IgG Antibodies Mediate Protective Immunity of Inactivated Vaccine for Highly Pathogenic H5N1 Influenza Viruses in Ferrets

ABSTRACT Wild waterfowl are the natural reservoir of all influenza A viruses, and these viruses are usually nonpathogenic in these birds. However, since late 2002, H5N1 outbreaks in Asia have resulted in mortality among waterfowl in recreational parks, domestic flocks, and wild migratory birds. The evolutionary stasis between influenza virus and its natural host may have been disrupted, prompting us to ask whether waterfowl are resistant to H5N1 influenza virus disease and whether they can still act as a reservoir for these viruses. To better understand the biology of H5N1 viruses in ducks and attempt to answer this question, we inoculated juvenile mallards with 23 different H5N1 influenza viruses isolated in Asia between 2003 and 2004. All virus isolates replicated efficiently in inoculated ducks, and 22 were transmitted to susceptible contacts. Viruses replicated to higher levels in the trachea than in the cloaca of both inoculated and contact birds, suggesting that the digestive tract is not the main site of H5N1 influenza virus replication in ducks and that the fecal-oral route may no longer be the main transmission path. The virus isolates' pathogenicities varied from completely nonpathogenic to highly lethal and were positively correlated with tracheal virus titers. Nevertheless, the eight virus isolates that were nonpathogenic in ducks replicated and transmitted efficiently to naïve contacts, suggesting that highly pathogenic H5N1 viruses causing minimal signs of disease in ducks can propagate silently and efficiently among domestic and wild ducks in Asia and that they represent a serious threat to human and veterinary public health.

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Highly Pathogenic H5N1 Influenza Viruses Carry Virulence Determinants beyond the Polybasic Hemagglutinin Cleavage Site

PLoS ONE ◽

10.1371/journal.pone.0011826 ◽

2010 ◽

Vol 5 (7) ◽

pp. e11826 ◽

Cited By ~ 41

Author(s):

Jessica Bogs ◽

Jutta Veits ◽

Sandra Gohrbandt ◽

Jana Hundt ◽

Olga Stech ◽

...

Keyword(s):

Cleavage Site ◽

Influenza Viruses ◽

Virulence Determinants ◽

Highly Pathogenic ◽

H5n1 Influenza ◽

Pathogenic H5n1

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Competitive Fitness of Oseltamivir-Sensitive and -Resistant Highly Pathogenic H5N1 Influenza Viruses in a Ferret Model

Journal of Virology ◽

10.1128/jvi.00689-10 ◽

2010 ◽

Vol 84 (16) ◽

pp. 8042-8050 ◽

Cited By ~ 30

Author(s):

Elena A. Govorkova ◽

Natalia A. Ilyushina ◽

Bindumadhav M. Marathe ◽

Jennifer L. McClaren ◽

Robert G. Webster

Keyword(s):

Influenza Viruses ◽

Viral Fitness ◽

Upper Respiratory Tract ◽

Drug Pressure ◽

Highly Pathogenic ◽

Enzyme Active Site ◽

Enzymatic Function ◽

H5n1 Influenza ◽

Novel Approach ◽

Pathogenic H5n1

ABSTRACT The fitness of oseltamivir-resistant highly pathogenic H5N1 influenza viruses has important clinical implications. We generated recombinant human A/Vietnam/1203/04 (VN; clade 1) and A/Turkey/15/06 (TK; clade 2.2) influenza viruses containing the H274Y neuraminidase (NA) mutation, which confers resistance to NA inhibitors, and compared the fitness levels of the wild-type (WT) and resistant virus pairs in ferrets. The VN-H274Y and VN-WT viruses replicated to similar titers in the upper respiratory tract (URT) and caused comparable disease signs, and none of the animals survived. On days 1 to 3 postinoculation, disease signs caused by oseltamivir-resistant TK-H274Y virus were milder than those caused by TK-WT virus, and all animals survived. We then studied fitness by using a novel approach. We coinoculated ferrets with different ratios of oseltamivir-resistant and -sensitive H5N1 viruses and measured the proportion of clones in day-6 nasal washes that contained the H274Y NA mutation. Although the proportion of VN-H274Y clones increased consistently, that of TK-H274Y virus decreased. Mutations within NA catalytic (R292K) and framework (E119A/K, I222L, H274L, and N294S) sites or near the NA enzyme active site (V116I, I117T/V, Q136H, K150N, and A250T) emerged spontaneously (without drug pressure) in both pairs of viruses. The NA substitutions I254V and E276A could exert a compensatory effect on the fitness of VN-H274Y and TK-H274Y viruses. NA enzymatic function was reduced in both drug-resistant H5N1 viruses. These results show that the H274Y NA mutation affects the fitness of two H5N1 influenza viruses differently. Our novel method of assessing viral fitness accounts for both virus-host interactions and virus-virus interactions within the host.

