Proteolytic cleavage of influenza virus hemagglutinins: primary structure of the connecting peptide between HA1 and HA2 determines proteolytic cleavability and pathogenicity of avian influenza viruses

H7 low pathogenic avian influenza viruses (LPAIVs) can mutate into highly pathogenic avian influenza viruses (HPAIVs). In addition to avian species, H7 avian influenza viruses (AIVs) also infect humans. In this study, two AIVs, H7N9 (20X-20) and H7N7 (34X-2), isolated from the feces of wild birds in South Korea in 2021, were genetically analyzed. The HA cleavage site of the two H7 Korean viruses was confirmed to be ELPKGR/GLF, indicating they are LPAIVs. There were no amino acid substitutions at the receptor-binding site of the HA gene of two H7 Korean viruses compared to that of A/Anhui/1/2013 (H7N9), which prefer human receptors. In the phylogenetic tree analysis, the HA gene of the two H7 Korean viruses shared the highest nucleotide similarity with the Korean H7 subtype AIVs. In addition, the HA gene of the two H7 Korean viruses showed high nucleotide similarity to that of the A/Jiangsu/1/2018(H7N4) virus, which is a human influenza virus originating from avian influenza virus. Most internal genes (PB2, PB1, PA, NP, NA, M, and NS) of the two H7 Korean viruses belonged to the Eurasian lineage, except for the M gene of 34X-2. This result suggests that active reassortment occurred among AIVs. In pathogenicity studies of mice, the two H7 Korean viruses replicated in the lungs of mice. In addition, the body weight of mice infected with 34X-2 decreased 7 days post-infection (dpi) and inflammation was observed in the peribronchiolar and perivascular regions of the lungs of mice. These results suggest that mammals can be infected with the two H7 Korean AIVs. Our data showed that even low pathogenic H7 AIVs may infect mammals, including humans, as confirmed by the A/Jiangsu/1/2018(H7N4) virus. Therefore, continuous monitoring and pathogenicity assessment of AIVs, even of LPAIVs, are required.

Download Full-text

Development of a duplex TaqMan real-time RT-PCR assay for simultaneous detection of newly emerged H5N6 influenza viruses

Virology Journal ◽

10.1186/s12985-019-1229-2 ◽

2019 ◽

Vol 16 (1) ◽

Cited By ~ 1

Author(s):

Lin Liu ◽

Ying Zhang ◽

Pengfei Cui ◽

Congcong Wang ◽

Xianying Zeng ◽

...

Keyword(s):

Influenza Virus ◽

Avian Influenza ◽

Simultaneous Detection ◽

Reaction System ◽

Taxonomic Status ◽

Human Infection ◽

Influenza Viruses ◽

Rt Pcr ◽

Avian Influenza Viruses ◽

Pcr Assay

Abstract Background In 2017–2018, a new highly pathogenic H5N6 avian influenza virus (AIV) variant appeared in poultry and wild birds in Asian and European countries and caused multiple outbreaks. These variant strains are different from the H5N6 virus associated with human infection in previous years, and their genetic taxonomic status and antigenicity have changed. Therefore, revision of the primers and probes of fluorescent RT-PCR is important to detect the new H5N6 subtype AIV in poultry and reduce the risk of an epidemic in birds or humans. Methods In this study, the primers and probes including three groups of HA and four groups of NA for H5N6 influenza virus were evaluated. Then a set of ideal primer and probes were selected to further optimize the reaction system and established a method of double rRT-PCR assay. The specificity of this method was determined by using H1~H16 subtype AIV. Results The results showed that fluorescence signals were obtained for H5 virus in FAM channel and N6 virus in VIC channel, and no fluorescent signal was observed in other subtypes of avian influenza viruses. The detection limit of this assay was 69 copies for H5 and 83 copies for N6 gene. And, the variability tests of intra- and inter-assay showed excellent reproducibility. Moreover, this assay showed 100% agreement with virus isolation method in detecting samples from poultry. Conclusion The duplex rRT-PCR assay presented here has high specificity, sensitivity and reproducibility, and can be used for laboratory surveillance and rapid diagnosis of newly emerged H5N6 subtype avian influenza viruses.

