scholarly journals The Endogenous RIG-I Ligand Is Generated in Influenza A-Virus Infected Cells

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1564
Author(s):  
Julia Steinberg ◽  
Timo Wadenpohl ◽  
Stephanie Jung
Keyword(s):  
Influenza A ◽  
Hek293 Cells ◽  
Rnase L ◽  
Internal Transcribed Spacer Region ◽  
Specific Sequence ◽  
Viral Genomes ◽  
Specific Pcr ◽  
Reporter Systems ◽  
Infected Cells

As a result of a viral infection, viral genomes are not only recognized by RIG-I, but also lead to the activation of RNase L, which cleaves cellular RNA to generate the endogenous RIG-I ligand (eRL). The eRL was previously identified as a specific sequence derived from the internal transcribed spacer region 2, which bears a 2′3′ cyclic phosphate instead of the common 5′ triphosphate. By now, the generation of the eRL and its immunostimulatory effect were shown both in vitro and in reporter systems. In this work, we aimed to elucidate whether the eRL is also generated in Influenza A (IAV) and vesicular stomatitis virus (VSV) infected cells. RNA was extracted from virus-infected cells and used for immunostimulations as well as specific PCR-strategies to detect eRL cleavage. We show that the eRL is generated in IAV infected HEK293 cells, but we could not detect specific eRL fragments in VSV infected cells. Further, RIG-I mediated IFN-response depends not only on viral genomes but also on the eRL, as immunostimulatory properties remain present under 5′triphosphate degrading conditions. In summary, we prove the IAV infection induced eRL generation in HEK293 cells, amplifying the innate immune response.

scholarly journals Volatile scents of influenza A and S. pyogenes (co-)infected cells

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Selina Traxler ◽  
Gina Barkowsky ◽  
Radost Saß ◽  
Ann-Christin Klemenz ◽  
Nadja Patenge ◽  
...  
Keyword(s):  
Influenza A ◽  
Early Recognition ◽  
Bacterial Species ◽  
Bacterial Load ◽  
Gas Chromatography Mass Spectrometry ◽  
Sampling System ◽  
Infected Cells ◽  
Infection Processes ◽  
Non Destructive

AbstractInfluenza A is a serious pathogen itself, but often leads to dangerous co-infections in combination with bacterial species such as Streptococcus pyogenes. In comparison to classical biochemical methods, analysis of volatile organic compounds (VOCs) in headspace above cultures can enable destruction free monitoring of metabolic processes in vitro. Thus, volatile biomarkers emitted from biological cell cultures and pathogens could serve for monitoring of infection processes in vitro. In this study we analysed VOCs from headspace above (co)-infected human cells by using a customized sampling system. For investigating the influenza A mono-infection and the viral-bacterial co-infection in vitro, we analysed VOCs from Detroit cells inoculated with influenza A virus and S. pyogenes by means of needle-trap micro-extraction (NTME) and gas chromatography mass spectrometry (GC-MS). Besides the determination of microbiological data such as cell count, cytokines, virus load and bacterial load, emissions from cell medium, uninfected cells and bacteria mono-infected cells were analysed. Significant differences in emitted VOC concentrations were identified between non-infected and infected cells. After inoculation with S. pyogenes, bacterial infection was mirrored by increased emissions of acetaldehyde and propanal. N-propyl acetate was linked to viral infection. Non-destructive monitoring of infections by means of VOC analysis may open a new window for infection research and clinical applications. VOC analysis could enable early recognition of pathogen presence and in-depth understanding of their etiopathology.

scholarly journals Activation and Antagonism of the OAS–RNase L Pathway

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 14
Author(s):  
Susan R. Weiss
Keyword(s):  
Cell Death ◽  
Antiviral Activity ◽  
Apoptotic Cell ◽  
Early Response ◽  
Apoptotic Cell Death ◽  
Rnase L ◽  
Oligoadenylate Synthetase ◽  
Viral Genomes ◽  
L System ◽  
Infected Cells

