolar value and diluted to a final concentration of 10 nM using 10 mM Tris pH 8.5, 0.1% Tween 20. The average size of the final library was verified with a Bioanalyzer 2100. The amplicon library was then sequenced on an Illumina MiSeq. Insertions and deletions were identified by comparison of expected versus actual amplicon size. Clones with frameshift indels in all alleles were selected for expansion in shake flasks (shaking at 120 rpm, 25 mm throw), banking and characterization.

Results and Discussion

CHO-K1 cells fail to prevent infection by RNA viruses despite possessing functional type I IFN-inducible anti- viral mechanisms.

[0077] To evaluate the response of CHO cells to the three different RNA viruses

(VSV, EMCV and Reo; see Table 1), CHO cells were infected and monitored for cytopathic effects and gene expression changes related to the type I IFN response (see Materials and Methods). All three viruses induced a cytopathic effect (Figure 1A, right panels) and measured a modest increase in IRNb transcript levels in CHO cells (Figure 1B). Through its cellular receptor, IFNa/b can further activate downstream interferon- stimulated genes known to limit viral infection both in cell culture and in vivo (Katze et al. 2002; McNab et al. 2015; Schneider et al. 2014; Seo and Hahm 2010). Indeed, the results indicate that CHO cells have a functional IFNa/b receptor and that its activation with exogenous IFN confers resistance of CHO cells to VSV infection (see Figure 8). Interestingly, CHO cells expressed high levels of the antiviral gene Mxl when infected with Reo, but not VSV and EMCV (Figure 1C). Nevertheless, the virus-induced IFN response in the host cell was insufficient to prevent cell culture destruction. These data suggest a possible inhibition of the antiviral type I IFN response that varies across viruses, as previously reported (Ahmed et al. 2003; Ng et al. 2013; Rieder and Conzelmann 2009; Sherry 2009).

[0078] To explore why the induced type I IFN failed to mount a productive antiviral response in CHO cells, RNA-Seq and pathway analysis was conducted using GSEA (see EXAMPLE 2) GSEA analysis that compared control vs. infected CHO cells (m vs. Vm) revealed the modulation of several immune-related gene sets and pathways activated by the vims (Figure 1D). Unlike VSV and EMCV, Reo induced the‘interferon alpha response’ and‘RIG-I and MDA5-mediated induction of IFNa’ pathways ((p-value, NES) = (9.05xl0 ³ , 3.68) and (1.12xl0 ² , 2.74), respectively). These findings were consistent with observations that the reovirus genome (dsRNA) can stimulate TLR3 and RIG-I to induce innate immune responses in other organisms (Goubau et al. 2014; Jensen and Thomsen 2012; Loo et al. 2008), but the observed response diverged markedly from the VSV infection, which is also sensed by RIG-I but nonetheless failed to induce an interferon alpha response.

[0079] As observed for Mxl, only Reo-infected cells showed a significant enrichment of differentially expressed genes involved in the type I IFN response (FDR- adjusted p-value = 9.05xlQ ^~3 ; normalized enrichment score, NES = 3.68). These genes contain the consensus transcription factor binding sites in the promoters that are mainly regulated by the transcription factor STAT1 and the interferon regulatory factors (IRF) family, such as IRF1, IRF3, IRF7 and IRF8 (Figure IE). These results are consistent with observations that the IRF family transcription factors activate downstream immune responses in vims -infected mammalian cells (Honda and Taniguchi 2006; Ivashkiv and Donlin 2014). In contrast, VSV and EMCV failed to trigger anti-viral related mechanisms (e.g., type I IFN responses) downstream of IENb (Figure ID). Examples of a few pathways that were stimulated included ‘immune system’ (including adaptive/innate immune system and cytokine signaling in immune system) in VSV (FDR-adjusted p-value = l.49xl0 ² ; normalized enrichment score, NES= 1.99) and the‘G2M checkpoint’ in EMCV (p-value = 8.95x10 ³ ; NES = 2.64). However, neither VSV nor EMCV infection activated known upstream activators (Figure IE) of type I IFN pathways when analyzed with Ingenuity Pathway Analysis (IPA) (Kramer et al. 2014).

