Virus resilient genetically engineered animals

Abstract

The disclosure provides genetic modification that result in animals and cells being unable to support replication of viruses of the Flaviviridae family, including, for example the non- cytopathic pestiviruses.

Description

[0001]Virus resilient genetically engineered animals FIELDThe present disclosure relates to genetically engineered animals which are tolerantand / or resistant to viral infections.BACKGROUNDInfections caused by viruses belonging to the Flaviviridae family, such as pestiviruses,result in severe economic losses to global livestock production systems. In order toidentify means to reduce or prevent the spread of such viral infections, in vitroexperiments using cell lines to alter the cellular chaperone DNAJC14 (DnaJ Heat ShockProtein Family (Hsp40) Member C14) have previously been used to demonstrate theimportance of this molecule for pestiviral replication.However, cells used in in vitro experiments are typically monocultures of a single celltype, such as an immortalised cell line, and therefore do not reflect the majority of thecells in an animal’s body. Even the best in vitro models often lack interactions that arenecessary for the robust function of an animal. As a consequence, discoveries made in cultured cells often fail to fully translate in the context of an animal, or negatively impactthe health and / or viability of the animal.For example, CD169 had been identified as a potential cell surface receptor for PorcineReproductive and Respiratory Syndrome Virus (PRRSV), and An et al. (2010) showedthat by overexpressing CD169 in PK15 cells that they became permissive to PRRSV entry. De Baere et al. (2012) further demonstrated that by incubating porcine macrophages with mouse antibodies directed against CD169, PRRSV infection wasblocked. Based on these cellular data, Prather et al. (2013) produced pigs lacking CD169– however, when the findings were translated to in vivo models, these pigs remainedfully susceptible to PRRSV infection.Animals are more than a conglomeration of the cells from which they are formed, withinterplay between adjacent cells and between different organ systems being key in bothhomeostasis and response to disease. Many genes have important functions, andaltering their expression profile in vitro and in vivo can have different outcomes. As such,demonstration of a desired phenotype in immortalised cell lines often does not translateto a useful phenotype in animals. Furthermore, viruses often have preferred cellular trophism. Indeed, some viruses exclusively replicate in a specific and narrow range of cell types. The pestivirusesClassical Swine Fever Virus (CSFV) and Bovine Viral Diarrhoea Virus (BVDV)predominantly replicate in cells of the immune system. Other members of the Flaviviridae such as Yellow Fever Virus or Japanese Encephalitis Virus have preferred trophism for the liver and nervous system respectively. Lab experiments that have demonstrated interaction of these viruses with DNAJC14 have been performed largely in cultured cell lines that may not fully recapitulate the natural trophism of the virus, and as such may provide an environment which is potentially suboptimal for the virus to replicate. Modulation of an interacting partner in such an environment may thus be more consequential than would be the case in thenatural target cell of the virus. Thus, it is difficult to extrapolate data generated in vitro toaccurately predict the importance of DNAJC14 to replication of viruses of the Flaviviridae family in vivo, where viral access to the niche in which it has optimal performance is readily available. It is amongst one of the objectives of the present disclosure to provide further strategies to combat Flaviviruses, such as pestiviruses, which render animals resistant to infection. SUMMARYThe present disclosure is based on the finding that by modulating the expression of eitherthe gene encoding the cellular chaperone DNAJC14 (the DNAJC14 gene) and / or theexpression of the DNAJC14 protein (encoded by the DNAJC14 gene), it is possible torender animals tolerant and / or resistant to viral infections caused by members of theFlaviviridae family. Indeed, the inventors have noted that cells lacking a functionalDNAJC14 gene or a functional DNAJC14 protein are unable to support replication ofviruses of the Flaviviridae family, including, for example the non-cytopathic pestiviruses.Expression of the DNAJC14 gene and / or DNAJC14 protein may be modulated (e.g.increased or decreased) by modification of the DNAJC14 gene sequence and / or theDNAJC14 amino acid sequence encoded thereby.Additionally, the inventors have noted that despite modifications to the DNAJC14 geneor its expression profile and / or modifications to the DNAJC14 protein or its expressionprofile and / or function in a cell, animals, for example pigs, remain viable with substantiallyno adverse or detrimental impact on their health. The findings are all the moreunexpected given that knocking out the DNAJC14 gene in mice results in a number ofadverse phenotypes as compared to control mice (harbouring a wild-type DNAJC14gene with a ‘normal’ level of expression). Examples of these adverse phenotypes mayinclude, significant (adverse) effects on homeostasis / metabolism (including circulating electrolyte levels) and / or growth and / or behaviour. It should be noted that the variousanimals, genetically modified animals and / or genetically modified cloven hoovedlivestock (as defined below) / pigs, are healthy and show no obvious deviations fromnormal health. The term ‘healthy’ means that the animal exhibits no, or substantially no,adverse phenotypes – for example adverse phenotypes associated with disease statesand / or growth and / or behaviour. The health of an animal or the effect / presence orabsence of any specific phenotype may be assessed relative to an animal which doesnot contain or comprise a modified DNAJC14 gene / protein.Accordingly, in a first aspect of the present disclosure, there is provided an animalcomprising a modified DNAJC14 gene.An animal of this type may be referred to as a genetically modified animal (the geneticmodification being in the DNAJC14 gene).The DNAJC14 gene may be modified (in any way as described herein) so as to alter theexpression, function and / or activity of the gene and / or its protein product (DNAJC14). As stated, the overall effect of the modification is to confer a tolerance and / or resistance toa viral infection, especially a Flavivirus infection, including members of the Pestivirusgenus. Without wishing to be bound by theory, in one teaching, the modification detailedherein may confer a tolerance and / or resistance to viral infection, especially of aFlavivirus, including pestiviruses, without (substantially) any associated detrimentaleffect on animal viability and / or health).It should be noted that the term “DNAJC14” embraces all forms (including all mammalian,avian and / or piscine forms) of the DNAJ Heat Shock Protein Family Member C14 geneand / or its protein product. Throughout the present disclosure, reference is made to a number of terms, which are to be understood to have the meanings provided below, unless a context indicates to the contrary.The terms “comprising”, “comprise” and / or “comprises” are used to denote aspects andembodiments of this disclosure that “comprise” a particular feature or features. It should be understood that these terms may also encompass aspects and / or embodiments which “consists of” or “consists essentially of” the relevant feature(s). The term “tolerance” as used herein refers to situation in which an animal has becomeinfected with a virus but the infection does not cause a significant negative effect on theanimal, nor negatively affect its health and / or viability. Tolerance to a viral infection may also be used to describe a protective host response, which response prevents, inhibits and / or reduces pathogenesis of virus infections.The term “resistant” (or “resistance”) refers to animals which are not susceptible to a viralinfection. Without wishing to be bound by theory, in a resistant animal, a virus, forexample a virus belonging to the Flaviviridae family or the Flavivirus, Pestivirus,Hepacivirus and Pegivirus genus, may not be able to enter and / or replicate in its usualhost cell. For example, a resistant animal may generate cells which cannot be infected by a specific virus and / or which do not support viral replication. Animals of this disclosure (which animals may be genetically modified animalscomprising a modified DNAJC14 gene) may also exhibit one or more of the followingtraits or characteristics: (a) following challenge with a virus belonging to the Flaviviridae family or theFlavivirus, Pestivirus, Hepacivirus and Pegivirus genus, there is no, or substantially no,viraemia; and / or (b) following challenge with a virus belonging to the Flaviviridae family or theFlavivirus, Pestivirus, Hepacivirus and Pegivirus genus, there is no, or substantially no,detectable leukopenia; and / or (c) following challenge with a virus belonging to the Flaviviridae family or theFlavivirus, Pestivirus, Hepacivirus and Pegivirus genus, there is no, or substantially nodetectable change in body temperature; and / or(d) following challenge with a virus belonging to the Flaviviridae family or theFlavivirus, Pestivirus, Hepacivirus and Pegivirus genus, there is no, or substantially no,detectable (anti-Flaviviridae / Flavivirus / Pestivirus / Hepacivirus / Pegivirus) antibodyresponse. The term “Flaviviridae” refers to a family of enveloped positive-strand RNA viruses, whichtypically infect mammals and birds. Examples of genera belonging to the Flaviviridaefamily include Flavivirus, Pestivirus, Hepacivirus and Pegivirus.The term “Flavivirus” encompasses, but is not limited to, Avian Tembusu virus (whichtypically infects birds, human), Dengue virus (which typically infects human, pig,marsupial, bat, bird, horse, bovid, rodent and dog), Japanese encephalitis virus (whichtypically infects human, bird, pig), Louping ill virus (which typically infects