Effect of Neuraminidase Inhibitor–Resistant Mutations on Pathogenicity of Clade 2.2 A/Turkey/15/06 (H5N1) Influenza Virus in Ferrets

English version České info

The acquisition of neuraminidase (NA) inhibitor resistance by H5N1 influenza viruses has serious clinical implications, as this class of drugs can be an essential component of pandemic control measures. The continuous evolution of the highly pathogenic H5N1 influenza viruses results in the emergence of natural NA gene variations whose impact on viral fitness and NA inhibitor susceptibility are poorly defined. We generated seven genetically stable recombinant clade 2.2 A/Turkey/15/06-like (H5N1) influenza viruses carrying NA mutations located either in the framework residues (E119A, H274Y, N294S) or in close proximity to the NA enzyme active site (V116A, I117V, K150N, Y252H). NA enzyme inhibition assays showed that NA mutations at positions 116, 117, 274, and 294 reduced susceptibility to oseltamivir carboxylate (IC₅₀s increased 5- to 940-fold). Importantly, the E119A NA mutation (previously reported to confer resistance in the N2 NA subtype) was stable in the clade 2.2 H5N1 virus background and induced cross-resistance to oseltamivir carboxylate and zanamivir. We demonstrated that Y252H NA mutation contributed for decreased susceptibility of clade 2.2 H5N1 viruses to oseltamivir carboxylate as compared to clade 1 viruses. The enzyme kinetic parameters (V_max, K_m and K_i) of the avian-like N1 NA glycoproteins were highly consistent with their IC₅₀ values. None of the recombinant H5N1 viruses had attenuated virulence in ferrets inoculated with 10⁶ EID₅₀ dose. Most infected ferrets showed mild clinical disease signs that differed in duration. However, H5N1 viruses carrying the E119A or the N294S NA mutation were lethal to 1 of 3 inoculated animals and were associated with significantly higher virus titers (P<0.01) and inflammation in the lungs compared to the wild-type virus. Our results suggest that highly pathogenic H5N1 variants carrying mutations within the NA active site that decrease susceptibility to NA inhibitors may possess increased virulence in mammalian hosts compared to drug-sensitive viruses. There is a need for novel anti-influenza drugs that target different virus/host factors and can limit the emergence of resistance.

Vyšlo v časopise: Effect of Neuraminidase Inhibitor–Resistant Mutations on Pathogenicity of Clade 2.2 A/Turkey/15/06 (H5N1) Influenza Virus in Ferrets. PLoS Pathog 6(5): e32767. doi:10.1371/journal.ppat.1000933
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1000933

Souhrn

Zdroje

1. World Health Organization 2010 Cumulative number of confirmed human cases of avian influenza A/(H5N1) reported to WHO. World Health Organization. Available: http://www.who.int/csr/disease/avian_influenza/country/cases_table_2010_03_04/en/index.html. Accessed March 8 2010.

2. WebsterRG

GovorkovaEA

2006 H5N1 influenza – continuing evolution and spread. N Engl J Med 355 2174 2177

3. Abdel-GhafarAN

ChotpitayasunondhT

GaoZ

HaydenFG

NguyenDH

2008 Update on avian influenza A (H5N1) virus infection in humans. N Engl J Med 358 261 273

4. StephensonI

NicholsonKG

WoodJM

ZambonMC

KatzJM

2004 Confronting the avian influenza target: vaccine development for a potential pandemic. Lancet Infect Dis 4 499 509

5. MosconaA

2008 Medical management of influenza infection. Annul Rev Med 59 397 413

6. von ItzsteinM

WuWY

KokGB

PeggMS

DyasonJC

1993 Rational design of potent sialidase-based inhibitors of influenza virus replication. Nature 363 418 423

7. LiW

EscarpePA

EisenbergEJ

CundyKC

SweetC

1998 Identification of GS 4104 as an orally bioavailable prodrug of the influenza virus neuraminidase inhibitor GS 4071. Antimicrob Agents Chemother 42 647 653