Poly I:C induces a robust type I interferon response in CHO cells

[0080] Type I IFN responses limit viral infection (Perry et al. 2005; Sadler and

Williams 2008; Schoggins and Rice 2011; Taniguchi and Takaoka 2002), and innate immune modulators (Bohlson 2008; Mutwiri et al. 2007; Olive 2012) mimic pathogenic signals and stimulate pattern recognition receptors (PRRs), leading to the activation of downstream immune -related pathways. Intracellular PRRs, including toll-like receptors (TLR) 7, 8 and 9, and cytosolic receptors RIG-I or MDA5, can sense viral nucleic acids and trigger the production of type I IFN. This example sought to determine whether CHO cell viral resistance could be improved by innate immune modulators.

[0081] CHO PRRs have not been studied extensively, so the ability of synthetic ligands to stimulate their cognate receptors to induce a type I IFN response was first assessed. CHO cells were incubated with LPS (TLR4 ligand), CpG-oligodeoxynucleotide (ODN) type D (activates TLR9 on human cells), ODN-1555 (activates TLR9 on murine cells), imidazoquinoline R837 (TLR7/8 ligand) and poly I:C-Low molecular weight/LyoVec (poly I:C) (activates the RIG- I/M DA-5 pathway), and subsequently tested for changes in expression of IFN stimulated genes with anti- viral properties. After 24h of culture, gene expression levels of IRF7 and Mxl increased significantly in cells treated with poly I:C but not in those treated with any of the other innate immune modulators (Figure 2A). Furthermore, STAT1 and STAT2 phosphorylation and Mxl protein levels were elevated following treatment with poly I:C or exogenous interferon-alpha (IFNa), which was used as a control (Figure 2B and 2C). By monitoring changes in the gene expression levels of IRNb and Mxl in the cells, it was established that 16-20 h would be an adequate time interval for treating cells with poly I:C prior to infection (Figures 10A- 10B).

[0082] Next, the type I IFN response induced by poly I:C was characterized by analyzing the transcriptome of untreated vs. treated CHO cells. Cells were cultured with poly I:C in the media for 30, 54 and 78 h after an initial 16 h pre-incubation period. GSEA of the RNA-Seq data demonstrated that poly I:C induced a strong ‘innate immune response’ in comparison to untreated cultures (media) (m vs. p; (p-value, NES, Enrichment strength) = (8.08xl0 ^~3 , 2.98, 73%), (l.57xl0 ^~2 , 3.95, 70%) and (3.91xl0 ^~3 , 3.58, 78%)) evident at all the tested time points (Figures 2D and 9B). In addition, it activated several upstream regulators of the type I IFN pathways (Figure 2E). It was noted that the strength of the gene set enrichment (see EXAMPLE 2) of the innate immune response induced by poly I:C (m vs. p) was stronger than the innate immune response seen for Reo infection alone (m vs. Vm in Figures 9A-9B). Thus, CHO cells can activate the type I IFN signaling (JAK-STAT) pathway in response to poly I:C and display an anti viral gene signature, which was sustained for at least 4 days. Poly I:C-induced type I interferon response protect CHO cells from RNA virus infections

