PB1-mediated virulence attenuation of H5N1 influenza virus in mice is associated with PB2

H5N1 avian influenza viruses demonstrate different phenotypes, such as pathogenicity after one or serial passages in mammalian hosts or cells. To establish the molecular basis of these phenotypes, we cloned isolates from the lungs of mice infected with human A/Vietnam/1194/2004 (H5N1) influenza virus. Large-plaque isolates were less pathogenic to mice than small-plaque isolates. Genome sequencing revealed that the small-plaque and large-plaque isolates differed in several amino acids. In order to assess their effects on pathogenicity in mice, two amino acid changes common to attenuated isolates, one in PB2 (I63T) and the other in PB1 (T677M), were inserted into a wild-type recombinant virus construct. The PB2 (I63T) or PB1 (T677M) mutations alone did not alter the phenotype of H5N1 virus, whereas recombinant virus with both mutations was less pathogenic than the wild-type recombinant virus. Furthermore, the PB1 (T677M) mutation showed a lower replication efficiency, although it had higher polymerase activity. The recombinant virus with the PB2 (63T) mutation replicated as well as the wild-type recombinant virus. These results suggest that the C terminus of PB1 of H5N1 influenza virus mediates virulence attenuation of H5N1 influenza virus in mice, associating with the N terminus of PB2. However, the role of the N terminus of PB2 in virulence attenuation in mice remains unclear.

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Molecular Changes in the Polymerase Genes (PA and PB1) Associated with High Pathogenicity of H5N1 Influenza Virus in Mallard Ducks

Journal of Virology ◽

10.1128/jvi.00435-07 ◽

2007 ◽

Vol 81 (16) ◽

pp. 8515-8524 ◽

Cited By ~ 132

Author(s):

D. J. Hulse-Post ◽

J. Franks ◽

K. Boyd ◽

R. Salomon ◽

E. Hoffmann ◽

...

Keyword(s):

Influenza Virus ◽

Gene Mutations ◽

Influenza Viruses ◽

H5n1 Influenza Virus ◽

Wild Type ◽

Small Plaque ◽

Highly Pathogenic ◽

Large Plaque ◽

Ha Gene ◽

H5n1 Influenza

ABSTRACT The highly pathogenic (HP) influenza viruses H5 and H7 are usually nonpathogenic in mallard ducks. However, the currently circulating HP H5N1 viruses acquired a different phenotype and are able to cause mortality in mallards. To establish the molecular basis of this phenotype, we cloned the human A/Vietnam/1203/04 (H5N1) influenza virus isolate that is highly pathogenic in ferrets, mice, and mallards and found it to be a heterogeneous mixture. Large-plaque isolates were highly pathogenic to ducks, mice, and ferrets, whereas small-plaque isolates were nonpathogenic in these species. Sequence analysis of the entire genome revealed that the small-plaque and the large-plaque isolates differed in the coding of five amino acids. There were two differences in the hemagglutinin (HA) gene (K52T and A544V), one in the PA gene (T515A), and two in the PB1 gene (K207R and Y436H). We inserted the amino acid changes into the wild-type reverse genetic virus construct to assess their effects on pathogenicity in vivo. The HA gene mutations and the PB1 gene K207R mutation did not alter the HP phenotype of the large-plaque virus, whereas constructs with the PA (T515A) and PB1 (Y436H) gene mutations were nonpathogenic in orally inoculated ducks. The PB1 (Y436H) construct was not efficiently transmitted in ducks, whereas the PA (T515A) construct replicated as well as the wild-type virus did and was transmitted efficiently. These results show that the PA and PB1 genes of HP H5N1 influenza viruses are associated with lethality in ducks. The mechanisms of lethality and the perpetuation of this lethal phenotype in ducks in nature remain to be determined.