sheep, human,cattle, goat, horse, dog, pig), Tick-borne encephalitis virus (which typically infects goat,sheep, cow, human) and Zika virus (which typically infects humans, pigs).The term “Pestivirus” encompasses, but is not limited to, Pestivirus A (bovine viraldiarrhea virus 1 [BVDV-1]), Pestivirus B (bovine viral diarrhea virus 2 [BVDV-2]),Pestivirus C (porcine Classical swine fever virus [CSFV]), Pestivirus D (ruminant Borderdisease virus [BDV]) and Pestivirus K (porcine atypical porcine pestivirus). The term“Hepacivirus” encompasses, but is not limited to, Hepacivirus N (bovine hepacivirus).It should be noted that within the context of this disclosure the tolerance or resistance toany of (i) a member of the Flaviviridae family, (ii) a Flavivirus or (iii) a Pestivirus, embracea tolerance and / or resistance to any of the specific viral types / species / strains described herein. Within the context of this disclosure, the term ‘animal’ embraces livestock which is a term applied to any type of domesticated or farmed animal. In this disclosure, the terms ‘animal’ and ‘livestock’ embrace at least those animals classed as mammals, ungulates, cloven hooved livestock, ovine, bovine, swine and caprine, but also non-ungulatelivestock such as farmed avian and piscine species. As such, the terms ‘animals’ and / or‘livestock’ includes, for example, pigs, wild boar, cattle, sheep, buffalo, goats, deer, birds,and fish. The term ‘animal’ may not include rodents, for example murine (mouse)animals. The term ‘sequence’ may embrace a nucleic acid or peptide / protein sequence. Forexample, the term ‘DNAJC14 sequence’ may embrace a nucleic acid sequence whichencodes or provides a DNAJC14 gene sequence, and / or any amino acid / peptidesequence encoded thereby or which provides a DNAJC14 protein. As stated, thisdisclosure may further extend to homologous or orthologous DNAJC14 sequences. Theterm “sequence” may embrace, for example, mammalian, avian, ungulate, ovine,porcine, piscine, equine and / or canine DNAJC14 (gene or nucleic acid / amino acid) sequences. A “reference sequence” may be any wild-type DNAJC14 sequence. For example, a reference sequence may comprise, consist essentially of or consist of a wild-typeDNAJC14 gene or nucleic acid sequence or an amino acid sequence of a wild typeDNAJC14 protein.In one teaching, the ‘reference sequence’ may comprise a wild-type (e.g. pig, cattle, sheep, goat) DNAJC14 nucleic acid sequence or an amino acid / peptide sequence. It should be appreciated that DNAJC14 sequences which are similar or homologous to the specific DNAJC14 sequences described herein are to be encompassed within the scope of the term ‘DNAJC14’ and / or as ‘DNAJC14 reference sequences’.The term “modified sequence” embraces a sequence which contains one or moremutations relative to a reference sequence. As used herein, a modified sequencetypically refers to a modified DNAJC14 gene or nucleic acid sequence (e.g. a modifiedDNAJC14 gene sequence) or a modified DNAJC14 amino acid sequence.The term “genetically modified animal” may refer to an animal comprising an altered ormodified gene sequence. By way of example, a genetically modified animal maycomprise a gene modified to include any one or more of the following modifications (or mutations): an insertion; a deletion; an inversion; a substitution; or a combination thereof. A genetically modified animal of this disclosure may exhibit modulated expression,function and / or activity of either the DNAJC14 gene and / or its protein product (theDNAJC14 protein itself). It should be noted that any modulated expression may be dueto modifications made to either the DNAJC14 gene sequence and / or the sequence of itsprotein product. For convenience, the term ‘DNAJC14’ should be taken to embrace eitherthe DNAJC14 gene (including its regulatory elements, exons and introns) or theDNAJC14 protein. As such, a reference to DNAJC14 expression, function or activityshould be taken to embrace either the expression, function and / or activity of theDNAJC14 gene or the expression, function and / or activity of the DNAJC14 protein.As stated, any of the modifications or mutations of this disclosure may result in somemodulation of DNAJC14 expression, function and / or activity. In this regard, the terms“modulation”, “modulate” or “modulating” may refer to an alteration (for example a decrease) in the level of DNAJC14 expression. Any modulation of DNAJC14 expression,function and / or activity may be determined relative to DNAJC14 expression, functionand / or activity in a wild type animal, cell or system. For example, the level of DNAJC14gene expression or protein expression observed in a wild-type animal. In an animal according to this disclosure (which animal comprises a modified DNAJC14gene / protein), DNAJC14 expression, function and / or activity may be lower as comparedto DNAJC14 expression, function and / or activity in a wild type animal (wherein theDNAJC14 gene / protein does not contain any modifications).The term “mutation” may include any alteration to a wild-type DNAJC14 nucleicacid / amino acid sequence.As used herein, the term “mutation” encompasses any modification to a DNAJC14nucleic acid / amino acid sequence, which mutation effects some aspect of theexpression, activity and / or function of the DNAJC14 gene / protein.For example, the term “mutation” may embrace: (i) one or more nucleotide or amino acid substitution(s) (where one or more of thewild-type nucleotide(s) or amino acid(s) is / are swapped or changed for another(different) nucleotide or amino acid – the term “substitutions” would includeconservative amino acid substitutions); and / or (ii) one or more nucleotide or amino acid deletion(s) (where one or more of thewild type nucleotide(s) or amino acid residue(s) are removed); and / or(iii) one or more nucleotide or amino acid addition(s) / insertion(s) (where additionalamino acid residue(s) are added to a wild type (or reference) primary sequence); and / or(iv) one or more amino acid / sequence inversions (usually where two or moreconsecutive amino acids in a primary sequence are reversed; and / or (v) one or more amino acid / sequence duplications (where an amino acid or a partof the primary amino acid sequence (for example a stretch of 5-10 amino acids) is repeated).Nucleotide mutations as described above may result in a frameshift mutation. A“frameshift mutation” typically refers to a mutation caused by a deletion and / or an insertion in a DNA sequence that results in a shift in the reading frame. By way of example, a frameshift mutation may be the result of an insertion or deletion of nucleotides, wherein the number of nucleotides inserted or deleted is not a multiple of three.This disclosure may provide modified DNAJC14 sequences, and the modifiedsequences of this disclosure (either DNAJC14 nucleic acid sequences or DNAJC14amino acid sequences) may be derived from a specific wild type DNAJC14 sequence. Inthis regard, a modified DNAJC14 sequence may comprise a wild type DNAJC14sequence modified to comprise one or more mutations. As such, a genetically modified animal of this disclosure may comprise or express any of the modified sequences described herein.The term “coding sequence” or “encoding nucleic acid” refers to the nucleic acidsequence that encodes a peptide or a protein. The coding sequence can further comprise initiation and / or termination signals operably linked to regulatory elements,such as a promoter and a polyadenylation signal capable of directing expression in ananimal or a cell of an animal to which the nucleic acid is provided.Exemplary wild-type (or reference) DNAJC14 sequences are provided below as SEQ IDNOS: 1-8. Any of these sequences may be used as a reference sequence from which amodified DNAJC14 sequence of this disclosure may be derived.Pig mRNA coding sequence (SEQ ID NO: 1) atggcccagaagcaccccggagaaagagggttgtgtggagcccaccacagtggtggtgcctccc tcaggactctaggaccctctgtggaccctgaaatactttcattctcaggactcagggactcagc cgggcctgctcctaatggtgcccgctgcctcacagagcactctagtcctaagtacacacagccc ccaaatccagctcactggtcggatccaagccatggccccccaaggggtccaggaccccctagga atggagaggaccctgatcagagtgaggcatcttcagaagaagagtcaggagtggaccaggaact ctcaaaagagaatgaggctgggtaccaggaggatgggaacccttcctttctttccattccatct gcttgtgactgccagggaatccctggaattcctgaagggccttattctcagggaggagatagct cttctagcaacttttgccaccattgtaccactccagctttgggggaagatgaagagttggaaga ggaatatgatgatgaggaacctcttaagttccccaatgatttttcacgtgtgcccagtggaaag aaacctccaccgaggagacagcggcaccgcattccagccaaggaggatactcgggagggtggac gcagagatcctaggtctcctggtcgacatcggcttggtcggaaacggagtcaggcagataagcg cagaggactgggattgtggggagcagaggaactgtgtcagcttggacaggcaggcttctggtgg ctgatcgaactgctggtattagtgggggagtacgtggaaacttgtggccatctcatctatgcat gcaggcagctgaaaggcagtgatctggacctttttcgtgtttgggtgggagtctgggcagggcg gctggggacttgggcccagctgatgttccagtttctgagccaggggttttcctgtggggcaggg ctgttcatccgttttcttaggctattgggtgctttgctgctctgggttctggcccttttgttgg gctgtctacagttgggctggtggtttctggtaggactgggtgaccgattaggctggaggggtaa agctgcgtggctcttctcttggctagcttctcccacctggcagcggtgcctgattctgctgaaa gatagcaggccatggcagcagctggtaagaatggttcagcggggttggctggagttgccttggg ttaagcagaggactgataggcaggggaatgcacctgtagctagtggtcgctactgccagcctga agaggaagtggctcgactcttgaccatggctggggttcctgaggatgagctaaaccctttccat gtgttgggagttgaagccacagcatcagatgttgagctgaagaaggcctataggcagctggcag tgatggttcatcctgacaaaaatcgtcatccccgggctgaggaggccttcaaggttttgagggc agcttgggacattgtcagcaaccctgaaagacggaaggaatatgagatgaaacgaatggcagag aatgagctgagccggtcagtgaatgagtttctgtccaagctgcaggatgaccttaaagaggcaa tgaatactatgatgtgcagccgatgccagggaaagcataggaggtttgaaatggaccgggaacc taagaatgccagatactgtgctgagtgtaagaggctgcatcccgctgaggaaggagacttttgg gcagagtcaagcatgtttggcctcaagatcacctactttgcactgatggatggaaaggtgtatg acatcacagagtgggctggatgccagcgtgtggggatatccccagatactcacagagtccccta tcacatctcatttggttcgaggatgccaggcaccagtgggcgtcagagagctacccctgatgcc cctcctgctgaccttcaagatttcctgagccggatctttcaagtacccccaggccagatgtcca atgggaacttctttgcagggcctcagccaggctctggggccactgcagcctccaagcccaacag cacagtacccaagggagaagctaaaccgaagcggcggaagaaagtgaggaggcccttccaacgt tga Pig protein sequence (SEQ ID NO: 2) MAQKHPGERGLCGAHHSGGASLRTLGPSVDPEILSFSGLRDSAGPAPNGARCLTEHSSPKYTQP PNPAHWSDPSHGPPRGPGPPRNGEDPDQSEASSEEESGVDQELSKENEAGYQEDGNPSFLSIPS