Download Full-text

Swine ANP32A Supports Avian Influenza Virus Polymerase

Journal of Virology ◽

10.1128/jvi.00132-20 ◽

2020 ◽

Vol 94 (12) ◽

Cited By ~ 2

Author(s):

Thomas P. Peacock ◽

Olivia C. Swann ◽

Hamish A. Salvesen ◽

Ecco Staller ◽

P. Brian Leung ◽

...

Keyword(s):

Influenza Virus ◽

Avian Influenza ◽

Avian Influenza Virus ◽

Virus Replication ◽

Mammalian Species ◽

Gene Mutations ◽

Influenza Viruses ◽

Avian Influenza Viruses ◽

2009 H1n1 ◽

Mixing Vessels

ABSTRACT Avian influenza viruses occasionally infect and adapt to mammals, including humans. Swine are often described as “mixing vessels,” being susceptible to both avian- and human-origin viruses, which allows the emergence of novel reassortants, such as the precursor to the 2009 H1N1 pandemic. ANP32 proteins are host factors that act as influenza virus polymerase cofactors. In this study, we describe how swine ANP32A, uniquely among the mammalian ANP32 proteins tested, supports the activity of avian-origin influenza virus polymerases and avian influenza virus replication. We further show that after the swine-origin influenza virus emerged in humans and caused the 2009 pandemic, it evolved polymerase gene mutations that enabled it to more efficiently use human ANP32 proteins. We map the enhanced proviral activity of swine ANP32A to a pair of amino acids, 106 and 156, in the leucine-rich repeat and central domains and show these mutations enhance binding to influenza virus trimeric polymerase. These findings help elucidate the molecular basis for the mixing vessel trait of swine and further our understanding of the evolution and ecology of viruses in this host. IMPORTANCE Avian influenza viruses can jump from wild birds and poultry into mammalian species such as humans or swine, but they only continue to transmit if they accumulate mammalian adapting mutations. Pigs appear uniquely susceptible to both avian and human strains of influenza and are often described as virus “mixing vessels.” In this study, we describe how a host factor responsible for regulating virus replication, ANP32A, is different between swine and humans. Swine ANP32A allows a greater range of influenza viruses, specifically those from birds, to replicate. It does this by binding the virus polymerase more tightly than the human version of the protein. This work helps to explain the unique properties of swine as mixing vessels.

Download Full-text

Generation of Live Attenuated Influenza Virus by Using Codon Usage Bias

Journal of Virology ◽

10.1128/jvi.01443-15 ◽

2015 ◽

Vol 89 (21) ◽

pp. 10762-10773 ◽

Cited By ~ 20

Author(s):

Rebecca L. Y. Fan ◽

Sophie A. Valkenburg ◽

Chloe K. S. Wong ◽

Olive T. W. Li ◽

John M. Nicholls ◽

...

Keyword(s):

