scholarly journals A Mechanism Underlying Attenuation of Recombinant Influenza A Viruses Carrying Reporter Genes

Viruses ◽  
2018 ◽  
Vol 10 (12) ◽  
pp. 679 ◽  
Author(s):  
Xiujuan Zhao ◽  
Lin Wang ◽  
Qinghua Cui ◽  
Ping Li ◽  
Yanyan Wang ◽  
...  
Keyword(s):  
Genetic Instability ◽  
Influenza A ◽  
Essential Gene ◽  
Reporter Genes ◽  
Influenza A Viruses ◽  
New Approach ◽  
Infected Cells ◽  
Rdrp Assay ◽  
Infectious Unit

Influenza A viruses (IAV) carrying reporter genes provide a powerful tool to study viral infection and pathogenesis in vivo, however, incorporating a non-essential gene into the IAV genome often results in virus attenuation and genetic instability. Very few studies have systematically compared different reporter IAVs, and most optimization attempts seem to lack authentic directions. In this study, we evaluated the ratio of genome copies to the number of infectious unit of two reporter IAVs, PR8-NS1-Gluc and PR8-PB2-Gluc. As a result, PR8-NS1-Gluc and PR8-PB2-Gluc produced 41.4 and 3.8 genomes containing noninfectious particles respectively for every such particle produced by parental PR8 virus. RdRp assay demonstrated that modification of segment NS by inserting reporter genes can interfere with the replication competitive property of the corresponding vRNAs, and the balance of the 8 segments of the reporter IAVs were drastically impaired in infected cells. As a consequence, large amounts of NS-null noninfectious particles were produced during the PR8-NS1-Gluc packaging. In summary, we unravel a mechanism underlying attenuation of reporter IAVs, which suggests a new approach to restore infectivity and virulence by introducing extra mutations compensating for the impaired replication property of corresponding segments.

scholarly journals Efficient Generation of Recombinant Influenza A Viruses Employing a New Approach to Overcome the Genetic Instability of HA Segments

PLoS ONE ◽  
2015 ◽  
Vol 10 (1) ◽  
pp. e0116917 ◽  
Author(s):  
Ahmed Mostafa ◽  
Pumaree Kanrai ◽  
Henning Petersen ◽  
Sherif Ibrahim ◽  
Silke Rautenschlein ◽  
...  
Keyword(s):  
Genetic Instability ◽  
Influenza A ◽  
Influenza A Viruses ◽  
New Approach ◽  
Efficient Generation

scholarly journals Generation of Replication-Competent Recombinant Influenza A Viruses Carrying a Reporter Gene Harbored in the Neuraminidase Segment

2010 ◽  
Vol 84 (22) ◽  
pp. 12075-12081 ◽  
Author(s):  
Feng Li ◽  
Liqiang Feng ◽  
Weiqi Pan ◽  
Zhenyuan Dong ◽  
Chufang Li ◽  
...  
Keyword(s):  
Influenza A ◽  
Genome Stability ◽  
Fluorescent Protein ◽  
Basic Research ◽  
Reporter Genes ◽  
Influenza Viruses ◽  
Peptide Sequence ◽  
Egfp Expression ◽  
Influenza A Viruses

ABSTRACT Replication-competent influenza viruses carrying reporter genes are of great use for basic research, screening of antiviral drugs, and neutralizing of antibodies. In this study, two recombinant influenza A viruses with a neuraminidase (NA) segment harboring enhanced green fluorescent protein (EGFP) in the background of A/PR/8/34 (PR8) were generated. The viral RNA (vRNA)-specific packaging signals for NA were largely retained for efficient packaging. An “autocleave” 2A peptide sequence, which was inserted at the N terminus or the COOH terminus of NA to link with EGFP, enabled NA and EGFP to be expressed monocistronically. Further analysis demonstrated that both viruses, named rPR8-EGFP+NA and rPR8-NA+EGPF, although with some characteristic differences in growth and EGFP expression, could replicate in noncomplementary cells and propagate to large quantities while maintaining genome stability after multiple passages in embryonated eggs. These replication-competent influenza viruses carrying reporter genes are a great addition to the tool set for developing antiviral therapeutics and vaccines and for in vivo studies of viral dissemination and pathogenicity.