ACDCQGIPGIPEGPYSQGGDSSSSNFCHHCTTPALGEDEELEEEYDDEEPLKFPNDFSRVPSGK KPPPRRQRHRIPAKEDTREGGRRDPRSPGRHRLGRKRSQADKRRGLGLWGAEELCQLGQAGFWW LIELLVLVGEYVETCGHLIYACRQLKGSDLDLFRVWVGVWAGRLGTWAQLMFQFLSQGFSCGAG LFIRFLRLLGALLLWVLALLLGCLQLGWWFLVGLGDRLGWRGKAAWLFSWLASPTWQRCLILLK DSRPWQQLVRMVQRGWLELPWVKQRTDRQGNAPVASGRYCQPEEEVARLLTMAGVPEDELNPFH VLGVEATASDVELKKAYRQLAVMVHPDKNRHPRAEEAFKVLRAAWDIVSNPERRKEYEMKRMAE NELSRSVNEFLSKLQDDLKEAMNTMMCSRCQGKHRRFEMDREPKNARYCAECKRLHPAEEGDFW AESSMFGLKITYFALMDGKVYDITEWAGCQRVGISPDTHRVPYHISFGSRMPGTSGRQRATPDA PPADLQDFLSRIFQVPPGQMSNGNFFAGPQPGSGATAASKPNSTVPKGEAKPKRRKKVRRPFQR Sheep mRNA coding sequence (SEQ ID NO: 3) atggcccagaagcaccccggagaaggagggttgtgtggagcccaccacagtggtggtgcctccc tcaggacttcaggaccctctgtggaccctgacatactttcattctcaggactcagggactcagc ggggcctgctcctaatggtacccgctgcctcacagagcactctagtcctaagtacacacagccc ccaaatccagcccactggtcggatccaagccatggacccccaaggggtccaggaccccctctgg ctgaagaggaccctgatcagagtgaggcatcttcagaagagtcaggagtggaccaggaactctc aagagagaatgaggctgggtaccaggatgatgggaactcttctttctttcccattccatctact tgtaactgccagggaacccctggaatccctgaagggccttactctgagggaggagatagctctt ctagcaacttttgccaccattgtacctctccagctttgggggaagatgaagagttggaagggga atatgatgaagaggaacctcttaagtttcccagtgatctttcacgtgtgcacagtgaaaagaaa cctgcaccccggagacaacggcaccgtgttccagccaaggaggacactcgggagggtggacgaa gagatcccagatcccctggtcgacatcggctgggccggaaacggagtcaggcagataaacgcag aggactgggattgtggggagcagaggaactgtgtcagcttggacaggcaggcttctggtggctg atcgaactgctagtattagtgggggagtacgtggagacttgtggccatctcatctatgcatgca ggcagctgaaaggcagtgatctggaccttttacgtgtctgggtgggagtgtgggcagggcggct ggggggctgggcccaggtgatgttccagtttctgagccaggggttttgctatggggcagggctg ttcacccgttttcttaggcttgtgggtgctttgctgctcctggctctggcccttttgttgggct gtttacagttgggctggcggtttctggtaggattgagtgaccggctaggctggaggggtaaagc cacctggctcttctcttggctggcttctcccacctggcagcgttgcctgattctgctgagagag agcaggccctggcagcagctggtaagaatagttcagttaggttggctggagttaccttgggtca aacagaggaccaataggcaggggaatgcacctgtagctggtggtcgttactgccagcctgaaga ggaagtggctcgactcttgaccatggctggggttcctgaggatgagctaaacccttttcacgtg ttgggggttgaagccacggcatcagatgttgaactgaagaaggcctataggcagctggcagtga tggttcatcctgacaaaaatcatcatcctcgggctgaggaggccttcaaggttttgcgggcagc ttgggacattgtcagcaaccctgaaagacggaaggaatatgagatgaaacgaatggctgaaaat gagctgagccggtcagtgaatgagtttctgtccaagctgcaagaagcaatgaatactatgatgt gcaaccgatgccagggaaagcataggaggttcgaaatggaccgggaacctaagagtgccagata ctgtgctgagtgtaataggctgcatcctgctgaggaaggtgacttttgggcagagtcaagcatg ttgggcctcaaaatcacctactttgcgctgatggatggaaaggtgtatgatatcacagagtggg ctggatgccagcgtgtgggaatctccccagatacccacagagtcccttatcacatctcatttgg ttcacggatgccaggcaccagtgggcggcagagagctactccagatgcccctcctgctgacctt caggatttcttgagccggatctttcaagtacccccaggccagatgtccaacgggaacttctttg cagctcctcagcccagccctggggccactgcagcctccaagcccaacagcacagtacccaaggg agacgccaaaccgaagcggcggaagaaagtgaggaggcccttccaacgttgaSheep protein sequence (SEQ ID NO: 4)MAQKHPGEGGLCGAHHSGGASLRTSGPSVDPDILSFSGLRDSAGPAPNGTRCLTEHSSPKYTQP PNPAHWSDPSHGPPRGPGPPLAEEDPDQSEASSEESGVDQELSRENEAGYQDDGNSSFFPIPST CNCQGTPGIPEGPYSEGGDSSSSNFCHHCTSPALGEDEELEGEYDEEEPLKFPSDLSRVHSEKK PAPRRQRHRVPAKEDTREGGRRDPRSPGRHRLGRKRSQADKRRGLGLWGAEELCQLGQAGFWWL IELLVLVGEYVETCGHLIYACRQLKGSDLDLLRVWVGVWAGRLGGWAQVMFQFLSQGFCYGAGL FTRFLRLVGALLLLALALLLGCLQLGWRFLVGLSDRLGWRGKATWLFSWLASPTWQRCLILLRE SRPWQQLVRIVQLGWLELPWVKQRTNRQGNAPVAGGRYCQPEEEVARLLTMAGVPEDELNPFHV LGVEATASDVELKKAYRQLAVMVHPDKNHHPRAEEAFKVLRAAWDIVSNPERRKEYEMKRMAEN ELSRSVNEFLSKLQEAMNTMMCNRCQGKHRRFEMDREPKSARYCAECNRLHPAEEGDFWAESSM LGLKITYFALMDGKVYDITEWAGCQRVGISPDTHRVPYHISFGSRMPGTSGRQRATPDAPPADL QDFLSRIFQVPPGQMSNGNFFAAPQPSPGATAASKPNSTVPKGDAKPKRRKKVRRPFQR Cattle mRNA coding sequence (SEQ ID NO: 5) atggcccagaagcaccccggagaaggagggttgtgtggagcccaccacagtggtggtgcctccc tcaggactttaggaccctctgtggaccctgacatactttcattctcaggactcagggactcagc ggggtctgctcctaatggtacccgctgcctcacagagcactctagtcctaagtacacacagccc ccaaatccagcccactggtcggatccaagccatggacccccaaggggtccaggaccccctctgg ctgaagaggaccctgatcagagtgaggcatcttcagaagagtcaggagtggaccaggaactctc aagagagaatgaaactgggtaccaggatgatgggaactcttctttcctttccattccatctact tgtaactgccagggaacccctggaatccctgaagggccttactctgagggaagagatagctctt ctagcaacttttgccaccattgtacctctccagctttgggggaagatgaagagttggaagggga atatgatgaagaggaacctcttaagtttcccagtgatgtttcacgtgtgcccagtgaaaagaaa cctgcaccccggagacaacggcaccgtgttccagccaaggaggacactcgggagggtggacgaa gagatcccagatcccctggtagacatcggctgggccggaaacggagtcaggcagataaacgcag aggactgggattgtggggagcagaggaactgtgtcagcttggacaggcaggcttctggtggctg atcgaactgctagtattagtaggggagtacgtggagacttgtggccatctcatctatgcatgca ggcagctgaaaggcagtgatctggaccttttacgtgtctgggtgggagtgtgggcagggcggct gcggggctgggcgcaggtgatgttccagtttctgagccaggggttttgctatggggcagggctg ttcacccgttttcttaggcttgtgggtgctttgctgctcctggctctggcccttttgttgggct gtttacagttgggctggcggtttctggtaggattgagtgaccggctaggctggagggataaagc cacctggatcttctcttggctggcttctcccacctggcagcgttgcctgattctgctgagagag agcaggccctggcagcagctggtaagaatagttcagtggggttggctggagttaccttgggtca aacagaggaccaataggcaggcgaatgcacctgtagctggtggtcgttactgccagcctgaaga ggaagtggctcgactcttgaccatggctggggttcctgaggatgagctaaacccttttcacgtg ttgggggttgaagccacagcatcagatgttgaactgaagaaggcctataggcagctggcagtga tggttcatcctgacaaaaatcatcatcctcgtgctgaggaagccttcaaggttttgcgggcagc ttgggacattgtcagcaaccctgaaagacggaaggaatatgagatgaaacgaatggcagaaaat gagctgagccggtcagtgaatgagtttctgtccaagctgcaagaagcaatgaatacgatgatgt gcagccgatgccagggaaagcataggaggtttgaaatggaccgggaacctaagagtgccagata ctgtgctgagtgtaataggctgcatcctgctgaggaaggtgacttttgggcagagtcaagcatg ttgggcctcaaaatcacctactttgcgctgatggatggaaaggtgtatgatatcacagagtggg ctggatgccagcgtgtgggaatctccccagatacccacagagtcccttatcacatctcatttgg ttcacggatgccaggcaccagtgggcggcagagagctactccagatgcccctcctgctgacctt caggatttcttgagccggatctttcaagtacccccaggccagatgtccaacgggaacttctttg cagctcctcagcccggccctggggccactgcagcctccaagcccaacagcacagtacccaaggg agaagccaaaccgaagcggcggaagaaagtgaggaggcccttccaacgttga Cattle protein sequence (SEQ ID NO: 6) MAQKHPGEGGLCGAHHSGGASLRTLGPSVDPDILSFSGLRDSAGSAPNGTRCLTEHSSPKYTQP PNPAHWSDPSHGPPRGPGPPLAEEDPDQSEASSEESGVDQELSRENETGYQDDGNSSFLSIPST CNCQGTPGIPEGPYSEGRDSSSSNFCHHCTSPALGEDEELEGEYDEEEPLKFPSDVSRVPSEKK PAPRRQRHRVPAKEDTREGGRRDPRSPGRHRLGRKRSQADKRRGLGLWGAEELCQLGQAGFWWL IELLVLVGEYVETCGHLIYACRQLKGSDLDLLRVWVGVWAGRLRGWAQVMFQFLSQGFCYGAGL FTRFLRLVGALLLLALALLLGCLQLGWRFLVGLSDRLGWRDKATWIFSWLASPTWQRCLILLRE SRPWQQLVRIVQWGWLELPWVKQRTNRQANAPVAGGRYCQPEEEVARLLTMAGVPEDELNPFHV LGVEATASDVELKKAYRQLAVMVHPDKNHHPRAEEAFKVLRAAWDIVSNPERRKEYEMKRMAEN ELSRSVNEFLSKLQEAMNTMMCSRCQGKHRRFEMDREPKSARYCAECNRLHPAEEGDFWAESSM LGLKITYFALMDGKVYDITEWAGCQRVGISPDTHRVPYHISFGSRMPGTSGRQRATPDAPPADL QDFLSRIFQVPPGQMSNGNFFAAPQPGPGATAASKPNSTVPKGEAKPKRRKKVRRPFQR Goat mRNA coding sequence (SEQ ID NO: 7) atggcccagaagcaccccggagaaggagggttgtgtggagcccaccacagtggtggtgcctccc tcaggactttaggaccctctgtggaccctgacatactttcattctcaggactcagggactcagc ggggcctgctcctaatggtacccgctgcctcacagagcactctagtcctaagtatacacagccc ccaaatccagcccactggtcggatccaagccatggacccccaaggggtccaggaccccctctgg ctgaagaggaccctgatcagagtgaggcatcttcagaagagtcaggagtggaccaggaactctc aagagagaatgaggctgggtaccaggatgatgggaactcttctttcctttccattccatctact tgtaactgccagggaacccctggaatccctgaagggccttactctgagggaggagataactctt ctagcaacttttgccaccattgtacctctccagctttgggggaagatgaagagttggaagggga atatgatgaagaggaacctcttaagtttcccagtgatctttcacgtgtgcacagtgaaaagaaa cctgcaccccggagacaacggcaccgtgttccagccaaggaggacactcgggagggtggacgaa gagatcccagatcccctggtcgacatcggctgggccggaaacggagtcaggcagataaacgcag aggactgggattgtggggagcagaggaactgtgtcagcttggacaggcaggcttctggtggctg attgaactgctagtattagtgggggagtacgtggagacttgtggccatctcatctatgcatgca ggcagctgaaaggcagtgatctggaccttttacgtgtctgggtgggagtgtgggcagggcggct ggggggctgggcccaggcgatgttccagtttctgagccaggggttttgctatggggcagggctg ttcacccgttttcttaggcttgtgggtgctttgctgctcctggctctggccctgttgttgggct gtttacagttgggctggcggtttctggtaggattgagtgaccggctaggctggaggggtaaagc tacctggctcttctcttggctggcttctcccacctggcagcgttgtctgattctgctgagagag agcaggccctggcagcagctggtaagaatagttcagttgggttggctggagttaccttgggtca aacagaggaccaataggcaggggaatgcacctgtagctggtgctcgttactgccagcctgaaga ggaagtggctcgactcttgaccatggctggggttcctgaggatgagctaaacccttttcacgtg ttgggggttgaagccacagcatcagatgttgaactgaagaaggcctataggcagctggcagtga tggttcatcctgacaaaaatcatcatcctcgggctgaggaggccttcaaggttttgcgggcagc ttgggatattgtcagcaaccctgaaagacggaaggaatatgagatgaaacgaatggctgaaaat gagctgagccggtcagtgaatgagtttctgtccaagctgcaagaagcaatgaatactatgatgt gcagccgatgccagggaaagcataggaggttcgaaatggaccgggaacctaagagtgccagata ctgtgctgagtgtaataggctgcatcctgctgaggaaggtgacttttgggcagagtcaagcatg tttggcctcaaaatcacctactttgcgctgatggatggaaaggtgtatgatatcacagagtggg ctggatgccagcgtgtgggaatctccccagatacccacagagtcccttatcacatctcatttgg ttcacggatgccaggcaccagtgggcggcagagagctactccagatgcccctcctgctgacctt caggatttcttgagccggatctttcaagtacccccaggccagatgtccaacgggaacttctttg cagctcctcagcccagccctggggccactgcagcctccaagcccaacagcacagtacccaaggg agacgccaaaccgaagcggcggaagaaagtgaggaggcccttccaacgttga Goat protein sequence (SEQ ID NO: 8) MAQKHPGEGGLCGAHHSGGASLRTLGPSVDPDILSFSGLRDSAGPAPNGTRCLTEHSSPKYTQP PNPAHWSDPSHGPPRGPGPPLAEEDPDQSEASSEESGVDQELSRENEAGYQDDGNSSFLSIPST CNCQGTPGIPEGPYSEGGDNSSSNFCHHCTSPALGEDEELEGEYDEEEPLKFPSDLSRVHSEKK PAPRRQRHRVPAKEDTREGGRRDPRSPGRHRLGRKRSQADKRRGLGLWGAEELCQLGQAGFWWL IELLVLVGEYVETCGHLIYACRQLKGSDLDLLRVWVGVWAGRLGGWAQAMFQFLSQGFCYGAGL FTRFLRLVGALLLLALALLLGCLQLGWRFLVGLSDRLGWRGKATWLFSWLASPTWQRCLILLRE SRPWQQLVRIVQLGWLELPWVKQRTNRQGNAPVAGARYCQPEEEVARLLTMAGVPEDELNPFHV LGVEATASDVELKKAYRQLAVMVHPDKNHHPRAEEAFKVLRAAWDIVSNPERRKEYEMKRMAEN ELSRSVNEFLSKLQEAMNTMMCSRCQGKHRRFEMDREPKSARYCAECNRLHPAEEGDFWAESSM FGLKITYFALMDGKVYDITEWAGCQRVGISPDTHRVPYHISFGSRMPGTSGRQRATPDAPPADL QDFLSRIFQVPPGQMSNGNFFAAPQPSPGATAASKPNSTVPKGDAKPKRRKKVRRPFQR As such, the present disclosure provides modified forms of any of SEQ ID NOS: 1-8, wherein the modified form comprises (relative to the relevant sequence of SEQ ID NOS: 1-8) one or more mutations. In a further teaching, the disclosure provides a genetically modified animal comprisingor expressing a modified form of any one of SEQ ID NOS: 1-8 (which modified formcomprises one or more mutations). The disclosure provides modified forms of SEQ ID NO: 1 or SEQ ID NO: 2. A modified form of SEQ ID NO: 1 and / or SEQ ID NO: 2 may comprise any one or more of the mutations described herein. As stated, the mutation(s) may comprise, nucleic acid or amino acid substitutions, additions / insertions, duplications, deletions and / or inversions relative to the sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2.The disclosure