Replication and transmission of an influenza A(H3N2) virus harboring the polymerase acidic I38T substitution, in guinea pigs.

2021 ◽  
Vol 102 (10) ◽  
Author(s):  
Zeineb Mhamdi ◽  
Julie Carbonneau ◽  
Marie-Christine Venable ◽  
Mariana Baz ◽  
Yacine Abed ◽  
...  
Keyword(s):  
Guinea Pigs ◽  
Influenza A ◽  
Reverse Genetics ◽  
Mdck Cells ◽  
A549 Cells ◽  
Viral Fitness ◽  
Viral Population ◽  
H3n2 Virus ◽  
The Impact

The polymerase acidic (PA) I38T substitution is a dominant marker of resistance to baloxavir. We evaluated the impact of I38T on the fitness of a contemporary influenza A(H3N2) virus. Influenza A/Switzerland/9715293/2013 (H3N2) wild-type (WT) virus and its I38T mutant were rescued by reverse genetics. Replication kinetics were compared using ST6GalI-MDCK and A549 cells and infectivity/contact transmissibility were evaluated in guinea pigs. Nasal wash (NW) viral titres were determined by TCID50 ml−1 in ST6GalI-MDCK cells. Competition experiments were performed and the evolution of viral population was assessed by droplet digital RT-PCR. I38T did not alter in vitro replication. I38T induced comparable titres vs the WT in guinea pigs NWs and the two viruses transmitted equally by direct contact. However, a 50 %:50 % mixture inoculum evolved to mean WT/I38T ratios of 71 %:29 % and 66.4 %:33.6 % on days 4 and 6 p.i., respectively. Contemporary influenza A(H3N2)-I38T PA variants may conserve a significant level of viral fitness.

scholarly journals In Vitro Properties and Virulence of Contemporary Recombinant Influenza B Viruses Harboring Mutations of Cross-Resistance to Neuraminidase Inhibitors

Viruses ◽  
2018 ◽  
Vol 11 (1) ◽  
pp. 6 ◽  
Author(s):  
Clément Fage ◽  
Yacine Abed ◽  
Liva Checkmahomed ◽  
Marie-Christine Venable ◽  
Guy Boivin
Keyword(s):  
Influenza A ◽  
Mdck Cells ◽  
Viral Fitness ◽  
World Health ◽  
Cross Resistance ◽  
Influenza B ◽  
Neuraminidase Inhibitors ◽  
Clinical Markers ◽  
The Impact

Three neuraminidase inhibitors (NAIs: Oseltamivir, zanamivir and peramivir) are currently approved in many countries for the treatment of influenza A and B infections. The emergence of influenza B viruses (IBVs) containing mutations of cross-resistance to these NAIs constitutes a serious clinical threat. Herein, we used a reverse genetics system for the current B/Phuket/3073/2013 vaccine strain to investigate the impact on in vitro properties and virulence of H136N, R152K, D198E/N, I222T and N294S NA substitutions (N2 numbering), reported by the World Health Organization (WHO) as clinical markers of reduced or highly-reduced inhibition (RI/HRI) to multiple NAIs. Recombinant viruses were tested by NA inhibition assays. Their replicative capacity and virulence were evaluated in ST6GalI-MDCK cells and BALB/c mice, respectively. All NA mutants (excepted D198E/N) showed RI/HRI phenotypes against ≥ 2 NAIs. These mutants grew to comparable titers of the recombinant wild-type (WT) IBV in vitro, and some of them (H136N, I222T and N294S mutants) induced more weight loss and mortality in BALB/c mice in comparison to the recombinant WT IBV. These results demonstrate that, in contemporary IBVs, some NA mutations may confer RI/HRI phenotypes to existing NAIs without altering the viral fitness. This reinforces the need for development of novel antiviral strategies with different mechanisms of action.