8. YenHL

MontoAS

WebsterRG

GovorkovaEA

2005 Virulence may determine the necessary duration and dosage of oseltamivir treatment for highly pathogenic A/Vietnam/1203/04 influenza virus in mice. J Infect Dis 192 665 672

9. IlyushinaNA

HayA

YilmazN

BoonAC

WebsterRG

2008 Oseltamivir-ribavirin combination therapy for highly pathogenic H5N1 influenza virus infection in mice. Antimicrob Agents Chemother 52 3889 3897

10. GovorkovaEA

IlyushinaNA

BoltzDA

DouglasA

YilmazN

2007 Efficacy of oseltamivir therapy in ferrets inoculated with different clades of H5N1 influenza virus. Antimicrob Agents Chemother 51 1414 1424

11. McKimm-BreschkinJL

SelleckPW

UsmanTB

JohnsonMA

2007 Reduced sensitivity of influenza A (H5N1) to osletamivir. Emerg Inf Dis 13 1354 1357

12. HurtAC

SelleckP

KomadinaN

ShawR

BrownL

2007 Susceptibility of highly pathogenic A (H5N1) avian influenza viruses to the neuraminidase inhibitors and adamantanes. Antivir Res 73 228 231

13. GovorkovaEA

IlyushinaNA

McClarenJL

NaiposposTS

DouangngeunB

2009 Susceptibility of highly pathogenic H5N1 influenza viruses to the neuraminidase inhibitor osletamivir differs in vitro and in mouse model. Antimicrob Agents Chemother 53 3088 3096

14. AokiFY

BoivinG

RobertsNA

2007 Influenza virus susceptibility and resistance to oseltamivir. Antiv Therapy 12 603 616

15. McKimm-BreschkinJL

2002 Neuraminidase inhibitors for the treatment and prevention of influenza. Expert Opin Pharmacother 3 103 112

16. HurtAC

HolienJK

ParkerM

KelsoA

BarrIG

2009 Zanamivir-resistant influenza viruses with a novel neuraminidase mutation. J Virol 83 10366 10373

17. McKimm-BreschkinJ

TriverdiT

HampsonA

HayA

KlimovA

2003 Neuraminidase sequence analysis and susceptibilities of influenza virus clinical isolates to zanamivir and oseltamivir. Antimicrob Agents Chemother 47 2264 2272

18. Rameix-WeltiMA

AgouF

BuchyP

MardyS

AubinJT

2006 Natural variation can significantly alter the sensitivity of influenza A (H5N1) viruses to oseltamivir. Antimicrob Agents Chemother 50 3809 3815

19. DeydeVM

NguyenT

BrightRA

BalishA

ShuB

2010 Detection of molecular markers of antiviral resistance in influenza A (H5N1) viruses using pyrosequencing method. Antimicrob Agents Chemother 54 1102 1110

20. CarrJ

IvesJ

KellyL

LambkinR

OxfordJ

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. Antivir Res 54 79 88

21. IvesJAL

CarrJA

MendelDB

TaiCY

LambkinR

2002 The H274Y mutation in the influenza A/H1N1 neuraminidase active site following oseltamivir phosphate treatment leaves virus severely compromised both in vitro and in vivo. Antivir Res 55 307 317

22. ColacinoJM

ChirgadzeNY

GarmanE

MurtiKG

LoncharichRJ

1997 A single sequence change destabilizes the influenza virus neuraminidase tetramer. Virology 236 66 75

23. LeQM

KisoM

SomeyaK

SakaiYT

NguyenTH

2005 Avian flu: isolation of drug-resistant H5N1 virus. Nature 437 1108

24. YenHL

IlyushinaNA

SalomonR

HoffmannE

WebsterRG

2007 Neuraminidase inhibitor-resistant recombinant A/Vietnam/1203/04 (H5N1) influenza viruses retain their replication efficiency and pathogenicity in vitro and in vivo. J Virol 81 12418 12426

