Influenza A virus production in a single-use orbital shaken bioreactor with ATF or TFF perfusion systems

In mammals the interferon (IFN) system is a central innate antiviral defence mechanism, while the involvement of RNA interference (RNAi) in antiviral response against RNA viruses is uncertain. Here, we tested whether RNAi is involved in the antiviral response in mammalian cells. To investigate the role of RNAi in influenza A virus-infected cells in the absence of IFN, we used Vero cells that lack IFN-α and IFN-β genes. Our results demonstrate that knockdown of a key RNAi component, Dicer, led to a modest increase of virus production and accelerated apoptosis of influenza A virus-infected cells. These effects were much weaker in the presence of IFN. The results also show that in both Vero cells and the IFN-producing alveolar epithelial A549 cell line influenza A virus targets Dicer at mRNA and protein levels. Thus, RNAi is involved in antiviral response, and Dicer is important for protection against influenza A virus infection.

Download Full-text

Influenza A Virus Replication Induces Cell Cycle Arrest in G0/G1 Phase

Journal of Virology ◽

10.1128/jvi.01216-10 ◽

2010 ◽

Vol 84 (24) ◽

pp. 12832-12840 ◽

Cited By ~ 85

Author(s):

Yuan He ◽

Ke Xu ◽

Bjoern Keiner ◽

Jianfang Zhou ◽

Volker Czudai ◽

...

Keyword(s):

Cell Cycle ◽

Cell Cycle Arrest ◽

Influenza A Virus ◽

Influenza A ◽

Cell Cycle Progression ◽

Virus Production ◽

Phase Cell ◽

Cycle Progression ◽

Cycle Arrest ◽

Infected Cells

ABSTRACT Many viruses interact with the host cell division cycle to favor their own growth. In this study, we examined the ability of influenza A virus to manipulate cell cycle progression. Our results show that influenza A virus A/WSN/33 (H1N1) replication results in G0/G1-phase accumulation of infected cells and that this accumulation is caused by the prevention of cell cycle entry from G0/G1 phase into S phase. Consistent with the G0/G1-phase accumulation, the amount of hyperphosphorylated retinoblastoma protein, a necessary active form for cell cycle progression through late G1 into S phase, decreased after infection with A/WSN/33 (H1N1) virus. In addition, other key molecules in the regulation of the cell cycle, such as p21, cyclin E, and cyclin D1, were also changed and showed a pattern of G0/G1-phase cell cycle arrest. It is interesting that increased viral protein expression and progeny virus production in cells synchronized in the G0/G1 phase were observed compared to those in either unsynchronized cells or cells synchronized in the G2/M phase. G0/G1-phase cell cycle arrest is likely a common strategy, since the effect was also observed in other strains, such as H3N2, H9N2, PR8 H1N1, and pandemic swine H1N1 viruses. These findings, in all, suggest that influenza A virus may provide favorable conditions for viral protein accumulation and virus production by inducing a G0/G1-phase cell cycle arrest in infected cells.

Download Full-text

The Influenza A Virus M2 Cytoplasmic Tail Is Required for Infectious Virus Production and Efficient Genome Packaging

Journal of Virology ◽

10.1128/jvi.79.6.3595-3605.2005 ◽

2005 ◽

Vol 79 (6) ◽

pp. 3595-3605 ◽

Cited By ~ 127

Author(s):

Matthew F. McCown ◽

Andrew Pekosz

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Infectious Virus ◽

Virus Production ◽

Cytoplasmic Tail ◽

Host Cells ◽

Wild Type Virus ◽

Type Virus ◽

Wild Type ◽

Virus Particles

ABSTRACT The M2 integral membrane protein encoded by influenza A virus possesses an ion channel activity that is required for efficient virus entry into host cells. The role of the M2 protein cytoplasmic tail in virus replication was examined by generating influenza A viruses encoding M2 proteins with truncated C termini. Deletion of 28 amino acids (M2Stop70) resulted in a virus that produced fourfold-fewer particles but >1,000-fold-fewer infectious particles than wild-type virus. Expression of the full-length M2 protein in trans restored the replication of the M2 truncated virus. Although the M2Stop70 virus particles were similar to wild-type virus in morphology, the M2Stop70 virions contained reduced amounts of viral nucleoprotein and genomic RNA, indicating a defect in vRNP packaging. The data presented indicate the M2 cytoplasmic tail plays a role in infectious virus production by coordinating the efficient packaging of genome segments into influenza virus particles.