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Neuraminidase Inhibitor-Resistant Recombinant A/Vietnam/1203/04 (H5N1) Influenza Viruses Retain Their Replication Efficiency and Pathogenicity In Vitro and In Vivo

Journal of Virology ◽

10.1128/jvi.01067-07 ◽

2007 ◽

Vol 81 (22) ◽

pp. 12418-12426 ◽

Cited By ~ 130

Author(s):

Hui-Ling Yen ◽

Natalia A. Ilyushina ◽

Rachelle Salomon ◽

Erich Hoffmann ◽

Robert G. Webster ◽

...

Keyword(s):

Influenza Virus ◽

Influenza Pandemic ◽

Influenza Virus Infection ◽

Influenza Viruses ◽

Wild Type Virus ◽

H5n1 Influenza Virus ◽

Wild Type ◽

Replication Efficiency ◽

H5n1 Influenza

ABSTRACT Effective antiviral drugs are essential for early control of an influenza pandemic. It is therefore crucial to evaluate the possible threat posed by neuraminidase (NA) inhibitor-resistant influenza viruses with pandemic potential. Four NA mutations (E119G, H274Y, R292K, and N294S) that have been reported to confer resistance to NA inhibitors were each introduced into recombinant A/Vietnam/1203/04 (VN1203) H5N1 influenza virus. For comparison, the same mutations were introduced into recombinant A/Puerto Rico/8/34 (PR8) H1N1 influenza virus. The E119G and R292K mutations significantly compromised viral growth in vitro, but the H274Y and N294S mutations were stably maintained in VN1203 and PR8 viruses. In both backgrounds, the H274Y and N294S mutations conferred resistance to oseltamivir carboxylate (50% inhibitory concentration [IC50] increases, >250-fold and >20-fold, respectively), and the N294S mutation reduced susceptibility to zanamivir (IC50 increase, >3.0-fold). Although the H274Y and N294S mutations did not compromise the replication efficiency of VN1203 or PR8 viruses in vitro, these mutations slightly reduced the lethality of PR8 virus in mice. However, the VN1203 virus carrying either the H274Y or N294S mutation exhibited lethality similar to that of the wild-type VN1203 virus. The different enzyme kinetic parameters (V max and Km ) of avian-like VN1203 NA and human-like PR8 NA suggest that resistance-associated NA mutations can cause different levels of functional loss in NA glycoproteins of the same subtype. Our results suggest that NA inhibitor-resistant H5N1 variants may retain the high pathogenicity of the wild-type virus in mammalian species. Patients receiving NA inhibitors for H5N1 influenza virus infection should be closely monitored for the emergence of resistant variants.

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Cross-Reactive, Cell-Mediated Immunity and Protection of Chickens from Lethal H5N1 Influenza Virus Infection in Hong Kong Poultry Markets

Journal of Virology ◽

10.1128/jvi.75.6.2516-2525.2001 ◽

2001 ◽

Vol 75 (6) ◽

pp. 2516-2525 ◽

Cited By ~ 161

Author(s):

Sang Heui Seo ◽

Robert G. Webster

Keyword(s):

T Cells ◽

Influenza Virus ◽

Hong Kong ◽

Virus Infection ◽

Cellular Immunity ◽

Influenza Virus Infection ◽

Influenza Viruses ◽

H5n1 Influenza Virus ◽

H9n2 Influenza Virus ◽

H5n1 Influenza

ABSTRACT In 1997, avian H5N1 influenza virus transmitted from chickens to humans resulted in 18 confirmed infections. Despite harboring lethal H5N1 influenza viruses, most chickens in the Hong Kong poultry markets showed no disease signs. At this time, H9N2 influenza viruses were cocirculating in the markets. We investigated the role of H9N2 influenza viruses in protecting chickens from lethal H5N1 influenza virus infections. Sera from chickens infected with an H9N2 influenza virus did not cross-react with an H5N1 influenza virus in neutralization or hemagglutination inhibition assays. Most chickens primed with an H9N2 influenza virus 3 to 70 days earlier survived the lethal challenge of an H5N1 influenza virus, but infected birds shed H5N1 influenza virus in their feces. Adoptive transfer of T lymphocytes or CD8+ T cells from inbred chickens (B2/B2) infected with an H9N2 influenza virus to naive inbred chickens (B2/B2) protected them from lethal H5N1 influenza virus. In vitro cytotoxicity assays showed that T lymphocytes or CD8+ T cells from chickens infected with an H9N2 influenza virus recognized target cells infected with either an H5N1 or H9N2 influenza virus in a dose-dependent manner. Our findings indicate that cross-reactive cellular immunity induced by H9N2 influenza viruses protected chickens from lethal infection with H5N1 influenza viruses in the Hong Kong markets in 1997 but permitted virus shedding in the feces. Our findings are the first to suggest that cross-reactive cellular immunity can change the outcome of avian influenza virus infection in birds in live markets and create a situation for the perpetuation of H5N1 influenza viruses.

