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Resistance - Animal Studies

Studies in animals (in addition to use in pre-clinical development) provide an important adjunct to in vitro assessment of the effects of ‘resistance’ mutations on antiviral efficacy and fitness of the virus to replicate, transmit and spread, in assessing potential clinical impact and epidemiological significance of sporadic cases of resistance.

Recently, three animal (mammalian) models, ferrets, guinea pigs and mice have been used to investigate the consequences of neuraminidase inhibitor (NAI) resistance of influenza viruses in vivo.

The ferret is well established as the animal of choice in influenza virus research because of its natural susceptibility to infection, human-like distribution of sialic-acid receptors in the respiratory tract, and the manifestation of influenza symptoms (e.g. fever, sneezing, coughing, inactivity) similar to those in humans.  Notably, ferrets are susceptible to both contact and respiratory droplet routes of influenza virus transmission.  They are the most frequently used animal model to study fitness deficit of influenza viruses caused by NA mutations located either in framework residues (E119A, H275Y, N294S) or catalytic residues (R292K) or in close proximity to the enzyme active site (V116A, I117V, K150N, Y252H) (Baz et al, 2010; Kiso et al, 2010; Govorkova et al, 2010; Ilyushina et al., 2010).

The guinea pig was developed relatively recently as an alternative animal model for human influenza virus transmission studies (Lowen et al, 2006).  The Hartley strain has been used to study direct contact and respiratory droplet transmission of NAI-resistant H3N2 viruses with either an E119V mutation or E119V+I222V NA mutations (Bouvier et al., 2008) and oseltamivir-resistant A(H1N1)pdm09 viruses carrying the H275Y NA mutation (Seibert et al., 2010). The lack of clinical signs of infection limits the usefulness of guinea pigs for evaluating pathogenicity of influenza viruses.

The usefulness of the BALB/c mouse model is limited by a number of factors: mice are not a natural host for influenza and thus generally not susceptible to infection by unadapted viruses; avian-like sialic acid receptor distribution in the lung may restrict evaluation of human influenza viruses; disease symptoms do not match human clinical signs; lack of virus transmission from one animal to another makes this model unsuitable for transmissibility studies.  However, the ability to use large numbers of animals, and the availability of reagents, affords the use of the mouse model to study pathogenicity of e.g. NAI-resistant seasonal H1N1 and H3N2 influenza viruses (Hamelin et al., 2010) and highly pathogenic H5N1 influenza viruses (Yen et al., 2007).

Studies which addressed concern about the potential spread of oseltamivir-resistant (H275Y) A(H1N1)pdm09 viruses have yielded contradictory results, emphasising the difficulty in reaching definitive conclusions from animal studies as to the epidemic potential of such viruses (Govorkova, 2012).

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Baz M, Abed Y, Simon P, Hamelin ME, Boivin G: Effect of the neuraminidase mutation H274Y conferring resistance to oseltamivir on the replicative capacity and virulence of old and recent human influenza A(H1N1) viruses. J Infect Dis. 2010;201:740-5.

Bouvier NM, Lowen AC, Palese P: Oseltamivir-resistant influenza A viruses are transmitted efficiently among guinea pigs by direct contact but not by aerosol. J Virol. 2008; 82: 10052-10058.

Carr J, Ives J, Kelly L, Lambkin R, Oxford J, Mendel D, Tai L, Roberts N (2002).  Influenza virus carrying neuraminidase with reduced sensitivity to oseltamivir carboxylate has altered properties in vitro and is compromised for infectivity and replicative ability in vivo.  Antiviral Res; 54: 79-88.

Govorkova EA: Consequences of resistance:in vitro fitness, in vivo infectivity and transmissibility of oseltamivir-resistant influenza A viruses. Influenza Other Respir Viruses 2012: in press.

Govorkova EA, Ilyushina NA, Marathe BM, McClaren JL, Webster RG: Competitive fitness of oseltamivir-sensitive and -resistant highly pathogenic H5N1 influenza viruses in a ferret model. J Virol. 2010;84:8042-50.

Hamelin ME, Baz M, Abed Y, Couture C, Joubert P, Beaulieu E, Bellerose N, Plante M, Mallett C, Schumer G, et al.: Oseltamivir-resistant pandemic A/H1N1 virus is as virulent as its wild-type counterpart in mice and ferrets. PLoS Pathog. 2010;6:e1001015.

Ilyushina NA, Seiler JP, Rehg JE, Webster RG, Govorkova EA: Effect of neuraminidase inhibitor-resistant mutations on pathogenicity of clade 2.2 A/Turkey/15/06 (H5N1) influenza virus in ferrets. PLoS Pathog. 2010;6:e1000933.

Ives JA, Carr JA, Mendel DB, et al.  The H274Y mutation in the influenza A/H1N1 neuraminidase active site following oseltamivir phosphate treatment leave virus severely compromised both in vitro and in vivo. Antiviral Res. 2002 Aug;55(2):307-17.

Kiso M, Shinya K, Shimojima M, Takano R, Takahashi K, Katsura H, Kakugawa S, Le MT, Yamashita M, Furuta Y, et al.: Characterization of oseltamivir-resistant 2009 H1N1 pandemic influenza A viruses. PLoS Pathog. 2010;6:e1001079.

Lowen AC, Mubareka S, Tumpey TM, Garcia-Sastre A, Palese P: The guinea pig as a transmission model for human influenza viruses. Proc Natl Acad Sci U S A. 2006; 103: 9988-9992.

Seibert CW, Kaminski M, Philipp J, Rubbenstroth D, Albrecht RA, Schwalm F, Stertz S, Medina RA, Kochs G, García-Sastre A, et al.: Oseltamivir-resistant variants of the 2009 pandemic H1N1 influenza a virus are not attenuated in the guinea pig and ferret transmission models. J Virol. 2010;84:11219-11226.

Yen H-L, Herlocher LM, Hoffman E, et al.  Neuraminidase inhibitor-resistant influenza viruses may differ substantially in fitness and transmissibility.  Antimicrob Agents Chemother 2005; 49: 4075-4084.

Yen HL, Ilyushina NA, Salomon R, Hoffmann E, Webster RG, Govorkova EA: Neuraminidase inhibitor-resistant recombinant A/Vietnam/1203/04 (H5N1) influenza viruses retain their replication efficiency and pathogenicity in vitro and in vivo. J Virol. 2007; 81: 12418-12426.

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