scholarly journals Influenza A and B viruses with reduced baloxavir susceptibility display attenuated in vitro fitness but retain ferret transmissibility

2020 ◽  
Vol 117 (15) ◽  
pp. 8593-8601 ◽  
Author(s):  
Jeremy C. Jones ◽  
Philippe Noriel Q. Pascua ◽  
Thomas P. Fabrizio ◽  
Bindumadhav M. Marathe ◽  
Patrick Seiler ◽  
...  
Keyword(s):  
Influenza A ◽  
Clinical Trial Data ◽  
Drug Susceptibility ◽  
Influenza Viruses ◽  
Endonuclease Activity ◽  
Influenza A Viruses ◽  
Complex Activity ◽  
Minimal Impact

Baloxavir marboxil (BXM) was approved in 2018 for treating influenza A and B virus infections. It is a first-in-class inhibitor targeting the endonuclease activity of the virus polymerase acidic (PA) protein. Clinical trial data revealed that PA amino acid substitutions at residue 38 (I38T/F/M) reduced BXM potency and caused virus rebound in treated patients, although the fitness characteristics of the mutant viruses were not fully defined. To determine the fitness impact of the I38T/F/M substitutions, we generated recombinant A/California/04/2009 (H1N1)pdm09, A/Texas/71/2017 (H3N2), and B/Brisbane/60/2008 viruses with I38T/F/M and examined drug susceptibility in vitro, enzymatic properties, replication efficiency, and transmissibility in ferrets. Influenza viruses with I38T/F/M substitutions exhibited reduced baloxavir susceptibility, with 38T causing the greatest reduction. The I38T/F/M substitutions impaired PA endonuclease activity as compared to that of wild-type (I38-WT) PA. However, only 38T/F A(H3N2) substitutions had a negative effect on polymerase complex activity. The 38T/F substitutions decreased replication in cells among all viruses, whereas 38M had minimal impact. Despite variable fitness consequences in vitro, all 38T/M viruses disseminated to naive ferrets by contact and airborne transmission, while 38F-containing A(H3N2) and B viruses failed to transmit via the airborne route. Reversion of 38T/F/M to I38-WT was rare among influenza A viruses in this study, suggesting stable retention of 38T/F/M genotypes during these transmission events. BXM reduced susceptibility-associated mutations had variable effects on in vitro fitness of influenza A and B viruses, but the ability of these viruses to transmit in vivo indicates a risk of their spreading from BXM-treated individuals.

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

Viruses ◽  
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.

scholarly journals Attenuation and immunogenicity in mice of temperature-sensitive influenza viruses expressing truncated NS1 proteins

2005 ◽  
Vol 86 (10) ◽  
pp. 2817-2821 ◽  
Author(s):  
Ana M. Falcón ◽  
Ana Fernandez-Sesma ◽  
Yurie Nakaya ◽  
Thomas M. Moran ◽  
Juan Ortín ◽  
...  
Keyword(s):  
Influenza A ◽  
Influenza Viruses ◽  
Wild Type Virus ◽  
Temperature Sensitive ◽  
Ns1 Protein ◽  
Wild Type ◽  
Influenza A Viruses ◽  
Lethal Challenge

It was previously shown that two mutant influenza A viruses expressing C-terminally truncated forms of the NS1 protein (NS1-81 and NS1-110) were temperature sensitive in vitro. These viruses contain HA, NA and M genes derived from influenza A/WSN/33 H1N1 virus (mouse-adapted), and the remaining five genes from human influenza A/Victoria/3/75 virus. Mice intranasally infected with the NS1 mutant viruses showed undetectable levels of virus in lungs at day 3, whereas those infected with the NS1 wild-type control virus still had detectable levels of virus at this time. Nevertheless, the temperature-sensitive mutant viruses induced specific cellular and humoral immune responses similar to those induced by the wild-type virus. Mice immunized with the NS1 mutant viruses were protected against a lethal challenge with influenza A/WSN/33 virus. These results indicate that truncations in the NS1 protein resulting in temperature-sensitive phenotypes in vitro correlate with attenuation in vivo without compromising viral immunogenicity, an ideal characteristic for live attenuated viral vaccines.

scholarly journals Cellular Immune Recognition of Influenza A Viruses in Equines: in vitro and in vivo Studies on the Immunogenicity of Equine Influenza Viruses

