Comparison of infectious influenza A virus quantification methods employing immuno-staining

Hollý, Jaroslav; Fogelová, Margaréta; Jakubcová, Lucia; Tomčíková, Karolína; Vozárová, Mária; Varečková, E; Kostolanský, F

doi:10.1016/j.jviromet.2017.06.004

Cited by 17 publications

(26 citation statements)

References 22 publications

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“…The ultra-centrifuged lentiviral particles were re-suspended in sterile PBS and analyzed via TCID50, western blotting and luciferase activity assay. TCID50 assay was carried out to determine the viral titer and cytopathic effects of infected cells (40). Briefly, MDCK cells (1 × 10 5 ) were infected with either purified pH1N1 and H3N2 viral parties or pseudotyped lentiviral particles, incubated at 37 • C for 3 days under 5% v/v CO 2 , until a cytopathic effect was observed in terms of structural changes in MDCK cells caused by viral invasion.…”

Section: Cell Culture and Treatmentsmentioning

confidence: 99%

Complement-Independent Modulation of Influenza A Virus Infection by Factor H

et al. 2020

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The complement system is an ancient innate immune defense mechanism that can recognize molecular patterns on the invading pathogens. Factor H, as an inhibitor of the alternative pathway, down-regulates complement activation on the host cell surface. Locally synthesized factor H at the site of infection/injury, including lungs, can act as a pattern recognition molecule without involving complement activation. Here, we report that factor H, a sialic acid binder, interacts with influenza A virus (IAV) and modulates IAV entry, as evident from down-regulation of matrix protein 1 (M1) in H1N1 subtype-infected cells and up-regulation of M1 expression in H3N2-infected A549 cells. Far-western blot revealed that factor H binds hemagglutinin (HA, ∼70 kDa), neuraminidase (NA, ∼60 kDa), and M1 (∼25 kDa). IAV-induced transcriptional levels of IFN-α, TNF-α, IL-12, IL-6, IFN-α, and RANTES were reduced following factor H treatment for the H1N1 subtype at 6 h post-infection. However, for the H3N2 subtype, mRNA levels of these pro-inflammatory cytokines were enhanced. A recombinant form of vaccinia virus complement control protein (VCP), which like factor H, contains CCP modules and has complement-regulatory activity, mirrored the results obtained with factor H. Both factor H (25%), and VCP (45%) were found to reduce luciferase reporter activity in MDCK cells transduced with H1N1 pseudotyped lentiviral particles. Factor H (50%) and VCP (30%) enhanced the luciferase reporter activity for H3N2, suggesting an entry inhibitory role of factor H and VCP against H1N1, but not H3N2. Thus, factor H can modulate IAV infection and inflammatory responses, independent of its complement-related functions.

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Section: Cell Culture and Treatmentsmentioning

confidence: 99%

Complement-Independent Modulation of Influenza A Virus Infection by Factor H

et al. 2020

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“…We evaluated the acid stability of the HA2 mutant viruses after their exposure to different pH, ranging from 4.8 to 6.8 (Δ 0.2) for 10 min at 37 °C. The infectivity of all viruses was determined by a modified plaque assay on MDCK cells [16]. Compared with the low pH stability of the wt and mutant viruses, we observed that the wt and mutants retained the highest infectivity at pH 6.4-6.8 (Fig.…”

Section: Acid Stability Of Ha2 Mutant Virusesmentioning

confidence: 88%

“…After this interval monolayers were washed and incubated at 37 °C for 12, 24, 36, 48, 60 and 72 h in MEM with 0.5 % FBS. The infectious virus titres in the supernatant of infected cells at each time-point were measured by a plaque assay [ 16–18 ].…”

Section: Methodsmentioning

confidence: 99%

“…The bound mAb was detected with goat anti-mouse IgG conjugated with horseradish peroxidase (GAM IgG-Px; Bio-Rad) and the reaction was visualized by the addition of a substrate solution containing 3-amino-9-ethylcarbazole (Sigma) in 0.03 % H 2 O 2 . The visible immune-stained viral plaques were evaluated to determine infectious virus titre [16]. The experiment was repeated twice in quadruplicate for each virus.…”