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Efficacy of Oseltamivir Therapy in Ferrets Inoculated with Different Clades of H5N1 Influenza Virus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01312-06 ◽

2007 ◽

Vol 51 (4) ◽

pp. 1414-1424 ◽

Cited By ~ 118

Author(s):

Elena A. Govorkova ◽

Natalia A. Ilyushina ◽

David A. Boltz ◽

Alan Douglas ◽

Neziha Yilmaz ◽

...

Keyword(s):

H5n1 Virus ◽

Direct Sequencing ◽

Influenza Viruses ◽

Upper Respiratory Tract ◽

Virus Inoculation ◽

Highly Pathogenic ◽

H5n1 Influenza ◽

Homologous Virus ◽

Ha1 Gene ◽

Pathogenic H5n1

ABSTRACT Highly pathogenic H5N1 influenza viruses have infected an increasing number of humans in Asia, with high mortality rates and the emergence of multiple distinguishable clades. It is not known whether antiviral drugs that are effective against contemporary human influenza viruses will be effective against systemically replicating viruses, such as these pathogens. Therefore, we evaluated the use of the neuraminidase (NA) inhibitor oseltamivir for early postexposure prophylaxis and for treatment in ferrets exposed to representatives of two clades of H5N1 virus with markedly different pathogenicities in ferrets. Ferrets were protected from lethal infection with the A/Vietnam/1203/04 (H5N1) virus by oseltamivir (5 mg/kg of body weight/day) given 4 h after virus inoculation, but higher daily doses (25 mg/kg) were required for treatment when it was initiated 24 h after virus inoculation. For the treatment of ferrets inoculated with the less pathogenic A/Turkey/15/06 (H5N1) virus, 10 mg/kg/day of oseltamivir was sufficient to reduce the lethargy of the animals, significantly inhibit inflammation in the upper respiratory tract, and block virus spread to the internal organs. Importantly, all ferrets that survived the initial infection were rechallenged with homologous virus after 21 days and were completely protected from infection. Direct sequencing of the NA or HA1 gene segments in viruses isolated from ferret after treatment showed no amino acid substitutions known to cause drug resistance in conserved residues. Thus, early oseltamivir treatment is crucial for protection against highly pathogenic H5N1 viruses and the higher dose may be needed for the treatment of more virulent viruses.

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Host Genetic Variation Affects Resistance to Infection with a Highly Pathogenic H5N1 Influenza A Virus in Mice

Journal of Virology ◽

10.1128/jvi.00514-09 ◽

2009 ◽

Vol 83 (20) ◽

pp. 10417-10426 ◽

Cited By ~ 123

Author(s):

Adrianus C. M. Boon ◽

Jennifer deBeauchamp ◽

Anna Hollmann ◽

Jennifer Luke ◽

Malak Kotb ◽

...

Keyword(s):

Influenza Virus ◽

Influenza A ◽

H5n1 Virus ◽

Lethal Dose ◽

Influenza Virus Infection ◽

Virus Titer ◽

Influenza Viruses ◽

Highly Pathogenic ◽

H5n1 Influenza ◽

Pathogenic H5n1

ABSTRACT Despite the prevalence of H5N1 influenza viruses in global avian populations, comparatively few cases have been diagnosed in humans. Although viral factors almost certainly play a role in limiting human infection and disease, host genetics most likely contribute substantially. To model host factors in the context of influenza virus infection, we determined the lethal dose of a highly pathogenic H5N1 virus (A/Hong Kong/213/03) in C57BL/6J and DBA/2J mice and identified genetic elements associated with survival after infection. The lethal dose in these hosts varied by 4 logs and was associated with differences in replication kinetics and increased production of proinflammatory cytokines CCL2 and tumor necrosis factor alpha in susceptible DBA/2J mice. Gene mapping with recombinant inbred BXD strains revealed five loci or Qivr (quantitative trait loci for influenza virus resistance) located on chromosomes 2, 7, 11, 15, and 17 associated with resistance to H5N1 virus. In conjunction with gene expression profiling, we identified a number of candidate susceptibility genes. One of the validated genes, the hemolytic complement gene, affected virus titer 7 days after infection. We conclude that H5N1 influenza virus-induced pathology is affected by a complex and multigenic host component.

