scholarly journals Modeling Amantadine Treatment of Inuenza A Virus In Vitro

2021 ◽  
Author(s):  
Catherine A. A. Beauchemin ◽  
James J. McSharry ◽  
George L. Drusano ◽  
Jack T. Nguyen ◽  
Gregory T. Went ◽  
...  
Keyword(s):  
Experimental Data ◽  
Viral Infection ◽  
Mathematical Models ◽  
Influenza A ◽  
Mdck Cells ◽  
Rapid Development ◽  
Human Model ◽  
Parameter Estimates

We analyzed the dynamics of an influenza A/Albany/1/98 (H3N2) viral infection, using a set of mathematical models highlighting the differences between in vivo and in vitro infection. For example, we found that including virion loss due to cell entry was critical for the in vitro model but not for the in vivo model. Experiments were performed on influenza virus-infected MDCK cells in vitro inside a hollow-fiber (HF) system, which was used to continuously deliver the drug amantadine. The HF system captures the dynamics of an influenza infection, and is a controlled environment for producing experimental data which lend themselves well to mathematical modeling. The parameter estimates obtained from fitting our mathematical models to the HF experimental data are consistent with those obtained earlier for a primary infection in a human model. We found that influenza A/Albany/1/98 (H3N2) virions under normal experimental conditions at 37°C rapidly lose infectivity with a half-life of ~ 6.6 ± 0.2 h, and that the lifespan of productively infected MDCK cells is ~ 13 h. Finally, using our models we estimated that the maximum efficacy of amantadine in blocking viral infection is ~ 74%, and showed that this low maximum efficacy is likely due to the rapid development of drug resistance.

scholarly journals Modeling Amantadine Treatment of Inuenza A Virus In Vitro

2021 ◽  
Author(s):  
Catherine A. A. Beauchemin ◽  
James J. McSharry ◽  
George L. Drusano ◽  
Jack T. Nguyen ◽  
Gregory T. Went ◽  
...  
Keyword(s):  
Experimental Data ◽  
Viral Infection ◽  
Mathematical Models ◽  
Influenza A ◽  
Mdck Cells ◽  
Rapid Development ◽  
Human Model ◽  
Parameter Estimates

We analyzed the dynamics of an influenza A/Albany/1/98 (H3N2) viral infection, using a set of mathematical models highlighting the differences between in vivo and in vitro infection. For example, we found that including virion loss due to cell entry was critical for the in vitro model but not for the in vivo model. Experiments were performed on influenza virus-infected MDCK cells in vitro inside a hollow-fiber (HF) system, which was used to continuously deliver the drug amantadine. The HF system captures the dynamics of an influenza infection, and is a controlled environment for producing experimental data which lend themselves well to mathematical modeling. The parameter estimates obtained from fitting our mathematical models to the HF experimental data are consistent with those obtained earlier for a primary infection in a human model. We found that influenza A/Albany/1/98 (H3N2) virions under normal experimental conditions at 37°C rapidly lose infectivity with a half-life of ~ 6.6 ± 0.2 h, and that the lifespan of productively infected MDCK cells is ~ 13 h. Finally, using our models we estimated that the maximum efficacy of amantadine in blocking viral infection is ~ 74%, and showed that this low maximum efficacy is likely due to the rapid development of drug resistance.

scholarly journals Innate triggering and antiviral effector functions of activin A

2021 ◽  
Author(s):  
Kinda Al-Hourani ◽  
Narayan Ramamurthy ◽  
Emanuele Marchi ◽  
Ruth M Eichinger ◽  
Lian N Lee ◽  
...  
Keyword(s):  
Viral Infection ◽  
Influenza A ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Activin A ◽  
Type I ◽  
Type I Ifn ◽  
Effector Functions