[0083] It was next examined if the type I IFN response, induced by poly I:C, could protect CHO cells from RNA virus infections. It was found that poly I:C pre-treatment protected CHO cells against viral infection through the I RNb- mediated pathway (Figures 11A-11B), and that poly I:C protected against all three viruses tested (Figures 3A-C). Cell morphology differed notably between cultures infected with virus (Vm), control uninfected cells (m), and poly I:C pre-treated cultures (p and Vp) (Figures 3A-3C). These morphological changes correlated with the cytopathic effect observed in the cell monolayers (Figures 3A-3C, right panels). At 78h, the extent of cell culture damage by Reo, however, was milder than by VSV and EMCV at a shorter incubation times (30h and 54h, respectively) (Figures 3A-3C), possibly since Reo induced higher levels of anti-viral related genes in the CHO cells but VSV and EMCV did not (Figures 1C, 1D and 1E). Notably, although poly I:C pre-treatment conferred protection of CHO cells to all three viral infections (Figure 3A-3C), striking transcriptomic differences were observed. Poly I:C pre-treatment significantly activated immune-related pathways and up-regulated type I IFN-related gene expression in CHO cells infected with VSV and EMCV when compared to non-poly I:C pre-treated cells that were infected (Vm vs. Vp) (Figures 3D-3E). Poly I:C pre-treatment was sufficient to induce a protective type I IFN response to VSV and EMCV. For Reo infection, however, pre-treatment with poly I:C did not further increase the levels of expression of IFN associated genes over those observed in poly I:C-untreated, infected cells. The lack of enhanced expression of antiviral genes in Reo Vm vs. Vp observed in the GSEA was further confirmed by Taqman analysis. A similar level of expression of anti-viral Mxl and IITMP3 genes (Diamond and Farzan 2013; Li et al. 2013; Pillai et al. 2016; Verhelst et al. 2013) was obtained for CHO cells independently infected with Reo (Vm) treated with poly I:C (p) or pre-treated with poly I:C and infected (Vp), which resulted in no differences in transcript levels when we compared Vm vs. Vp (Figure S5C). Nevertheless, the outcome of infection was surprisingly different in Vm or Vp samples. To understand these differences, genes that were differently modulated by poly I:C treatment in the context of Reo infection were identified. Indeed, 30 genes (Figures 13A-13B) that were significantly up regulated (adjusted p-value <0.05, fold change >1.5) in the comparisons of m vs. Vp and m vs. p but not in the comparison of m vs. Vm. These genes are significantly enriched in 11 KEGG pathways related to host- immune response (e.g., antigen processing and presentation, p-value=3.4xl0 ³ ) and processes important to virus infection (e.g., endocytosis, p-value=2.5xlQ ² ). It was also observed that many of these genes significantly enriched molecular functions: 1) RNA polymerase II transcription factor activity (11 genes; G0:000098l FDR-adjusted p- value < 1.30xl0 ¹⁵ ) and 2) nucleic acid binding transcription factor activity (12 genes G0:000l07l FDR-adjusted p-value < 3.54x10 ¹⁵ ) by gene set enrichment analysis (see EXAMPLE 2). This suggests that poly I:C treatment, 16 hours prior to virus infection, pre disposes the cell to adopt an antiviral state and might restore the host transcription machinery subverted by Reo vims resulting in the protection of the CHO cells.

[0084] The results revealed other processes that are differentially activated or repressed between Vm and Vp (Figure 3D). For example, the top down-regulated Reactome pathways in the virus-infected cells are protein translational related processes: ‘nonsense mediated decay enhanced by the exon junction complex’ (p-value = 3.32xl0 ² , NES = -3.50),‘peptide chain elongation’ (p-value = 3.32xl0 ² , NES = -3.59), and‘3’-UTR mediated translational regulation’ (p-value = 3.38x10 ² , NES = -3.61). These results agree with studies showing viral hijacking of the host protein translation machinery during infection (Walsh et al. 2013), and that the activation of interferon-stimulated genes restrain vims infections by inhibiting viral transcription and/or translation (Schoggins and Rice 2011). All these results suggest that poly I:C treatment provides the cell with an advantageous immune state that counteracts viral escape mechanisms and results in cell survival.

A STATl-dependent regulatory network governs viral resistance in CHO cells.

[0085] GSEA revealed that several transcriptional regulators were activated or repressed during different viral infections and poly EC-treated cells (Figures 1C- IE, 2E, and 3E). Among these, six were consistently and significantly activated across different vims and media conditions (highlighted in dash rectangles; Figures 1C, 2E and 3E). These included NFATC2, STAT1, IRF3, IRF5, and IRF7, which were all activated in poly I:C pretreatment of CHO cells (m vs. p and Vm vs. Vp). These transcription factors are involved in TLR-signaling (IRF3, IRF5, and IRF7; (Honda and Taniguchi 2006)) and JAK/STAT signaling (NFATC2, STAT1, and TRIM24). The TLR signaling pathway is a downstream mediator in virus recognition/response and in activating downstream type-I interferon immune responses (Arpaia and Barton 2011; Kawai and Akira 2009; Thompson and Locamini 2007). Meanwhile, the JAK/STAT pathway contributes to the antiviral responses by up-regulating interferon simulated genes to rapidly kill virus within infected cells (Aaronson and Horvath 2002; Au- Yeung et al. 2013; Li and Watowich 2014). Importantly, one mechanism by which STAT1 expression and activity may be enhanced is via the poly I:C induced repression of TRIM24, which inhibits STAT1. The crosstalk between TLR- and JAK/STAT-signaling pathways plays essential roles in virus clearance of the virus infected host cells (Hu and Ivashkiv 2009).