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Protective Efficacy of the H5 Inactivated Vaccine Against Different Highly Pathogenic H5N1 Avian Influenza Viruses Isolated in China and Vietnam

Avian Diseases ◽

10.1637/8707-031709-resnote.1 ◽

2010 ◽

Vol 54 (s1) ◽

pp. 287-289 ◽

Cited By ~ 27

Author(s):

Guobin Tian ◽

Xianying Zeng ◽

Yanbing Li ◽

Jianzhong Shi ◽

Hualan Chen

Keyword(s):

Avian Influenza ◽

Protective Efficacy ◽

Influenza Viruses ◽

Inactivated Vaccine ◽

Avian Influenza Viruses ◽

Highly Pathogenic ◽

H5n1 Avian Influenza ◽

Pathogenic H5n1

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Oseltamivir-Ribavirin Combination Therapy for Highly Pathogenic H5N1 Influenza Virus Infection in Mice

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01579-07 ◽

2008 ◽

Vol 52 (11) ◽

pp. 3889-3897 ◽

Cited By ~ 90

Author(s):

Natalia A. Ilyushina ◽

Alan Hay ◽

Neziha Yilmaz ◽

Adrianus C. M. Boon ◽

Robert G. Webster ◽

...

Keyword(s):

Influenza Virus ◽

Influenza Virus Infection ◽

Antiviral Effect ◽

Influenza Viruses ◽

Oseltamivir Carboxylate ◽

Highly Pathogenic ◽

H5n1 Influenza ◽

Pathogenic H5n1

ABSTRACT We studied the effects of a neuraminidase inhibitor (oseltamivir) and an inhibitor of influenza virus polymerases (ribavirin) against two highly pathogenic H5N1 influenza viruses. In vitro, A/Vietnam/1203/04 virus (clade 1) was highly susceptible to oseltamivir carboxylate (50% inhibitory concentration [IC50] = 0.3 nM), whereas A/Turkey/15/06 virus (clade 2.2) had reduced susceptibility (IC50 = 5.5 nM). In vivo, BALB/c mice were treated with oseltamivir (1, 10, 50, or 100 mg/kg of body weight/day), ribavirin (37.5, 55, or 75 mg/kg/day), or the combination of both drugs for 8 days, starting 4 h before virus inoculation. Monotherapy produced a dose-dependent antiviral effect against the two H5N1 viruses in vivo. Three-dimensional analysis of the drug-drug interactions revealed that oseltamivir and ribavirin interacted principally in an additive manner, with several exceptions of marginal synergy or marginal antagonism at some concentrations. The combination of ribavirin at 37.5 mg/kg/day and oseltamivir at 1 mg/kg/day and the combination of ribavirin at 37.5 mg/kg/day and oseltamivir at 10 mg/kg/day were synergistic against A/Vietnam/1203/04 and A/Turkey/15/06 viruses, respectively. These optimal oseltamivir-ribavirin combinations significantly inhibited virus replication in mouse organs, prevented the spread of H5N1 viruses beyond the respiratory tract, and abrogated the cytokine response (P < 0.01). Importantly, we observed clear differences between the efficacies of the drug combinations against two H5N1 viruses: higher doses were required for the protection of mice against A/Turkey/15/06 virus than for the protection of mice against A/Vietnam/1203/04 virus. Our preliminary results suggest that oseltamivir-ribavirin combinations can have a greater or lesser antiviral effect than monotherapy, depending on the H5N1 virus and the concentrations used.

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Susceptibility of Highly Pathogenic H5N1 Influenza Viruses to the Neuraminidase Inhibitor Oseltamivir Differs In Vitro and in a Mouse Model

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01667-08 ◽

2009 ◽

Vol 53 (7) ◽

pp. 3088-3096 ◽

Cited By ~ 35

Author(s):

Elena A. Govorkova ◽

Natalia A. Ilyushina ◽

Jennifer L. McClaren ◽

Tri S. P. Naipospos ◽

Bounlom Douangngeun ◽

...

