H5N1 Influenza Virus Pathogenesis in Genetically Diverse Mice Is Mediated at the Level of Viral Load

ABSTRACTThe genotype of the host is one of several factors involved in the pathogenesis of an infectious disease and may be a key parameter in the epidemiology of highly pathogenic H5N1 influenza virus infection in humans. Gene polymorphisms may affect the viral replication rate or alter the host’s immune response to the virus. In humans, it is unclear which aspect dictates the severity of H5N1 virus disease. To identify the mechanism underlying differential responses to H5N1 virus infection in a genetically diverse population, we assessed the host responses and lung viral loads in 21 inbred mouse strains upon intranasal inoculation with A/Hong Kong/213/03 (H5N1). Resistant mouse strains survived large inocula while susceptible strains succumbed to infection with 1,000- to 10,000-fold-lower doses. Quantitative analysis of the viral load after inoculation with an intermediate dose found significant associations with lethality as early as 2 days postinoculation, earlier than any other disease indicator. The increased viral titers in the highly susceptible strains mediated a hyperinflamed environment, indicated by the distinct expression profiles and increased production of inflammatory mediators on day 3. Supporting the hypothesis that viral load rather than an inappropriate response to the virus was the key severity-determining factor, we performed quantitative real-time PCR measuring the cytokine/viral RNA ratio. No significant differences between susceptible and resistant mouse strains were detected, confirming that it is the host genetic component controlling viral load, and therefore replication dynamics, that is primarily responsible for a host’s susceptibility to a given H5N1 virus.IMPORTANCEHighly pathogenic H5N1 influenza virus has circulated in Southeast Asia since 2003 but has been confirmed in relatively few individuals. It has been postulated that host genetic polymorphisms increase the susceptibility to infection and severe disease. The mechanisms and host proteins affected during severe disease are unknown. Inbred mouse strains vary considerably in their ability to resist H5N1 virus and were used to identify the primary mechanism determining disease severity. After inoculation with H5N1, resistant mouse strains had reduced amounts of virus in their lungs, which subsequently resulted in lower production of proinflammatory mediators and less pathology. We therefore conclude that the host genetic component controlling disease severity is primarily influencing viral replication. This is an important concept, as it emphasizes the need to limit virus replication through antiviral therapies and it shows that the hyperinflammatory environment is simply a reflection of more viral genetic material inducing a response.

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Epitope Mapping of the Hemagglutinin Molecule of a Highly Pathogenic H5N1 Influenza Virus by Using Monoclonal Antibodies

Journal of Virology ◽

10.1128/jvi.01522-07 ◽

2007 ◽

Vol 81 (23) ◽

pp. 12911-12917 ◽

Cited By ~ 136

Author(s):

Nikolai V. Kaverin ◽

Irina A. Rudneva ◽

Elena A. Govorkova ◽

Tatyana A. Timofeeva ◽

Aleksandr A. Shilov ◽

...

Keyword(s):