[0086] The roles of upstream regulators were further investigated by examining the expression of their downstream target genes. Table 2 shows the results of the regulatory pathways emanating from poly I:C treatment and their expected effects on the downstream phenotypes. Regulatory networks were identified that capture the anti- viral response of the cells (Figures 4 A and 4B for VSV and EMCV respectively). The networks are predominantly regulated by these same 6 transcription factors (NFATC2, STAT1, IRF3, IRF5, IRF7, and TRIM24), which can regulate many genes that together inhibit VSV and EMCV virus replication in poly I:C pretreated cells (Table 2). The activation of this STATl-dependent regulatory network by poly I:C-treated media leads to the induction of several immune-related responses (e.g., recruitment for leukocytes; Figure 14). The induction of the STATl-dependent regulatory network with poly I:C pretreatment, and the subsequent viral resistance suggests that the network may have protective power against virus infection. While the STATl-dependent regulatory network did not apparently emerge when comparing the poly I:C pre-treatment compared to the untreated Reo infected cells (Vm vs. Vp), because those pathways are natively activated by Reo since poly X:C is a structural analog of double-stranded RNA (Fortier et al. 2004). For example, NFATC2-dependent network (Figures 15A-15B) and IRF3 -dependent network (Figures 16A-16B) are two example networks that presented in both of the comparisons m vs. p and m vs. Vm.

Deletion of Trim24 and Gfil induced CHO cell innate immunity and viral resistance

[0087] With the STAT1 network potentially contributing to viral resistance, upstream regulators were sought that could be modulated to naturally induce STAT1. That identified sixteen statistically significant (p < 0.05) upstream regulators, including 13 positive and 3 negative regulators of Statl using IPA (Figure 5). It was hypothesized that the deletion of the most active repressors of Statl could improve vims resistance by inducing Statl expression and the downstream type I IFN antiviral response (Figure 5). Three Statl repressors (Trim24, Gfil and Cbl) with a negative regulatory score were identified and therefore having potential for inhibiting Statl based on the RNA-Seq differential expression data (see details in Figures 19A-19B). However, Cbl did not present in samples involving Reo virus infection. Therefore, the two negative regulators, Gfil (Sharif-Askari et al. 2010) and Trim24 (Tisserand et al. 2011), of Statl were selected as targets for genetic engineering (Figure 5 and Table 4) and subsequently tested their susceptibility to Reo and EMCV. To evaluate the impact of gene editing on the engineered CHO-S cells, RNA-Seq was conducted in uninfected single (Gfil or Trim 24) or double (Gfil + Trim 24) KO cell lines (Figures 6A-6C). The results revealed that these cells had increased transcript levels of a number of genes involved in innate immunity pathways, such as those mediated by interleukins (ILs) (e.g. IL-33 pathway (IL-1R, IL-5, IL-13, IL- 33) and IL-18) (Figure 6A) and STAT (e.g., STAT1 , 3, 5B and 6)-related genes (Figure 6B), leading to the upregulation of several immune functions (Figure 6C) that could limit vims infection. Subsequently and as a proof of concept, the vims susceptibility of the cells was evaluated using Reo-3 and EMCV. It was found that the that the Trim24 and Gfil single knockout clones (Figure 7A-7C) show resistance to Reo but moderate or no resistance against EMCV, compared to positive controls (Figures 17A-17B). However, the Gfil and Trim24 double knockout (Figure 1C) showed resistance to both vimses tested, even when cultured with virus for a second week (Figures 18A-18B). Together these results show that the regulatory network contributes to antiviral mechanisms of CHO cells, which could possibly be harnessed to obtain virus resistant CHO bioprocesses.

[0088] These results suggest that the genomes of these RNA vimses are sensed by the same RIG-I/TLR3 receptors of the host cell, even if these RNA viruses of different families have found mechanisms to overcome the innate immune mechanisms of the CHO cells (Fig. 1). Activation of RIG-I/TLR3 with the ligand Poly I:C prior to virus infection gives an advantage to the host cell over the virus by inducing a robust type I IFN response allowing its survival. A similar outcome appears to be reached by deleting two of the type I IFN pathway negative regulators. The systems biology approach to identifying transcription factors impacting RNA virus infection can be replicated in the future for other vims classes, such as DNA viruses (e.g. MVM) which use other mechanisms for viral sensing such as TLR9, which is not expressed in CHO cells, therefore making CHO susceptible to MVM infection. Thus, using the present invention approach, regulators of innate immunity can be provided to make DNA virus resistance cells by simulating TLR9 or its downstream activities in CHO cells with the use of CpG ODN to induce a TLR9- driven type I IFN response on the cell.