1997 ◽  
Vol 66 (1) ◽  
pp. 39-49
Author(s):  
C. A. O. Adeyefa ◽  
J. W. McCauley ◽  
A. I. Daneji ◽  
O. A. Kalejaiye ◽  
A. Bakare ◽  
...  
Keyword(s):  
Influenza A ◽  
Influenza Viruses ◽  
In Vivo Studies ◽  
Immune Recognition ◽  
Influenza A Viruses ◽  
Equine Influenza ◽  
Cellular Immune

scholarly journals Inhibition of Influenza Virus Infection by a Novel Antiviral Peptide That Targets Viral Attachment to Cells

2006 ◽  
Vol 80 (24) ◽  
pp. 11960-11967 ◽  
Author(s):  
Jeremy C. Jones ◽  
Elizabeth A. Turpin ◽  
Hermann Bultmann ◽  
Curtis R. Brandt ◽  
Stacey Schultz-Cherry
Keyword(s):  
Influenza A ◽  
Signal Sequence ◽  
Influenza Virus Infection ◽  
Influenza Viruses ◽  
Influenza A Viruses ◽  
Viral Attachment ◽  
H5n1 Influenza ◽  
Eb Peptide

ABSTRACT Influenza A viruses continue to cause widespread morbidity and mortality. There is an added concern that the highly pathogenic H5N1 influenza A viruses, currently found throughout many parts of the world, represent a serious public health threat and may result in a pandemic. Intervention strategies to halt an influenza epidemic or pandemic are a high priority, with an emphasis on vaccines and antiviral drugs. In these studies, we demonstrate that a 20-amino-acid peptide (EB, for entry blocker) derived from the signal sequence of fibroblast growth factor 4 exhibits broad-spectrum antiviral activity against influenza viruses including the H5N1 subtype in vitro. The EB peptide was protective in vivo, even when administered postinfection. Mechanistically, the EB peptide inhibits the attachment to the cellular receptor, preventing infection. Further studies demonstrated that the EB peptide specifically binds to the viral hemagglutinin protein. This novel peptide has potential value as a reagent to study virus attachment and as a future therapeutic.

scholarly journals Viruses harness YxxØ motif to interact with host AP2M1 for replication: A vulnerable broad-spectrum antiviral target

2020 ◽  
Vol 6 (35) ◽  
pp. eaba7910
Author(s):  
Shuofeng Yuan ◽  
Hin Chu ◽  
Jingjing Huang ◽  
Xiaoyu Zhao ◽  
Zi-Wei Ye ◽  
...  
Keyword(s):  
Broad Spectrum ◽  
Influenza A ◽  
Host Protein ◽  
Influenza A Viruses ◽  
Protein Protein Interaction ◽  
Enterovirus A71 ◽  
Evolutionarily Conserved ◽  
Immunodeficiency Virus

Targeting a universal host protein exploited by most viruses would be a game-changing strategy that offers broad-spectrum solution and rapid pandemic control including the current COVID-19. Here, we found a common YxxØ-motif of multiple viruses that exploits host AP2M1 for intracellular trafficking. A library chemical, N-(p-amylcinnamoyl)anthranilic acid (ACA), was identified to interrupt AP2M1-virus interaction and exhibit potent antiviral efficacy against a number of viruses in vitro and in vivo, including the influenza A viruses (IAVs), Zika virus (ZIKV), human immunodeficiency virus, and coronaviruses including MERS-CoV and SARS-CoV-2. YxxØ mutation, AP2M1 depletion, or disruption by ACA causes incorrect localization of viral proteins, which is exemplified by the failure of nuclear import of IAV nucleoprotein and diminished endoplasmic reticulum localization of ZIKV-NS3 and enterovirus-A71-2C proteins, thereby suppressing viral replication. Our study reveals an evolutionarily conserved mechanism of protein-protein interaction between host and virus that can serve as a broad-spectrum antiviral target.

scholarly journals Genetically and Antigenically Divergent Influenza A(H9N2) Viruses Exhibit Differential Replication and Transmission Phenotypes in Mammalian Models

2020 ◽  
Vol 94 (17) ◽  
Author(s):  
Jessica A. Belser ◽  
Xiangjie Sun ◽  
Nicole Brock ◽  
Claudia Pappas ◽  
Joanna A. Pulit-Penaloza ◽  
...  
Keyword(s):  
Influenza A ◽  
Human Infection ◽  
Influenza Viruses ◽  
Human Populations ◽  
Human Respiratory Tract ◽  
Live Bird Markets ◽  
Human Infections ◽  
Live Bird