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Mutations in the NS1 Protein of Swine Influenza Virus Impair Anti-Interferon Activity and Confer Attenuation in Pigs

Journal of Virology ◽

10.1128/jvi.79.12.7535-7543.2005 ◽

2005 ◽

Vol 79 (12) ◽

pp. 7535-7543 ◽

Cited By ~ 176

Author(s):

Alicia Solórzano ◽

Richard J. Webby ◽

Kelly M. Lager ◽

Bruce H. Janke ◽

Adolfo García-Sastre ◽

...

Keyword(s):

Influenza Virus ◽

Nonstructural Protein ◽

Swine Influenza Virus ◽

Swine Influenza ◽

Influenza Viruses ◽

Model Systems ◽

Ns1 Protein ◽

Wild Type ◽

Virulence Attenuation

ABSTRACT It has been shown previously that the nonstructural protein NS1 of influenza virus is an alpha/beta interferon (IFN-α/β) antagonist, both in vitro and in experimental animal model systems. However, evidence of this function in a natural host has not yet been obtained. Here we investigated the role of the NS1 protein in the virulence of a swine influenza virus (SIV) isolate in pigs by using reverse genetics. The virulent wild-type A/Swine/Texas/4199-2/98 (TX/98) virus and various mutants encoding carboxy-truncated NS1 proteins were rescued. Growth properties of TX/98 viruses with mutated NS1, induction of IFN in tissue culture, and virulence-attenuation in pigs were analyzed and compared to those of the recombinant wild-type TX/98 virus. Our results indicate that deletions in the NS1 protein decrease the ability of the TX/98 virus to prevent IFN-α/β synthesis in pig cells. Moreover, all NS1 mutant viruses were attenuated in pigs, and this correlated with the amount of IFN-α/β induced in vitro. These data suggest that the NS1 protein of SIV is a virulence factor. Due to their attenuation, NS1-mutated swine influenza viruses might have a great potential as live attenuated vaccine candidates against SIV infections of pigs.

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Are Ducks Contributing to the Endemicity of Highly Pathogenic H5N1 Influenza Virus in Asia?

Journal of Virology ◽

10.1128/jvi.79.17.11269-11279.2005 ◽

2005 ◽

Vol 79 (17) ◽

pp. 11269-11279 ◽

Cited By ~ 325

Author(s):

K. M. Sturm-Ramirez ◽

D. J. Hulse-Post ◽

E. A. Govorkova ◽

J. Humberd ◽

P. Seiler ◽

...

Keyword(s):

Influenza Virus ◽

Influenza A ◽

Virus Disease ◽

Influenza Viruses ◽

H5n1 Influenza Virus ◽

Highly Pathogenic ◽

Main Site ◽

H5n1 Influenza ◽

Pathogenic H5n1 ◽

Virus Isolates

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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Host DNA released by NETosis in neutrophils exposed to seasonal H1N1 and highly pathogenic H5N1 influenza viruses

10.21203/rs.2.24164/v1 ◽

2020 ◽

Author(s):

Louisa L.Y. Chan ◽

John M. Nicholls ◽

J.S. Malik Peiris ◽

Yu Lung Lau ◽

Michael C.W. Chan ◽

...