Download Full-text

Distinct Domains of the Influenza A Virus M2 Protein Cytoplasmic Tail Mediate Binding to the M1 Protein and Facilitate Infectious Virus Production

Journal of Virology ◽

10.1128/jvi.00627-06 ◽

2006 ◽

Vol 80 (16) ◽

pp. 8178-8189 ◽

Cited By ~ 134

Author(s):

Matthew F. McCown ◽

Andrew Pekosz

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Reverse Genetics ◽

Infectious Virus ◽

Virus Assembly ◽

Virus Production ◽

Cytoplasmic Tail ◽

M2 Protein ◽

Virus Particles ◽

M1 Protein

ABSTRACT The cytoplasmic tail of the influenza A virus M2 protein is highly conserved among influenza A virus isolates. The cytoplasmic tail appears to be dispensable with respect to the ion channel activity associated with the protein but important for virus morphology and the production of infectious virus particles. Using reverse genetics and transcomplementation assays, we demonstrate that the M2 protein cytoplasmic tail is a crucial mediator of infectious virus production. Truncations of the M2 cytoplasmic tail result in a drastic decrease in infectious virus titers, a reduction in the amount of packaged viral RNA, a decrease in budding events, and a reduction in budding efficiency. The M1 protein binds to the M2 cytoplasmic tail, but the M1 binding site is distinct from the sequences that affect infectious virus particle formation. Influenza A virus strains A/Udorn/72 and A/WSN/33 differ in their requirements for M2 cytoplasmic tail sequences, and this requirement maps to the M1 protein. We conclude that the M2 protein is required for the formation of infectious virus particles, implicating the protein as important for influenza A virus assembly in addition to its well-documented role during virus entry and uncoating.

Download Full-text

Covalent Functionalization of Melt-Blown Polypropylene Filters with Diazirine–Photosensitizer Conjugates Producing Visible Light Driven Virus Inactivating Materials.

10.26434/chemrxiv.14423672.v1 ◽

2021 ◽

Author(s):

Tyler Cuthbert ◽

Siobhan Ennis ◽

Stefania F. Musolino ◽

Heather L. Buckley ◽

Masahiro Niikura ◽

...

Keyword(s):

Visible Light ◽

Influenza A Virus ◽

Influenza A ◽

Bond Activation ◽

Rna Virus ◽

Filter Material ◽

Pathogen Inactivation ◽

Insertion Method ◽

Visible Light Driven ◽

Single Use

The SARS-CoV-2 pandemic has highlighted the weaknesses of relying on single-use mask and respirator personal protective equipment (PPE) and the global supply chain that supports this market. There have been no major innovations in filter technology for PPE in the past two decades. Non-woven textiles used for filtering PPE are single-use products in the healthcare environment; use and protection is focused on preventing infection from airborne or aerosolized pathogens such as Influenza A virus SARS-CoV-2. Recently, C–H bond activation under mild and controllable conditions was reported for crosslinking commodity aliphatic polymers such as polyethylene and polypropylene. Significantly, these are the same types of polymers used in PPE filtration systems. In this report, we take advantage of this C–H insertion method to covalently attach a photosensitizing zinc-porphyrin to the surface of a melt-blow non-woven textile filter material. With the photosensitizer covalently attached to the surface of the textile, illumination with visible light was expected to produce oxidizing 1O2/ROS at the surface of the material that would result in pathogen inactivation. The filter was tested for its ability to inactivate Influenza A virus, an enveloped RNA virus similar to SARS-CoV-2, over a period of four hours with illumination of high intensity visible light. The photosensitizer-functionalized polypropylene filter inactivated our model virus by 99.99% in comparison to a control.

Download Full-text

Cellular co-infection can modulate the efficiency of influenza A virus production and shape the interferon response

10.1101/752329 ◽

2019 ◽

Cited By ~ 1

Author(s):

Brigitte E. Martin ◽

Jeremy D. Harris ◽

Jiayi Sun ◽

Katia Koelle ◽

Christopher B. Brooke

Keyword(s):

Influenza A Virus ◽

Cell Lines ◽

Influenza A ◽

Mdck Cells ◽

A549 Cells ◽

Virus Production ◽

Innate Immune ◽

Type I ◽

Functional Forms ◽

A Cell

ABSTRACTDuring viral infection, the numbers of virions infecting individual cells can vary significantly over time and space. The functional consequences of this variation in cellular multiplicity of infection (MOI) remain poorly understood. Here, we rigorously quantify the phenotypic consequences of cellular MOI during influenza A virus (IAV) infection over a single round of replication in terms of cell death rates, viral output kinetics, interferon and antiviral effector gene transcription, and superinfection potential. By statistically fitting mathematical models to our data, we precisely define specific functional forms that quantitatively describe the modulation of these phenotypes by MOI at the single cell level. To determine the generality of these functional forms, we compare two distinct cell lines (MDCK cells and A549 cells), both infected with the H1N1 strain A/Puerto Rico/8/1934 (PR8). We find that a model assuming that infected cell death rates are independent of cellular MOI best fits the experimental data in both cell lines. We further observe that a model in which the rate and efficiency of virus production increase with cellular co-infection best fits our observations in MDCK cells, but not in A549 cells. In A549 cells, we also find that induction of type III interferon, but not type I interferon, is highly dependent on cellular MOI, especially at early timepoints. This finding identifies a role for cellular co-infection in shaping the innate immune response to IAV infection. Finally, we show that higher cellular MOI is associated with more potent superinfection exclusion, thus limiting the total number of virions capable of infecting a cell. Overall, this study suggests that the extent of cellular co-infection by influenza viruses may be a critical determinant of both viral production kinetics and cellular infection outcomes in a host cell type-dependent manner.AUTHOR SUMMARYDuring influenza A virus (IAV) infection, the number of virions to enter individual cells can be highly variable. Cellular co-infection appears to be common and plays an essential role in facilitating reassortment for IAV, yet little is known about how cellular co-infection influences infection outcomes at the cellular level. Here, we combine quantitative in vitro infection experiments with statistical model fitting to precisely define the phenotypic consequences of cellular co-infection in two cell lines. We reveal that cellular co-infection can increase and accelerate the efficiency of IAV production in a cell line-dependent fashion, identifying it as a potential determinant of viral replication kinetics. We also show that induction of type III, but not type I, interferon is highly dependent upon the number of virions that infect a given cell, implicating cellular co-infection as an important determinant of the host innate immune response to infection. Altogether, our findings show that cellular co-infection plays a crucial role in determining infection outcome. The integration of experimental and statistical modeling approaches detailed here represents a significant advance in the quantitative study of influenza virus infection and should aid ongoing efforts focused on the construction of mathematical models of IAV infection.

