scholarly journals QuantIF: An ImageJ Macro to Automatically Determine the Percentage of Infected Cells after Immunofluorescence

Viruses ◽  
2019 ◽  
Vol 11 (2) ◽  
pp. 165 ◽  
Author(s):  
Lynda Handala ◽  
Tony Fiore ◽  
Yves Rouillé ◽  
Francois Helle
Keyword(s):  
Viral Infections ◽  
Java Virtual Machine ◽  
High Content Screening ◽  
Supplementary Data ◽  
Reporter Protein ◽  
Fluorescent Reporter ◽  
Processing Program ◽  
Infected Cells ◽  
Number Of Cells

Counting labeled cells, after immunofluorescence or expression of a genetically fluorescent reporter protein, is frequently used to quantify viral infection. However, this can be very tedious without a high content screening apparatus. For this reason, we have developed QuantIF, an ImageJ macro that automatically determines the total number of cells and the number of labeled cells from two images of the same field, using DAPI- and specific-stainings, respectively. QuantIF can automatically analyze hundreds of images, taking approximately one second for each field. It is freely available as supplementary data online at MDPI.com and has been developed using ImageJ, a free image processing program that can run on any computer with a Java virtual machine, which is distributed for Windows, Mac, and Linux. It is routinely used in our labs to quantify viral infections in vitro, but can easily be used for other applications that require quantification of labeled cells.

scholarly journals Transcriptional role of p53 in interferon-mediated antiviral immunity

2008 ◽  
Vol 205 (8) ◽  
pp. 1929-1938 ◽  
Author(s):  
César Muñoz-Fontela ◽  
Salvador Macip ◽  
Luis Martínez-Sobrido ◽  
Lauren Brown ◽  
Joseph Ashour ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Viral Replication ◽  
Viral Infections ◽  
Suppressor Gene ◽  
Central Component ◽  
Type I ◽  
Infected Cells

Tumor suppressor p53 is activated by several stimuli, including DNA damage and oncogenic stress. Previous studies (Takaoka, A., S. Hayakawa, H. Yanai, D. Stoiber, H. Negishi, H. Kikuchi, S. Sasaki, K. Imai, T. Shibue, K. Honda, and T. Taniguchi. 2003. Nature. 424:516–523) have shown that p53 is also induced in response to viral infections as a downstream transcriptional target of type I interferon (IFN) signaling. Moreover, many viruses, including SV40, human papillomavirus, Kaposi's sarcoma herpesvirus, adenoviruses, and even RNA viruses such as polioviruses, have evolved mechanisms designated to abrogate p53 responses. We describe a novel p53 function in the activation of the IFN pathway. We observed that infected mouse and human cells with functional p53 exhibited markedly decreased viral replication early after infection. This early inhibition of viral replication was mediated both in vitro and in vivo by a p53-dependent enhancement of IFN signaling, specifically the induction of genes containing IFN-stimulated response elements. Of note, p53 also contributed to an increase in IFN release from infected cells. We established that this p53-dependent enhancement of IFN signaling is dependent to a great extent on the ability of p53 to activate the transcription of IFN regulatory factor 9, a central component of the IFN-stimulated gene factor 3 complex. Our results demonstrate that p53 contributes to innate immunity by enhancing IFN-dependent antiviral activity independent of its functions as a proapoptotic and tumor suppressor gene.

scholarly journals More than efficacy revealed by single-cell analysis of antiviral therapeutics

2019 ◽  
Vol 5 (10) ◽  
pp. eaax4761 ◽  
Author(s):  
Wu Liu ◽  
Mehmet U. Caglar ◽  
Zhangming Mao ◽  
Andrew Woodman ◽  
Jamie J. Arnold ◽  
...  
Keyword(s):  
Viral Infection ◽  
Single Cell ◽  
Single Cell Analysis ◽  
Principal Component ◽  
Cell Analysis ◽  
Infection Cycle ◽  
Reporter Protein ◽  
Fluorescent Reporter ◽  
Infection Dynamics ◽  
Infected Cells

Because many aspects of viral infection dynamics and inhibition are governed by stochastic processes, single-cell analysis should provide more information than approaches using population averaging. We have developed a microfluidic device composed of ~6000 wells, with each well containing a microstructure to capture single, infected cells replicating an enterovirus expressing a fluorescent reporter protein. We have used this system to characterize enterovirus inhibitors with distinct mechanisms of action. Single-cell analysis reveals that each class of inhibitor interferes with the viral infection cycle in a manner that can be distinguished by principal component analysis. Single-cell analysis of antiviral candidates not only reveals efficacy but also facilitates clustering of drugs with the same mechanism of action and provides some indication of the ease with which resistance will develop.

scholarly journals Antiviral Activity of Exopolysaccharides Produced by Lactic Acid Bacteria of the Genera Pediococcus, Leuconostoc and Lactobacillus against Human Adenovirus Type 5