EXAMPLE 2

Gene set enrichment analysis (GSEA) and Upstream regulator (transcriptional factor) analysis

Gene set enrichment analysis (GSEA) and enrichment strength analysis

[0089] GSEA was performed using the Broad Institute GSEA software

(Subramanian et al. 2005). A ranked list of genes (adjusted p-values < 0.05) was made using the differential expression values (Fold change in the log ₂ scale) from differential gene expression analysis were ran through the GSEA pre-ranked protocol. GSEA-pre-rank analysis was processed to detect significant molecular signature terms (‘Hallmark’ (50) and‘Reactome’ (674) gene sets from the MSigDB were used here) for the differential expressed genes. Note that, the criteria for considering a molecular signature term as significant are: 1) after Benjamini-Hochberg false discovery correction, molecular signature terms with adjusted p-values less than 0.05; and 2) there are >30 genes presented in the gene list of this molecular signature terms.

[0090] The leading edge analysis allows for the GSEA to determine which subsets

(referred to as the leading edge subset) of genes contributed the most to the enrichment signal of a given gene set's leading edge or core enrichment (Subramanian et al. 2005). The leading edge analysis is determined from the enrichment score (ES), which is defined as the maximum deviation from zero. The enrichment strength describes the strength of the leading-edge subset of a gene set (i.e., the interferon-alpha response in this study) (Subramanian et al. 2005). Specifically, if the gene set is entirely within the first N positions in the ranked differentially expressed gene list, then the signal strength is maximal or 100%. If the gene set is spread throughout the list, then the signal strength decreases towards 0%.

Upstream regulator (transcriptional factor) analysis

[0091] The upstream regulators were predicted using the Ingenuity IPA Upstream

Regulator Analysis Tool by calculating a regulation Z-score and an overlap p-value (Kramer et al. 2014), which were based on the number of known target genes of interest pathway/function, expression changes of these target genes and their agreement with literature findings. It was considered significantly activated (or inhibited) with an overlap p-value less than 0.05 and an IPA activation IZ-scorel > 1.96. Note that, the criteria for generating the resulting table (Table 2) from IPA are: 1) Total nodes >= 10, and 2) Consistency score >= 5.00. Consistency score is an IPA measurement (Kramer et al. 2014) for measuring the consistency of a predicted network (capturing regulator-target-function relationships) from RNA-Seq data with literature knowledge. The higher consistency scores of the predicted regulatory networks denote better consistency with literature support than the predicted regulatory networks with lower consistency scores.

Type I IFN protects CHO cells from VSV infection

[0092] CHO cells failed to make a significant IFN response when infected with vims. It is well documented that type I IFN response is necessary to limit the extent of viral infection both in a cell culture and in vivo. Thus, this analysis sought to determine if the susceptibility to the vims was due to unresponsiveness of the cells to IFN rather than lack of ability to generate such a response. In order to simplify the screening, we first concentrated on VSV. Cells were seeded in 96- well plates and treated with human or murine type I IFN protein preparations for 24h, prior to the addition of serially diluted VSV (1:10) (Figure 8). Infection progressed for 24h and cultures were stained with crystal violet (CV) to assess the extent of the protection by cytopathic effect. All IFN preparations limited viral cytopathic effect (Figure 8). Of note, human IRNb had the most potent anti- VSV effect of all the interferons tested, at least at the dose used in the experiment (Figure 8). These results indicate that CHO cells have a functional IFNa/b receptor and that its activation confers resistance of CHO cells to VSV infection. Poly I:C pre-treatment of CHO cells protects against viral infection through the IFNp-mediated pathway.

[0093] It was next examined if the type I IFN response induced by poly I:C could protect CHO cells from RNA vims infections by evaluating effect of poly I:C on CHO susceptibility to VSV infection. Cells were cultured with lpg/ml of poly I:C for 24h prior to infection with VSV (MOI of 0.1). As in previous experiments, the control poly I:C- treated CHO cell monolayer remained intact during the length of the experiment (48h) indicating that poly I:C per se was not toxic for the cells (Figure 11 A). In contrast, disruption of the CHO cell monolayer was evident in wells where VSV was added, but not in wells where CHO cells were pre-incubated with poly I:C (Figures 11A and 11B). Moreover, the poly FC-induced anti-viral response of the cell was IRNb-dependent, as demonstrated by addition of a neutralizing antibody to IFN-b (Figure 11B). These results suggest that poly I:C treatment provides the cell with an advantageous immune state by activating the IFN -mediated pathway that counteracts viral escape mechanisms and results in cell survival.