Download Full-text

Application of an Inclined Settler for Cell Culture-Based Influenza A Virus Production in Perfusion Mode

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2020.00672 ◽

2020 ◽

Vol 8 ◽

Author(s):

Juliana Coronel ◽

Gwendal Gränicher ◽

Volker Sandig ◽

Thomas Noll ◽

Yvonne Genzel ◽

...

Keyword(s):

Cell Culture ◽

Influenza A Virus ◽

Influenza A ◽

Virus Production ◽

Perfusion Mode

Download Full-text

Single-Cell Analysis Uncovers a Vast Diversity in Intracellular Viral Defective Interfering RNA Content Affecting the Large Cell-to-Cell Heterogeneity in Influenza A Virus Replication

Viruses ◽

10.3390/v12010071 ◽

2020 ◽

Vol 12 (1) ◽

pp. 71 ◽

Cited By ~ 2

Author(s):

Sascha Young Kupke ◽

Lam-Ha Ly ◽

Stefan Thomas Börno ◽

Alexander Ruff ◽

Bernd Timmermann ◽

...

Keyword(s):

Single Cell ◽

Influenza A Virus ◽

Virus Replication ◽

Influenza A ◽

Single Cell Analysis ◽

Virus Production ◽

Large Cell ◽

Cell Heterogeneity ◽

Viral Mrnas ◽

Cell To Cell Variability

Virus replication displays a large cell-to-cell heterogeneity; yet, not all sources of this variability are known. Here, we study the effect of defective interfering (DI) particle (DIP) co-infection on cell-to-cell variability in influenza A virus (IAV) replication. DIPs contain a large internal deletion in one of their eight viral RNAs (vRNA) and are, thus, defective in virus replication. Moreover, they interfere with virus replication. Using single-cell isolation and reverse transcription polymerase chain reaction, we uncovered a large between-cell heterogeneity in the DI vRNA content of infected cells, which was confirmed for DI mRNAs by single-cell RNA sequencing. A high load of intracellular DI vRNAs and DI mRNAs was found in low-productive cells, indicating their contribution to the large cell-to-cell variability in virus release. Furthermore, we show that the magnitude of host cell mRNA expression (some factors may inhibit virus replication), but not the ribosome content, may further affect the strength of single-cell virus replication. Finally, we show that the load of viral mRNAs (facilitating viral protein production) and the DI mRNA content are, independently from one another, connected with single-cell virus production. Together, these insights advance single-cell virology research toward the elucidation of the complex multi-parametric origin of the large cell-to-cell heterogeneity in virus infections.

Download Full-text

Thapsigargin at Non-Cytotoxic Levels Induces a Potent Host Antiviral Response that Blocks Influenza A Virus Replication

Viruses ◽

10.3390/v12101093 ◽

2020 ◽

Vol 12 (10) ◽

pp. 1093

Author(s):

Leah V. Goulding ◽

Jiayun Yang ◽

Zhimin Jiang ◽

Hongyu Zhang ◽

Daniel Lea ◽

...

Keyword(s):

Virus Infection ◽

Influenza A Virus ◽

Influenza A ◽

Unmet Need ◽

Virus Production ◽

Antiviral Response ◽

Interferon Response ◽

Attractive Alternative ◽

Type I ◽

Host Antiviral Response

Influenza A virus is a major global pathogen of humans, and there is an unmet need for effective antivirals. Current antivirals against influenza A virus directly target the virus and are vulnerable to mutational resistance. Harnessing an effective host antiviral response is an attractive alternative. We show that brief exposure to low, non-toxic doses of thapsigargin (TG), an inhibitor of the sarcoplasmic/endoplasmic reticulum (ER) Ca2+ ATPase pump, promptly elicits an extended antiviral state that dramatically blocks influenza A virus production. Crucially, oral administration of TG protected mice against lethal virus infection and reduced virus titres in the lungs of treated mice. TG-induced ER stress unfolded protein response appears as a key driver responsible for activating a spectrum of host antiviral defences that include an enhanced type I/III interferon response. Our findings suggest that TG is potentially a viable host-centric antiviral for the treatment of influenza A virus infection without the inherent problem of drug resistance.

Download Full-text