Medicina ◽  
2019 ◽  
Vol 55 (9) ◽  
pp. 519 ◽  
Author(s):  
Biliavska ◽  
Pankivska ◽  
Povnitsa ◽  
Zagorodnya
Keyword(s):  
Cell Cycle ◽  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Flow Cytometric Analysis ◽  
Human Adenovirus ◽  
Adenovirus Type ◽  
Infected Cells ◽  
Adenovirus Type 5 ◽  
Number Of Cells

Background and objectives: The use of antagonistic probiotic microorganisms and their byproducts represents a promising approach for the treatment of viral diseases. In the current work, the effect of exopolysaccharides (EPSs) produced by lactic acid bacteria from different genera on the structural and functional characteristics of cells and the development of adenoviral infection in vitro was studied. Materials and Methods: Cytotoxicity of six EPSs of lactic acid bacteria of the genera Lactobacillus, Leuconostoc and Pediococcus was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. The influence of the EPSs on the infectivity of human adenovirus type 5 (HAdV-5) and on the cell cycle under a condition of adenovirus infection was studied using plaque reduction assay and flow cytometric analysis, respectively. Results: It was shown that exopolysaccharides were non-toxic to Madin-Darby bovine kidney cells (MDBK) as they reduced their viability by 3–17%. A change in the distribution of the cell cycle phases in the non-infected cell population treated with EPSs was observed. The analysis demonstrated an increase in the number of cells in the S phase by 47% when using EPSs 15a and a decrease in the number of cells in the G1 phase by 20–27% when treated with the EPSs 15a, 33a, and 19s. The use of EPSs did not led to the normalization of the life cycle of HAdV-5 infected cells to the level of non-infected cells. The EPSs showed low virucidal activity and reduced the HAdV-5 infectivity to 85%. Among the studied exopolysaccharides, anti-adenovirus activity was found for EPS 26a that is produced by Lactobacillus spp. strain. The treatment of cells with the EPS following virus adsorption completely (100%) suppressed the formation and release of HAdV-5 infectious. Conclusions: EPS 26a possessed distinct anti-HAdV-5 activity and the obtained data demonstrate the potential of using exopolysaccharides as anti-adenoviral agents.

scholarly journals Evaluation of in vitro activity of copper gluconate against SARS-CoV-2 using confocal microscopy-based high content screening

2020 ◽  
Author(s):  
Killian Rodriguez ◽  
Josselin Rigaill ◽  
Estelle Audoux ◽  
Florian Saunier ◽  
Elisabeth Botelho-Nevers ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Screening Method ◽  
Etiologic Agent ◽  
Vitro Activity ◽  
High Content Screening ◽  
In Vitro Activity ◽  
Infection Level ◽  
Xtt Assay ◽  
Infected Cells

Context: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) that emerged late in 2019 is the etiologic agent of coronavirus disease 2019 (Covid-19). There is an urgent need to develop curative and preventive therapeutics to limit the current pandemic and to prevent the re-emergence of Covid-19. This study aimed to assess the in vitro activity of copper gluconate against SRAS-CoV-2. Methods: Vero E6 cells were treated with copper gluconate 18 hours before infection. Cells were infected with a recombinant GFP expressing SARS-CoV-2. Infected cells were maintained in fresh medium containing copper gluconate for an additional 48-hour period. The infection level was measured by the confocal microscopy-based high content screening method. The cell viability in presence of copper gluconate was assessed by XTT assay. Results: The viability of Vero E6 cells treated with copper gluconate up to 200 μM was found to be similar to that of untreated cells, but it dropped below 40% with 400 μM of this agent. The infection rate was 23.8%, 18.9%, 20.6%, 6.9%, 5.3%,5.2% in cells treated with 0, 2, 10, 25, 50 and 100 μM of copper gluconate respectively. As compared to untreated cells, the number of infected cells was reduced by 71%, 77%, and 78% with 25, 50, and 100 μM of copper gluconate respectively (p < 0.05). Conclusion: Copper gluconate was found to mitigate SARS-CoV-2 infection in Vero E6 cells. Furthers studies are needed to determine whether copper homeostasis could play a role in SARS-CoV-2 infection.

scholarly journals Gamma Delta T Cells and Their Involvement in COVID-19 Virus Infections

2021 ◽  
Vol 12 ◽  
Author(s):  
Georg von Massow ◽  
Steve Oh ◽  
Alan Lam ◽  
Kenth Gustafsson
Keyword(s):  
T Cells ◽  
Viral Infections ◽  
Cell Subset ◽  
Γδ T Cells ◽  
Common Symptom ◽  
Virus Infections ◽  
Infected Cells ◽  
The Relationship