First-line defence against viral infection is contingent upon rapid detection of conserved viral structural and genomic motifs by germline-encoded pattern recognition receptors, followed by activation of the type I IFN system and establishment of an intracellular antiviral state. Novel antiviral functions of bone morphogenetic protein and related activin cytokines, acting in conjunction with, and independently of, type I IFN, have recently been described. Activin A mediates multiple innate and adaptive immune functions, including antiviral effects. However, how such effects are mediated and how activin might be triggered by viral infection have not been defined. Here we addressed this in vivo and in vitro, in humans and mice. Transcriptomic analyses delineated strikingly congruent patterns of gene regulation in hepatocytes stimulated with recombinant activin A and IFNα in vitro. Activin A mRNA, encoded by INHBA, is induced upon activation of RIG-I, MDA5 and TLR7/8 viral nucleic acid sensors in vitro, across multiple cell lines and in human peripheral blood mononuclear cells. In vivo, infection of mice with influenza A also upregulated Inhba mRNA in the lung; this local upregulation of Inhba is retained in MAVS knockout mice, indicating a role for non-RIG-I-like receptors in its induction. Activin induction and signalling were also detectable in patients with chronic viral hepatitis. Together, these data suggest Activin A is triggered in parallel with type I IFN responses and can trigger related antiviral effector functions. This model has implications for the development of targeted antiviral therapies, in addition to revealing novel facets of activin biology.

scholarly journals Zanamivir Susceptibility Monitoring and Characterization of Influenza Virus Clinical Isolates Obtained during Phase II Clinical Efficacy Studies

2000 ◽  
Vol 44 (1) ◽  
pp. 78-87 ◽  
Author(s):  
J. M. Barnett ◽  
A. Cadman ◽  
D. Gor ◽  
M. Dempsey ◽  
M. Walters ◽  
...  
Keyword(s):  
Phase Ii ◽  
Clinical Efficacy ◽  
Influenza A ◽  
Mdck Cells ◽  
Virus Infections ◽  
Virus Shedding ◽  
Efficacy Trials ◽  
Natural Variants

ABSTRACT Zanamivir is a highly selective neuraminidase (NA) inhibitor with demonstrated clinical efficacy against influenza A and B virus infections. In phase II clinical efficacy trials (NAIB2005 and NAIB2008), virological substudies showed mean reductions in virus shedding after 24 h of treatment of 1.5 to 2.0 log1050% tissue culture infective doses compared to a placebo, with no reemergence of virus after the completion of therapy. Paired isolates (n = 41) obtained before and during therapy with zanamivir demonstrated no shifts in susceptibility to zanamivir when measured by NA assays, although for a few isolates NA activity was too low to evaluate. In plaque reduction assays in MDCK cells, the susceptibility of isolates to zanamivir was extremely variable even at baseline and did not correlate with the speed of resolution of virus shedding. Isolates with apparent limited susceptibility to zanamivir by plaque reduction proved highly susceptible in vivo in the ferret model. Further sequence analysis of paired isolates revealed no changes in the hemagglutinin and NA genes in the majority of isolates. The few changes observed were all natural variants. No amino acid changes that had previously been identified in vitro as being involved with reduced susceptibility to zanamivir were observed. These studies highlighted problems associated with monitoring susceptibility to NA inhibitors in the clinic, in that no reliable cell-based assay is available. At present the NA assay is the best available predictor of susceptibility to NA inhibitors in vivo, as measured in the validated ferret model of infection.

scholarly journals Extending the Cytoplasmic Tail of the Influenza A Virus M2 Protein Leads to Reduced Virus Replication In Vivo but Not In Vitro

2007 ◽  
Vol 82 (2) ◽  
pp. 1059-1063 ◽  
Author(s):  
Wai-Hong Wu ◽  
Andrew Pekosz
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Mdck Cells ◽  
Cytoplasmic Tail ◽  
M2 Protein ◽  
Coding Region ◽  
Epitope Tag ◽  
Carboxy Terminal

ABSTRACT A carboxy-terminal epitope tag introduced into the coding region of the A/WSN/33 M2 protein resulted in a recombinant virus (rWSN M2myc) which replicated to titers similar to those of the parental virus (rWSN) in MDCK cells. The rWSN M2myc virus was attenuated in its ability to induce mortality and weight loss after the intranasal inoculation of BALB/c mice, indicating that the M2 cytoplasmic tail plays a role in virus virulence. Mice infected with rWSN M2myc were completely protected from subsequent challenge with rWSN, suggesting that epitope tagging of the M2 protein may be a useful way of attenuating influenza A virus strains.