The oligoadenylate synthetase–ribonuclease L (OAS–RNase L) system is a potent antiviral pathway that severely limits the pathogenesis of many viruses. Upon sensing dsRNA, OASs produce 2′,5′-oligoadenylates (2-5A) that activate RNase L to cleave both host and viral single-stranded RNA, thereby limiting protein production, virus replication and spread, leading to apoptotic cell death. Endogenous host dsRNA, which accumulates in the absence of adenosine deaminase acting on RNA (ADAR)1, can also activate RNase L and lead to apoptotic cell death. RNase L activation and antiviral activity during infections with several types of viruses in human and bat cells is dependent on OAS3 but independent of virus-induced interferon (IFN) and, thus, RNase L can be activated even in the presence of IFN antagonists. Differently from other human viruses examined, Zika virus is resistant to the antiviral activity of RNase L and instead utilizes RNase L to enhance its replication factories to produce more infectious virus. Some betacoronaviruses antagonize RNase L activation by expressing 2′,5′-phosphodiesterases (PDEs) that cleave 2-5A and thereby antagonize activation of RNase L. The best characterized of these PDEs is the murine coronavirus (MHV) NS2 accessory protein. Enzymatically active NS2 is required for replication in myeloid cells and in the liver. Interestingly, while wild type mice clear MHV from the liver by 7–10 days post-infection, RNase L knockout mice fail to effectively clear MHV, probably due to diminished apoptotic death of infected cells. We suggest that RNase L antiviral activity stems from direct cleavage of viral genomes and cessation of protein synthesis as well as through promoting death of infected cells, limiting the spread of virus. Importantly, OASs are pattern recognition receptors and the OAS–RNase L pathway is a primary innate response pathway to viruses, capable of early response, coming into play before IFN is induced or when the virus shuts down IFN signaling.

scholarly journals Tilapia Lake Virus Does Not Hemagglutinate Avian and Piscine Erythrocytes and NH4Cl Does Not Inhibit Viral Replication In Vitro

Viruses ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 1152 ◽  
Author(s):  
Augustino Alfred Chengula ◽  
Stephen Mutoloki ◽  
Øystein Evensen ◽  
Hetron Mweemba Munang’andu
Keyword(s):  
Atlantic Salmon ◽  
Influenza A ◽  
Meleagris Gallopavo ◽  
Host Cells ◽  
Pcr Analysis ◽  
Infectious Salmon Anemia ◽  
Influenza C Virus ◽  
Infected Cells ◽  
Anemia Virus

Tilapia lake virus (TiLV) is a negative-sense single-stranded RNA (-ssRNA) icosahedral virus classified to be the only member in the family Amnoonviridae. Although TiLV segment-1 shares homology with the influenza C virus PB1 and has four conserved motifs similar to influenza A, B, and C polymerases, it is unknown whether there are other properties shared between TiLV and orthomyxovirus. In the present study, we wanted to determine whether TiLV agglutinated avian and piscine erythrocytes, and whether its replication was inhibited by lysosomotropic agents, such as ammonium chloride (NH4Cl), as seen for orthomyxoviruses. Our findings showed that influenza virus strain A/Puerto Rico/8 (PR8) was able to hemagglutinate turkey (Meleagris gallopavo), Atlantic salmon (Salmo salar L), and Nile tilapia (Oreochromis niloticus) red blood cells (RBCs), while infectious salmon anemia virus (ISAV) only agglutinated Atlantic salmon, but not turkey or tilapia, RBCs. In contrast to PR8 and ISAV, TiLV did not agglutinate turkey, Atlantic salmon, or tilapia RBCs. qRT-PCR analysis showed that 30 mM NH4Cl, a basic lysosomotropic agent, neither inhibited nor enhanced TiLV replication in E-11 cells. There was no difference in viral quantities in the infected cells with or without NH4Cl treatment during virus adsorption or at 1, 2, and 3 h post-infection. Given that hemagglutinin proteins that bind RBCs also serve as ligands that bind host cells during virus entry leading to endocytosis in orthomyxoviruses, the data presented here suggest that TiLV may use mechanisms that are different from orthomyxoviruses for entry and replication in host cells. Therefore, future studies should seek to elucidate the mechanisms used by TiLV for entry into host cells and to determine its mode of replication in infected cells.