Keyword(s):

Antiviral Treatment ◽

Influenza Viruses ◽

Oseltamivir Carboxylate ◽

In Vitro Susceptibility ◽

Highly Pathogenic ◽

Proinflammatory Mediators ◽

H5n1 Influenza ◽

Pathogenic H5n1

ABSTRACT While the neuraminidase (NA) inhibitor oseltamivir is currently our first line of defense against a pandemic threat, there is little information about whether in vitro testing can predict the in vivo effectiveness of antiviral treatment. Using a panel of five H5N1 influenza viruses (H5 clades 1 and 2), we determined that four viruses were susceptible to the drug in vitro (mean 50% inhibitory concentration [IC50], 0.1 to 4.9 nM), and A/Turkey/65-1242/06 virus was slightly less susceptible (mean IC50, 10.8 nM). Two avian viruses showed significantly greater NA enzymatic activity (V max) than the human viruses, and the five viruses varied in their affinity for the NA substrate MUNANA (Km , 64 to 300 μM) and for oseltamivir carboxylate (Ki , 0.1 to 7.9 nM). The protection of mice provided by a standard oseltamivir regimen (20 mg/kg/day for 5 days) also varied among the viruses used. We observed (i) complete protection against the less virulent A/chicken/Jogjakarta/BBVET/IX/04 virus; (ii) moderate protection (60 to 80% survival) against three viruses, two of which are neurotropic; and (iii) no protection against A/Turkey/65-1242/06 virus, which induced high pulmonary expression of proinflammatory mediators (interleukin-1α [IL-1α], IL-6, alpha interferon, and monocyte chemotactic protein 1) and contained a minor subpopulation of drug-resistant clones (I117V and E119A NA mutations). We found no correlation between in vitro susceptibility and in vivo protection (Spearman rank correlation coefficient ρ = −0.1; P > 0.05). Therefore, the in vivo efficacy of oseltamivir against highly pathogenic H5N1 influenza viruses cannot be reliably predicted by susceptibility testing, and more prognostic ways to evaluate anti-influenza compounds must be developed. Multiple viral and host factors modulate the effectiveness of NA inhibitor regimens against such viruses and new, more consistently effective treatment options, including combination therapies, are needed.

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Novel Approach to the Development of Effective H5N1 Influenza A Virus Vaccines: Use of M2 Cytoplasmic Tail Mutants

Journal of Virology ◽

10.1128/jvi.01899-07 ◽

2007 ◽

Vol 82 (5) ◽

pp. 2486-2492 ◽

Cited By ~ 47

Author(s):

Tokiko Watanabe ◽

Shinji Watanabe ◽

Jin Hyun Kim ◽

Masato Hatta ◽

Yoshihiro Kawaoka

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Cytoplasmic Tail ◽

Influenza Viruses ◽

Wild Type Virus ◽

Highly Pathogenic ◽

Live Attenuated Vaccines ◽

H5n1 Influenza ◽

Pathogenic H5n1

ABSTRACT Outbreaks of highly pathogenic H5N1 influenza viruses in avian species began in Asia and have since spread to other continents. Concern regarding the pandemic potential of these viruses in humans is clearly warranted, and there is an urgent need to develop effective vaccines against them. Previously, we and others demonstrated that deletions of the M2 cytoplasmic tail caused a growth defect in A/WSN/33 (H1N1) influenza A virus in vitro (K. Iwatsuki-Horimoto, T. Horimoto, T. Noda, M. Kiso, J. Maeda, S. Watanabe, Y. Muramoto, K. Fujii, and Y. Kawaoka, J. Virol. 80:5233-5240, 2006; M. F. McCown and A. Pekosz, J. Virol. 79:3595-3605, 2005; M. F. McCown and A. Pekosz, J. Virol. 80:8178-8189, 2006). We therefore tested the feasibility of using M2 tail mutants as live attenuated vaccines against H5N1 virus. First we generated a series of highly pathogenic H5N1 (A/Vietnam/1203/04 [VN1203]) M2 cytoplasmic tail deletion mutants and examined their growth properties in vitro and in vivo. We found that one mutant, which contains an 11-amino-acid deletion from the C terminus (M2del11 virus), grew as well as the wild-type virus but replicated in mice less efficiently. We then generated a recombinant VN1203M2del11 virus whose hemagglutinin (HA) gene was modified by replacing sequences at the cleavage site with those of an avirulent type of HA (M2del11-HAavir virus). This M2del11-HAavir virus protected mice against challenge with lethal doses of homologous (VN1203; clade 1) and antigenically distinct heterologous (A/Indonesia/7/2005; clade 2) H5N1 viruses. Our results suggest that M2 cytoplasmic tail mutants have potential as live attenuated vaccines against H5N1 influenza viruses.

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