A cautionary perspective regarding the isolation and serial propagation of SARS-CoV-2 in Vero cells

AbstractAn array of SARS-CoV-2 virus variants have been isolated, propagated and used in in vitro assays, in vivo animal studies and human clinical trials. Observations of working stocks of SARS-CoV-2 suggest that sequential propagation in Vero cells leads to critical changes in the region of the furin cleavage site, which significantly reduce the value of the working stock for critical research studies. Serially propagating SARS-CoV-2 in Vero E6 cells leads to rapid increases in genetic variants while propagation in other cell lines (e.g. Vero/hSLAM) appears to mitigate this risk thereby improving the overall genetic stability of working stocks. From these observations, investigators are urged to monitor genetic variants carefully when propagating SARS-CoV-2 in Vero cells.

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Modulation of Matrix Metalloproteinases by Plant-Derived Products

Current Cancer Drug Targets ◽

10.2174/1568009620666201120144838 ◽

2020 ◽

Vol 20 ◽

Author(s):

Nur Najmi Mohamad Anuar ◽

Nurul Iman Natasya Zulkafali ◽

Azizah Ugusman

Keyword(s):

Clinical Trials ◽

Natural Products ◽

Matrix Metalloproteinases ◽

Animal Studies ◽

Current Knowledge ◽

In Vitro Models ◽

In Vivo Studies ◽

Extracellular Matrix Components

: Matrix metalloproteinases (MMPs) are a group of zinc-dependent metallo-endopeptidase that are responsible towards the degradation, repair and remodelling of extracellular matrix components. MMPs play an important role in maintaining a normal physiological function and preventing diseases such as cancer and cardiovascular diseases. Natural products derived from plants have been used as traditional medicine for centuries. Its active compounds, such as catechin, resveratrol and quercetin, are suggested to play an important role as MMPs inhibitors, thereby opening new insights into their applications in many fields, such as pharmaceutical, cosmetic and food industries. This review summarises the current knowledge on plant-derived natural products with MMP-modulating activities. Most of the reviewed plant-derived products exhibit an inhibitory activity on MMPs. Amongst MMPs, MMP-2 and MMP-9 are the most studied. The expression of MMPs is inhibited through respective signalling pathways, such as MAPK, NF-κB and PI3 kinase pathways, which contribute to the reduction in cancer cell behaviours, such as proliferation and migration. Most studies have employed in vitro models, but a limited number of animal studies and clinical trials have been conducted. Even though plant-derived products show promising results in modulating MMPs, more in vivo studies and clinical trials are needed to support their therapeutic applications in the future.

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Mesenchymal stromal cells: a novel therapy for the treatment of chronic obstructive pulmonary disease?

Thorax ◽

10.1136/thoraxjnl-2017-210672 ◽

2018 ◽

Vol 73 (6) ◽

pp. 565-574 ◽

Cited By ~ 32

Author(s):

Winifred Broekman ◽

Padmini P S J Khedoe ◽

Koen Schepers ◽

Helene Roelofs ◽

Jan Stolk ◽

...

Keyword(s):

Clinical Trials ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Animal Studies ◽

Chronic Obstructive ◽

Tissue Destruction ◽

Novel Therapy ◽

Model Studies

COPD is characterised by tissue destruction and inflammation. Given the lack of curative treatments and the progressive nature of the disease, new treatments for COPD are highly relevant. In vitro cell culture and animal studies have demonstrated that mesenchymal stromal cells (MSCs) have the capacity to modify immune responses and to enhance tissue repair. These properties of MSCs provided a rationale to investigate their potential for treatment of a variety of diseases, including COPD. Preclinical models support the hypothesis that MSCs may have clinical efficacy in COPD. However, although clinical trials have demonstrated the safety of MSC treatment, thus far they have not provided evidence for MSC efficacy in the treatment of COPD. In this review, we discuss the rationale for MSC-based cell therapy in COPD, the main findings from in vitro and in vivo preclinical COPD model studies, clinical trials in patients with COPD and directions for further research.

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Inclusion of a Furin Cleavage Site Enhances Antitumor Efficacy against Colorectal Cancer Cells of Ribotoxin α-Sarcin- or RNase T1-Based Immunotoxins

Toxins ◽

10.3390/toxins11100593 ◽

2019 ◽

Vol 11 (10) ◽

pp. 593 ◽

Cited By ~ 5

Author(s):

Javier Ruiz-de-la-Herrán ◽

Jaime Tomé-Amat ◽

Rodrigo Lázaro-Gorines ◽

José G. Gavilanes ◽

Javier Lacadena

Keyword(s):