Influenza Virus ◽

Amino Acid ◽

H5n1 Virus ◽

Antigenic Site ◽

H5n1 Influenza Virus ◽

Highly Pathogenic ◽

H5n1 Influenza ◽

Antigenic Sites ◽

Escape Mutants ◽

Pathogenic H5n1

ABSTRACT We mapped the hemagglutinin (HA) antigenic epitopes of a highly pathogenic H5N1 influenza virus on the three-dimensional HA structure by characterizing escape mutants of a recombinant virus containing A/Vietnam/1203/04 (H5N1) ΔHA and neuraminidase genes in the genetic background of A/Puerto Rico/8/34 (H1N1) virus. The mutants were selected with a panel of eight anti-HA monoclonal antibodies (MAbs), seven to A/Vietnam/1203/04 (H5N1) virus and one to A/Chicken/Pennsylvania/8125/83 (H5N2) virus, and the mutants’ HA genes were sequenced. The amino acid changes suggested three MAb groups: four MAbs reacted with the complex epitope comprising parts of the antigenic site B of H3 HA and site Sa of H1 HA, two MAbs reacted with the epitope corresponding to the antigenic site A in H3 HA, and two MAbs displayed unusual behavior: each recognized amino acid changes at two widely separate antigenic sites. Five changes were detected in amino acid residues not previously reported as changed in H5 escape mutants, and four others had substitutions not previously described. The HA antigenic structure differs substantially between A/Vietnam/1203/04 (H5N1) virus and the low-pathogenic A/Mallard/Pennsylvania/10218/84 (H5N2) virus we previously characterized (N. V. Kaverin et al., J. Gen. Virol. 83:2497-2505, 2002). The hemagglutination inhibition reactions of the MAbs with recent highly pathogenic H5N1 viruses were consistent with the antigenic-site amino acid changes but not with clades and subclades based on H5 phylogenetic analysis. These results provide information on the recognition sites of the MAbs widely used to study H5N1 viruses and demonstrate the involvement of the HA antigenic sites in the evolution of highly pathogenic H5N1 viruses, findings that can be critical for characterizing pathogenesis and vaccine design.

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A single dose of a vesicular stomatitis virus-based influenza vaccine confers rapid protection against H5 viruses from different clades

npj Vaccines ◽

10.1038/s41541-019-0155-z ◽

2020 ◽

Vol 5 (1) ◽

Cited By ~ 12

Author(s):

Wakako Furuyama ◽

Pierce Reynolds ◽

Elaine Haddock ◽

Kimberly Meade-White ◽

Mai Quynh Le ◽

...

Keyword(s):

Influenza Virus ◽

Single Dose ◽

Vesicular Stomatitis Virus ◽

Severe Disease ◽

Lethal Dose ◽

H5n1 Influenza Virus ◽

Lethal Challenge ◽

H5n1 Influenza ◽

Vesicular Stomatitis ◽

Fast Acting

AbstractThe avian influenza virus outbreak in 1997 highlighted the potential of the highly pathogenic H5N1 virus to cause severe disease in humans. Therefore, effective vaccines against H5N1 viruses are needed to counter the potential threat of a global pandemic. We have previously developed a fast-acting and efficacious vaccine against Ebola virus (EBOV) using the vesicular stomatitis virus (VSV) platform. In this study, we generated recombinant VSV-based H5N1 influenza virus vectors to demonstrate the feasibility of this platform for a fast-acting pan-H5 influenza virus vaccine. We chose multiple approaches regarding antigen design and genome location to define a more optimized vaccine approach. After the VSV-based H5N1 influenza virus constructs were recovered and characterized in vitro, mice were vaccinated by a single dose or prime/boost regimen followed by challenge with a lethal dose of the homologous H5 clade 1 virus. We found that a single dose of VSV vectors expressing full-length hemagglutinin (HAfl) were sufficient to provide 100% protection. The vaccine vectors were fast-acting as demonstrated by uniform protection when administered 3 days prior to lethal challenge. Moreover, single vaccination induced cross-protective H5-specific antibodies and protected mice against lethal challenge with various H5 clade 2 viruses, highlighting the potential of the VSV-based HAfl as a pan-H5 influenza virus emergency vaccine.

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Characterization of a highly pathogenic H5N1 influenza virus derived from bar-headed geese in China

Journal of General Virology ◽

10.1099/vir.0.81800-0 ◽

2006 ◽

Vol 87 (7) ◽

pp. 1823-1833 ◽

Cited By ~ 70

Author(s):

Ji-Yong Zhou ◽

Hui-Gang Shen ◽

Hong-Xun Chen ◽

Guang-Zhi Tong ◽

Ming Liao ◽

...

