Neutralization activity of influenza A virus humanized antibodies against new subtypes of influenza viruses

The long-term control strategy of SARS-CoV-2 and other major respiratory viruses needs to include antivirals to treat acute infections, in addition to the judicious use of effective vaccines. Whilst COVID-19 vaccines are being rolled out for mass vaccination, the modest number of antivirals in use or development for any disease bears testament to the challenges of antiviral development. We recently showed that non-cytotoxic levels of thapsigargin (TG), an inhibitor of the sarcoplasmic/endoplasmic reticulum (ER) Ca2+ ATPase pump, induces a potent host innate immune antiviral response that blocks influenza A virus replication. Here we show that TG is also highly effective in blocking the replication of respiratory syncytial virus (RSV), common cold coronavirus OC43, SARS-CoV-2 and influenza A virus in immortalized or primary human cells. TG’s antiviral performance was significantly better than remdesivir and ribavirin in their respective inhibition of OC43 and RSV. Notably, TG was just as inhibitory to coronaviruses (OC43 and SARS-CoV-2) and influenza viruses (USSR H1N1 and pdm 2009 H1N1) in separate infections as in co-infections. Post-infection oral gavage of acid-stable TG protected mice against a lethal influenza virus challenge. Together with its ability to inhibit the different viruses before or during active infection, and with an antiviral duration of at least 48 h post-TG exposure, we propose that TG (or its derivatives) is a promising broad-spectrum inhibitor against SARS-CoV-2, OC43, RSV and influenza virus.

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Functional Analysis of PA Binding by Influenza A Virus PB1: Effects on Polymerase Activity and Viral Infectivity

Journal of Virology ◽

10.1128/jvi.75.17.8127-8136.2001 ◽

2001 ◽

Vol 75 (17) ◽

pp. 8127-8136 ◽

Cited By ~ 78

Author(s):

Daniel R. Perez ◽

Ruben O. Donis

Keyword(s):

Amino Acids ◽

Influenza Virus ◽

Amino Acid ◽

Viral Replication ◽

Influenza A Virus ◽

Influenza A ◽

Amino Acid Sequences ◽

Influenza Viruses ◽

Virus Growth ◽

Transcription And Replication

ABSTRACT Influenza A virus expresses three viral polymerase (P) subunits—PB1, PB2, and PA—all of which are essential for RNA and viral replication. The functions of P proteins in transcription and replication have been partially elucidated, yet some of these functions seem to be dependent on the formation of a heterotrimer for optimal viral RNA transcription and replication. Although it is conceivable that heterotrimer subunit interactions may allow a more efficient catalysis, direct evidence of their essentiality for viral replication is lacking. Biochemical studies addressing the molecular anatomy of the P complexes have revealed direct interactions between PB1 and PB2 as well as between PB1 and PA. Previous studies have shown that the N-terminal 48 amino acids of PB1, termed domain α, contain the residues required for binding PA. We report here the refined mapping of the amino acid sequences within this small region of PB1 that are indispensable for binding PA by deletion mutagenesis of PB1 in a two-hybrid assay. Subsequently, we used site-directed mutagenesis to identify the critical amino acid residues of PB1 for interaction with PA in vivo. The first 12 amino acids of PB1 were found to constitute the core of the interaction interface, thus narrowing the previous boundaries of domain α. The role of the minimal PB1 domain α in influenza virus gene expression and genome replication was subsequently analyzed by evaluating the activity of a set of PB1 mutants in a model reporter minigenome system. A strong correlation was observed between a functional PA binding site on PB1 and P activity. Influenza viruses bearing mutant PB1 genes were recovered using a plasmid-based influenza virus reverse genetics system. Interestingly, mutations that rendered PB1 unable to bind PA were either nonviable or severely growth impaired. These data are consistent with an essential role for the N terminus of PB1 in binding PA, P activity, and virus growth.