Influenza type A virus M protein expression on infected cells is responsible for cross-reactive recognition by cytotoxic thymus-derived lymphocytes

1980 ◽  
Vol 29 (2) ◽  
pp. 719-723 ◽  
Author(s):  
C S Reiss ◽  
J L Schulman
Keyword(s):  
T Cell ◽  
Influenza A Virus ◽  
Influenza A ◽  
Rabbit Antiserum ◽  
Type A ◽  
M Protein ◽  
Cytotoxic T Cell ◽  
Influenza A Viruses ◽  
Infected Cells ◽  
Cytotoxic T Cell Responses

M protein of influenza A virus was detected with rabbit antiserum by both indirect immunofluorescence and by antibody plus complement-mediated cytolysis on the cell surfaces of both productively and nonproductively infected cells. In contrast, antiserum to nucleoprotein failed to react with unfixed infected cells, but did bind to fixed infected cells, especially in the perinuclear area. Incorporation of antiserum to M protein in a T-cell-mediated cytotoxicity assay produced almost complete abrogation of lysis of H-2-compatible cells infected with an influenza A virus of a subtype which differed from that used to elicit the cytotoxic T cells. However, the antibody did not significantly block 51Cr release from cells infected with the homotypic type A influenza virus. These observations are in accord with the hypothesis that the cross-reactive cytotoxic T-cell responses seen with cells infected by heterotypic influenza A viruses are due to recognition of a common M protein.

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 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 In vivo analysis of influenza A mRNA secondary structures identifies critical regulatory motifs

2019 ◽  
Vol 47 (13) ◽  
pp. 7003-7017 ◽  
Author(s):  
Lisa Marie Simon ◽  
Edoardo Morandi ◽  
Anna Luganini ◽  
Giorgio Gribaudo ◽  
Luis Martinez-Sobrido ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Influenza A ◽  
Host Cells ◽  
Messenger Rnas ◽  
Infected Cells ◽  
In Vivo Analysis ◽  
First Time ◽  
Negative Sense

AbstractThe influenza A virus (IAV) is a continuous health threat to humans as well as animals due to its recurring epidemics and pandemics. The IAV genome is segmented and the eight negative-sense viral RNAs (vRNAs) are transcribed into positive sense complementary RNAs (cRNAs) and viral messenger RNAs (mRNAs) inside infected host cells. A role for the secondary structure of IAV mRNAs has been hypothesized and debated for many years, but knowledge on the structure mRNAs adopt in vivo is currently missing. Here we solve, for the first time, the in vivo secondary structure of IAV mRNAs in living infected cells. We demonstrate that, compared to the in vitro refolded structure, in vivo IAV mRNAs are less structured but exhibit specific locally stable elements. Moreover, we show that the targeted disruption of these high-confidence structured domains results in an extraordinary attenuation of IAV replicative capacity. Collectively, our data provide the first comprehensive map of the in vivo structural landscape of IAV mRNAs, hence providing the means for the development of new RNA-targeted antivirals.

scholarly journals In VitroAssessment of the Immunological Significance of a Human Monoclonal Antibody Directed to the Influenza A Virus Nucleoprotein

2013 ◽  
Vol 20 (8) ◽  
pp. 1333-1337 ◽  
Author(s):  
Rogier Bodewes ◽  
Martina M. Geelhoed-Mieras ◽  
Jens Wrammert ◽  
Rafi Ahmed ◽  
Patrick C. Wilson ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Influenza A Virus ◽  
Influenza A ◽  
Viral Antigen ◽  
Human Monoclonal Antibody ◽  
Cell Cytotoxicity ◽  
Influenza A Viruses ◽  
Dependent Cell ◽  
Infected Cells

ABSTRACTInfluenza A viruses cause annual epidemics and occasionally pandemics. Antibodies directed to the conserved viral nucleoprotein (NP) may play a role in immunity against various influenza A virus subtypes. Here, we assessed the immunological significance of a human monoclonal antibody directed to NPin vitro. This antibody bound to virus-infected cells but did not display virus-neutralizing activity, complement-dependent cell cytotoxicity, or opsonization of viral antigen for improved antigen presentation to CD8+T cells by dendritic cells.

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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