ABSTRACT Low-pathogenicity avian influenza A(H9N2) viruses, enzootic in poultry populations in Asia, are associated with fewer confirmed human infections but higher rates of seropositivity compared to A(H5) or A(H7) subtype viruses. Cocirculation of A(H5) and A(H7) viruses leads to the generation of reassortant viruses bearing A(H9N2) internal genes with markers of mammalian adaptation, warranting continued surveillance in both avian and human populations. Here, we describe active surveillance efforts in live poultry markets in Vietnam in 2018 and compare representative viruses to G1 and Y280 lineage viruses that have infected humans. Receptor binding properties, pH thresholds for HA activation, in vitro replication in human respiratory tract cells, and in vivo mammalian pathogenicity and transmissibility were investigated. While A(H9N2) viruses from both poultry and humans exhibited features associated with mammalian adaptation, one human isolate from 2018, A/Anhui-Lujiang/39/2018, exhibited increased capacity for replication and transmission, demonstrating the pandemic potential of A(H9N2) viruses. IMPORTANCE A(H9N2) influenza viruses are widespread in poultry in many parts of the world and for over 20 years have sporadically jumped species barriers to cause human infection. As these viruses continue to diversify genetically and antigenically, it is critical to closely monitor viruses responsible for human infections, to ascertain if A(H9N2) viruses are acquiring properties that make them better suited to infect and spread among humans. In this study, we describe an active poultry surveillance system established in Vietnam to identify the scope of influenza viruses present in live bird markets and the threat they pose to human health. Assessment of a recent A(H9N2) virus isolated from an individual in China in 2018 is also reported, and it was found to exhibit properties of adaptation to humans and, importantly, it shows similarities to strains isolated from the live bird markets of Vietnam.

scholarly journals Cross-Protection of Influenza A Virus Infection by a DNA Aptamer Targeting the PA Endonuclease Domain

2015 ◽  
Vol 59 (7) ◽  
pp. 4082-4093 ◽  
Author(s):  
Shuofeng Yuan ◽  
Naru Zhang ◽  
Kailash Singh ◽  
Huiping Shuai ◽  
Hin Chu ◽  
...  
Keyword(s):  
Virus Infection ◽  
Influenza A Virus ◽  
Influenza A ◽  
H5n1 Virus ◽  
Influenza Viruses ◽  
Cross Protection ◽  
Dna Aptamer ◽  
Endonuclease Activity ◽  
Endonuclease Domain

ABSTRACTAmino acid residues in the N-terminal of the PA subunit (PAN) of the influenza A virus polymerase play critical roles in endonuclease activity, protein stability, and viral RNA (vRNA) promoter binding. In addition, PANis highly conserved among different subtypes of influenza virus, which suggests PANto be a desired target in the development of anti-influenza agents. We selected DNA aptamers targeting the intact PA protein or the PANdomain of an H5N1 virus strain using systematic evolution of ligands by exponential enrichment (SELEX). The binding affinities of selected aptamers were measured, followed by an evaluation ofin vitroendonuclease inhibitory activity. Next, the antiviral effects of enriched aptamers against influenza A virus infections were examined. A total of three aptamers targeting PA and six aptamers targeting PANwere selected. Our data demonstrated that all three PA-selected aptamers neither inhibited endonuclease activity nor exhibited antiviral efficacy, whereas four of the six PAN-selected aptamers inhibited both endonuclease activity and H5N1 virus infection. Among the four effective aptamers, one exhibited cross-protection against infections of H1N1, H5N1, H7N7, and H7N9 influenza viruses, with a 50% inhibitory concentration (IC50) of around 10 nM. Notably, this aptamer was identified at the 5th round but disappeared after the 10th round of selection, suggesting that the identification and evaluation of aptamers at early rounds of selection may be highly helpful for screening effective aptamers. Overall, our study provides novel insights for screening and developing effective aptamers for use as anti-influenza drugs.

scholarly journals The influenza A virus nucleoprotein gene controls the induction of both subtype specific and cross-reactive cytotoxic T cells.