provides modified forms of SEQ ID NO: 3 or SEQ ID NO: 4. A modifiedform of SEQ ID NO: 3 and / or SEQ ID NO: 4 may comprise any one or more of themutations described herein. As stated, the mutation(s) may comprise, nucleic acid or amino acid substitutions, additions / insertions, duplications, deletions and / or inversionsrelative to the sequence of SEQ ID NO: 3 and / or SEQ ID NO: 4.The disclosure provides modified forms of SEQ ID NO: 5 or SEQ ID NO: 6. A modifiedform of SEQ ID NO: 5 and / or SEQ ID NO: 6 may comprise any one or more of themutations described herein. As stated, the mutation(s) may comprise, nucleic acid or amino acid substitutions, additions / insertions, duplications, deletions and / or inversionsrelative to the sequence of SEQ ID NO: 5 and / or SEQ ID NO: 6.The disclosure provides modified forms of SEQ ID NO: 7 or SEQ ID NO: 8. A modifiedform of SEQ ID NO: 7 and / or SEQ ID NO: 8 may comprise any one or more of themutations described herein. As stated, the mutation(s) may comprise, nucleic acid or amino acid substitutions, additions / insertions, duplications, deletions and / or inversionsrelative to the sequence of SEQ ID NO: 7 and / or SEQ ID NO: 8.In one embodiment, a genetically modified animal of the present disclosure maycomprise or express a modified DNAJC14 protein, wherein, relative to a DNAJC14reference sequence (for example any of the reference sequences disclosed herein), themodified DNAJC14 protein comprises a mutation to one or more of the residues of theDNAJC14 Jiv90 domain It should be noted that the position of the Jiv90 domain may vary within different DNAJC14 protein sequences (namely DNAJC14 protein sequences from different animals). Furthermore, depending on the species (pig, cattle, goat, deer, sheep etc.) the precise sequence of the Jiv90 domain may vary. By way of example in a pig DNAJC14 protein sequence (e.g. as represented by SEQ ID NO:2) the position of the Jiv90 domain is from residue 538-627. In this case, the pig Jiv90 domain has the following sequence (SEQ ID NO: 9): MCSRCQGKHRRFEMDREPKNARYCAECKRLHPAEEGDFWAESSMFGLKITYFALMDGKVYDITE WAGCQRVGISPDTHRVPYHISFGSRM In a sheep DNAJC14 protein sequence (e.g. as represented by SEQ ID NO:4) the position of the Jiv90 domain is from residue 533-622. In this case, the sheep Jiv90 domain has the following sequence (SEQ ID NO: 10): MCNRCQGKHRRFEMDREPKSARYCAECNRLHPAEEGDFWAESSMLGLKITYFALMDGKVYDITE WAGCQRVGISPDTHRVPYHISFGSRM In a cattle DNAJC14 protein sequence (e.g. as represented by SEQ ID NO:6) the position of the Jiv90 domain is from residue 533-622. In this case, the sheep Jiv90 domain has the following sequence (SEQ ID NO: 11): MCSRCQGKHRRFEMDREPKSARYCAECNRLHPAEEGDFWAESSMLGLKITYFALMDGKVYDITE WAGCQRVGISPDTHRVPYHISFGSRM In a goat DNAJC14 protein sequence (e.g. as represented by SEQ ID NO:8) the position of the Jiv90 domain is from residue 533-622. In this case, the sheep Jiv90 domain has the following sequence (SEQ ID NO: 12): MCSRCQGKHRRFEMDREPKSARYCAECNRLHPAEEGDFWAESSMFGLKITYFALMDGKVYDITE WAGCQRVGISPDTHRVPYHISFGSRM In one teaching, the disclosure provides a genetically modified animal of the presentdisclosure which may comprise or express a modified DNAJC14 protein in which at leastresidue 39 of the Jiv90 (a tryptophan residue (W)) is modified or mutated – for example,substituted. In one teaching, the tryptophan (W) residue at position 39 of the Jiv90 domain may be substituted with an alanine (A) residue. In one teaching, a genetically modified animal of the present disclosure may compromise or express a modified DNAJC14 protein, wherein: (i) relative to the DNAJC14 reference sequence of SEQ ID NO: 2, themodified DNAJC14 protein comprises a substitution at position 576; (ii) relative to the DNAJC14 reference sequence of SEQ ID NO: 2, themodified DNAJC14 protein comprises a W576A substitution; (iii) relative to the DNAJC14 reference sequences of SEQ ID NOS: 4, 6 or 8the modified DNAJC14 protein comprises a substitution at position 571; and (iv) relative to the DNAJC14 reference sequences of SEQ ID NOS: 4, 6 or 8the modified DNAJC14 protein comprises a W571A substitution. In addition to any of the mutations described above as (i)-(iv), a genetically modified animal of the present disclosure may comprise or express a DNAJC14 gene / protein in which one or more other residues of the DNAJC14 gene / protein sequence have beenmodified or mutated. For example, in addition to the Jiv90 domain modifications detailedabove, a genetically modified animal may express a DNAJC14 gene / protein sequence comprising a number, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30, additional sequence modifications / mutations. All or some of those additional modifications / mutations may be present in the Jiv90 domain. By way of example, in addition to any of the sequence modifications / mutations described herein (including the Jiv90 domain mutations described herein) any one or more of thefollowing restudies of the Jiv90 domain may also be modified or mutated:(a) the residue (C: cysteine) at position 24; and / or(b) the residue (L: leucine) at position 30; and / or(c) the residue (H: histidine) at position 31; and / or(d) the residue (G: glycine) at position 36; and / or(e) the residue (D: aspartic acid) at position 37; and / or(f) the residue (E: glutamic acid) at position 41; and / or(g) the residue (S: serine) at position 42.In some embodiments, the genetically modified animal of the present disclosurecomprises a modified DNAJC14 gene, which modification results in (or from) a frameshiftmutation. In one teaching, the modification made to the DNAJC14 gene results in aframeshift mutation in at least a portion of the Jiv90 domain. In a further teaching, theframeshift mutation may occur in an earlier exon that then results in a frame shift to theJiv90 domain. In one embodiment, the modification in the DNAJC14 gene results in aframeshift mutation in the N-terminal region of the Jiv90 domain.Any of the mutations disclosed herein may be functional – that is to say they mayindividually (and / or independently) or collectively (for example, synergistically) modulate (alter, improve or suppress / inhibit) one or more of the wild-type functions, activitiesand / or physiological, biological immunological and / or pharmacological propertiescharacteristic of a wild-type DNAJC14 (for example the wild-type DNAJC14 from whichthe modified DNAJC14 is derived). Without wishing to be bound by theory, the various modifications disclosed herein result in DNAJC14 having altered function, expression and / or activity. The altered function, expression and / or activity of a modified DNAJC14 according to this disclosure reducesor inhibits the interaction of DNAJC14 with a Flaviviridae non-structural protein, therebyinhibiting or reducing viral replication. By way of example, a modified DNAJC14 asdisclosed herein, exhibits a reduced or inhibited ability to interact with the non-structuralproteins NS2 and / or NS2-3 of any of the Flaviviridae disclosed herein (including, forexample Pestivirus).In one teaching the one or more mutation(s) may:(i) alter or inhibit / suppress a DNAJC14 function; and / or(ii) reduce DNAJC14 gene / protein expression in a cell (especially a cellwhich is usually a host for a member of the Flaviviridae); and / or (iii) inhibit, supress or prevent transcription and / or replication of a genome ofa member of the Flaviviridae in a host cell; and / or(iv) inhibit or suppress Flaviviridae RNA replicase; and / or(v) inhibit, suppress or prevent the transcription and / or replication ofPestivirus genomes in a host cell; and / or(vi) prevent Pestivirus infection of a host cell.It should be noted that this disclosure encompasses not only the fully modified or mutatedsequences described herein, but also fragments, variants and / or derivatives of any ofthose sequences. In one embodiment a variant, derivative or fragment of any of the DNAJC14 sequences described herein, may comprise a sequence which is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical or homologous to all or part of any of a modified DNAJC14 sequence of thisdisclosure. In a further teaching a modified DNAJC14 variant, derivative or fragment may comprisea sequence which is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical or homologous to the modified Jiv90 domain of the DNAJC14 sequencedisclosed herein.Any of such fragments, variants and / or derivatives may be functional and retainsubstantially all of the functional properties of the described modified DNAJC14.In this context, the term ‘functional’ also encompasses a modification wherein theDNAJC14 gene (or DNAJC14 protein) is knocked out (so that it cannot express therelevant DNAJC14 protein), functionally deleted (so that it cannot express a functionalDNAJC14 protein) and / or it is altered so that the resulting DNAJC14 protein does notsupport viral replication. In a preferred embodiment, the genetically modified animalscomprise or express a functional fragment, variant and / or derivative of DNAJC14 whichinhibits, supresses or prevents transcription and / or replication of a Flaviviridae virusgenome in a host cell.As such, a genetically modified animal according to this disclosure may be modified byremoval (knockout) of the gene encoding DNAJC14. A genetically modified animal of this type may be referred to as an ‘DNAJC14 knock out (KO) animal’. In one teaching, a genetically modified animal according to this disclosure may be modified by functional deletion of the gene encoding DNAJC14. In a further teaching, a genetically modified animal according to this disclosure mayexpress a modified DNAJC14 gene sequence in which there is one or more modifications(as described herein) in any one or more of the exons, for example in any exon after exon 2 (which contains the start codon). As described herein, such mutations may bringabout intentional (for example the targeted introduction of a stop codon) or random (forexample by way of a frameshift mutations that results in altered coding sequence and / or the introduction of a stop codon) changes to the coding sequence, with any changesafter the start codon being