Identification of the STAT1 upstream regulators.

[0094] The upstream regulators of STAT1 were identified by the three steps. First, collect all the upstream regulators predicted using the Ingenuity IPA Upstream Regulator Analysis Tool in the RNA-Seq data of the comparisons: m vs. p (media vs poly I:C treated media) and Vm vs. Vp (virus+media vs virus-i- poly I:C treated media)). Second, further select those IPA predicted upstream regulators that can regulate STAT1 gene with literature evidences (Table 4). Third, define the negative regulatory score as below.

Negative regulatory score = — logl0(P— value) xReg latmon Direction

[0095] The p-value (Table 4) here is calculated using Fisher’s Exact Test for measuring whether there is a statistically significant overlap between the differentially expressed genes in our dataset genes and the genes that are regulated by a TF, as reported in IPA. The higher negative regulatory score of a TF represents the larger potential in inhibiting STAT1 based on the RNA-Seq differential expression data (Figures 19A-19B).

TABLES

Table 1. Study prototype viruses and MOI on CHO-K1 cells.

Referenced

Genomic

CHO cell

Vims Virus family nucleic acid MOI

culture

nature

infection

Vesicular stomatitis

Rabdoviridae ss (-) RNA Potts, 2008 0.003 virus (VSV)

Encephalomyocarditis

Picornaviridae ss (+) RNA Potts, 2008 0.007 vims (EMCV)

Wisher, 2005;

Reovims 3 (Reo) Reoviridae ds RNA 0.0013

Rabenau 1993

Table 2A. The downstream effects of the upstream regulators from the comparison

of m vs. p.

Total

Consis Target

nodes Biological

Vims tency TF ^' a gene ^*b Relations^

(TF, TG, Process ^*0 score (TG)

BP)

CASP1,

CXCF10, Inhibit

DDX58, Replication

EIF2AK2, of vims.

IFIH1,

IF15, Activate

21 STAT1, IRF3, IRF5, IRF7,

VSV 5.82 ISG15, Activation of 6/15 (40%)

(5, 13, 3) NFATC2

Mxl/Mx2, phagocytes;

OASF2, Apoptosis of

PEFI1, antigen

PMF, presenting

SOCS1, cells.

TNFSF10

Inhibit

BST2, C3, Replication

CASP1, of vims;

48 STAT1, IRF3, IRF5, IRF7,

EMCV 22.47 CXCF10, Infection by 21/84 (25%)

(7.29.12) NFATC2, TRIM24, NCOA2 DDX58, RNA vims;

EGR2, Infection of

EIF2AK2, central Total

Consis Target

nodes Biological

Vims tency TF a gene *b Relations ^*01

(TF, TG, Process ^*0 score ^" (TG)

BP)

GBP2, nervous

IFIH1, system.

IFIT1B,

Activate

IFIT2,

Antiviral

IFITM3

response;

(IITMP3),

Clearance of

Igtp, IL15,

vims;

ISG15,

Immune

Mxl/Mx2,

response of

MYC,

antigen

OASL2,

presenting

PML,

cells;

PSMB10,

Immune

PSMB8,

response of

PSME2,

phagocytes;

PTGS2,

Cytotoxicity

SPP1,

of

STAT2,

leukocytes;

TAPI,

Function of

TLR3,

leukocytes;

TNFSF10,

Infiltration

TRAFD1

by T

lymphocytes;

Quantity of

MHC Class I

of cell

surface; Cell

death of

myeloid

cells.

Activate

Activation of

C3, macrophages;

CCL2, Apoptosis of

CCL7, myeloid

CD36, cells; Cell

CXCL10, movement of

CXCL9, T

STAT1, IRF5, NFATC2,

30 DDX58, lymphocytes;

REO 27.80 NR3C1, PPARD, 11/64 (17%)

(8, 14, 8) EIF2AK2, Cellular

ZBTB16, CDKN2A, EBF1

ISG15, infiltration by

MYC, leukocytes;

THBS1, Damage of

TLR3, lung;

TNFSF10, Recruitment

VEGFA of

leukocytes;

Response of Total

Consis Target

nodes Biological

Vims tency TF gene *b ReIations ^*d

(TF, TG, Process ^*0

score ^" (TG)

BP)

myeloid

cells;

Response of

phagocytes.