Cleavage Site ◽

Tumor Antigens ◽

Functional Characterization ◽

Target Cells ◽

Furin Cleavage ◽

Ribonucleolytic Activity ◽

Furin Cleavage Site ◽

Potential Therapeutic Application

Immunotoxins are chimeric molecules that combine the specificity of an antibody to recognize and bind tumor antigens with the potency of the enzymatic activity of a toxin, thus, promoting the death of target cells. Among them, RNases-based immunotoxins have arisen as promising antitumor therapeutic agents. In this work, we describe the production and purification of two new immunoconjugates, based on RNase T1 and the fungal ribotoxin α-sarcin, with optimized properties for tumor treatment due to the inclusion of a furin cleavage site. Circular dichroism spectroscopy, ribonucleolytic activity studies, flow cytometry, fluorescence microscopy, and cell viability assays were carried out for structural and in vitro functional characterization. Our results confirm the enhanced antitumor efficiency showed by these furin-immunotoxin variants as a result of an improved release of their toxic domain to the cytosol, favoring the accessibility of both ribonucleases to their substrates. Overall, these results represent a step forward in the design of immunotoxins with optimized properties for potential therapeutic application in vivo.

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QTQTN motif upstream of the furin-cleavage site plays key role in SARS-CoV-2 infection and pathogenesis.

10.1101/2021.12.15.472450 ◽

2021 ◽

Author(s):

Michelle N Vu ◽

Kumari Lokugamage ◽

Jessica A Plante ◽

Dionna Scharton ◽

Bryan A Johnson ◽

...

Keyword(s):

Cleavage Site ◽

Unusual Feature ◽

Spike Protein ◽

Loop Length ◽

Dependent Manner ◽

Furin Cleavage ◽

Furin Cleavage Site ◽

Respiratory Cells

The furin cleavage site (FCS), an unusual feature in the SARS-CoV-2 spike protein, has been spotlighted as a factor key to facilitating infection and pathogenesis by increasing spike processing 1,2. Similarly, the QTQTN motif directly upstream of the FCS is also an unusual feature for group 2B coronaviruses (CoVs). The QTQTN deletion has consistently been observed in in vitro cultured virus stocks and some clinical isolates 3. To determine whether the QTQTN motif is critical to SARS-CoV-2 replication and pathogenesis, we generated a mutant deleting the QTQTN motif (ΔQTQTN). Here we report that the QTQTN deletion attenuates viral replication in respiratory cells in vitro and attenuates disease in vivo. The deletion results in a shortened, more rigid peptide loop that contains the FCS, and is less accessible to host proteases, such as TMPRSS2. Thus, the deletion reduced the efficiency of spike processing and attenuates SARS-CoV-2 infection. Importantly, the QTQTN motif also contains residues that are glycosylated4, and disruption its glycosylation also attenuates virus replication in a TMPRSS2-dependent manner. Together, our results reveal that three aspects of the S1/S2 cleavage site (the FCS, loop length, and glycosylation) are required for efficient SARS-CoV-2 replication and pathogenesis.

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Quantitative proteomic analysis of the tizoxanide effect in vero cells

Scientific Reports ◽

10.1038/s41598-020-71634-2 ◽

2020 ◽

Vol 10 (1) ◽

Cited By ~ 1

Author(s):

K. A. Yamamoto ◽

K. Blackburn ◽

E. Migowski ◽

M. B. Goshe ◽

D. T. Brown ◽

...

Keyword(s):

Clinical Trials ◽

Rna Processing ◽

Cell Metabolism ◽

Antimicrobial Effect ◽

Vero Cells ◽

Label Free ◽

Proteomic Approach ◽

The Usa

Abstract Nitazoxanide (NTZ) is effective against helminths and numerous microorganisms, including bacteria and viruses. In vivo, NTZ is metabolized into Tizoxanide (TIZ), which is the active circulating metabolite. With the emergence of SARS-Cov-2 as a Pandemic agent, NTZ became one of the molecules already approved for human use to engage clinical trials, due to results in vitro showing that NTZ was highly effective against the SARS-Cov-2, agent of COVID-19. There are currently several ongoing clinical trials mainly in the USA and Brazil involving NTZ due not only to the in vitro results, but also for its long-known safety. Here, we study the response of Vero cells to TIZ treatment and unveil possible mechanisms for its antimicrobial effect, using a label-free proteomic approach (LC/MS/MS) analysis to compare the proteomic profile between untreated- and TIZ-treated cells. Fifteen differentially expressed proteins were observed related to various biological processes, including translation, intracellular trafficking, RNA processing and modification, and signal transduction. The broad antimicrobial range of TIZ points towards its overall effect in lowering cell metabolism and RNA processing and modification. The decreased levels of FASN, HNRNPH and HNRNPK with the treatment appear to be important for antiviral activity.

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COMPUTATIONAL FLUID DYNAMIC ANALYSIS OF A CELL-BASED MICROFLUIDIC DRUG SCREENING PLATFORM

Nano LIFE ◽

10.1142/s1793984410000201 ◽

2010 ◽

Vol 01 (03n04) ