A view and overview on the control of avian influenza outbreaks in poultry: (6-6) Host genetic selection and transgenic chickens

The host genetics play a pivotal role in susceptibility to influenza including the highly pathogenic avian influenza virus (HPAIV) H5N1 which is frequently studied in mice models as reviewed by Horby et al. [23]. Indeed, the impact of host genetic selection on resistance to AIV infections in poultry has not yet been fully determined. Emergence of panzootic H5N1 virus since the last decade has raised concerns in respect to influenza-resistant chickens either by selective breeding or genetic modification. 

It has been supposed that fast-growing domestic birds have reduced immune competence against several viral diseases and resistant breeds are mostly poor producers [62]. Natural resistance or less susceptibility of some species/breeds of birds to AIV is not uncommon. In an experiment, five chicken lines were infected with an HPAIV H7N1. Three lines showed high susceptibility to the virus while two lines showed some resistance and survived the infection [48]. Swayne et al. [52] observed that an low pathogenic (LPAIV) H4N8 produced more severe lesions in commercial and SPF White Leghorn (WL) chickens than in 5 week-old commercial broiler chickens suggesting that SPF WL chickens are more susceptible than broilers to this strain. Thomas et al. [53] suggested that WL chickens may be more susceptible to an H3N2 virus of swine origin than White Plymouth Rock broiler-type chickens. On the contrary, severe lesions in commercial broiler chickens compared to SPF was observed after experimental infection with a Jordanian H9N2 isolate [18]. Some wild duck species, particularly mallards, are more resistance to HPAIV H5N1 than others [26]. Conversely, dabbling ducks and white fronted goose were more frequently infected with AIV than other wild ducks and geese, respectively [36]. Wood ducks were the only species to exhibit illness or death between different species of experimentally infected wild ducks in a study conducted by Brown and others [10]. 

Myxovirus resistance gene is an interferon-stimulated gene encodes Mx1 protein that able to interfere with AIV replication by inhibiting viral polymerases in the nucleus and by binding viral components in the cytoplasm. The role of the Mx gene in resistance against influenza viruses including the HPAIV H5N1 in mammals is well defined [11, 20, 35, 38, 40, 41, 50, 51]. However, the contribution of avian Mx proteins as antiviral elements in AIV infection in birds is contradictory and worth further exploration. Although intra- and inter-breed/-species Mx variations have been frequently reported [8, 14, 27, 29, 30, 43, 48, 58, 61], however commercial chicken lines have lower frequencies of the resistant allele compared to the indigenous chicken breeds [27, 29, 47] probably due to intensive modern breeding techniques [2]. Duck Mx was the first avian Mx protein to be characterized but no antiviral activity against an HPAIV H7N7 when transfected in chicken and mouse cells was obtained [4]. On the contrary, chickens have a single Mx1 gene [44] with multiple alleles [29] encoding a deduced protein with 705 amino acids in length. Notably, results of anti-influenza activity of the Mx1 protein in chickens are contradictory likely due to using variable experimental setups and different AIV strains. Also, a similar disparity has been noted between in-vitro and in-vivo experiments [17, 48].

Phenotypic variation in the antiviral activity of Mx gene has been linked to a single amino acid substitution of asparagine (Asn) at position 631 in resistant breeds or serine (Ser) in sensitive ones [27] probably due to inhibition of the PB2-NP interaction decreasing the viral polymerase activity [54]. The 631Asn identified mostly in Japanese native chicken breeds screened by Ko et al. [27] was associated with enhanced antiviral activity to H5N1 virus in transfected mouse fibroblast 3T3 cells. Conversely, results obtained by Benfield et al. [6], Benfield, et al. [7] and Schusser et al. [45] indicated that neither the 631Asn nor the 631Ser genotypes of chicken Mx1 was able to confer protection against several LPAIV and HPAIV including H5N1 subtype in chicken embryo fibroblasts or ECE. Similarly, Mx1 631Asn had no effect on viral replication after in-vitro infection of chicken embryo kidney cells with an LPAIV H5N9 [17]. Moreover, transfected chicken cells expressing chicken Mx protein did not induce resistance to HPAIV H7N7 [9]. In-vivo, following intranasal infection with an HPAIV H5N2, chickens carry Asn631 allele showed delayed mortality, milder morbidity and lesser virus excretion than 631Ser homozygotes [17]. Conversely, no correlation was observed between Mx-631 genotypes and susceptibility of chickens either to an HPAIV H7N1 [48] or after infection with H5N3 [56] as indicated by clinical status and time course of infection. Although, one out of six chicken lines infected with an HPAIV H7N1 had lower mortality, the Mx gene was not involved in this variations among tested chicken lines [49]. Additionally, chickens carry the homozygous Mx resistant allele genotype augmented the lowest HI titer after vaccination with an inactivated H5N2 vaccine compared with chickens that carry the sensitive allele [39].