likely to impact protein function.This disclosure also provides a method of producing a genetically modified animal(otherwise referred to as a genetically edited animal), wherein said method comprises introducing a genetic modification into an animal’s genome, which modification alters theexpression and / or activity of the DNAJC14 gene or DNAJC14 protein. As stated the term‘animal’ may embrace any of the animal types described herein and the term ‘modification’ may embrace any of the nucleic acid or amino acid mutations describedherein. The term ‘genetically modified animal’ may include, for example, geneticallymodified cloven hooved livestock, cattle, pigs, wild boar, sheep, buffalo, goats, deer and the like.The genetically modified animals of this disclosure may be resistant to infection bymembers of the Flaviviridae family or the Flavivirus, Pestivirus, Hepacivirus andPegivirus genus. In one teaching, cattle which have been genetically modified accordingto this disclosure (for example to comprise a modified DNAJC14 gene or to express a modified DNAJC14 protein) may be resistant to BVDV infection. In a further teaching,pigs which have been genetically modified according to this disclosure (for example to comprise a modified DNAJC14 gene or to express a modified DNAJC14 protein) may beresistant to CSFV infection.Additionally, primary cells from any of the genetically modified animals of this disclosuremay be resistant to infection by or with members of the Flaviviridae family or theFlavivirus, Pestivirus, Hepacivirus and Pegivirus genus. For example, cells (primarycells) from genetically modified pigs of this disclosure may be resistant to CSFV infectionand BVDV infection. Also, cells (primary cells) from genetically modified cattle of thisdisclosure may also be resistant to CSFV and / or BVDV infection.Accordingly, this disclosure provides a method of rendering a cell resistant to infectionby or with members of the Flaviviridae family or the Flavivirus, Pestivirus, Hepacivirusand Pegivirus genus, said method comprising modifying the DNAJC14 gene as set outin this disclosure. Any gene editing method known in the art may be employed to introduce a geneticmodification as disclosed herein which knocks-out, alters or modifies the expression,function and / or activity of DNAJC14. As stated, such methods are useful as they resultin animals which are tolerant and / or resistant to viral infections caused by members ofthe Flaviviridae family. Moreover, animals generated by these methods are healthyand / or viable. Genetically modified animals of this disclosure may be made using standard published techniques. This includes, but is not restricted to, genetic modification (as described herein) using site-specific nucleases and / or DNA templates to direct sequence specific changes in the target genetic locus of animal germ cells, zygotes, primary cells or stem cells which can subsequently contribute to an embryo. In mammalian species the modified embryos may be re-introduced into a surrogate host animal and the resulting offspring of this host animal will carry the genetic change in its chromosomes, including in the chromosomes of its gametes. In avian and piscine species, a surrogate host may not be required. Standard selective breeding techniques can be used to increase the frequency of this genetic change in the future descendants. These (and / or similar) methods may be used to create genetically modified embryos, which harbour modified genetic sequences. As stated, these modified genetic sequences may result in the expression of a DNAJC14 protein with altered function and an inability to support replication / infection by members of the Flaviviridae. The introduced genetic modifications may result in the functional deletion of the DNAJC14 protein. Without wishing to be bound by theory, the method of producing a genetically editedanimal may comprise genome editing using site-specific nucleases, such as zinc fingernucleases (ZFNs), transcription activator-like effector nucleases (TALENs) or theclustered regularly interspaced short palindromic repeat (CRISPR) / Cas system, forexample. In one example, CRISPR / Cas9 system may be employed to modify or alter theDNAJC14 gene of an animal.Site-specific nucleases are nucleases that can bind to and cleave designated genomicDNA, which results in double-strand breaks. The double-strand breaks in eukaryotic systems are typically repaired by homology-directed repair (HDR) or non-homologousend-joining (NHEJ). Alternative methods of gene editing that do not require the creationof double-stranded breaks in the genome could also be applied, such as site-specificnickases, prime editors, base editors, or site-specific recombinases.Typically, in the context of a CRISPR system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence. Without wishing to be bound by theory, the tracr sequence, which may comprise or consist of all or a portion of a wild-type tracr sequence (e.g. about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of a wild-type tracr sequence), may also form part of a CRISPR complex, such as by hybridization along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence that is operably linked to the guide sequence. In some examples, the tracr sequence has sufficient complementarity to a tracr mate sequence to hybridize and participate in formation of a CRISPR complex. As with the target sequence, it is believed that complete complementarity is not needed, provided there is sufficient to be functional. In some examples, the tracr sequence has at least 50%, 60%, 70%, 80%, 90%, 95% or 99% of sequence complementarity along the length of the tracr mate sequence when optimally aligned. In some examples, one or morevectors driving expression of one or more elements of a CRISPR system are introducedinto a host cell such that expression of the elements of the CRISPR system direct formation of a CRISPR complex at one or more target sites. For example, a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence could each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more of the elements expressed from the same or different regulatory elements, maybe combined in a single vector, with one or more additional vectors providing anycomponents of the CRISPR system not included in the first vector. CRISPR system elements that are combined in a single vector may be arranged in any suitable orientation, such as one element located 5’ with respect to (“upstream” of) or 3’ with respect to (“downstream” of) a second element. The coding sequence of one element may be located on the same or opposite strand of the coding sequence of a second element, and oriented in the same or opposite direction. In some examples, a single promoter drives expression of a transcript encoding a CRISPR enzyme and one or more of the guide sequence, tracr mate sequence (optionally operably linked to the guide sequence), and a tracr sequence embedded within one or more intron sequences (e.g. each in a different intron, two or more in at least one intron, or all in a single intron). In some embodiments, a vector comprises a regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, such as a Cas protein. Non- limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1,Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof. Theseenzymes are known; for example, the amino acid sequence of S. pyogenes Cas9 proteinmay be found in the SwissProt database under accession number Q99ZW2. In some embodiments, the unmodified CRISPR enzyme has DNA cleavage activity, such as Cas9. In some embodiments the CRISPR enzyme is Cas9, and may be Cas9 from S.pyogenes or S. pneumoniae. In some embodiments, the CRISPR enzyme directscleavage of one or both strands at the location of a target sequence, such as within the target sequence and / or within the complement of the target sequence. In someembodiments, the CRISPR enzyme directs cleavage of one or both strands within about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the firstor last nucleotide of a target sequence of the DNAJC14 gene.In some embodiments, a Cas9 nickase may be used in combination with guide sequence(s), e.g., two guide sequences, which target respectively sense and antisense strands of the DNA target. This combination allows both strands to be nicked and used to induce DNA changes at the target site. In some embodiments, a Cas protein and guide RNA may be used in combination witha HDR donor template, wherein the HDR donor template comprises a desired genomicedit and sequences homologous to the sequence flanking the cut site of a Cas protein.As used herein, the term “gRNA” refers to single guide RNA used in conjunction with CRISPR associated systems (Cas). gRNAs are a fusion of crRNA and tracrRNA and contain nucleotides of sequence complementary to the desired target site. Jinek et al., “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity”Science 337(6096):816-821 (2012). Watson-Crick pairing of the gRNA with the targetsite permits R-loop formation, which in conjunction with a functional PAM permits DNAcleavage or in the case of nuclease-deficient Cas9 allows binding to the DNA at thatlocus.The disclosure further provides vectors comprising nucleic acid sequences encoding anyof the guide RNA of this disclosure, including those provided as SEQ ID NO: 15-18, orany functional fragments or variants thereof.Also provided is a cell comprising a modified DNAJC14 of this disclosure. Such a cellmay be obtained from a genetically modified animal according to any of the embodimentsprovided herein. The cell may be a fibroblast. In one embodiment, the cell may be usedin in vitro methods for testing the effects of a compound or a drug, such as an anti-viraldrug. Additionally or alternatively, the in vitro method may be used to conduct viralchallenge studies.Also provided is a method of culturing a cell transformed with a vector of this disclosureunder conditions suitable to induce the expression of the relevant DNAJC14 protein and purifying said protein. The step of purifying may involve lysing the cell and using centrifugation and / or affinity