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Broad neutralization of H1 and H3 viruses by adjuvanted influenza HA stem vaccines in nonhuman primates

Science Translational Medicine ◽

10.1126/scitranslmed.abe5449 ◽

2021 ◽

Vol 13 (583) ◽

pp. eabe5449

Author(s):

Nicole Darricarrère ◽

Yu Qiu ◽

Masaru Kanekiyo ◽

Adrian Creanga ◽

Rebecca A. Gillespie ◽

...

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Nonhuman Primates ◽

Vaccine Development ◽

Protective Efficacy ◽

Neutralizing Antibody ◽

Gene Families ◽

Influenza Viruses ◽

Public Health Importance ◽

Water Emulsion

Seasonal influenza vaccines confer protection against specific viral strains but have restricted breadth that limits their protective efficacy. The H1 and H3 subtypes of influenza A virus cause most of the seasonal epidemics observed in humans and are the major drivers of influenza A virus–associated mortality. The consequences of pandemic spread of COVID-19 underscore the public health importance of prospective vaccine development. Here, we show that headless hemagglutinin (HA) stabilized-stem immunogens presented on ferritin nanoparticles elicit broadly neutralizing antibody (bnAb) responses to diverse H1 and H3 viruses in nonhuman primates (NHPs) when delivered with a squalene-based oil-in-water emulsion adjuvant, AF03. The neutralization potency and breadth of antibodies isolated from NHPs were comparable to human bnAbs and extended to mismatched heterosubtypic influenza viruses. Although NHPs lack the immunoglobulin germline VH1-69 residues associated with the most prevalent human stem-directed bnAbs, other gene families compensated to generate bnAbs. Isolation and structural analyses of vaccine-induced bnAbs revealed extensive interaction with the fusion peptide on the HA stem, which is essential for viral entry. Antibodies elicited by these headless HA stabilized-stem vaccines neutralized diverse H1 and H3 influenza viruses and shared a mode of recognition analogous to human bnAbs, suggesting that these vaccines have the potential to confer broadly protective immunity against diverse viruses responsible for seasonal and pandemic influenza infections in humans.

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Clinical Investigations. Comparison of Directigen Flu A+B with Real Time PCR in the Diagnosis of Influenza / Сравнение Иммунохроматографического Метода (Directigen Flu A+B) И Теста RT-PCR При Инфекциях Гриппа

Folia Medica ◽

10.1515/folmed-2015-0027 ◽

2015 ◽

Vol 57 (2) ◽

pp. 104-110 ◽

Cited By ~ 4

Author(s):

Golubinka Bosevska ◽

Nikola Panovski ◽

Elizabeta Janceska ◽

Vladimir Mikik ◽

Irena Kondova Topuzovska ◽

...

Keyword(s):

Real Time ◽

Influenza A Virus ◽

Real Time Pcr ◽

Influenza A ◽

Age Groups ◽

Influenza Viruses ◽

Influenza B Virus ◽

Influenza B ◽

B Virus ◽

Nose And Throat

AbstractEarly diagnosis and treatment of patients with influenza is the reason why physicians need rapid high-sensitivity influenza diagnostic tests that require no complex lab equipment and can be performed and interpreted within 15 min. The Aim of this study was to compare the rapid Directigen Flu A+B test with real time PCR for detection of influenza viruses in the Republic of Macedonia. MATERIALS AND METHODS: One-hundred-eight respiratory samples (combined nose and throat swabs) were routinely collected for detection of influenza virus during influenza seasons. Forty-one patients were pediatric cases and 59 were adult. Their mean age was 23 years. The patients were allocated into 6 age groups: 0 - 4 yrs, 5 - 9 yrs, 10 - 14 yrs, 15 - 19 yrs, 20-64 yrs and > 65 yrs. Each sample was tested with Directigen Flu A+B and CDC real time PCR kit for detection and typisation/subtypisation of influenza according to the lab diagnostic protocol. RESULTS: Directigen Flu A+B identified influenza A virus in 20 (18.5%) samples and influenza B virus in two 2 (1.9%) samples. The high specificity (100%) and PPV of Directigen Flu A+B we found in our study shows that the positive results do not need to be confirmed. The overall sensitivity of Directigen Flu A+B is 35.1% for influenza A virus and 33.0% for influenza B virus. The sensitivity for influenza A is higher among children hospitalized (45.0%) and outpatients (40.0%) versus adults. CONCLUSION: Directigen Flu A+B has relatively low sensitivity for detection of influenza viruses in combined nose and throat swabs. Negative results must be confirmed.