Keyword(s):

Influenza Virus ◽

Virus Infection ◽

H1n1 Virus ◽

Influenza Virus Infection ◽

Alveolar Epithelium ◽

Influenza Viruses ◽

H5n1 Influenza Virus ◽

H5n1 Influenza ◽

Influenza H1n1

Abstract Background Neutrophil (Nϕ) is of the most abundant number in human immune system. During acute influenza virus infection, Nϕs are already active in the early phase of inflammation-a time in which clinical biopsy or autopsy material is not readily available. However, the role of Nϕ in virus infection is not well understood. Here, we studied the role of Nϕ in host defense during influenza A virus infection, specifically assessing if it contributes to the differential pathogenesis in H5N1 disease. Methods Nϕs were freshly isolated from healthy volunteers and subjected to direct influenza H1N1 and H5N1 virus infection in vitro . The ability of the naïve Nϕs to infiltrate from the basolateral to the apical phase of the influenza virus infected alveolar epithelium was assessed. The viral replication, innate immune responses and Neutrophil extracellular trap (NET) formation of Nϕs upon influenza virus infection were evaluated. Results Our results demonstrated that influenza virus infected alveolar epithelium allowed more Nϕs transmigration. Significantly more Nϕs migrated across the H5N1 influenza virus infected the epithelium than the counterpart infected by the seasonal influenza H1N1 virus infected. Nϕs were equally susceptible to H5N1 and H1N1 virus infection with similar viral gene transcription. Productive replication was observed in H5N1 infected Nϕs. Both H5N1 and H1N1 infected Nϕs induced cytokines and chemokines including TNF-α, IFN-β, CXCL10, MIP-1α and IL-8. This inferred a more intense inflammatory response posed by H5N1 than H1N1 virus. Strikingly, NADPH oxidase-independent NET formation was observed in H1N1 infected Nϕs at 6 hpi while no NET formation was observed upon H5N1 infection. Conclusion Our data is the first to demonstrate that NET formation is abrogated in H5N1 influenza virus infection. Its contribution to the differential severity of H5N1 disease requires further investigation.

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The pH of Activation of the Hemagglutinin Protein Regulates H5N1 Influenza Virus Pathogenicity and Transmissibility in Ducks

Journal of Virology ◽

10.1128/jvi.02069-09 ◽

2009 ◽

Vol 84 (3) ◽

pp. 1527-1535 ◽

Cited By ~ 91

Author(s):

Mark L. Reed ◽

Olga A. Bridges ◽

Patrick Seiler ◽

Jeong-Ki Kim ◽

Hui-Ling Yen ◽

...

Keyword(s):

Weight Loss ◽

Influenza Virus ◽

Membrane Fusion ◽

Influenza Viruses ◽

Wild Type Virus ◽

Type Virus ◽

Wild Type ◽

H5n1 Influenza ◽

Ha Protein

ABSTRACT While the molecular mechanism of membrane fusion by the influenza virus hemagglutinin (HA) protein has been studied extensively in vitro, the role of acid-dependent HA protein activation in virus replication, pathogenesis, and transmission in vivo has not been characterized. To investigate the biological significance of the pH of activation of the HA protein, we compared the properties of four recombinant viruses with altered HA protein acid stability to those of wild-type influenza virus A/chicken/Vietnam/C58/04 (H5N1) in vitro and in mallards. Membrane fusion by wild-type virus was activated at pH 5.9. Wild-type virus had a calculated environmental persistence of 62 days and caused extensive morbidity, mortality, shedding, and transmission in mallards. An N114K mutation that increased the pH of HA activation by 0.5 unit resulted in decreased replication, genetic stability, and environmental stability. Changes of +0.4 and −0.5 unit in the pH of activation by Y23H and K58I mutations, respectively, reduced weight loss, mortality, shedding, and transmission in mallards. An H24Q mutation that decreased the pH of activation by 0.3 unit resulted in weight loss, mortality, clinical symptoms, and shedding similar to those of the wild type. However, the HA-H241Q virus was shed more extensively into drinking water and persisted longer in the environment. The pH of activation of the H5 HA protein plays a key role in the propagation of H5N1 influenza viruses in ducks and may be a novel molecular factor in the ecology of influenza viruses. The data also demonstrate that H5N1 neuraminidase activity increases the pH of activation of the HA protein in vitro.