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.

scholarly journals Zanamivir-resistant influenza viruses with Q136K or Q136R neuraminidase residue mutations can arise during MDCK cell culture creating challenges for antiviral susceptibility monitoring

2015 ◽  
Vol 20 (45) ◽  
Author(s):  
Karen Little ◽  
Sook-Kwan Leang ◽  
Jeff Butler ◽  
Chantal Baas ◽  
Bruce Harrower ◽  
...  
Keyword(s):  
Cell Culture ◽  
Influenza A ◽  
Treatment Guidelines ◽  
Mdck Cells ◽  
Influenza Viruses ◽  
Viral Fitness ◽  
Patient Treatment ◽  
Close Monitoring ◽  
Clinical Specimens

Surveillance of circulating influenza strains for antiviral susceptibility is important to ensure patient treatment guidelines remain appropriate. Influenza A(H3N2) and A(H1N1)pdm09 virus isolates containing mutations at the Q136 residue of the neuraminidase (NA) that conferred reduced susceptibility to the NA inhibitor (NAI) zanamivir were detected during antiviral susceptibility monitoring. Interestingly, the mutations were not detectable in the viruses from respective clinical specimens, only in the cultured isolates. We showed that variant viruses containing the Q136K and Q136R NA mutations were preferentially selected in Madin-Darby canine kidney epithelial (MDCK) cells, but were less well supported in MDCK-SIAT1 cells and embryonated eggs. The effect of Q136K, Q136R, Q136H and Q136L substitutions in NA subtypes N1 and N2 on NAI susceptibility and in vitro viral fitness was assessed. This study highlights the challenges that cell culture derived mutations can pose to the NAI susceptibility analysis and interpretation and reaffirms the need to sequence viruses from respective clinical specimens to avoid misdiagnosis. However, we also demonstrate that NA mutations at residue Q136 can confer reduced zanamivir, peramivir or laninamivir susceptibility, and therefore close monitoring of viruses for mutations at this site from patients being treated with these antivirals is important.

scholarly journals Replication of a Dog-Origin H6N1 Influenza Virus in Cell Culture and Mice

Viruses ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 704
Author(s):  
Shou-Kuan Tsai ◽  
Cheng-Hsin Shih ◽  
Hui-Wen Chang ◽  
Kuang-Huan Teng ◽  
Wei-En Hsu ◽  
...  
Keyword(s):  
Influenza A ◽  
Mdck Cells ◽  
Plaque Assay ◽  
A549 Cells ◽  
Interspecies Transmission ◽  
Virus Shedding ◽  
Transmission Potential ◽  
Relative Viability ◽  
Avian Origin ◽  
The Impact

The world’s first natural avian-origin H6N1 influenza A virus infection case in dogs was confirmed in Taiwan in 2014. The H6N1 virus in chickens has been endemic in Taiwan since 1972. Whether the dog H6N1 virus has interspecies transmission potential is the key issue we aim to understand. Following one virus passage in embryonated eggs and two further passages in MDCK cells, we obtained two virus derivatives, E01EE (PB1 739E and PB2 627E) and E01GK (PB1 739G and PB2 627K), respectively. The pathogenicity of E01EE and E01GK was investigated using plaque assay, growth dynamic analysis and cell viability quantification in cells from different animal species. The impact of amino acid mutation on PB1 739 and PB2 627 on viral ribonucleoprotein (RNP) activity was also analyzed. Further mouse infection experiments were performed. The results showed that both E01EE and E01GK decreased cell relative viability of canine MDCK cells, human A549 cells and chicken DF1 cells. E01Gk caused greater cellular harm in MDCK and A549 cells and had significantly higher virus titers in all of the cells compared to E01EE. The PB2 627K but not PB1 739G was the critical mutation that influenced the viral RNP activity. Both E01EE and E01GK caused mice pneumonia and considerable virus shedding, especially E01GK. This report verifies PB2 E627K mutation in virulence and spotlights the potential for the dog H6N1 virus to extend interspecies transmission.