Taken together, although few breeds of chickens and ducks can survive challenge with HPAIV in nature, resistance or susceptibility to a disease is usually multifactorial in nature and greatly influenced by both the host and the virus. To elucidate the role of Mx1 gene in the resistance of poultry to AIV more in-depth investigation [14, 45] and in-vivo comparative studies using several native breeds from different countries are highly required [17]. Also, interrelation of disease-resistance and production should be weighed. 

Apart from the Mx1 gene, resistance or less susceptibility of ducks to AIV infections compared with chickens has been linked to an influenza virus sensor known as retinoic acid-inducible gene I “RIG-I” (a cytoplasmic RNA sensor contribute to AIV detection and IFN production) which is absent in chickens [3, 25, 32]. This RIG-I gene as a natural AIV resistance gene in ducks could be a promising candidate for creation of transgenic chickens [3]. Likewise, different genes and cytokines have been expressed after infection of chicken and duck cells with several AIV subtypes including HPAIV H5N1 [1, 28, 31, 42]. Development of new drugs which modulate the expression of those cytokines may be a new target to control AIV replication [15]. Additional genetic candidates that contribute to inhibition of AIV replication such as cyclophilin A [60], ISG15 [24], viperin [55], heat shock cognate protein 70 (Hsc70) [57] or Ebp1 and/or ErbB3-binding protein [22] could be useful in creation of genetically modified chickens. It is worth mentioning that genome manipulation technologies have been actively developed and researched in the last decade to design hosts-on-demand. A number of techniques including transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced palindromic repeats (CRISPR) can modulate desired target genes in poultry in the near future to control AIV infections. For more information the readers are referred to other reviews [5, 13, 19, 33, 37, 59]. 

Current advance in molecular biology and genetic manipulation can facilitate the development of influenza-resistant poultry. Increase resistance of cell lines to influenza virus infection using RNA interfering (RNAi) molecules expressed by a lentiviral vector is more efficient transgenic tool than direct DNA injection or oncoretroviral vectors infection [12, 21, 46]. Recently, creation of AIV built-in resistant chickens by genetic modification has been experimentally proven by Lyall and colleagues [34]. Chickens equipped with a short-hairpin RNA targets the AIV polymerase binding sites have been created and infected with HPAIV H5N1. Although all infected transgenic birds succumbed to the infection however the virus did not spread to the in-contact transgenic and non-transgenic cagemates [34].

The most important challenges facing the development of genetically modified chickens are the applicability in food production, safety regulations and consumer’s preferences [16, 34]. Moreover, AIV is a “master of mutability” and global production of the resistant chickens must be equipped with many decoys target different genes to avoid rapid generation of AIV resistance. In addition, replacement of the commercial flocks with the newly flu-resistant birds is expected to occur within short period due to globalization of the poultry industry however replacement of backyard birds seems to be more complicated [16].

Finally, although a proof-of-principle to produce transgenic chickens has been reported, technical, logistic and social constraints are facing development of chicken resistant to AIV. Stable transmission and expression of the transgene from generation to generation require extensive studies. Regulatory approval, mass production, costs and marketing of commercial AIV resistant pedigree lines, consumer preferences and food safety issues need to be carefully and fully addressed. Overall, mutation of the virus in the face of any control approach remains the real challenge. 

1.         Adams SC, Xing Z, Li J, Cardona CJ (2009) Immune-related gene expression in response to H11N9 low pathogenic avian influenza virus infection in chicken and Pekin duck peripheral blood mononuclear cells. Molecular immunology 46:1744-1749

2.         Balkissoon D, Staines K, McCauley J, Wood J, Young J, Kaufman J, Butter C (2007) Low frequency of the Mx allele for viral resistance predates recent intensive selection in domestic chickens. Immunogenetics 59:687-691

3.         Barber MR, Aldridge JR, Jr., Webster RG, Magor KE (2010) Association of RIG-I with innate immunity of ducks to influenza. Proceedings of the National Academy of Sciences of the United States of America 107:5913-5918

4.         Bazzigher L, Schwarz A, Staeheli P (1993) No enhanced influenza virus resistance of murine and avian cells expressing cloned duck Mx protein. Virology 195:100-112