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Rewiring the RNAs of influenza virus to prevent reassortment

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0908897106 ◽

2009 ◽

Vol 106 (37) ◽

pp. 15891-15896 ◽

Cited By ~ 52

Author(s):

Qinshan Gao ◽

Peter Palese

Keyword(s):

Influenza Virus ◽

Influenza A Virus ◽

Influenza A ◽

Nonstructural Protein ◽

Influenza Viruses ◽

Ha Gene ◽

Ns Gene ◽

Virus Rna ◽

Reassortant Viruses ◽

Packaging Signals

Influenza viruses contain segmented, negative-strand RNA genomes. Genome segmentation facilitates reassortment between different influenza virus strains infecting the same cell. This phenomenon results in the rapid exchange of RNA segments. In this study, we have developed a method to prevent the free reassortment of influenza A virus RNAs by rewiring their packaging signals. Specific packaging signals for individual influenza virus RNA segments are located in the 5′ and 3′ noncoding regions as well as in the terminal regions of the ORF of an RNA segment. By putting the nonstructural protein (NS)-specific packaging sequences onto the ORF of the hemagglutinin (HA) gene and mutating the packaging regions in the ORF of the HA, we created a chimeric HA segment with the packaging identity of an NS gene. By the same strategy, we made an NS gene with the packaging identity of an HA segment. This rewired virus had the packaging signals for all eight influenza virus RNAs, but it lost the ability to independently reassort its HA or NS gene. A similar approach can be applied to the other influenza A virus segments to diminish their ability to form reassortant viruses.

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Unseasonal Transmission of H3N2 Influenza A Virus During the Swine-Origin H1N1 Pandemic

Journal of Virology ◽

10.1128/jvi.00018-10 ◽

2010 ◽

Vol 84 (11) ◽

pp. 5715-5718 ◽

Cited By ~ 12

Author(s):

Elodie Ghedin ◽

David E. Wentworth ◽

Rebecca A. Halpin ◽

Xudong Lin ◽

Jayati Bera ◽

...

Keyword(s):

New York ◽

Influenza A Virus ◽

Influenza A ◽

New York State ◽

The United States ◽

Influenza Viruses ◽

Influenza A Viruses ◽

York State ◽

Low Incidence ◽

H3n2 Influenza

ABSTRACT The initial wave of swine-origin influenza A virus (pandemic H1N1/09) in the United States during the spring and summer of 2009 also resulted in an increased vigilance and sampling of seasonal influenza viruses (H1N1 and H3N2), even though they are normally characterized by very low incidence outside of the winter months. To explore the nature of virus evolution during this influenza “off-season,” we conducted a phylogenetic analysis of H1N1 and H3N2 sequences sampled during April to June 2009 in New York State. Our analysis revealed that multiple lineages of both viruses were introduced and cocirculated during this time, as is typical of influenza virus during the winter. Strikingly, however, we also found strong evidence for the presence of a large transmission chain of H3N2 viruses centered on the south-east of New York State and which continued until at least 1 June 2009. These results suggest that the unseasonal transmission of influenza A viruses may be more widespread than is usually supposed.

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Antiviral Activity of Benzoic Acid Derivative NC-5 Against Influenza A Virus and Its Neuraminidase Inhibition

International Journal of Molecular Sciences ◽

10.3390/ijms20246261 ◽

2019 ◽

Vol 20 (24) ◽

pp. 6261

Author(s):

Min Guo ◽

Jiawei Ni ◽

Jie Yu ◽

Jing Jin ◽

Lingman Ma ◽

...