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An Anti-H5N1 Influenza Virus FcDART Antibody Is a Highly Efficacious Therapeutic Agent and Prophylactic against H5N1 Influenza Virus Infection

Journal of Virology ◽

10.1128/jvi.00078-15 ◽

2015 ◽

Vol 89 (8) ◽

pp. 4549-4561 ◽

Cited By ~ 6

Author(s):

Mark Zanin ◽

Zhen-Yong Keck ◽

G. Jonah Rainey ◽

Chia-Ying Kao Lam ◽

Adrianus C. M. Boon ◽

...

Keyword(s):

Influenza Virus ◽

Therapeutic Agent ◽

Influenza Viruses ◽

H5n1 Influenza Virus ◽

Avian Influenza Viruses ◽

Highly Pathogenic ◽

H5n1 Influenza ◽

Prophylactic Dose ◽

H5n1 Avian Influenza ◽

Pathogenic H5n1

ABSTRACTHighly pathogenic H5N1 avian influenza viruses are associated with severe disease in humans and continue to be a pandemic threat. While vaccines are available, other approaches are required for patients that typically respond poorly to vaccination, such as the elderly and the immunocompromised. To produce a therapeutic agent that is highly efficacious at low doses and is broadly specific against antigenically drifted H5N1 influenza viruses, we developed two neutralizing monoclonal antibodies and combined them into a single bispecific Fc fusion protein (the Fc dual-affinity retargeting [FcDART] molecule). In mice, a single therapeutic or prophylactic dose of either monoclonal antibody at 2.5 mg/kg of body weight provided 100% protection against challenge with A/Vietnam/1203/04 (H5N1) or the antigenically drifted strain A/Whooper swan/Mongolia/244/05 (H5N1). In ferrets, a single 1-mg/kg prophylactic dose provided 100% protection against A/Vietnam/1203/04 challenge. FcDART was also effective, as a single 2.5-mg/kg therapeutic or prophylactic dose in mice provided 100% protection against A/Vietnam/1203/04 challenge. Antibodies bound to conformational epitopes in antigenic sites on the globular head of the hemagglutinin protein, on the basis of analysis of mutants with antibody escape mutations. While it was possible to generate escape mutantsin vitro, they were neutralized by the antibodiesin vivo, as mice infected with escape mutants were 100% protected after only a single therapeutic dose of the antibody used to generate the escape mutantin vitro. In summary, we have combined the antigen specificities of two highly efficacious anti-H5N1 influenza virus antibodies into a bispecific FcDART molecule, which represents a strategy to produce broadly neutralizing antibodies that are effective against antigenically diverse influenza viruses.IMPORTANCEHighly pathogenic H5N1 avian influenza viruses are associated with severe disease in humans and are a pandemic threat. A vaccine is available, but other approaches are required for patients that typically respond poorly to vaccination, such as the elderly and the immunocompromised. The variability of the virus means that such an approach must be broad spectrum. To achieve this, we developed two antibodies that neutralize H5N1 influenza viruses. In mice, these antibodies provided complete protection against a spectrum of H5N1 influenza viruses at a single low dose. We then combined the two antibodies into a single molecule, FcDART, which combined the broad-spectrum activity and protective efficacy of both antibodies. This treatment provides a novel and effective therapeutic agent or prophylactic with activity against highly pathogenic H5N1 avian influenza viruses.

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Cross-Recognition of Avian H5N1 Influenza Virus by Human Cytotoxic T-Lymphocyte Populations Directed to Human Influenza A Virus

Journal of Virology ◽

10.1128/jvi.02694-07 ◽

2008 ◽

Vol 82 (11) ◽

pp. 5161-5166 ◽

Cited By ~ 166

Author(s):

J. H. C. M. Kreijtz ◽

G. de Mutsert ◽

C. A. van Baalen ◽

R. A. M. Fouchier ◽

A. D. M. E. Osterhaus ◽

...