Influenza Virus ◽

Avian Influenza ◽

Codon Usage ◽

Codon Bias ◽

Seasonal Influenza ◽

Viral Proteins ◽

Influenza Viruses ◽

Donor Strain ◽

Avian Influenza Viruses ◽

Attenuated Viruses

ABSTRACTSeasonal influenza epidemics and occasional pandemics threaten public health worldwide. New alternative strategies for generating recombinant viruses with vaccine potential are needed. Interestingly, influenza viruses circulating in different hosts have been found to have distinct codon usage patterns, which may reflect host adaptation. We therefore hypothesized that it is possible to make a human seasonal influenza virus that is specifically attenuated in human cells but not in eggs by converting its codon usage so that it is similar to that observed from avian influenza viruses. This approach might help to generate human live attenuated viruses without affecting their yield in eggs. To test this hypothesis, over 300 silent mutations were introduced into the genome of a seasonal H1N1 influenza virus. The resultant mutant was significantly attenuated in mammalian cells and mice, yet it grew well in embryonated eggs. A single dose of intranasal vaccination induced potent innate, humoral, and cellular immune responses, and the mutant could protect mice against homologous and heterologous viral challenges. The attenuated mutant could also be used as a vaccine master donor strain by introducing hemagglutinin and neuraminidase genes derived from other strains. Thus, our approach is a successful strategy to generate attenuated viruses for future application as vaccines.IMPORTANCEVaccination has been one of the best protective measures in combating influenza virus infection. Current licensed influenza vaccines and their production have various limitations. Our virus attenuation strategy makes use of the codon usage biases of human and avian influenza viruses to generate a human-derived influenza virus that is attenuated in mammalian hosts. This method, however, does not affect virus replication in eggs. This makes the resultant mutants highly compatible with existing egg-based vaccine production pipelines. The viral proteins generated from the codon bias mutants are identical to the wild-type viral proteins. In addition, our massive genome-wide mutational approach further minimizes the concern over reverse mutations. The potential use of this kind of codon bias mutant as a master donor strain to generate other live attenuated viruses is also demonstrated. These findings put forward a promising live attenuated influenza vaccine generation strategy to control influenza.

Download Full-text

A review of H5Nx avian influenza viruses

Therapeutic Advances in Vaccines and Immunotherapy ◽

10.1177/2515135518821625 ◽

2019 ◽

Vol 7 ◽

pp. 251513551882162 ◽

Cited By ~ 9

Author(s):

Ivette A. Nuñez ◽

Ted M. Ross

Keyword(s):

Influenza Virus ◽

Avian Influenza ◽

Avian Influenza Virus ◽

Highly Pathogenic Avian Influenza ◽

Influenza Viruses ◽

Avian Influenza Viruses ◽

Highly Pathogenic ◽

Bird Populations ◽

Pathogenic Avian Influenza ◽

Pathogenic Avian Influenza Virus

Highly pathogenic avian influenza viruses (HPAIVs), originating from the A/goose/Guangdong/1/1996 H5 subtype, naturally circulate in wild-bird populations, particularly waterfowl, and often spill over to infect domestic poultry. Occasionally, humans are infected with HPAVI H5N1 resulting in high mortality, but no sustained human-to-human transmission. In this review, the replication cycle, pathogenicity, evolution, spread, and transmission of HPAIVs of H5Nx subtypes, along with the host immune responses to Highly Pathogenic Avian Influenza Virus (HPAIV) infection and potential vaccination, are discussed. In addition, the potential mechanisms for Highly Pathogenic Avian Influenza Virus (HPAIV) H5 Reassorted Viruses H5N1, H5N2, H5N6, H5N8 (H5Nx) viruses to transmit, infect, and adapt to the human host are reviewed.

Download Full-text

Constitutively Expressed IFITM3 Protein in Human Endothelial Cells Poses an Early Infection Block to Human Influenza Viruses

Journal of Virology ◽

10.1128/jvi.01254-16 ◽

2016 ◽

Vol 90 (24) ◽

pp. 11157-11167 ◽

Cited By ~ 15

Author(s):

Xiangjie Sun ◽

Hui Zeng ◽

Amrita Kumar ◽

Jessica A. Belser ◽

Taronna R. Maines ◽

...

Keyword(s):