5.         Belhaj K, Chaparro-Garcia A, Kamoun S, Nekrasov V (2013) Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system. Plant methods 9:39

6.         Benfield CT, Lyall JW, Kochs G, Tiley LS (2008) Asparagine 631 variants of the chicken Mx protein do not inhibit influenza virus replication in primary chicken embryo fibroblasts or in vitro surrogate assays. Journal of virology 82:7533-7539

7.         Benfield CT, Lyall JW, Tiley LS (2010) The cytoplasmic location of chicken mx is not the determining factor for its lack of antiviral activity. PloS one 5:e12151

8.         Berlin S, Qu L, Li X, Yang N, Ellegren H (2008) Positive diversifying selection in avian Mx genes. Immunogenetics 60:689-697

9.         Bernasconi D, Schultz U, Staeheli P (1995) The interferon-induced Mx protein of chickens lacks antiviral activity. Journal of interferon & cytokine research 15:47-53

10.       Brown JD, Stallknecht DE, Beck JR, Suarez DL, Swayne DE (2006) Susceptibility of North American ducks and gulls to H5N1 highly pathogenic avian influenza viruses. Emerging infectious diseases 12:1663-1670

11.       Chang KC, Goldspink G, Lida J (1990) Studies in the in vivo expression of the influenza resistance gene Mx by in-situ hybridisation. Archives of virology 110:151-164

12.       Chen J, Chen SC, Stern P, Scott BB, Lois C (2008) Genetic strategy to prevent influenza virus infections in animals. The Journal of infectious diseases 197 Suppl 1:S25-28

13.       Daimon T, Kiuchi T, Takasu Y (2014) Recent progress in genome engineering techniques in the silkworm, Bombyx mori. Dev Growth Differ 56:14-25

14.       Dillon D, Runstadler J (2010) Mx gene diversity and influenza association among five wild dabbling duck species (Anas spp.) in Alaska. Infect Genet Evol 10:1085-1093

15.       Ehrhardt C, Ruckle A, Hrincius ER, Haasbach E, Anhlan D, Ahmann K, Banning C, Reiling SJ, Kuhn J, Strobl S, Vitt D, Leban J, Planz O, Ludwig S (2013) The NF-kappaB inhibitor SC75741 efficiently blocks influenza virus propagation and confers a high barrier for development of viral resistance. Cellular microbiology 15:1198-1211

16.       Enserink M (2011) Avian influenza. Transgenic chickens could thwart bird flu, curb pandemic risk. Science 331:132-133

17.       Ewald SJ, Kapczynski DR, Livant EJ, Suarez DL, Ralph J, McLeod S, Miller C (2011) Association of Mx1 Asn631 variant alleles with reductions in morbidity, early mortality, viral shedding, and cytokine responses in chickens infected with a highly pathogenic avian influenza virus. Immunogenetics 63:363-375

18.       Gharaibeh S (2008) Pathogenicity of an avian influenza virus serotype H9N2 in chickens. Avian diseases 52:106-110

19.       Gratz SJ, Wildonger J, Harrison MM, O'Connor-Giles KM (2013) CRISPR/Cas9-mediated genome engineering and the promise of designer flies on demand. Fly 7

20.       Haller O, Staeheli P, Kochs G (2009) Protective role of interferon-induced Mx GTPases against influenza viruses. Rev Sci Tech 28:219-231

21.       Harvey AJ, Speksnijder G, Baugh LR, Morris JA, Ivarie R (2002) Consistent production of transgenic chickens using replication-deficient retroviral vectors and high-throughput screening procedures. Poult Sci 81:202-212

22.       Honda A, Okamoto T, Ishihama A (2007) Host factor Ebp1: selective inhibitor of influenza virus transcriptase. Genes to cells : devoted to molecular & cellular mechanisms 12:133-142

23.       Horby P, Nguyen NY, Dunstan SJ, Baillie JK (2012) The role of host genetics in susceptibility to influenza: a systematic review. PloS one 7:e33180

24.       Hsiang TY, Zhao C, Krug RM (2009) Interferon-induced ISG15 conjugation inhibits influenza A virus gene expression and replication in human cells. J Virol 83:5971-5977

25.       Karpala AJ, Stewart C, McKay J, Lowenthal JW, Bean AG (2011) Characterization of chicken Mda5 activity: regulation of IFN-beta in the absence of RIG-I functionality. J Immunol 186:5397-5405