Keyword(s):

Influenza Virus ◽

Avian Influenza ◽

Avian Influenza Virus ◽

H5n1 Virus ◽

H5n1 Influenza Virus ◽

Highly Pathogenic ◽

H5n1 Influenza ◽

H5n1 Avian Influenza ◽

Pathogenic H5n1 ◽

Migratory Waterbirds

Influenza A viruses are usually non-pathogenic in wild aquatic birds, their natural reservoir. However, from May to July 2005, at Qinghai Lake in China, an unprecedented outbreak of highly pathogenic H5N1 avian influenza virus caused the death of thousands of wild migratory waterbirds. Herein, H5N1 influenza virus from bar-headed geese collected during the outbreak was characterized. Genomic analysis showed that A/Bar-headed Goose/Qinghai/0510/05 (Bh H5N1 virus) is a reassortant virus. Amino acid residue (lysine) at position 627 in the PB2 gene of the Bh H5N1 virus was the same as that of the human H5N1 virus (A/HK/483/97) and different from that of H5N1 avian influenza viruses deposited in GenBank. Antigenic analysis showed that significant antigenic variation has occurred in the Bh H5N1 virus. The Bh H5N1 virus induced systemic infections and caused 100 % mortality in chickens and mice, and 80 % mortality in ducks and geese. Bh H5N1 virus titres were higher in multiple organs of chickens, ducks and geese than in mice, and caused more severe histological lesions in chickens, ducks and mice than in geese. These results support the need to pay close attention and create control programmes to prevent the transmission of highly pathogenic avian influenza virus from wild migratory waterbirds into domestic chickens, ducks, geese and mammalian hosts.

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Predicting Disease Severity and Viral Spread of H5N1 Influenza Virus in Ferrets in the Context of Natural Exposure Routes

Journal of Virology ◽

10.1128/jvi.01878-15 ◽

2015 ◽

Vol 90 (4) ◽

pp. 1888-1897 ◽

Cited By ~ 5

Author(s):

Kathryn M. Edenborough ◽

Suzanne Lowther ◽

Karen Laurie ◽

Manabu Yamada ◽

Fenella Long ◽

...

Keyword(s):

Influenza Virus ◽

Respiratory Tract ◽

Disease Severity ◽

Severe Disease ◽

Upper Respiratory Tract ◽

H5n1 Influenza Virus ◽

Viral Spread ◽

H5n1 Influenza ◽

Upper Respiratory ◽

The Brain

ABSTRACTAlthough avian H5N1 influenza virus has yet to develop the capacity for human-to-human spread, the severity of the rare cases of human infection has warranted intensive follow-up of potentially exposed individuals that may require antiviral prophylaxis. For countries where antiviral drugs are limited, the World Health Organization (WHO) has developed a risk categorization for different levels of exposure to environmental, poultry, or human sources of infection. While these take into account the infection source, they do not account for the likely mode of virus entry that the individual may have experienced from that source and how this could affect the disease outcome. Knowledge of the kinetics and spread of virus after natural routes of exposure may further inform the risk of infection, as well as the likely disease severity. Using the ferret model of H5N1 infection, we compared the commonly used but artificial inoculation method that saturates the total respiratory tract (TRT) with virus to upper respiratory tract (URT) and oral routes of delivery, those likely to be encountered by humans in nature. We show that there was no statistically significant difference in survival rate with the different routes of infection, but the disease characteristics were somewhat different. Following URT infection, viral spread to systemic organs was comparatively delayed and more focal than after TRT infection. By both routes, severe disease was associated with early viremia and central nervous system infection. After oral exposure to the virus, mild infections were common suggesting consumption of virus-contaminated liquids may be associated with seroconversion in the absence of severe disease.IMPORTANCERisks for human H5N1 infection include direct contact with infected birds and frequenting contaminated environments. We used H5N1 ferret infection models to show that breathing in the virus was more likely to produce clinical infection than swallowing contaminated liquid. We also showed that virus could spread from the respiratory tract to the brain, which was associated with end-stage disease, and very early viremia provided a marker for this. With upper respiratory tract exposure, infection of the brain was common but hard to detect, suggesting that human neurological infections might be typically undetected at autopsy. However, viral spread to systemic sites was slower after exposure to virus by this route than when virus was additionally delivered to the lungs, providing a better therapeutic window. In addition to exposure history, early parameters of infection, such as viremia, could help prioritize antiviral treatments for patients most at risk of succumbing to infection.