chromatography or other similar technique to extract orpurify the modified DNAJC14 protein.Detailed Description The present disclosure will now be further described by way of example and with reference to the Figures, which show:Figure 1. Phylogeny of the Flaviviridae family showing relatedness of Flaviviruses,Pestiviruses and HepacivirusesFigure 2. Conservation of DNAJC14 protein in evolutionarily diverse species (pig, cow,sheep, chicken and northern pike). The selected sequence is of the JIV90 interactiondomain, with the tryptophan at position 576 of the pig protein annotated with an arrow.Figure 3. Genomic organisation of the pig and chicken DNAJC14 genes. Both geneshave 7 exons with the transcription start site located in exon 2.Figure 4. Exemplary editing strategy of CRISPR-Cas9 sgRNAs targeting W576 in pigs.Represented is sequence of exon 5 of the pig DNAJC14 gene, the sequence encodingW576, two potential crRNA binding sites (JIV90 gRNA1 and JIV90 gRNA3) which could potentially be used as a component for CRISPR / Cas9 alteration of the sequence encoding the Jiv90 domain.Figure 5. Infection of primary fibroblasts from founder edited animals with CSFV AlfortTuebingen. Cells were fixed with paraformaldehyde and permeabilized with b-D-glucopyranoside. Indirect immunfluorescence staining was done with a monoclonalantibody directed against NS3 (C16) in combination with an α-mouse IgG-Cy3 secondaryantibody. Mock = no CSFV infection.Figure 6. Infection of primary fibroblasts, isolated from a second generation of editedpigs, with CSFV Alfort Tuebingen. Cells were fixed with paraformaldehyde,permeabilized with b-D-glucopyranoside. Indirect immunfluorescence staining was donewith a monoclonal antibody directed against NS3 (C16) in combination with an α-mouseIgG-Cy3 secondary antibody. Each group of cells was challenged in triplicate.Figure 7. Infection of primary fibroblasts, isolated from a second generation of editedpigs, with BVDV-1 NCP7. Cells were fixed with paraformaldehyde and permeabilizedwith b-D-glucopyranoside. Indirect immunfluorescence staining was done with amonoclonal antibody directed against NS3 (C16) in combination with an α-mouse IgG-Cy3 secondary antibody. Each group of cells was challenged in triplicate.Figure 8. CSF viraemia during the course of the challenge study. Viral RNA waspurified from whole blood collected on days 4, 7, 10, 14 and 17 post inoculation, and ateuthanasia (days 21 or 22 p.i.) and quantified for viral genome copy number by qRT-PCR.Figure 9. Leukocyte counts during the course of the challenge study. Leukocytes inwhole blood were labelled with anti-porcine CD45-FITC and quantified by flow cytometry.Figure 10. Core body temperatures during the course of the challenge study. Rectaltemperature was recorded daily in the morning by inserting a thermometer into the rectum of each animal.Figure 11. Clinical scores during the course of the challenge study. Clinical scoringwas performed daily during the course of the challenge study, including the liveliness,body tension, body shape, breathing, walking, skin colour, eye inflammation, appetite and faeces constitution of each animal.Figure 12. Pig antibodies to CSFV. Pig antibodies to the CSFV E2 protein wereassayed in serum collected from pig using a commercial blocking (competitive) assay. Materials and methodsDesign of sgRNAs targeting Jiv90The annotated gene locus and protein sequence for DNAJC14 was acquired from theSus Scrofa 11.1 assembly from Ensembl (26th April 2019). DNAJC14 is located on Susscrofa chromosome 5, and is predicted to be composed of 7 exons, with the transcriptionstart site located in exon 2. The Jiv90 domain of DNAJC14, which is believed to be pivotalfor interaction with the genomes of viruses belonging to the Flaviviridae family, spansexons 4-6. Tryptophan at amino acid position W576, identified in vitro as crucial forinteraction between DNAJC14 and Flaviviridae, is encoded within exon 5 of the gene. PCR primers flanking exon 5 were designed using NCBI primer blast: Jiv90 Pr-3 (fwd) –GGTAGGTTGTGTGTAGGCG (SEQ ID NO: 13), and Jiv90 Pr-4 (rev) –GTGCCTGGCATCCTCGAAC (SEQ ID NO: 14) produce a product of 471 bases whenusing porcine genomic DNA as template. Cas9 guide RNAs were designed using theonline design tool, CRISPOR (Haeussler et al. 2016) by entering exon 5 as a targetsequence, selecting the Sscrofa11.1 genome and selecting guides based on thefollowing criteria “20bp-NGG - Sp Cas9, SpCas9-HF1, eSpCas91.1”. Four guides werechosen based on their proximity to W576: Jiv90 gRNA 1 – TAAGAGGCTGCATCCCGCTG-AGG (SEQ ID NO: 15);Jiv90 gRNA 2 – AGGCTGCATCCCGCTGAGGA-AGG (SEQ ID NO: 16);Jiv90 gRNA 3 – CCGCTGAGGAAGGAGACTTT-TGG (SEQ ID NO: 17); andJiv90 gRNA 4 – CGCTGAGGAAGGAGACTTTT-GGG (SEQ ID NO: 18).The 20bp guide sequences were adapted (excluding the -NGG) and ordered as oligosto be cloned into a plasmid vector encoding an sgRNA under control of the human U6 promoter, and a Cas9-2A-GFP under control of a CAG promoter, using the methodoutlined by Ran et al (2013).Assessing Cas9 gRNA efficiency in vitro Each of the four plasmids (one for each of the designed gRNAs) were transfected individually into the porcine kidney cell line PK-15. Briefly, each plasmid was combined with Lipofectamine 3000 at a ratio of 1:3.125 according to manufacturer’s instructions, following which transfection complexes were applied to cells in a dropwise manner. To enrich for transfected cells a BD FACSAria III (BD Biosystems) was employed to isolatethose cells which expressed GFP at 24 hours post transfection. Selected cells werecultured for a further 5 days, following which genomic DNA was extracted using a DNeasy Blood and Tissue kit (Qiagen) according to manufacturer’s instructions. Theresulting DNA was quantified by nanodrop and 50 ng was used as a PCR template usingPhusion Master Mix with HF buffer (NEB) and Jiv90 Pr-3 and -4 primers. The reactions were performed according to manufacturer’s protocol for 40 cycles with an annealing temperature of 64.6oC. To assess purity of the PCR product, 2 µl of each PCR product was resolved on a TAE agarose gel alongside Generuler DNA Ladder Mix (ThermoFisher), resolving fragments with the predicted product size of 471 bp. PCR productswere purified in preparation for sequencing. Briefly, 5 μl of PCR product was combinedwith 0.2 μl of Exonuclease I (NEB), 0.4 μl of Shrimp Alkaline Phosphatase rSAP (NEB) and 0.4 μl of nuclease-free water and incubated at 37oC for 30 minutes. Reactions were then incubated at 80oC for 15 minutes to degrade excess dNTPs and primers. Sanger sequencing was outsourced to Edinburgh Genomics using the BigDye v3.1 Terminator Cycle Sequencing Kit (Thermofisher P / N AB0384 / 240; BS034042). Analysis of sequencing data was performed using the Inference of CRISPR Edits (ICE) tool (Synthego) by importing the sequences derived from transfected cells, a sequence derived from PCR performed on genomic DNA from negative control cells, and the individual guide RNA sequences. The output from the algorithm provided an ICE score, predictive of cutting efficiency at each target site. Jiv90 gRNA 1 was predicted to generate the highest cutting efficiency at 47%, followed by Jiv90 gRNA 3 at 9%. Jiv90 gRNA2 and Jiv90 gRNA4 demonstrated negligible activity. Figure 4 shows the position of W576 (highlighted in blue column) relative to guides Jiv90 gRNA1 and Jiv90 gRNA3. Jiv90 gRNA3 is predicted to cut the genomic DNA closer to W576 than JIV90 gRNA1. As one of our goals was to introduce a specific W576Amutation in pigs, we decided to proceed with Jiv90 gRNA3 despite the observation thatit had a lower targeted cutting efficiency than Jiv90 gRNA1 in PK-15 cells.An oligonucleotide template molecule oSL472, intended to induce a homology directed repair encoding the W576A change at the target site within the pig genome, was designed to be complementary to the sequence annotated in Figure 4. The sequence of oSL472 is AGTAGGTGATCTTGAGGCCAAACATGCTTGACTCTGCAGCAAAGTCTCCTTCCTCAGCGGGATGCAGCCT (SEQ ID NO: 19), with the three altered nucleotides conferringthe W576A change emboldened and underlined. This oligonucleotide was ordered from IDT, as was a crRNA based on JIV90 gRNA3 (SS Jiv90 W39A G3), tracrRNA and Alt- R™ S.p. Cas9 Nuclease V3.To produce edited pigs, donor gilts were superovulated and AI performed using standardprotocols. The following day the donor animals were culled, midline laparotomy performed and putative zygotes collected from the oviduct. A ribonucleoprotein solution composed of SS Jiv90 W39A G3 complexed with tracrRNA and Cas9 nuclease was combined with oSL472 and microinjected into the cytoplasm of zygotes. Midline laparotomy was performed on 7 synchronous gilts, and 30-40 microinjected zygotes transferred to each. Three pregnancies were established, from which 24 healthy piglets were weened. Surprisingly, there were no obvious health deficits in any of these animals at weening, or in the 6 edited animals retained for breeding purposes and reared to adults. Ear punches were collected from piglets as a by-product of identity tagging. Genomic DNA was isolated and PCR performed using primers Jiv90 Pr-3 and Jiv90 Pr-4. Sanger sequencing of PCR products was outsourced to SourceBioscience. Six selectedindividuals were retained for subsequent breeding to produce an F1 generation. Excesspiglets were culled. One of the culled piglets (DNAJC14-23) was not edited. Two of the culled piglets (DNAJC14-2 and DNAJC14-24) were edited at the target site, and in each instance had one allele in which there was a frameshift mutation predicted to result in loss of DNAJC14 function, with the other allele having the W576A change. A skin biopsy was collected from these 3 animals post mortem and primary fibroblasts isolated.Challenge of primary fibroblasts from founder pigs DNAJC14-2, DNAJC14-23 andDNAJC14-24 with the pestivirus Classical Swine Fever Virus (CSFV) Alfort Tuebingenwas performed. Figure 5 shows that while the cells isolated from piglet DNAJC14-23which was not edited were readily infected with CSFV, cells from the edited animalsDNAJC14-2 and DNAJC14-24 did not support viral replication. To our knowledge