Endothelial Cells ◽

Influenza Virus ◽

Virus Infection ◽

Avian Influenza ◽

Influenza Virus Infection ◽

Influenza Viruses ◽

Human Influenza ◽

Avian Influenza Viruses ◽

Human Influenza Virus ◽

Pulmonary Endothelial Cells

ABSTRACTA role for pulmonary endothelial cells in the orchestration of cytokine production and leukocyte recruitment during influenza virus infection, leading to severe lung damage, has been recently identified. As the mechanistic pathway for this ability is not fully known, we extended previous studies on influenza virus tropism in cultured human pulmonary endothelial cells. We found that a subset of avian influenza viruses, including potentially pandemic H5N1, H7N9, and H9N2 viruses, could infect human pulmonary endothelial cells (HULEC) with high efficiency compared to human H1N1 or H3N2 viruses. In HULEC, human influenza viruses were capable of binding to host cellular receptors, becoming internalized and initiating hemifusion but failing to uncoat the viral nucleocapsid and to replicate in host nuclei. Unlike numerous cell types, including epithelial cells, we found that pulmonary endothelial cells constitutively express a high level of the restriction protein IFITM3 in endosomal compartments. IFITM3 knockdown by small interfering RNA (siRNA) could partially rescue H1N1 virus infection in HULEC, suggesting IFITM3 proteins were involved in blocking human influenza virus infection in endothelial cells. In contrast, selected avian influenza viruses were able to escape IFITM3 restriction in endothelial cells, possibly by fusing in early endosomes at higher pH or by other, unknown mechanisms. Collectively, our study demonstrates that the human pulmonary endothelium possesses intrinsic immunity to human influenza viruses, in part due to the constitutive expression of IFITM3 proteins. Notably, certain avian influenza viruses have evolved to escape this restriction, possibly contributing to virus-induced pneumonia and severe lung disease in humans.IMPORTANCEAvian influenza viruses, including H5N1 and H7N9, have been associated with severe respiratory disease and fatal outcomes in humans. Although acute respiratory distress syndrome (ARDS) and progressive pulmonary endothelial damage are known to be present during severe human infections, the role of pulmonary endothelial cells in the pathogenesis of avian influenza virus infections is largely unknown. By comparing human seasonal influenza strains to avian influenza viruses, we provide greater insight into the interaction of influenza virus with human pulmonary endothelial cells. We show that human influenza virus infection is blocked during the early stages of virus entry, which is likely due to the relatively high expression of the host antiviral factors IFITMs (interferon-induced transmembrane proteins) located in membrane-bound compartments inside cells. Overall, this study provides a mechanism by which human endothelial cells limit replication of human influenza virus strains, whereas avian influenza viruses overcome these restriction factors in this cell type.

Download Full-text

Distinct Pathogenesis of Hong Kong-Origin H5N1 Viruses in Mice Compared to That of Other Highly Pathogenic H5 Avian Influenza Viruses

Journal of Virology ◽

10.1128/jvi.74.3.1443-1450.2000 ◽

2000 ◽

Vol 74 (3) ◽

pp. 1443-1450 ◽

Cited By ~ 97

Author(s):

Jody K. Dybing ◽

Stacey Schultz-Cherry ◽

David E. Swayne ◽

David L. Suarez ◽

Michael L. Perdue

Keyword(s):

Influenza Virus ◽

Hong Kong ◽

Avian Influenza ◽

Avian Influenza Virus ◽

Transforming Growth Factor ◽

Clinical Signs ◽

Influenza Viruses ◽

Rapid Weight Loss ◽

Avian Influenza Viruses ◽

Highly Pathogenic

ABSTRACT In 1997, an outbreak of virulent H5N1 avian influenza virus occurred in poultry in Hong Kong (HK) and was linked to a direct transmission to humans. The factors associated with transmission of avian influenza virus to mammals are not fully understood, and the potential risk of other highly virulent avian influenza A viruses infecting and causing disease in mammals is not known. In this study, two avian and one human HK-origin H5N1 virus along with four additional highly pathogenic H5 avian influenza viruses were analyzed for their pathogenicity in 6- to 8-week-old BALB/c mice. Both the avian and human HK H5 influenza virus isolates caused severe disease in mice, characterized by induced hypothermia, clinical signs, rapid weight loss, and 75 to 100% mortality by 6 to 8 days postinfection. Three of the non-HK-origin isolates caused no detectable clinical signs. One isolate, A/tk/England/91 (H5N1), induced measurable disease, and all but one of the animals recovered. Infections resulted in mild to severe lesions in both the upper and lower respiratory tracts. Most consistently, the viruses caused necrosis in respiratory epithelium of the nasal cavity, trachea, bronchi, and bronchioles with accompanying inflammation. The most severe and widespread lesions were observed in the lungs of HK avian influenza virus-infected mice, while no lesions or only mild lesions were evident with A/ck/Scotland/59 (H5N1) and A/ck/Queretaro/95 (H5N2). The A/ck/Italy/97 (H5N2) and the A/tk/England/91 (H5N1) viruses exhibited intermediate pathogenicity, producing mild to moderate respiratory tract lesions. In addition, infection by the different isolates could be further distinguished by the mouse immune response. The non-HK-origin isolates all induced production of increased levels of active transforming growth factor β following infection, while the HK-origin isolates did not.