Activate

Cell

C3,

movement of

CCL2,

T

CCL7,

12 lymphocytes;

REO 7.56 CDKN2A, ZBTB 16 CXCL10, 1/6 (17%)

(2, 7, 3) Recruitment

CXCL9,

of

MYC,

leukocytes;

VEGFA

Survival of

organism.

* ^a,b The upstream regulators (STAT1 is highlighted in bold face) and the antiviral relating genes.

* ^c The biological functions known to associated with the regulatory networks annotated by the IPA.

* ^d The number of identified relationships and the total relationships that represent the known regulatory relationships between regulators and functions supported by literatures annotated by the IPA.

Table 2B. The downstream effects of the upstream regulators from the comparison

of Vm vs. Vp.

Total

Consis- nodes

Biological Process

Virus tency (TF, TF Target genes (TG) Relations

(BP)

score TG,

BP)

CXCL10, DDX58, Inhibit

EIF2AK2, IFIH1, Replication of

22 IL15, ISG15, JUN, virus; Quantity of

STAT1, IRF3,

VSV 8.00 (4, Mxl/Mx2, OASL2, lesion. 2/12 (17%)

IRF5, IRF7

15, 3) PSMB10, PSMB8, Activate

PSMB9, SOCS1, Quantity of CD8+

TAPI, TNFSF10 T lymphocyte.

Inhibit

BST2, CXCL10,

Replication of

DDX58, EIF2AK2,

virus; Transport of

EIF4EBP1, IFIH1,

amino acids.

29 STAT1, IRF3, IL15, ISG15,

EMCV 12.16 (6, IRF5, IRF7, Mxl/Mx2, OASL2, Activate 3/24 (13%)

19, 4) TRIM24, ATF4 PSMB10, PSMB8, Quantity of CD8+

PSMB9, SLC1A5, T lymphocyte;

SLC3A2, SLC6A9, Quantity of MHC

SLC7A5, TAPI, Class I on cell

TNFSF10 surface.

Inhibit

Arthritis; Cell cycle

AREG, CCND2, progression; Cell

18 EREG, GJA1, viability; Growth of

EMCV 7.91 (2, CCND1, SMAD4 HSPA8, ITGAV, ovarian follicle; 7/12 (58%)

10, 6) NEKBIA, PTGS2, Proliferation of

SOX4, SPP1 cells.

Activate

Edema.

Inhibit

Cancer; Quantity of

interleukin;

Rheumatic Disease;

CAMP, CCL2, HLA-

19 Development of

A, ICAM1, IL6,

EMCV 6.96 (2, MKL1, VDR body trunk. 7/14 (50%)

MMP9, PTGS2,

10, 7)

RELB, SPP1, TNC Activate

Cell death of

connective tissue

cells; Nephritis;

Organismal death.

ACACB, CAV1,

21

GFI1, NR1H3, CD36, CSF3, ETS1, Inhibit

REO 5.61 (4, NRIP1, PPARG ID2, IL6, LDLR, Oxidation of 1/12 (8%)

14, 3)

LPL, NEKBIA, carbohydrate; Table 2B. The downstream effects of the upstream regulators from the comparison

of Vm vs. Vp.

Total

Consis- nodes

Biological Process

Virus tency (TF, TF Target genes (TG) Relations

(BP)

score TG,

PDK2, PDK4, Production of

PPARA, SFC2A1 leukocytes;

Quantity of vldl

triglyceride in

blood.

Table 3. Statistics of differentially expressed genes.

_* VSV EMCV REO

Comp. Differential expression -

Down Up Down Up Down Up

1 m vs. Vm 1 24 8 16 1688 1945

2 m vs. p 58 245 269 422 28 136

3 _ Vm vs. Vp _ 271 281 275 337 1859 1657 a. ^* m: untreated - uninfected (media control); p: poly I:C treated - uninfected; Vm: untreated - virus infected; Vp: poly I:C treated - virus infected. (Note that the criteria for identifying DEGs were: adjusted p-value < 0.05, and IFold Changel >

5 1.5.)

Table 4. Upstream regulators of STAT1 predicted by IPA.

Note that, the number in each vims column denote p-value of the enrichment (hypergeometric) of the differentially expressed TF target genes in that TF.

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