this isthe first time that cells isolated from an animal with edited DNAJC14 have beendemonstrated to be resistant to any member of the Flaviviridae family.The retained pigs were crossed to produce an F1 generation. The first 2 litters totalled 24 piglets, all of which had DNAJC14 edits on each allele. Some of these animals hadthe W576A change on both alleles, some had frameshift mutations predicted to result inloss of DNAJC14 function on both alleles, and some were a combination of both. Surprisingly, given the reported phenotypes associated with mice lacking DNAJC14,none of the edited F1 piglets showed any obvious evidence of health deficits.Ear biopsies were collected from F1 animals DNAJC14-31 (+1 / +1), DNAJC14-39(W576A / W576A) and DNAJC14-47 (W576A / W576A) and from a non-edited controlanimal. Primary fibroblasts were isolated and challenged with the pestivirus ClassicalSwine Fever Virus (CSFV) Alfort Tuebingen in triplicate. Figure 6 shows that while cellsisolated from the non-edited control pig were readily infected with CSFV, cells from the3 edited animals did not support viral replication. This result is consistent with theobservations made with cells isolated from the founder animals. Primary fibroblasts from the same animals (DNAJC14-31, DNAJC14-39, DNAJC14-47 and non-edited control) were challenged with pestivirus BVDV-1 NCP7 in triplicate.Figure 7 shows that while cells isolated from the control pig could be infected with BVDV- 1, cells from the three edited animals could not. It is worth noting that the overall reduction in level of BVDV-1 infection of control cells relative to CSFV infection of cells from the same animal likely reflects the fact that while CSFV is recognised as a disease primarily of pigs, BVDV-1 is recognised as a disease primarily of cattle. It is therefore of littlesurprise that BVDV-1 was able to infect pig cells less efficiently than CSFV.Edited F1 animals DNACJ14-55 (male, W576A / +1), DNAJC14-57 (male, +4 / +1),DNAJC14-58 (female W576A / +1) and DNAJC14-60 (female W576A / +1) and 4 agematched female controls were inoculated intranasally with CSFV Alfort 187.Venous blood samples were collected from animals at days 0, 4, 7, 10, 14, and 17 postinoculation (p.i.), and at euthanasia (either day 21 or 22 p.i.) into EDTA vacutainers. Atday 17, blood samples were only collected from control animals #1 and #4, and fromedited animals DNAJC14-55 and DNAJC14-58. Viral nucleic acid was extracted from140ul blood using a Viral RNA mini kit (Qiagen) and CSFV viral RNA levels (genomecopies) determined by qRT-PCR. The number of copies of viral genome per µl of bloodwere quantified by comparison to a standard of RNA transcribed in vitro from the plasmidpCRXLV234-6 encoding the target 5’UTR region of the CSFV Alfort 187 genome (Everettet al., 2010). Successful RNA extraction and PCR was monitored by detection of anexogenously added internal control RNA (intype IC-RNA, Indical) for EDTA bloodsamples. Viraemia (Figure 8) was evident at day 4 in samples collected from controlanimals, peaking at around day 10 p.i. and then declining to undetectable levels at theend of the three-week study. No virus was detected in blood samples collected from edited animals during the course of the challenge, consistent with edited animals being resistant to CSFV infection. Venous blood samples were also assessed for leukocyte counts (Figure 9). The number of leukocytes was monitored by labelling CD45+cells in EDTA blood with anti-porcine CD45-FITC (K252-1E4; BioRad) and quantification by flow cytometry using Cytek®Aurora flow cytometer. Leukopenia was evident in control animals, remaining steadybetween days 4 and 7 p.i. and then returning to normal levels over the following week.This is consistent with control animals becoming infected with CSFV and subsequentlyclearing the infection. Leukopenia was not observed in the edited animals during thecourse of the challenge study, consistent with these animals being resistant to infection by CSFV.Core body temperature was recorded daily in the morning both 2 days before and thenthroughout the course of the challenge study by inserting a thermometer into the rectum of each animal (Figure 10). A mild fever was observed in the control animals betweendays 4 and 8 post infection, which resolved thereafter. This is consistent with CSFVinfection of the control animals. A single spike in temperature in control-3 on day 14 wasassociated with the development of petechial spots (often associated with CSFV re- occurrence) but this also swiftly resolved. No notable deviation in body temperature was observed in the edited animals, consistent with resistance to CSFV infection.Clinical scoring was performed daily during the course of the challenge study aspreviously described (Mittelholzer et al., 2000), with the exception of parameter #10(Figure 11). In control animals, mild clinical signs manifesting as lethargy andinappetence were recorded from day 5 post infection and sporadically thereafter. Theseclinical signs are consistent with control animals being infected with CSFV. In the editedgroup only DNAJC14-55 displayed clinical signs over a prolonged period, but this wasdue to mild lameness on a rear leg. Lameness is not an anticipated clinical signassociated with a CSFV infection, but is more likely to be a husbandry issue which isrelatively common in pigs. Overall, clinical signs are consistent with the control groupbecoming infected with CSFV while the edited animals were resistant to infection.Venous blood samples were collected from animals at days 0, 7, 14, and 17 post infection(p.i.), and at euthanasia (either day 21 or 22 p.i.) into BD separation vacutainers. At day17, blood samples were only collected from control animals #1 and #4, and from editedanimals DNAJC14-55 and DNAJC14-58. Serum was prepared and an IDEXX CSFV AbTest performed as per the manufacturer’s instructions (Figure 12). This assay involvescompetitive binding of pig antibodies raised against the CSFV E2 protein, present in the serum of the pigs, with a labelled antibody for the same epitope provided in the assay.The greater the degree of blocking of the labelled antibody, the higher the antibody titreto CSFV in the pig serum. Pig antibodies to CSFV were detected in the control animalsfrom day 14 post inoculation, consistent with an active CSFV infection. Pigs with editedDNAJC14 did not raise antibodies to CSFV, consistent with these animals being resistant to infection by CSFV.Overall, these data are consistent with animals that have edited DNAJC14 being healthyand fully resistant to CSFV infection. In summary, we have made the surprising discovery that pigs with edited DNAJC14 showno obvious deviations from full health in a farm environment, and that at the level of thewhole organism changes to the sequence of this gene confer full resistance to infection with the Pestivirus CSFV. Cells isolated from edited animals are similarly non-permissiveto both replication of CSFV, and the related Pestivirus BVDV-1 in vitro. Taken togetherthis strongly suggests that livestock, and particularly ungulates, with edited DNAJC14are highly likely to be both healthy and resistant to infection by any Pestivirus (with the potential exception of atypical porcine pestivirus [APPV], which has been shown byothers to replicate in vitro in the absence of DNAJC14). For example, our data suggestthat cattle or buffalo with edited DNAJC14 are likely to be healthy and are highly likely tobe fully resistant to infection with BVDV. Similarly, sheep or goats with edited DNAJC14are likely to be healthy and are highly likely to be fully resistant to BDV. As previouslyreported, in vitro data indicate that cellular DNAJC14 is also a key component of thereplication mechanism of other members of the Flaviviridae. As such, any animal withedited DNAJC14 may also be resistant to other members of the Flaviviridae such asFlaviviruses or Hepaciviruses. References Porcine reproductive and respiratory syndrome virus attachment is mediated by the N- terminal domain of the sialoadhesin receptor. An TQ, Tian ZJ, He YX, Xiao Y, Jiang YF, Peng JM, Zhou YJ, Liu D, Tong GZ. Vet Microbiol. 2010 Jul 14;143(2-4):371-8. doi: 10.1016 / j.vetmic.2009.11.006. Epub 2009 Dec 6. PMID: 19969429 Interaction of the European genotype porcine reproductive and respiratory syndrome virus (PRRSV) with sialoadhesin (CD169 / Siglec-1) inhibits alveolar macrophage phagocytosis. De Baere MI, Van Gorp H, Delputte PL, Nauwynck HJ. Vet Res.2012 May 25;43(1):47. doi: 10.1186 / 1297-9716-43-47. PMID: 22630829 Evaluation of off-target and on-target scoring algorithms and integration into the guideRNA selection tool CRISPOR. Haeussler M, Schönig K, Eckert H, Eschstruth A, MiannéJ, Renaud JB, Schneider-Maunoury S, Shkumatava A, Teboul L, Kent J, Joly JS, Concordet JP. Genome Biol. 2016 Jul 5;17(1):148. doi: 10.1186 / s13059-016-1012-2. PMID: 27380939 An intact sialoadhesin (Sn / SIGLEC1 / CD169) is not required for attachment / internalization of the porcine reproductive and respiratory syndrome virus. Prather RS, Rowland RR, Ewen C, Trible B, Kerrigan M, Bawa B, Teson JM, Mao J, Lee K, Samuel MS, Whitworth KM, Murphy CN, Egen T, Green JA. J Virol. 2013 Sep;87(17):9538-46. doi: 10.1128 / JVI.00177-13. Epub 2013 Jun 19. PMID: 23785195Genome engineering using the CRISPR-Cas9 system. Ran FA, Hsu PD, Wright J,Agarwala V, Scott DA, Zhang F. Nat Protoc. 2013 Nov;8(11):2281-2308. doi: 10.1038 / nprot.2013.143. Epub 2013 Oct 24. PMID: 24157548 Characterisation of experimental infections of domestic pigs with genotype 2.1 and 3.3 isolates of classical swine fever virus. Everett H, Salguero FJ, Graham SP, Haines F, Johns H, Clifford D, Nunez A, La Rocca SA, Parchariyanon S, Steinbach F, Drew T,Crooke H. Vet Microbiol. 2010 Apr 21;142(1-2):26-33. doi:10.1016 / j.vetmic.2009.09.039. Epub 2009 Sep 30. PMID: 19875252Analysis of classical swine fever virus replication kinetics allows differentiation of highlyvirulent from avirulent strains. Mittelholzer C, Moser C, Tratschin JD, Hofmann MA. VetMicrobiol. 2000 Jun 12;74(4):293-308. doi: 10.1016 / s0378-1135(00)00195-4. PMID:10831853