The global outbreak of the SARS-Cov-2 virus in 2020 has killed millions of people worldwide and forced large parts of the world into lockdowns. While multiple vaccine programs are starting to immunize the global population, there is no direct cure for COVID-19, the disease caused by the SARS-Cov-2 infection. A common symptom in patients is a decrease in T cells, called lymphopenia. It is as of yet unclear what the exact role of T cells are in the immune response to COVID-19. The research so far has mainly focused on the involvement of classical αβ T cells. However, another subset of T cells called γδ T cells could have an important role to play. As part of the innate immune system, γδ T cells respond to inflammation and stressed or infected cells. The γδ T cell subset appears to be particularly affected by lymphopenia in COVID-19 patients and commonly express activation and exhaustion markers. Particularly in children, this subset of T cells seems to be most affected. This is interesting and relevant because γδ T cells are more prominent and active in early life. Their specific involvement in this group of patients could indicate a significant role for γδ T cells in this disease. Furthermore, they seem to be involved in other viral infections and were able to kill SARS infected cells in vitro. γδ T cells can take up, process and present antigens from microbes and human cells. As e.g. tumour-associated antigens are presented by MHC on γδ T cells to classical T-cells, we argue here that it stands to reason that also viral antigens, such as SARS-Cov-2-derived peptides, can be presented in the same way. γδ T cells are already used for medical purposes in oncology and have potential in cancer therapy. As γδ T cells are not necessarily able to distinguish between a transformed and a virally infected cell it could therefore be of great interest to investigate further the relationship between COVID-19 and γδ T cells.

Molecular and biochemical characterisation of a novel type II peroxiredoxin (XvPrx2) from the resurrection plant Xerophyta viscosa

2016 ◽  
Vol 43 (7) ◽  
pp. 669 ◽  
Author(s):  
Kershini Govender ◽  
Jennifer A. Thomson ◽  
Sagadevan Mundree ◽  
Abdelaleim Ismail ElSayed ◽  
Mohammed Suhail Rafudeen
Keyword(s):  
Type Ii ◽  
Dna Protection ◽  
Reporter Protein ◽  
Significant Similarity ◽  
Fluorescent Reporter ◽  
E Coli ◽  
Escherichia Coli Cells ◽  
Biochemical Characterisation

A type II peroxiredoxin gene (XvPrx2) was isolated from a Xerophyta viscosa (Baker) cDNA cold-stress library. The polypeptide displayed significant similarity to other plant type II peroxiredoxins, with the conserved amino acid motif (PGAFTPTCS) proposed to constitute the active site of the enzyme. Northern blot analyses showed that XvPrx2 gene was stress-inducible in response to abiotic stresses while gel analyses revealed that XvPrx2 homologues exist within the X. viscosa proteome. Using a yellow fluorescent reporter protein, the XvPrx2 protein localised to the cytosol. A mutated protein (XvV7) was generated by converting the valine at position 76 to a cysteine and an in vitro DNA protection assay showed that, in the presence of either XvPrx2 or XvV7, DNA protection occurred. In addition, an in vivo assay showed that increased protection was conferred to Escherichia coli cells overexpressing either XvPrx2 or XvV7. The XvPrx2 activity was maximal with DTT as electron donor and H2O2 as substrate. Using E. coli thioredoxin, a 2–15-fold lower enzyme activity was observed. The XvPrx2 activity with glutathione was significantly lower and glutaredoxin had no measurable effect on this reaction. The XvV7 protein displayed significantly lower activity compared with XvPrx2 for all substrates assessed.

scholarly journals Translation from the 5′ Untranslated Region (UTR) of mRNA 1 Is Repressed, but That from the 5′ UTR of mRNA 7 Is Stimulated in Coronavirus-Infected Cells

1999 ◽  
Vol 73 (10) ◽  
pp. 8003-8009 ◽  
Author(s):  
Savithra D. Senanayake ◽  
David A. Brian
Keyword(s):  
Viral Gene ◽  
Protein Accumulation ◽  
Regulation Of Transcription ◽  
Reporter Protein ◽  
Virus Growth ◽  
Regulation Of Translation ◽  
Infected Cells ◽  
Made In

ABSTRACT Viral gene products are generally required in widely differing amounts for successful virus growth and assembly. For coronaviruses, regulation of transcription is a major contributor to these differences, but regulation of translation may also be important. Here, we examine the possibility that the 5′ untranslated regions (UTRs), unique for each of the nine species of mRNA in the bovine coronavirus and ranging in length from 70 nucleotides (nt) to 210 nt (inclusive of the common 5′-terminal 65-nt leader), can differentially affect the rate of protein accumulation. When the natural 77-nt 5′ UTR on synthetic transcripts of mRNA 7 (mRNA for N and I proteins) was replaced with the 210-nt 5′ UTR from mRNA 1 (genomic RNA, mRNA for viral polymerase), approximately twofold-less N, or (N) CAT fusion reporter protein, was made in vitro. Twofold less was also made in vivo in uninfected cells when a T7 RNA polymerase-driven transient-transfection system was used. In coronavirus-infected cells, this difference surprisingly became 12-fold as the result of both a stimulated translation from the 77-nt 5′ UTR and a repression of translation from the 210-nt 5′ UTR. These results reveal that a differential 5′ UTR-directed regulation of translation can occur in coronavirus-infected cells and lead us to postulate that the direction and degree of regulation is carried out by viral or virally induced cellular factors acting in trans on cis-acting elements within the 5′ UTR.