25. de JongMD

TranTT

TruongHK

VoMH

SmithGJ

2005 Oseltamivir resistance during treatment of influenza A (H5N1) infection. N Engl J Med 353 2667 2672

26. SaadMD

BoyntonBR

EarhartKC

MansourMM

NimanHL

2007 Detection of oseltamivir resistanct mutation N294S in humans with influenza A H5N1. Options for the Control of Influenza VI Conference. Toronto, Canada

27. LackenbyA

HungnesO

DudmanSG

MeijerA

Paget WJ

2008 Emergence of resistance to oseltamivir among influenza A(H1N1) viruses in Europe Euro Surveill 13

28. DharanNJ

GubarevaLV

MeyerJJ

Okomo-AdhiamboM

McClintonRC

2009 Infections with oseltamivir-resistant influenza A (H1N1) virus in the United States. JAMA 301 1034 1041

29. RussellRJ

HaireLF

StevensDJ

CollinsPJ

LinYP

2006 The structure of H5N1 avian influenza neuraminidase suggests new opportunities for drug design. Nature 443 45 49

30. GubarevaLV

RobinsonMJ

BethellRC

WebsterRG

1997 Catalytic and framework mutations in the neuraminidase active site of influenza viruses that are resistant to 4-Guanidino-Neu5Ac2en. J Virol 71 3385 3390

31. GubarevaLV

BethellR

HartGJ

MurtiKG

PennCR

1996 Characterization of mutants of influenza A virus selected with the neuraminidase inhibitor 4-guanidino-Neu5Ac2en. J Virol 70 1818 1827

32. AbedY

GayetteN

BoivinG

2004 A reverse genetics study of resistance to neuraminidase inhibitors in an influenza A/H1N1 virus. Antivir Therapy 9 577 581

33. AbedY

NehmeB

BazM

BoivinG

2008 Activity of the neuraminidase inhibitor A-315675 against oseltamivir-resistant influenza neuraminidases of N1 and N2 subtypes. Antivir Res 77 163 166

34. CollinsPJ

HaireLF

LinYP

LiuJ

RussellRJ

2008 Crystal structures of oseltamivir-resistant influenza virus neuraminidase mutants. Nature 453 1258 1261

35. ReumanPD

KeelyS

SchiffGM

1989 Assessment of signs of influenza illness in the ferret model. J Virol Methods 24 27 34

36. MainesTR

ChenLM

MatsuokaY

ChenH

RoweT

2006 Lack of transmission of H5N1 avian-human reassortant influenza viruses in a ferret model. Proc Natl Acad Sci USA 103 12121 12126

37. HoffmannE

NeumannG

KawaokaY

HobomG

WebsterR

2000 97 6108 6113 Proc Natl Acad Sci USA A DNA transfection system for generation of influenza A virus from eight plasmids

38. PotierM

MameliL

BelisleM

DallaireL

MelanxonSB

1979 Fluorometric assay of neuraminidase with a sodium (4-methylumbelliferyl-alpha-D-N-acetylneuraminate) substrate. Anal Biochem 94 287 296

39. MalakhovMP

AschenbrennerLM

SmeeDF

WanderseeMK

SidwellRW

2006 Sialidase fusion protein as a novel broad-spectrum inhibitor of influenza virus infection. Antimicrob Agents Chemother 50 1470 1479

40. PalmerDF

DowdleWR

ColemanMT

SchildGC

1975 Advanced laboratory techniques for influenza diagnosis. U.S. Department of Health, Education, and Welfare, immunology series no. 6. Centers for Disease Control, Atlanta, GA

41. HoffmannE

StechJ

GuanY

WebsterRG

PerezDR

2001 Universal primer set for the full-length amplification of all influenza A viruses. Arch Virol 146 2275 2289

42. VenablesWN

RipleyBD

1997 Modern Applied Statistics. New York, NY Springer. 223 242, 297-321, 345-350

43. EllemanTC

AzadAA

WardCW