1984 ◽  
Vol 160 (2) ◽  
pp. 552-563 ◽  
Author(s):  
A R Townsend ◽  
J J Skehel
Keyword(s):  
T Cells ◽  
Influenza A ◽  
Cytotoxic T Cells ◽  
Target Cells ◽  
Spleen Cells ◽  
Influenza A Viruses ◽  
Group A ◽  
Selection Of

Using genetically typed recombinant influenza A viruses that differ only in their genes for nucleoprotein, we have demonstrated that repeated stimulation in vitro of C57BL/6 spleen cells primed in vivo with E61-13-H17 (H3N2) virus results in the selection of a population of cytotoxic T lymphocytes (CTL) whose recognition of infected target cells maps to the gene for nucleoprotein of the 1968 virus. Influenza A viruses isolated between 1934 and 1979 fall into two groups defined by their ability to sensitize target cells for lysis by these CTL: 1934-1943 form one group (A/PR/8/34 related) and 1946-1979 form the second group (A/HK/8/68 related). These findings complement and extend our previous results with an isolated CTL clone with specificity for the 1934 nucleoprotein (27, 28). It is also shown that the same spleen cells derived from mice primed with E61-13-H17 virus in vivo, but maintained in identical conditions by stimulation with X31 virus (which differs from the former only in the origin of its gene for NP) in vitro, results in the selection of CTL that cross-react on target cells infected with A/PR/8/1934 (H1N1) or A/Aichi/1968 (H3N2). These results show that the influenza A virus gene for NP can play a role in selecting CTL with different specificities and implicate the NP molecule as a candidate for a target structure recognized by both subtype-directed and cross-reactive influenza A-specific cytotoxic T cells.

scholarly journals Use of influenza A viruses expressing reporter genes to assess the frequency of double infections in vitro

2012 ◽  
Vol 93 (8) ◽  
pp. 1645-1648 ◽  
Author(s):  
R. Bodewes ◽  
N. J. Nieuwkoop ◽  
R. J. Verburgh ◽  
R. A. M. Fouchier ◽  
A. D. M. E. Osterhaus ◽  
...  
Keyword(s):  
Influenza A ◽  
Single Cells ◽  
Reporter Genes ◽  
Influenza Viruses ◽  
Influenza A Viruses ◽  
Double Infection ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

Exchange of gene segments between mammalian and avian influenza A viruses may lead to the emergence of potential pandemic influenza viruses. Since co-infection of single cells with two viruses is a prerequisite for reassortment to take place, we assessed frequencies of double-infection in vitro using influenza A/H5N1 and A/H1N1 viruses expressing the reporter genes eGFP or mCherry. Double-infected A549 and Madin–Darby canine kidney cells were detected by confocal microscopy and flow cytometry.

The influenza virus PB1-F2 protein has interferon antagonistic activity

2011 ◽  
Vol 392 (12) ◽  
pp. 1135-1144 ◽  
Author(s):  
Sabine E. Dudek ◽  
Ludmilla Wixler ◽  
Carolin Nordhoff ◽  
Alexandra Nordmann ◽  
Darisuren Anhlan ◽  
...  
Keyword(s):  
Influenza A ◽  
Molecular Mechanisms ◽  
Nonstructural Protein ◽  
Gene Segment ◽  
Antagonistic Activity ◽  
Influenza Viruses ◽  
Type I ◽  
Influenza A Viruses ◽  
Reading Frame

Abstract PB1-F2 is a nonstructural protein of influenza viruses encoded by the PB1 gene segment from a +1 open reading frame. It has been shown that PB1-F2 contributes to viral pathogenicity, although the underlying mechanisms are still unclear. Induction of type I interferon (IFN) and the innate immune response are the first line of defense against viral infection. Here we show that influenza A viruses (IAVs) lacking the PB1-F2 protein induce an enhanced expression of IFN-β and IFN-stimulated genes in infected epithelial cells. Studying molecular mechanisms underlying the PB1-F2-mediated IFN antagonistic activity showed that PB1-F2 interferes with the RIG-I/MAVS protein complex thereby inhibiting the activation of the downstream transcription factor IFN regulatory factor 3. These findings were also reflected in in vivo studies demonstrating that infection with PR8 wild-type (wt) virus resulted in higher lung titers and a more severe onset of disease compared with infection with its PB1-F2-deficient counterpart. Accordingly, a much more pronounced infiltration of lungs with immune cells was detected in mice infected with the PB1-F2 wt virus. In summary, we demonstrate that the PB1-F2 protein of IAVs exhibits a type I IFN-antagonistic function by interfering with the RIG-I/MAVS complex, which contributes to an enhanced pathogenicity in vivo.

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