scholarly journals 3-Indoleacetonitrile Is Highly Effective in Treating Influenza A Virus Infection In Vitro and In Vivo

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1433
Author(s):  
Xuejin Zhao ◽  
Lianzhong Zhao ◽  
Ya Zhao ◽  
Kun Huang ◽  
Wenxiao Gong ◽  
...  
Keyword(s):  
Influenza A ◽  
Mdck Cells ◽  
A549 Cells ◽  
H1n1 Influenza ◽  
Indole Derivatives ◽  
Influenza A Viruses ◽  
Toxic Concentration ◽  
Influenza Activity

Influenza A viruses are serious zoonotic pathogens that continuously cause pandemics in several animal hosts, including birds, pigs, and humans. Indole derivatives containing an indole core framework have been extensively studied and developed to prevent and/or treat viral infection. This study evaluated the anti-influenza activity of several indole derivatives, including 3-indoleacetonitrile, indole-3-carboxaldehyde, 3-carboxyindole, and gramine, in A549 and MDCK cells. Among these compounds, 3-indoleacetonitrile exerts profound antiviral activity against a broad spectrum of influenza A viruses, as tested in A549 cells. Importantly, in a mouse model, 3-indoleacetonitrile with a non-toxic concentration of 20 mg/kg effectively reduced the mortality and weight loss, diminished lung virus titers, and alleviated lung lesions of mice lethally challenged with A/duck/Hubei/WH18/2015 H5N6 and A/Puerto Rico/8/1934 H1N1 influenza A viruses. The antiviral properties enable the potential use of 3-indoleacetonitrile for the treatment of IAV infection.

Kallikrein-related peptidase 5 and seasonal influenza viruses, limitations of the experimental models for activating proteases

2018 ◽  
Vol 399 (9) ◽  
pp. 1053-1064 ◽  
Author(s):  
Mélia Magnen ◽  
Brigitta Margit Elsässer ◽  
Olga Zbodakova ◽  
Petr Kasparek ◽  
Fabien Gueugnon ◽  
...  
Keyword(s):  
Serine Proteases ◽  
Influenza A ◽  
Mdck Cells ◽  
Experimental Models ◽  
Influenza Viruses ◽  
Virus Infectivity ◽  
Peptide Substrates ◽  
Viral Hemagglutinin

Abstract Every year, influenza A virus (IAV) affects and kills many people worldwide. The viral hemagglutinin (HA) is a critical actor in influenza virus infectivity which needs to be cleaved by host serine proteases to exert its activity. KLK5 has been identified as an activating protease in humans with a preference for the H3N2 IAV subtype. We investigated the origin of this preference using influenza A/Puerto Rico/8/34 (PR8, H1N1) and A/Scotland/20/74 (Scotland, H3N2) viruses. Pretreatment of noninfectious virions with human KLK5 increased infectivity of Scotland IAV in MDCK cells and triggered influenza pneumonia in mice. These effects were not observed with the PR8 IAV. Molecular modeling and in vitro enzymatic studies of peptide substrates and recombinant HAs revealed that the sequences around the cleavage site do not represent the sole determinant of the KLK5 preference for the H3N2 subtype. Using mouse Klk5 and Klk5-deficient mice, we demonstrated in vitro and in vivo that the mouse ortholog protease is not an IAV activating enzyme. This may be explained by unfavorable interactions between H3 HA and mKlk5. Our data highlight the limitations of some approaches used to identify IAV-activating proteases.

scholarly journals Conserved Structural Motifs of Two Distant IAV Subtypes in Genomic Segment 5 RNA