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.

scholarly journals Impact of the Baloxavir-Resistant Polymerase Acid I38T Substitution on the Fitness of Contemporary Influenza A(H1N1)pdm09 and A(H3N2) Strains

2019 ◽  
Vol 221 (1) ◽  
pp. 63-70 ◽  
Author(s):  
Liva Checkmahomed ◽  
Zeineb M’hamdi ◽  
Julie Carbonneau ◽  
Marie-Christine Venable ◽  
Mariana Baz ◽  
...  
Keyword(s):  
Influenza A ◽  
Mdck Cells ◽  
Influenza Viruses ◽  
Recombinant Viruses ◽  
Influenza A H1n1 ◽  
2009 H1n1 ◽  
Similar Weight ◽  
Viral Subtypes ◽  
The Impact

Abstract Background Baloxavir is a cap-dependent inhibitor of the polymerase acid (PA) protein of influenza viruses. While appearing virologically superior to oseltamivir, baloxavir exhibits a low barrier of resistance. We sought to assess the impact of the common baloxavir-resistant I38T PA substitution on in vitro properties and virulence. Methods Influenza A/Quebec/144147/2009 (H1N1)pdm09 and A/Switzerland/9715293/2013 (H3N2) recombinant viruses and their I38T PA mutants were compared in single and competitive infection experiments in ST6GalI-MDCK cells and C57/BL6 mice. Virus titers in cell culture supernatants and lung homogenates were determined by virus yield assays. Ratios of wild-type (WT) and I38T mutant were assessed by digital RT-PCR. Results I38T substitution did not alter the replication kinetics of A(H1N1)pdm09 and A(H3N2) viruses. In competition experiments, a 50%:50% mixture evolved to 70%:30% (WT/mutant) for A(H1N1) and 88%:12% for A(H3N2) viruses after a single cell passage. The I38T substitution remained stable after 4 passages in vitro. In mice, the WT and its I38T mutant induced similar weight loss with comparable lung titers in both viral subtypes. The mutant virus tended to predominate over the WT in mouse competition experiments. Conclusion The fitness of baloxavir-resistant I38T PA mutants appears relatively unaltered in seasonal subtypes warranting surveillance for its dissemination.

scholarly journals Generation of a Reassortant Influenza A Subtype H3N2 Virus Expressing Gaussia Luciferase

Viruses ◽  
2019 ◽  
Vol 11 (7) ◽  
pp. 665 ◽  
Author(s):  
Lin Wang ◽  
Qinghua Cui ◽  
Xiujuan Zhao ◽  
Ping Li ◽  
Yanyan Wang ◽  
...  
Keyword(s):  
Influenza A ◽  
Reverse Genetics ◽  
Mdck Cells ◽  
H3n2 Virus ◽  
Influenza A Viruses ◽  
Madin Darby Canine Kidney ◽  
Gaussia Luciferase ◽  
Ns Gene ◽  
Darby Canine Kidney ◽  
Canine Kidney

Reporter influenza A viruses (IAVs) carrying fluorescent or luminescent genes provide a powerful tool for both basic and translational research. Most reporter IAVs are based on the backbone of either subtype H1N1 viruses, A/Puerto Rico/8/1934 (PR8) or A/WSN/1933, but no reporter subtype H3N2 virus is currently available to our knowledge. Since the IAV subtype H3N2 co-circulates with H1N1 among humans causing annual epidemics, a reporter influenza A subtype H3N2 virus would be highly valuable. In this study, the segments of A/Wyoming/3/03 (NY, H3N2) virus encoding hemagglutinin and neuraminidase, respectively, were reassorted with the six internal genes of PR8 where the NS gene was fused with a Gaussia luciferase (Gluc) gene. Using reverse genetics, NY-r19-Gluc, a replication competent reassortant influenza A subtype H3N2 virus expressing reporter Gluc was successfully generated. This reporter virus is stable during replication in Madin-Darby canine kidney (MDCK) cells, and preliminary studies demonstrated it as a useful tool to evaluate antivirals. In addition, NY-r19-Gluc virus will be a powerful tool in other studies including the application of diagnostic and therapeutic antibodies as well as the evaluation of novel vaccines.

scholarly journals In Vivo and In Vitro Alterations in Influenza A/H3N2 Virus M2 and Hemagglutinin Genes: Effect of Passage in MDCK-SIAT1 Cells and Conventional MDCK Cells