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 Neuraminidase inhibition contributes to influenza A virus neutralization by anti-hemagglutinin stem antibodies

2019 ◽  
Vol 216 (2) ◽  
pp. 304-316 ◽  
Author(s):  
Ivan Kosik ◽  
Davide Angeletti ◽  
James S. Gibbs ◽  
Matthew Angel ◽  
Kazuyo Takeda ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Influenza A ◽  
Cell Activation ◽  
Immune Cell ◽  
Broadly Neutralizing Antibodies ◽  
Neutralization Activity ◽  
Immune Cell Activation ◽  
Influenza Virus Hemagglutinin ◽  
Infected Cells

Broadly neutralizing antibodies (Abs) that bind the influenza virus hemagglutinin (HA) stem may enable universal influenza vaccination. Here, we show that anti-stem Abs sterically inhibit viral neuraminidase (NA) activity against large substrates, with activity inversely proportional to the length of the fibrous NA stalk that supports the enzymatic domain. By modulating NA stalk length in recombinant IAVs, we show that anti-stem Abs inhibit virus release from infected cells by blocking NA, accounting for their in vitro neutralization activity. NA inhibition contributes to anti-stem Ab protection in influenza-infected mice, likely due at least in part to NA-mediated inhibition of FcγR-dependent activation of innate immune cells by Ab bound to virions. Food and Drug Administration–approved NA inhibitors enhance anti-stem–based Fc-dependent immune cell activation, raising the possibility of therapeutic synergy between NA inhibitors and anti-stem mAb treatment in humans.

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 Protective Role of Combined Polyphenols and Micronutrients against Influenza A Virus and SARS-CoV-2 Infection In Vitro

Biomedicines ◽  
2021 ◽  
Vol 9 (11) ◽  
pp. 1721
Author(s):  
Marta De Angelis ◽  
David Della-Morte ◽  
Gabriele Buttinelli ◽  
Angela Di Martino ◽  
Francesca Pacifici ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Viral Proteins ◽  
Antiviral Effect ◽  
Inhibitory Effect ◽  
Protective Role ◽  
Virus Infections ◽  
Infected Cells ◽  
Respiratory Virus Infections

Polyphenols have been widely studied for their antiviral effect against respiratory virus infections. Among these, resveratrol (RV) has been demonstrated to inhibit influenza virus replication and more recently, it has been tested together with pterostilbene against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In the present work, we evaluated the antiviral activity of polydatin, an RV precursor, and a mixture of polyphenols and other micronutrients, named A5+, against influenza virus and SARS-CoV-2 infections. To this end, we infected Vero E6 cells and analyzed the replication of both respiratory viruses in terms of viral proteins synthesis and viral titration. We demonstrated that A5+ showed a higher efficacy in inhibiting both influenza virus and SARS-CoV-2 infections compared to polydatin treatment alone. Indeed, post infection treatment significantly decreased viral proteins expression and viral release, probably by interfering with any step of virus replicative cycle. Intriguingly, A5+ treatment strongly reduced IL-6 cytokine production in influenza virus-infected cells, suggesting its potential anti-inflammatory properties during the infection. Overall, these results demonstrate the synergic and innovative antiviral efficacy of A5+ mixture, although further studies are needed to clarify the mechanisms underlying its inhibitory effect.

scholarly journals Attenuation of Influenza A Virus mRNA Levels by Promoter Mutations

1998 ◽  
Vol 72 (8) ◽  
pp. 6283-6290 ◽  
Author(s):  
Ervin Fodor ◽  
Peter Palese ◽  
George G. Brownlee ◽  
Adolfo García-Sastre
Keyword(s):  
Influenza Virus ◽  
Transcription Initiation ◽  
Influenza A ◽  
Reverse Genetics ◽  
Mrna Levels ◽  
Double Mutation ◽  
Ribonucleoprotein Complexes ◽  
Virus Rna ◽  
Infected Cells