Viruses ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 525
Author(s):  
Paula Michalak ◽  
Julita Piasecka ◽  
Barbara Szutkowska ◽  
Ryszard Kierzek ◽  
Ewa Biala ◽  
...  
Keyword(s):  
Experimental Data ◽  
Secondary Structure ◽  
Influenza A Virus ◽  
Influenza A ◽  
Chemical Mapping ◽  
Rna Structures ◽  
Structural Motifs ◽  
2009 H1n1

The functionality of RNA is fully dependent on its structure. For the influenza A virus (IAV), there are confirmed structural motifs mediating processes which are important for the viral replication cycle, including genome assembly and viral packaging. Although the RNA of strains originating from distant IAV subtypes might fold differently, some structural motifs are conserved, and thus, are functionally important. Nowadays, NGS-based structure modeling is a source of new in vivo data helping to understand RNA biology. However, for accurate modeling of in vivo RNA structures, these high-throughput methods should be supported with other analyses facilitating data interpretation. In vitro RNA structural models complement such approaches and offer RNA structures based on experimental data obtained in a simplified environment, which are needed for proper optimization and analysis. Herein, we present the secondary structure of the influenza A virus segment 5 vRNA of A/California/04/2009 (H1N1) strain, based on experimental data from DMS chemical mapping and SHAPE using NMIA, supported by base-pairing probability calculations and bioinformatic analyses. A comparison of the available vRNA5 structures among distant IAV strains revealed that a number of motifs present in the A/California/04/2009 (H1N1) vRNA5 model are highly conserved despite sequence differences, located within previously identified packaging signals, and the formation of which in in virio conditions has been confirmed. These results support functional roles of the RNA secondary structure motifs, which may serve as candidates for universal RNA-targeting inhibitory methods.

scholarly journals 97. Competition Experiments for the Baloxavir-Resistant I38T Influenza A Mutant

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S9-S9
Author(s):  
Liva Checkmahomed ◽  
Zeineb Mhamdi ◽  
Julie Carbonneau ◽  
Mariana Baz ◽  
Yacine Abed ◽  
...  
Keyword(s):  
Weight Loss ◽  
Influenza A ◽  
Mdck Cells ◽  
Digital Pcr ◽  
The United States ◽  
Influenza Viruses ◽  
Viral Fitness ◽  
Copy Numbers

Abstract Background Baloxavir marboxil (BXM), a cap-dependent endonuclease inhibitor, has been recently approved in the United States for the treatment of influenza infections. It is superior to oseltamivir for reducing the time of viral shedding but is reported to have a low barrier of resistance. We sought to evaluate the viral fitness of the predominant BXM-resistant I38T PA mutant in the A/H1N1 and A/H3N2 viral backgrounds. Methods Recombinant A/Quebec/144147/2009 (H1N1) and A/Switzerland/9715293/2013 (H3N2) influenza viruses and their respective I38T PA mutants were generated by reverse genetics. Standardized inoculums (500 PFUs) of wild-type (WT) and mutant mixtures were inoculated on α2,6 MDCK cells. On day 3 post-infection (pi), the supernatants were collected and the ratios of WT/mutant viruses were determined by droplet digital PCR using specific LNA probes. Single infections and competitive experiments were also performed in C56/BL6 mice with quantification of lung viral titers on days 3 and 6 pi. Results In vitro A/H1N1 studies showed similar total copy numbers for the WT and mutant viruses on day 3 pi (1.2 × 109 and 1.3 × 109 copies/mL, respectively). The initial 50%/50% mixture became 70%/30% (WT/mutant) after one passage in cells. For A/H3N2, the total copy numbers were 8.1 × 109 and 1.0 × 109 copies/mL for the WT and mutant viruses. The initial 50%/50% mixture became 94%/6% (WT/mutant) after one passage. The I38T mutants remained stable after 4 passages in α2,6 MDCK cells. In mice, the A/H1N1 WT and I38T mutant induced similar weight loss and generated comparable lung titers on days 3 and 6 pi. In contrast, the weight loss of the A/H3N2 mutant was greater than that of the WT between days 3 and 7 pi with comparable lung titers on days 3 and 6. Following infection with 50%/50% mixtures, the mutant virus predominated over the WT on day 3 pi (73% A/H1N1 and 58% A/H3N2). Conclusion The BXM-resistant I38T PA mutant replicates well both in vitro and in vivo in the A/H1N1 and A/H3N2 backgrounds. Surveillance for the emergence and transmission of such mutant in the community is required. Disclosures All Authors: No reported Disclosures.