2008 ◽  
Vol 47 (2) ◽  
pp. 466-468 ◽  
Author(s):  
D. Li ◽  
R. Saito ◽  
Y. Suzuki ◽  
I. Sato ◽  
H. Zaraket ◽  
...  
Keyword(s):  
Influenza A ◽  
Mdck Cells ◽  
H3n2 Virus

VDAC1 Mediated Anticancer Activity of Gallic Acid in Human Lung Adenocarcinoma A549 Cells

2018 ◽  
Vol 18 (2) ◽  
pp. 255-262 ◽  
Author(s):  
Aikebaier Maimaiti ◽  
Amier Aili ◽  
Hureshitanmu Kuerban ◽  
Xuejun Li
Keyword(s):  
Western Blot ◽  
Cell Viability ◽  
Gallic Acid ◽  
Molecular Mechanisms ◽  
A549 Cells ◽  
Dependent Manner ◽  
Lung Carcinoma Cells ◽  
Induced Apoptosis ◽  
The Impact

Aims: Gallic acid (GA) is generally distributed in a variety of plants and foods, and possesses cell growth-inhibiting activities in cancer cell lines. In the present study, the impact of GA on cell viability, apoptosis induction and possible molecular mechanisms in cultured A549 lung carcinoma cells was investigated. Methods: In vitro experiments showed that treating A549 cells with various concentrations of GA inhibited cell viability and induced apoptosis in a dose-dependent manner. In order to understand the mechanism by which GA inhibits cell viability, comparative proteomic analysis was applied. The changed proteins were identified by Western blot and siRNA methods. Results: Two-dimensional electrophoresis revealed changes that occurred to the cells when treated with or without GA. Four up-regulated protein spots were clearly identified as malate dehydrogenase (MDH), voltagedependent, anion-selective channel protein 1(VDAC1), calreticulin (CRT) and brain acid soluble protein 1(BASP1). VDAC1 in A549 cells was reconfirmed by western blot. Transfection with VDAC1 siRNA significantly increased cell viability after the treatment of GA. Further investigation showed that GA down regulated PI3K/Akt signaling pathways. These data strongly suggest that up-regulation of VDAC1 by GA may play an important role in GA-induced, inhibitory effects on A549 cell viability.

scholarly journals Multiple Influenza A (H3N2) Mutations Conferring Resistance to Neuraminidase Inhibitors in a Bone Marrow Transplant Recipient

2014 ◽  
Vol 58 (12) ◽  
pp. 7188-7197 ◽  
Author(s):  
Alireza Eshaghi ◽  
Sarah Shalhoub ◽  
Paul Rosenfeld ◽  
Aimin Li ◽  
Rachel R. Higgins ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Antiviral Therapy ◽  
Influenza A ◽  
Antiviral Treatment ◽  
Marrow Transplant ◽  
Viral Fitness ◽  
H3n2 Virus ◽  
Resistance Mutations ◽  
Depth Analysis ◽  
Resistant Strains

ABSTRACTImmunocompromised patients are predisposed to infections caused by influenza virus. Influenza virus may produce considerable morbidity, including protracted illness and prolonged viral shedding in these patients, thus prompting higher doses and prolonged courses of antiviral therapy. This approach may promote the emergence of resistant strains. Characterization of neuraminidase (NA) inhibitor (NAI)-resistant strains of influenza A virus is essential for documenting causes of resistance. In this study, using quantitative real-time PCR along with conventional Sanger sequencing, we identified an NAI-resistant strain of influenza A (H3N2) virus in an immunocompromised patient. In-depth analysis by deep gene sequencing revealed that various known markers of antiviral resistance, including transient R292K and Q136K substitutions and a sustained E119K (N2 numbering) substitution in the NA protein emerged during prolonged antiviral therapy. In addition, a combination of a 4-amino-acid deletion at residues 245 to 248 (Δ245-248) accompanied by the E119V substitution occurred, causing resistance to or reduced inhibition by NAIs (oseltamivir, zanamivir, and peramivir). Resistant variants within a pool of viral quasispecies arose during combined antiviral treatment. More research is needed to understand the interplay of drug resistance mutations, viral fitness, and transmission.

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