Keyword(s):

Antiviral Activity ◽

Benzoic Acid ◽

Influenza A Virus ◽

Influenza A ◽

Matrix Protein ◽

Influenza Viruses ◽

Drug Candidate ◽

Dependent Manner ◽

Drug Resistant

The currently available drugs against influenza A virus primarily target neuraminidase (NA) or the matrix protein 2 (M2) ion channel. The emergence of drug-resistant viruses requires the development of new antiviral chemicals. Our study applied a cell-based approach to evaluate the antiviral activity of a series of newly synthesized benzoic acid derivatives, and 4-(2,2-Bis(hydroxymethyl)-5-oxopyrrolidin-l-yl)-3-(5-cyclohexyl-4H-1,2,4-triazol-3-yl)amino). benzoic acid, termed NC-5, was found to possess antiviral activity. NC-5 inhibited influenza A viruses A/FM/1/47 (H1N1), A/Beijing/32/92 (H3N2) and oseltamivir-resistant mutant A/FM/1/47-H275Y (H1N1-H275Y) in a dose-dependent manner. The 50% effective concentrations (EC50) for H1N1 and H1N1-H275Y were 33.6 μM and 32.8 μM, respectively, which showed that NC-5 had a great advantage over oseltamivir in drug-resistant virus infections. The 50% cytotoxic concentration (CC50) of NC-5 was greater than 640 μM. Orally administered NC-5 protected mice infected with H1N1 and H1N1-H275Y, conferring 80% and 60% survival at 100 mg/kg/d, reducing body weight loss, and alleviating virus-induced lung injury. NC-5 could suppress NP and M1 protein expression levels during the late stages of viral biosynthesis and inhibit NA activity, which may influence virus release. Our study proved that NC-5 has potent anti-influenza activity in vivo and in vitro, meaning that it could be regarded as a promising drug candidate to treat infection with influenza viruses, including oseltamivir-resistant viruses.

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Influenza A Virus Inhibits RSV Infection via a Two-Wave Expression of IFIT Proteins

Viruses ◽

10.3390/v12101171 ◽

2020 ◽

Vol 12 (10) ◽

pp. 1171

Author(s):

Yaron Drori ◽

Jasmine Jacob-Hirsch ◽

Rakefet Pando ◽

Aharona Glatman-Freedman ◽

Nehemya Friedman ◽

...

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Influenza Viruses ◽

Mass Spectrometry Analysis ◽

Influenza A Viruses ◽

Rsv Infection ◽

Syncytial Virus ◽

Mouse Lungs

Influenza viruses and respiratory syncytial virus (RSV) are respiratory viruses that primarily circulate worldwide during the autumn and winter seasons. Seasonal surveillance has shown that RSV infection generally precedes influenza. However, in the last four winter seasons (2016–2020) an overlap of the morbidity peaks of both viruses was observed in Israel, and was paralleled by significantly lower RSV infection rates. To investigate whether the influenza A virus inhibits RSV, human cervical carcinoma (HEp2) cells or mice were co-infected with influenza A and RSV. Influenza A inhibited RSV growth, both in vitro and in vivo. Mass spectrometry analysis of mouse lungs infected with influenza A identified a two-wave pattern of protein expression upregulation, which included members of the interferon-induced protein with the tetratricopeptide (IFITs) family. Interestingly, in the second wave, influenza A viruses were no longer detectable in mouse lungs. In addition, knockdown and overexpression of IFITs in HEp2 cells affected RSV multiplicity. In conclusion, influenza A infection inhibits RSV infectivity via upregulation of IFIT proteins in a two-wave modality. Understanding the immune system involvement in the interaction between influenza A and RSV viruses will contribute to the development of future treatment strategies against these viruses.

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IFN-κ suppresses the replication of influenza A viruses through the IFNAR-MAPK-Fos-CHD6 axis

Science Signaling ◽

10.1126/scisignal.aaz3381 ◽

2020 ◽

Vol 13 (626) ◽

pp. eaaz3381 ◽

Cited By ~ 4

Author(s):

Yongquan He ◽

Weihui Fu ◽

Kangli Cao ◽

Qian He ◽

Xiangqing Ding ◽

...

