scholarly journals 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 ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 71 ◽  
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.

scholarly journals Single cell infection with influenza A virus using drop-based microfluidics

2021 ◽  
Author(s):  
Emma Kate Loveday ◽  
Humberto S. Sanchez ◽  
Mallory M. Thomas ◽  
Connie B. Chang
Keyword(s):  
Single Cell ◽  
Influenza A Virus ◽  
Influenza A ◽  
Single Cell Analysis ◽  
Rna Virus ◽  
Host Cells ◽  
Single Cell Level ◽  
Cell Infection ◽  
Cell Level ◽  
High Genetic Diversity

SummaryInfluenza A virus (IAV) is an RNA virus with high genetic diversity which necessitates the development of new vaccines targeting emerging mutations each year. As IAV exists in genetically heterogeneous populations, current studies focus on understanding population dynamics at the single cell level. These studies include novel methodology that can be used for probing populations at the single cell level, such as single cell sequencing and microfluidics. Here, we introduce a drop-based microfluidics method to study IAV infection at a single cell level by isolating infected host cells in microscale drops. Single human alveolar basal epithelial (A549), Madin-Darby Canine Kidney cells (MDCK) and MDCK + human siat7e gene (Siat7e) cells infected with the pandemic A/California/07/2009 (H1N1) strain were encapsulated within 50 μm radii drops and incubated at 37°C. We demonstrate that drops remain stable over 24 hours, that 75% of cells remain viable, and that IAV virus can propagate within the drops. Drop-based microfluidics therefore enables single cell analysis of viral populations produced from individually infected cells.

scholarly journals Single cell heterogeneity in influenza A virus gene expression shapes the innate antiviral response to infection

2020 ◽  
Vol 16 (7) ◽  
pp. e1008671 ◽  
Author(s):  
Jiayi Sun ◽  
J. Cristobal Vera ◽  
Jenny Drnevich ◽  
Yen Ting Lin ◽  
Ruian Ke ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Influenza A Virus ◽  
Influenza A ◽  
Antiviral Response ◽  
Cell Heterogeneity ◽  
Virus Gene ◽  
Virus Gene Expression ◽  
Innate Antiviral Response

scholarly journals Multiplexed profiling of single-cell extracellular vesicles secretion

2019 ◽  
Vol 116 (13) ◽  
pp. 5979-5984 ◽  
Author(s):  
Yahui Ji ◽  
Dongyuan Qi ◽  
Linmei Li ◽  
Haoran Su ◽  
Xiaojie Li ◽  
...  
Keyword(s):  
Single Cell ◽  
Cell Lines ◽  
Extracellular Vesicles ◽  
Single Cell Analysis ◽  
Comprehensive Evaluation ◽  
Single Cells ◽  
Deep Understanding ◽  
Principal Component ◽  
Cell Heterogeneity ◽  
Indispensable Tool

Extracellular vesicles (EVs) are important intercellular mediators regulating health and diseases. Conventional methods for EV surface marker profiling, which was based on population measurements, masked the cell-to-cell heterogeneity in the quantity and phenotypes of EV secretion. Herein, by using spatially patterned antibody barcodes, we realized multiplexed profiling of single-cell EV secretion from more than 1,000 single cells simultaneously. Applying this platform to profile human oral squamous cell carcinoma (OSCC) cell lines led to a deep understanding of previously undifferentiated single-cell heterogeneity underlying EV secretion. Notably, we observed that the decrement of certain EV phenotypes (e.g.,CD63+EV) was associated with the invasive feature of both OSCC cell lines and primary OSCC cells. We also realized multiplexed detection of EV secretion and cytokines secretion simultaneously from the same single cells to investigate the multidimensional spectrum of cellular communications, from which we resolved tiered functional subgroups with distinct secretion profiles by visualized clustering and principal component analysis. In particular, we found that different cell subgroups dominated EV secretion and cytokine secretion. The technology introduced here enables a comprehensive evaluation of EV secretion heterogeneity at single-cell level, which may become an indispensable tool to complement current single-cell analysis and EV research.

scholarly journals Dicer is involved in protection against influenza A virus infection

2007 ◽  
Vol 88 (10) ◽  
pp. 2627-2635 ◽  
Author(s):  
Alexey A. Matskevich ◽  
Karin Moelling
Keyword(s):  
Virus Infection ◽  
Influenza A Virus ◽  
Mammalian Cells ◽  
Influenza A ◽  
Virus Production ◽  
Vero Cells ◽  
Antiviral Response ◽  
Protein Levels ◽  
Infected Cells ◽  
Influenza A Virus Infection

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.

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

2010 ◽  
Vol 84 (24) ◽  
pp. 12832-12840 ◽  
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.

MC1568 inhibits HDAC6/8 activity and influenza A virus replication in lung epithelial cells: role of Hsp90 acetylation

2016 ◽  
Vol 8 (17) ◽  
pp. 2017-2031 ◽  
Author(s):  
Simona Panella ◽  
Maria Elena Marcocci ◽  
Ignacio Celestino ◽  
Sergio Valente ◽  
Clemens Zwergel ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Influenza A Virus ◽  
Virus Replication ◽  
Influenza A ◽  
Lung Epithelial Cells ◽  
Lung Epithelial

CRISPR-Cas9 mediated knockout of AnxA6 gene enhances influenza A virus replication in low-permissive HEK293FT cell line

Gene ◽  
2021 ◽  
pp. 146024
Author(s):  
Andrey Komissarov ◽  
Mariia Sergeeva ◽  
Evgenii Zhuravlev ◽  
Sergey Medvedev ◽  
Anastasia Malakhova ◽  
...  
Keyword(s):  
Cell Line ◽  
Influenza A Virus ◽  
Virus Replication ◽  
Influenza A ◽  
Hek293ft Cell
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