26.       Keawcharoen J, van Riel D, van Amerongen G, Bestebroer T, Beyer WE, van Lavieren R, Osterhaus AD, Fouchier RA, Kuiken T (2008) Wild ducks as long-distance vectors of highly pathogenic avian influenza virus (H5N1). Emerging infectious diseases 14:600-607

27.       Ko JH, Jin HK, Asano A, Takada A, Ninomiya A, Kida H, Hokiyama H, Ohara M, Tsuzuki M, Nishibori M, Mizutani M, Watanabe T (2002) Polymorphisms and the differential antiviral activity of the chicken Mx gene. Genome research 12:595-601

28.       Kuchipudi SV, Dunham SP, Nelli R, White GA, Coward VJ, Slomka MJ, Brown IH, Chang KC (2012) Rapid death of duck cells infected with influenza: a potential mechanism for host resistance to H5N1. Immunology and cell biology 90:116-123

29.       Li XY, Qu LJ, Yao JF, Yang N (2006) Skewed allele frequencies of an Mx gene mutation with potential resistance to avian influenza virus in different chicken populations. Poult Sci 85:1327-1329

30.       Li XY, Qu LJ, Hou ZC, Yao JF, Xu GY, Yang N (2007) Genomic structure and diversity of the chicken Mx gene. Poult Sci 86:786-789

31.       Liang QL, Luo J, Zhou K, Dong JX, He HX (2011) Immune-related gene expression in response to H5N1 avian influenza virus infection in chicken and duck embryonic fibroblasts. Molecular immunology 48:924-930

32.       Liniger M, Summerfield A, Zimmer G, McCullough KC, Ruggli N (2012) Chicken cells sense influenza A virus infection through MDA5 and CARDIF signaling involving LGP2. J Virol 86:705-717

33.       Lisa Li H, Nakano T, Hotta A (2014) Genetic correction using engineered nucleases for gene therapy applications. Dev Growth Differ 56:63-77

34.       Lyall J, Irvine RM, Sherman A, McKinley TJ, Nunez A, Purdie A, Outtrim L, Brown IH, Rolleston-Smith G, Sang H, Tiley L (2011) Suppression of avian influenza transmission in genetically modified chickens. Science 331:223-226

35.       Meier E, Kunz G, Haller O, Arnheiter H (1990) Activity of rat Mx proteins against a rhabdovirus. J Virol 64:6263-6269

36.       Munster VJ, Baas C, Lexmond P, Waldenstrom J, Wallensten A, Fransson T, Rimmelzwaan GF, Beyer WE, Schutten M, Olsen B, Osterhaus AD, Fouchier RA (2007) Spatial, temporal, and species variation in prevalence of influenza A viruses in wild migratory birds. PLoS Pathog 3:e61

37.       Pan Y, Xiao L, Li AS, Zhang X, Sirois P, Zhang J, Li K (2013) Biological and biomedical applications of engineered nucleases. Molecular biotechnology 55:54-62

38.       Pavlovic J, Zurcher T, Haller O, Staeheli P (1990) Resistance to influenza virus and vesicular stomatitis virus conferred by expression of human MxA protein. J Virol 64:3370-3375

39.       Qu LJ, Li XY, Xu GY, Ning ZH, Yang N (2009) Lower antibody response in chickens homozygous for the Mx resistant allele to avian influenza. Asian-Aust J Anim Sci 22:465 - 470

40.       Ruff M (1983) Interferon-mediated development of influenza virus resistance in hybrids between Mx gene-bearing and control mouse embryo fibroblasts. The Journal of general virology 64 (Pt 6):1291-1300

41.       Salomon R, Staeheli P, Kochs G, Yen HL, Franks J, Rehg JE, Webster RG, Hoffmann E (2007) Mx1 gene protects mice against the highly lethal human H5N1 influenza virus. Cell Cycle 6:2417-2421

42.       Sarmento L, Afonso CL, Estevez C, Wasilenko J, Pantin-Jackwood M (2008) Differential host gene expression in cells infected with highly pathogenic H5N1 avian influenza viruses. Veterinary immunology and immunopathology 125:291-302

43.       Sartika T, Sulandari S, Zein MS (2011) Selection of Mx gene genotype as genetic marker for Avian Influenza resistance in Indonesian native chicken. BMC proceedings 5 Suppl 4:S37

44.       Schumacher B, Bernasconi D, Schultz U, Staeheli P (1994) The chicken Mx promoter contains an ISRE motif and confers interferon inducibility to a reporter gene in chick and monkey cells. Virology 203:144-148