Specific Inhibition of Influenza Virus RNA Polymerase and Nucleoprotein Gene Expression by Liposomally Encapsulated Antisense Phosphorothioate Oligonucleotides in MDCK Cells

1998 ◽  
Vol 9 (3) ◽  
pp. 253-262 ◽  
Author(s):  
T Abe ◽  
S Suzuki ◽  
T Hatta ◽  
K Takai ◽  
T Yokota ◽  
...  
Keyword(s):  
Influenza A ◽  
Mdck Cells ◽  
Cationic Lipid ◽  
Inhibitory Effect ◽  
Specific Inhibition ◽  
Nucleoprotein Gene ◽  
Virus Rna ◽  
Endocytic Vesicles

We have demonstrated that antisense phosphorothioate oligonucleotides (S-ODNs) inhibit influenza A virus replication in MDCK cells. Liposomally encapsulated and free antisense S-ODNs with four target sites (PB1, PB2, PA and NP genes) were tested for their abilities to inhibit virus-induced cytopathogenic effects in a MTT assay using MDCK cells. The liposomally encapsulated S-ODN complementary to the site around the PB2 AUG initiation codon showed highly inhibitory effects. In contrast, the inhibitory effect of the liposomally encapsulated S-ODN targeted to PB1 was considerably decreased in comparison with that directed to the PB2 target site. The liposomally encapsulated antisense S-ODNs exhibited higher inhibitory activities than the free oligonucleotides, and showed sequence-specific inhibition, whereas free antisense S-ODNs were observed to inhibit viral adsorption to MDCK cells. Liposomal preparations of oligonucleotides facilitated their release from endocytic vesicles, and thus cytoplasmic and nuclear localization was observed. The activities of the antisense S-ODNs were effectively enhanced by using the liposomal carrier. Interestingly, the liposomally encapsulated FITC-S-ODN-PB2–as accumulated in the nuclear region of MDCK cells. However, weak fluorescence was observed within the endosomes and the cytoplasm of MDCK cells treated with the free antisense S-ODNs. The cationic lipid particles may thus be a potentially useful delivery vehicle for oligonucleotide-based therapeutics and transgenes, appropriate for use in vitro or in vivo.

scholarly journals Palmitoylation of the Influenza A Virus M2 Protein Is Not Required for Virus Replication In Vitro but Contributes to Virus Virulence

2009 ◽  
Vol 83 (17) ◽  
pp. 8655-8661 ◽  
Author(s):  
Michael L. Grantham ◽  
Wai-Hong Wu ◽  
Erin N. Lalime ◽  
Maria E. Lorenzo ◽  
Sabra L. Klein ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Virus Replication ◽  
Influenza A ◽  
Mdck Cells ◽  
Cysteine Residue ◽  
M2 Protein ◽  
Recombinant Viruses ◽  
Virus Particles

ABSTRACT The influenza A virus M2 protein has important roles during virus entry and in the assembly of infectious virus particles. The cytoplasmic tail of the protein can be palmitoylated at a cysteine residue, but this residue is not conserved in a number of human influenza A virus isolates. Recombinant viruses encoding M2 proteins with a serine substituted for the cysteine at position 50 were generated in the A/WSN/33 (H1N1) and A/Udorn/72 (H3N2) genetic backgrounds. The recombinant viruses were not attenuated for replication in MDCK cells, Calu-3 cells, or in primary differentiated murine trachea epithelial cell cultures, indicating there was no significant contribution of M2 palmitoylation to virus replication in vitro. The A/WSN/33 M2C50S virus displayed a slightly reduced virulence after infection of mice, suggesting that there may be novel functions for M2 palmitoylation during in vivo infection.

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