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Neurovirulence in Mice of H5N1 Influenza Virus Genotypes Isolated from Hong Kong Poultry in 2001

Journal of Virology ◽

10.1128/jvi.77.6.3816-3823.2003 ◽

2003 ◽

Vol 77 (6) ◽

pp. 3816-3823 ◽

Cited By ~ 53

Author(s):

Aleksandr S. Lipatov ◽

Scott Krauss ◽

Yi Guan ◽

Malik Peiris ◽

Jerold E. Rehg ◽

...

Keyword(s):

Influenza Virus ◽

Amino Acid ◽

H5n1 Virus ◽

Gene Products ◽

H5n1 Influenza Virus ◽

Highly Pathogenic ◽

H5n1 Influenza ◽

B Virus ◽

Virus Genotypes ◽

The Brain

ABSTRACT We studied the pathogenicity of five different genotypes (A to E) of highly pathogenic avian H5N1 viruses, which contained HA genes similar to those of the H5N1 virus A/goose/Guangdong/1/96 and five different combinations of “internal” genes, in a mouse model. Highly pathogenic, neurotropic variants of genotypes A, C, D, and E were isolated from the brain after a single intranasal passage in mice. Genotype B virus was isolated from lungs only. The mouse brain variants had amino acid changes in all gene products except PB1, NP, and NS1 proteins but no common sets of mutations. We conclude that the original H5N1/01 isolates of genotypes A, C, D, and E were heterogeneous and that highly pathogenic neurotropic variants can be rapidly selected in mice.

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Aerosolized Exposure to H5N1 Influenza Virus Causes Less Severe Disease Than Infection via Combined Intrabronchial, Oral, and Nasal Inoculation in Cynomolgus Macaques

Viruses ◽

10.3390/v13020345 ◽

2021 ◽

Vol 13 (2) ◽

pp. 345

Author(s):

Petra Mooij ◽

Marieke A. Stammes ◽

Daniella Mortier ◽

Zahra Fagrouch ◽

Nikki van Driel ◽

...

Keyword(s):

Influenza Virus ◽

Immune Activation ◽

Influenza Infection ◽

Severe Disease ◽

Fatal Outcome ◽

H5n1 Influenza Virus ◽

Highly Pathogenic ◽

H5n1 Influenza ◽

Route Of Infection ◽

High Level

Infection with highly pathogenic avian H5N1 influenza virus in humans often leads to severe respiratory disease with high mortality. Experimental infection in non-human primates can provide additional insight into disease pathogenesis. However, such a model should recapitulate the disease symptoms observed in humans, such as pneumonia and inflammatory cytokine response. While previous studies in macaques have demonstrated the occurrence of typical lesions in the lungs early after infection and a high level of immune activation, progression to severe disease and lethality were rarely observed. Here, we evaluated a routinely used combined route of infection via intra-bronchial, oral, and intra-nasal virus inoculation with aerosolized H5N1 exposure, with or without the regular collection of bronchoalveolar lavages early after infection. Both combined route and aerosol exposure resulted in similar levels of virus replication in nose and throat and similar levels of immune activation, cytokine, and chemokine release in the blood. However, while animals exposed to H5N1 by combined-route inoculation developed severe disease with high lethality, aerosolized exposure resulted in less lesions, as measured by consecutive computed tomography and less fever and lethal disease. In conclusion, not virus levels or immune activation, but route of infection determines fatal outcome for highly pathogenic avian H5N1 influenza infection.

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