Keyword(s):

Influenza Virus ◽

T Lymphocyte ◽

Influenza A ◽

Cytotoxic T Lymphocyte ◽

Cross Reactivity ◽

Influenza Viruses ◽

H5n1 Influenza Virus ◽

H5n1 Influenza ◽

Cell Immunity ◽

The Impact

ABSTRACT Since the number of human cases of infection with avian H5N1 influenza viruses is ever increasing, a pandemic outbreak caused by these viruses is feared. Therefore, in addition to virus-specific antibodies, there is considerable interest in immune correlates of protection against these viruses, which could be a target for the development of more universal vaccines. After infection with seasonal influenza A viruses of the H3N2 and H1N1 subtypes, individuals develop virus-specific cytotoxic T-lymphocyte responses, which are mainly directed against the relatively conserved internal proteins of the virus, like the nucleoprotein (NP). Virus-specific cytotoxic T lymphocytes (CTL) are known to contribute to protective immunity against infection, but knowledge about the extent of cross-reactivity with avian H5N1 influenza viruses is sparse. In the present study, we evaluated the cross-reactivity with H5N1 influenza viruses of polyclonal CTL obtained from a group of well-defined HLA-typed study subjects. To this end, the recognition of synthetic peptides representing H5N1 analogues of known CTL epitopes was studied. In addition, the ability of CTL specific for seasonal H3N2 influenza virus to recognize the NP of H5N1 influenza virus or H5N1 virus-infected cells was tested. It was concluded that, apart from some individual epitopes that displayed amino acid variation between H3N2 and H5N1 influenza viruses, considerable cross-reactivity exists with H5N1 viruses. This preexisting cross-reactive T-cell immunity in the human population may dampen the impact of a next pandemic.

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Intranasal Delivery of an IgA Monoclonal Antibody Effective against Sublethal H5N1 Influenza Virus Infection in Mice

Clinical and Vaccine Immunology ◽

10.1128/cvi.00002-10 ◽

2010 ◽

Vol 17 (9) ◽

pp. 1363-1370 ◽

Cited By ~ 21

Author(s):

Jianqiang Ye ◽

Hongxia Shao ◽

Danielle Hickman ◽

Matthew Angel ◽

Kemin Xu ◽

...

Keyword(s):

Monoclonal Antibody ◽

Influenza Virus ◽

Antiviral Treatment ◽

Influenza Virus Infection ◽

Influenza Viruses ◽

Sublethal Dose ◽

H5n1 Influenza Virus ◽

Highly Pathogenic ◽

H5n1 Influenza

ABSTRACT Highly pathogenic avian H5N1 influenza viruses are endemic in poultry in Asia and pose a pandemic threat to humans. Since the deployment of vaccines against a pandemic strain may take several months, adequate antiviral alternatives are needed to minimize the effects and the spread of the disease. Passive immunotherapy is regarded as a viable alternative. Here, we show the development of an IgA monoclonal antibody (DPJY01 MAb) specific to H5 hemagglutinin. The DPJY01 MAb showed a broad hemagglutination inhibition (HI) profile against Asian H5N1 viruses of clades 0, 1.0, 2.1, 2.2, and 2.3 and also against H5 wild bird influenza viruses of the North American and Eurasian lineages. DPJY01 MAb displayed also high neutralization activity in vitro and in vivo. In mice, DPJY01 MAb provided protection via a single dose administered intranasally before or after inoculation with a sublethal dose of H5N1 viruses of clades 1.0 and 2.2. Pretreatment with 50 mg of DPJY01 MAb kg of body weight at either 24, 48, or 72 h before highly pathogenic H5N1 virus (A/Vietnam/1203/2004 [H5N1]) inoculation resulted in complete protection. Treatment with 50 mg/kg at either at 24, 48, or 72 h after H5N1 inoculation provided 100%, 80%, and 60% protection, respectively. These studies highlight the potential use of DPJY01 MAb as an intranasal antiviral treatment for H5N1 influenza virus infections.

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