45.       Schusser B, Reuter A, von der Malsburg A, Penski N, Weigend S, Kaspers B, Staeheli P, Hartle S (2011) Mx is dispensable for interferon-mediated resistance of chicken cells against influenza A virus. J Virol 85:8307-8315

46.       Scott BB, Lois C (2005) Generation of tissue-specific transgenic birds with lentiviral vectors. Proc Natl Acad Sci U S A 102:16443-16447

47.       Seyama T, Ko JH, Ohe M, Sasaoka N, Okada A, Gomi H, Yoneda A, Ueda J, Nishibori M, Okamoto S, Maeda Y, Watanabe T (2006) Population research of genetic polymorphism at amino acid position 631 in chicken Mx protein with differential antiviral activity. Biochemical genetics 44:437-448

48.       Sironi L, Williams JL, Moreno-Martin AM, Ramelli P, Stella A, Jianlin H, Weigend S, Lombardi G, Cordioli P, Mariani P (2008) Susceptibility of different chicken lines to H7N1 highly pathogenic avian influenza virus and the role of Mx gene polymorphism coding amino acid position 631. Virology 380:152-156

49.       Sironi L, Williams JL, Stella A, Minozzi G, Moreno A, Ramelli P, Han J, Weigend S, Wan J, Lombardi G, Cordioli P, Mariani P (2011) Genomic study of the response of chicken to highly pathogenic avian influenza virus. BMC proceedings 5 Suppl 4:S25

50.       Song MS, Cho YH, Park SJ, Pascua PN, Baek YH, Kwon HI, Lee OJ, Kong BW, Kim H, Shin EC, Kim CJ, Choi YK (2013) Early regulation of viral infection reduces inflammation and rescues mx-positive mice from lethal avian influenza infection. The American journal of pathology 182:1308-1321

51.       Staeheli P, Haller O, Boll W, Lindenmann J, Weissmann C (1986) Mx protein: constitutive expression in 3T3 cells transformed with cloned Mx cDNA confers selective resistance to influenza virus. Cell 44:147-158

52.       Swayne DE, Radin MJ, Hoepf TM, Slemons RD (1994) Acute renal failure as the cause of death in chickens following intravenous inoculation with avian influenza virus A/chicken/Alabama/7395/75 (H4N8). Avian diseases 38:151-157

53.       Thomas C, Manin TB, Andriyasov AV, Swayne DE (2008) Limited susceptibility and lack of systemic infection by an H3N2 swine influenza virus in intranasally inoculated chickens. Avian diseases 52:498-501

54.       Verhelst J, Parthoens E, Schepens B, Fiers W, Saelens X (2012) Interferon-inducible protein Mx1 inhibits influenza virus by interfering with functional viral ribonucleoprotein complex assembly. J Virol 86:13445-13455

55.       Wang X, Hinson ER, Cresswell P (2007) The interferon-inducible protein viperin inhibits influenza virus release by perturbing lipid rafts. Cell host & microbe 2:96-105

56.       Wang Y, Brahmakshatriya V, Lupiani B, Reddy S, Okimoto R, Li X, Chiang H, Zhou H (2012) Associations of chicken Mx1 polymorphism with antiviral responses in avian influenza virus infected embryos and broilers. Poult Sci 91:3019-3024

57.       Watanabe K, Fuse T, Asano I, Tsukahara F, Maru Y, Nagata K, Kitazato K, Kobayashi N (2006) Identification of Hsc70 as an influenza virus matrix protein (M1) binding factor involved in the virus life cycle. FEBS letters 580:5785-5790

58.       Watanabe T (2007) Polymorphisms of the chicken antiviral MX gene. Cytogenetic and genome research 117:370-375

59.       Wei C, Liu J, Yu Z, Zhang B, Gao G, Jiao R (2013) TALEN or Cas9 - rapid, efficient and specific choices for genome modifications. Journal of genetics and genomics = Yi chuan xue bao 40:281-289

60.       Xu C, Meng S, Liu X, Sun L, Liu W (2010) Chicken cyclophilin A is an inhibitory factor to influenza virus replication. Virol J 7:372

61.       Yin CG, Zhang CS, Zhang AM, Qin HW, Wang XQ, Du LX, Zhao GP (2010) Expression analyses and antiviral properties of the Beijing-You and White Leghorn myxovirus resistance gene with different amino acids at position 631. Poult Sci 89:2259-2264

62.       Zekarias B, Ter Huurne AA, Landman WJ, Rebel JM, Pol JM, Gruys E (2002) Immunological basis of differences in disease resistance in the chicken. Veterinary research 33:109-125