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Influenza Viruses ◽

Mitogen Activated Protein Kinase ◽

Type I Interferons ◽

Type I ◽

Influenza A Viruses ◽

Human Lung Cells ◽

Avian Influenza A Virus ◽

Factor C

Type I interferons (IFNs) are the first line of defense against viral infection. Using a mouse model of influenza A virus infection, we found that IFN-κ was one of the earliest responding type I IFNs after infection with H9N2, a low-pathogenic avian influenza A virus, whereas this early induction did not occur upon infection with the epidemic-causing H7N9 virus. IFN-κ efficiently suppressed the replication of various influenza viruses in cultured human lung cells, and chromodomain helicase DNA binding protein 6 (CHD6) was the major effector for the antiviral activity of IFN-κ, but not for that of IFN-α or IFN-β. The induction of CHD6 required both of the type I IFN receptor subunits IFNAR1 and IFNAR2, the mitogen-activated protein kinase (MAPK) p38, and the transcription factor c-Fos but was independent of signal transducer and activator of transcription 1 (STAT1) activity. In addition, we showed that pretreatment with IFN-κ protected mice from lethal influenza viral challenge. Together, our findings identify an IFN-κ–specific pathway that constrains influenza A virus and provide evidence that IFN-κ may have potential as a preventative and therapeutic agent against influenza A virus.

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Cathepsin W Is Required for Escape of Influenza A Virus from Late Endosomes

mBio ◽

10.1128/mbio.00297-15 ◽

2015 ◽

Vol 6 (3) ◽

Cited By ~ 13

Author(s):

Thomas O. Edinger ◽

Marie O. Pohl ◽

Emilio Yángüez ◽

Silke Stertz

Keyword(s):

Influenza Virus ◽

Rna Interference ◽

Influenza A Virus ◽

Proteolytic Activity ◽

Influenza A ◽

Antiviral Drugs ◽

Influenza Viruses ◽

Late Endosomes ◽

Genome Wide ◽

Cathepsin W

ABSTRACT Human cathepsin W (CtsW) is a cysteine protease, which was identified in a genome-wide RNA interference (RNAi) screen to be required for influenza A virus (IAV) replication. In this study, we show that reducing the levels of expression of CtsW reduces viral titers for different subtypes of IAV, and we map the target step of CtsW requirement to viral entry. Using a set of small interfering RNAs (siRNAs) targeting CtsW, we demonstrate that knockdown of CtsW results in a decrease of IAV nucleoprotein accumulation in the nuclei of infected cells at 3 h postinfection. Assays specific for the individual stages of IAV entry further show that attachment, internalization, and early endosomal trafficking are not affected by CtsW knockdown. However, we detected impaired escape of viral particles from late endosomes in CtsW knockdown cells. Moreover, fusion analysis with a dual-labeled influenza virus revealed a significant reduction in fusion events, with no detectable impact on endosomal pH, suggesting that CtsW is required at the stage of viral fusion. The defect in IAV entry upon CtsW knockdown could be rescued by ectopic expression of wild-type CtsW but not by the expression of a catalytically inactive mutant of CtsW, suggesting that the proteolytic activity of CtsW is required for successful entry of IAV. Our results establish CtsW as an important host factor for entry of IAV into target cells and suggest that CtsW could be a promising target for the development of future antiviral drugs. IMPORTANCE Increasing levels of resistance of influenza viruses to available antiviral drugs have been observed. Development of novel treatment options is therefore of high priority. In parallel to the classical approach of targeting viral enzymes, a novel strategy is pursued: cell-dependent factors of the virus are identified with the aim of developing small-molecule inhibitors against a cellular target that the virus relies on. For influenza A virus, several genome-wide RNA interference (RNAi) screens revealed hundreds of potential cellular targets. However, we have only limited knowledge on how these factors support virus replication, which would be required for drug development. We have characterized cathepsin W, one of the candidate factors, and found that cathepsin W is required for escape of influenza virus from the late endosome. Importantly, this required the proteolytic activity of cathepsin W. We therefore suggest that cathepsin W could be a target for future host cell-directed antiviral therapies.

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