FluoroCalins: engineered lipocalins with novel binding functions fused to a fluorescent protein for applications in biomolecular imaging and detection

2019 ◽  
Vol 32 (6) ◽  
pp. 289-296 ◽  
Author(s):  
Evelyn Eggenstein ◽  
Antonia Richter ◽  
Arne Skerra
Keyword(s):  
Fluorescent Protein ◽  
Signal To Noise Ratio ◽  
Growth Factor Receptor ◽  
High Specificity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Reporter Protein ◽  
Fluorescent Reporter ◽  
Promising Alternative ◽  
Biomolecular Imaging

Abstract FluoroCalins represent novel bifunctional protein reagents derived from engineered lipocalins fused to a fluorescent reporter protein, here the enhanced green fluorescent protein (eGFP). We demonstrate the construction, facile bacterial production and broad applicability of FluoroCalins using two Anticalin® molecules directed against the tumor vasculature-associated extra domain B of fibronectin (ED-B) and the vascular endothelial growth factor receptor 3, a marker of tumor and lymphangiogenesis. FluoroCalins were prepared with two different spacers: (i) a short Ser3Ala linker and (ii) a long hydrophilic and conformationally unstructured PASylation® polypeptide comprising 200 Pro, Ala and Ser residues. These FluoroCalins were applied for direct target quantification in enzyme-linked immunosorbent assay as well as target detection by flow cytometry and fluorescence microscopy of live and fixed cells, respectively, demonstrating high specificity and signal-to-noise ratio. Hence, FluoroCalins offer a promising alternative to antibody-based reagents for state of the art fluorescent in vitro detection and biomolecular imaging.

scholarly journals Identification of a Novel Viral Protein Expressed from the PB2 Segment of Influenza A Virus

2015 ◽  
Vol 90 (1) ◽  
pp. 444-456 ◽  
Author(s):  
Seiya Yamayoshi ◽  
Mariko Watanabe ◽  
Hideo Goto ◽  
Yoshihiro Kawaoka
Keyword(s):  
Influenza Virus ◽  
Influenza A Virus ◽  
Viral Protein ◽  
Influenza A ◽  
Viral Infections ◽  
Viral Proteins ◽  
Wild Type Virus ◽  
Infected Cells

ABSTRACTOver the past 2 decades, several novel influenza virus proteins have been identified that modulate viral infectionsin vitroand/orin vivo. The PB2 segment, which is one of the longest influenza A virus segments, is known to encode only one viral protein, PB2. In the present study, we used reverse transcription-PCR (RT-PCR) targeting viral mRNAs transcribed from the PB2 segment to look for novel viral proteins encoded by spliced mRNAs. We identified a new viral protein, PB2-S1, encoded by a novel spliced mRNA in which the region corresponding to nucleotides 1513 to 1894 of the PB2 mRNA is deleted. PB2-S1 was detected in virus-infected cells and in cells transfected with a protein expression plasmid encoding PB2. PB2-S1 localized to mitochondria, inhibited the RIG-I-dependent interferon signaling pathway, and interfered with viral polymerase activity (dependent on its PB1-binding capability). The nucleotide sequences around the splicing donor and acceptor sites for PB2-S1 were highly conserved among pre-2009 human H1N1 viruses but not among human H1N1pdm and H3N2 viruses. PB2-S1-deficient viruses, however, showed growth kinetics in MDCK cells and virulence in mice similar to those of wild-type virus. The biological significance of PB2-S1 to the replication and pathogenicity of seasonal H1N1 influenza A viruses warrants further investigation.IMPORTANCETranscriptome analysis of cells infected with influenza A virus has improved our understanding of the host response to viral infection, because such analysis yields considerable information about bothin vitroandin vivoviral infections. However, little attention has been paid to transcriptomes derived from the viral genome. Here we focused on the splicing of mRNA expressed from the PB2 segment and identified a spliced viral mRNA encoding a novel viral protein. This result suggests that other, as yet unidentified viral proteins encoded by spliced mRNAs could be expressed in virus-infected cells. A viral transcriptome including the viral spliceosome should be evaluated to gain new insights into influenza virus infection.

Sign in / Sign up

Export Citation Format

Share Document