Download Full-text

H7N9 and Other Pathogenic Avian Influenza Viruses Elicit a Three-Pronged Transcriptomic Signature That Is Reminiscent of 1918 Influenza Virus and Is Associated with Lethal Outcome in Mice

Journal of Virology ◽

10.1128/jvi.00570-14 ◽

2014 ◽

Vol 88 (18) ◽

pp. 10556-10568 ◽

Cited By ~ 33

Author(s):

J. Morrison ◽

L. Josset ◽

N. Tchitchek ◽

J. Chang ◽

J. A. Belser ◽

...

Keyword(s):

Influenza Virus ◽

Avian Influenza ◽

Influenza Viruses ◽

Avian Influenza Viruses ◽

Lethal Outcome ◽

Pathogenic Avian Influenza ◽

Transcriptomic Signature ◽

1918 Influenza

Download Full-text

The PB1 Gene from H9N2 Avian Influenza Virus Showed High Compatibility and Increased Mutation Rate after Reassorting with a Human H1N1 Influenza Virus

10.21203/rs.3.rs-907889/v1 ◽

2021 ◽

Author(s):

Hongrui Cui ◽

Guangsheng Che ◽

Mart CM de Jong ◽

Xuesong Li ◽

Qinfang Liu ◽

...

Keyword(s):

Influenza Virus ◽

Avian Influenza ◽

H1n1 Virus ◽

Polymerase Activity ◽

Influenza Viruses ◽

Luciferase Reporter ◽

Avian Influenza Viruses ◽

Reassortant Virus ◽

Rnp Complex ◽

Reassortant Viruses

Abstract BackgroundReassortment between human and avian influenza viruses (AIV) may result in novel viruses with new characteristics that may threaten human health when causing the next flu pandemic. A particular risk may be posed by avian influenza viruses of subtype H9N2 that are currently massively circulating in domestic poultry in Asia and have been shown to infect humans. In this study, we investigate the characteristics and compatibility of a human H1N1 virus with avian H9N2 derived genes. MethodsThe polymerase activity of the viral ribonucleoprotein (RNP) complex from different reassortments was tested in luciferase reporter assays. Reassortant viruses were generated by reverse genetics in which genes of the human WSN-H1N1 virus (A/WSN/1933) were replaced by genes of the avian A2093-H9N2 virus (A/chicken/Jiangsu/A2093/2011). We replaced both the Hemagglutinin (HA) and Neuraminidase (NA) genes in combination with one of the genes involved in the RNP complex (either PB2, PB1, PA or NP). The growth kinetics and virulence of reassortant viruses were tested on cell lines and mice. The reassortant viruses were then passaged for five generations in MDCK cells and mice lungs. The HA gene of progeny viruses from different passaging paths was analyzed using Next Generation Sequencing (NGS). ResultsWe discovered that the avian PB1 gene increased the polymerase activity of the RNP complex. Reassortant viruses were able to replicate in MDCK and DF1 cells and mice. Analysis of the NGS data showed a higher substitution rate for the PB1-reassortant virus. In particular, for the PB1-reassortant virus, increased virulence for mice was measured by increased body weight loss after infection in mice. ConclusionsThe higher polymerase activity and increased mutation frequency measured for the PB1-reassortant virus suggests that the avian PB1 gene may drive the evolution and adaptation of novel reassortant viruses to the human host. This study provides novel insights in the characteristics of novel viruses that may arise by reassortment of human and avian influenza viruses. Surveillance for infections with H9N2 viruses and the emergence of novel reassortant viruses in humans is important for pandemic preparedness.

Download Full-text