ABSTRACT We have engineered influenza A/WSN/33 viruses which have viral RNA (vRNA) segments with altered base pairs in the conserved double-stranded region of their vRNA promoters. The mutations were introduced into the segment coding for the neuraminidase (NA) by using a reverse genetics system. Two of the rescued viruses which share a C-G→A-U double mutation at positions 11 and 12′ at the 3′ and 5′ ends of the NA-specific vRNA, respectively, showed approximately a 10-fold reduction of NA levels. The mutations did not dramatically affect the NA-specific vRNA levels found in virions or the NA-specific vRNA and cRNA levels in infected cells. In contrast, there was a significant decrease in the steady-state levels of NA-specific mRNAs in infected cells. Transcription studies in vitro with ribonucleoprotein complexes isolated from the two transfectant viruses indicated that transcription initiation of the NA-specific segment was not affected. However, the majority of NA-specific transcripts lacked poly(A) tails, suggesting that mutations in the double-stranded region of the influenza virus vRNA promoter can attenuate polyadenylation of mRNA molecules. This is the first time that a promoter mutation in an engineered influenza virus has shown a differential effect on influenza virus RNA transcription and replication.

scholarly journals A Novel Sulfonated Derivative of β-Cyclodextrin Effectively Inhibits Influenza A Virus Infection in vitro and in vivo

Acta Naturae ◽  
2019 ◽  
Vol 11 (3) ◽  
pp. 20-30 ◽  
Author(s):  
E. P. Goncharova ◽  
Y. A. Kostyro ◽  
A. V. Ivanov ◽  
M. A. Zenkova
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
High Efficiency ◽  
Influenza Infection ◽  
Lethal Infection ◽  
Novel Drugs ◽  
Infected Cells ◽  
Low Toxicity

The development of novel drugs against the influenza virus with high efficiency and low toxicity is an urgent and important task. Previous reports have demonstrated that compounds based on sulfo derivatives of oligo- and polysaccharides possess high antiviral activity. In this study, we have examined the ability of a novel sulfonated derivative of -cyclodextrin (KS-6469) to inhibit the influenza virus A/WSN/33 (H1N1) infection in vitro and in vivo. The antiviral potential of KS-6469 against the influenza virus was evaluated in Madin-Darby Canine Kidney epithelial cells treated with serially diluted KS-6469. We found out that KS-6469 completely inhibited viral reproduction after treatment of the infected cells with the compound for 48 h. Our data show that double intranasal treatment of mice with KS-6469 fully protected the animals from a lethal infection and significantly decreased the viral titers in the lungs of the infected animals. Thus, the novel sulfonated -cyclodextrin derivative KS-6469 is a promising candidate for the development of antiviral drugs for preventing and treating the influenza infection.

In-vitro anti Myxovirus Activity and Mechanism of Antiinfluenzavirus Activity of Polyoxometalates PM-504 and PM-523

1996 ◽  
Vol 7 (6) ◽  
pp. 346-352 ◽  
Author(s):  
S. Shigeta ◽  
S. Mori ◽  
J. Watanabe ◽  
T. Yamase ◽  
R. F. Schinazi
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Mdck Cells ◽  
Cellular Membrane ◽  
Facs Analysis ◽  
Virus Envelope ◽  
Cytotoxic Compounds ◽  
Infected Cells ◽  
Chicken Erythrocytes

Sixty polyoxometalates were examined for anti-influenza A virus (FluV-A) activity in vitro. Two of the most potent and least cytotoxic compounds, PM-504 K9H5(Ge2Ti6W18O77)16H2O] and PM-523 (iPrNH3)6H [PTi2W10O38(O2)9H2O2] were selected for further studies. Examination of the antiviral effects of PM-504 and PM-523 against other human ortho- and paramyxoviruses revealed that both compounds had broad spectrum antimyxovirus activities. From a time of addition study and FACS analysis for influenza A virus infected cells, the compounds were found not to inhibit binding of virus to MDCK cells. However, these compounds inhibited haemolysis of chicken erythrocytes by virus and also inhibited fluorescence dequenching of octadecylrhodamine B-labelled virus after binding to cells. This indicates that these polyoxometalates inhibited fusion of the virus envelope to the cellular membrane.

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