Section: Acid Stability Determination Of Viruses By Plaque Assaymentioning

confidence: 99%

“…Stained red coloured cells identified by light microscopy were considered as positive. The virus titre was expressed as the reciprocal value of the highest sample dilution positive for replicating virus in cell culture [16].…”

Section: Rapid Culture Assay (Rca)mentioning

confidence: 99%

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Biological properties of influenza A virus mutants with amino acid substitutions in the HA2 glycoprotein of the HA1/HA2 interaction region

Jakubcová

Vozárová

Hollý

et al. 2019

Journal of General Virology

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Influenza A viruses (IAVs) enter into cells by receptor-dependent endocytosis. Subsequently, conformational changes of haemagglutinin are triggered by low environmental pH and the N terminus of HA2 glycoprotein (gp) is inserted into the endosomal membrane, resulting in fusion pore formation and genomic vRNA release into the cytoplasm. However, the pH optimum of membrane fusion is host-and virus-specific and can have an impact on virus pathogenicity. We prepared mutants of neurotropic IAV A/WSN/33 (H1N1) with aa substitutions in HA2 gp at the site of HA1/HA2 interaction, namely T64 2 H (HA2 numbering position 64, H1 numbering position HA407; referred to as mutant '64'), V66 2 H ('66') (HA409); and a double mutant ('D') with two aa substitutions (T64 2 H, V66 2 H). These substitutions were hypothesized to influence the pH optimum of fusion. The pH optimum of fusion activity was measured by a luciferase assay and biological properties of viruses were monitored. The in vitro and in vivo replication ability and pathogenicity of mutants were comparable (64) or lower (66, D) than those of the wild-type virus. However, the HA2 mutation V66 2 H and double mutation T64 2 H, V66 2 H shifted the fusion pH maximum to lower values (ranging from 5.1 to 5.3) compared to pH from 5.4 to 5.6 for the wild-type and 64 mutant. The decreased replication ability and pathogenicity of 66 and D mutants was accompanied by higher titres in late intervals post-infection in lungs, and viral RNA in brains compared to wild-type virus-infected mice. These results have implications for understanding the pathogenicity of influenza viruses.

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Sterilization of Drug‐Resistant Influenza Virus through Genetic Interference Inspired by Unnatural Amino Acid‐Engineered Particles

Zheng

Chen

et al. 2022

Advanced Therapeutics

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The frequent emergence of drug resistance during the treatment of influenza A virus (IAV) infections highlights a need for effective antiviral countermeasures. Here, this work presents an antiviral method that utilizes unnatural amino acid-engineered drug-resistant (UAA-DR) virus generated through genetic code expansion to combat emerging drug-resistant viruses through genomic segment reassortment to produce sterilized progenies due to artificial amber codons in the viral genome. This work validates in vitro that UAA-DR can provide a broad-spectrum antiviral strategy for several H1N1 strains, different DR-IAV strains, multidrug-resistant (MDR) strains, and even antigenically distant influenza strains (e.g., H3N2). Moreover, a minimum dose of neuraminidase (NA) inhibitors for influenza virus can further enhance the sterilizing effect when combating inhibitor-resistant strains, partly due to the promoted superinfection of unnatural amino acid-modified virus in cellular and animal models. The fact that the antiviral effect of UAA-DR virus cannot be attributed to interferon stimulation and interferon stimulating genes is also investigated. It is anticipated that UAA-engineered virus-like particle can be extended to combat emerging drug-resistant influenza virus and other segmented RNA viruses.

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Comparison of infectious influenza A virus quantification methods employing immuno-staining

Cited by 17 publications

References 22 publications

Complement-Independent Modulation of Influenza A Virus Infection by Factor H

Complement-Independent Modulation of Influenza A Virus Infection by Factor H

Biological properties of influenza A virus mutants with amino acid substitutions in the HA2 glycoprotein of the HA1/HA2 interaction region

Sterilization of Drug‐Resistant Influenza Virus through Genetic Interference Inspired by Unnatural Amino Acid‐Engineered Particles

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