Claims

CLAIMS:

1. An animal, or genetically modified animal, comprising a modified DNAJC14 gene.

2. The animal of claim 1, wherein the animal is selected from the group consistingof: an ungulate; an ovine; a bovine; a caprine; a porcine / swine; an avian; or a piscine animal.

3. The animal of claim 1 or 2, wherein the animal is a pig.

4. A porcine / swine animal, or genetically modified porcine / swine animal, comprisinga modified DNAJC14 gene.

5. The animal of any preceding claim, wherein the modified DNAJC14 sequenceencodes a modified DNAJC14 protein sequence.

6. The animal of any preceding claim, wherein the animal is tolerant and / or resistantto a viral infection; an infection with a member of the Flaviviridae family; an infection withany one or more of a Pestivirus, Avian Tembusu virus, Dengue virus, Japaneseencephalitis virus, Louping ill virus, Tick-borne encephalitis virus, Zika virus or Hepacivius N.

7. The animal according to claim 6, wherein the Pestivirus is Pestivirus A, PestivirusB, Pestivirus C, Pestivirus D and / or Pestivirus K, or a combination thereof.

8. The animal according to any preceding claim, wherein the modified DNAJC14gene / protein reduces or inhibits the interaction of host cell DNAJC14 with a viral non-structural protein to inhibit or reduce viral replication.

9. The animal according to claim 8, wherein the viral non-structural protein is NS2-3 and / or NS2.

10. The animal according to any preceding claim, wherein the modified DNAJC14gene comprises a homozygous or heterozygous modification of the DNAJC14 gene.

11. The animal according to any preceding claim, wherein the modified DNAJC14gene or modified DNAJC14 protein comprises one or more mutation(s) selected fromthe group consisting of: (i) a substitution; (ii) a deletion; (iii) an insertion; and / or (iv) an inversion.

12. The animal according to any preceding claim, wherein the modified DNAJC14gene comprises one or more mutation(s) resulting in a frameshift mutation.

13. The animal according to any preceding claim, wherein the DNAJC14 gene isknocked out or functionally deleted.

14. The animal according to any preceding claim, wherein the modified DNAJC14gene comprises one or more mutation(s) in any one or more exons after exon 2 (whichcontains the start codon) of the DNAJC14 gene.

15. The animal according to any preceding claim, wherein the modified DNAJC14gene comprises one or more mutation(s) in exon 5 of the DNAJC14 gene.

16. The animal according to any preceding claim, wherein the modified DNAJC14gene comprises a modified Jiv90 domain.

17. The animal according to claim 16, wherein at least the codon for residue 39 ofthe Jiv90 domain or the tryptophan at position 39 of the Jiv90 domain is modified ormutated.

18. The animal according to claim 17, wherein the tryptophan (W) residue at position39 of the Jiv90 domain is substituted with an alanine (A) residue.

19. A method of producing a non-human animal with a modified DNAJC14 gene, saidmethod comprising modifying the DNAJC14 gene in a zygote and using said zygote toestablish a pregnancy in a non-human animal.

20. The method of claim 19, wherein the DNAJC14 gene in the zygote is modifiedusing: asite-specific nuclease and optionally a HDR donor template, wherein the HDRdonor template comprises a desired genomic edit and sequences homologous to the sequence flanking the cut site of the site-specific nuclease; or a CRISPR system.

21. An ungulate animal obtainable or obtained by the method of any one of claims19 or 20.

22. The ungulate of claim 21, wherein the ungulate is tolerant and / or resistant to aninfection caused by a member of the Flaviviridae.

23. The ungulate of claim 21 or 22, wherein the ungulate is an ovine animal, a bovineanimal, a caprine animal, a swine animal, a pig or a cattle-type animal.

24. The method of claim 20, wherein the CRISPR system uses a guide RNA selectedfrom SEQ ID NO: 15 to SEQ ID NO: 18.

25. The method according to any of one of claims 19, 20 or 24, wherein the geneticmodification encodes a modified DNAJC14 protein comprising a mutation or modificationin the Jiv90 domain; a mutation or modification at position 39 of the Jiv90 domain or atryptophan to alanine substitution at a position corresponding to residue 576 in the pigDNAJC14 protein.

26. A porcine, swine or pig cell or isolated cell comprising a modified, knocked out orfunctionally deleted DNAJC14 gene.

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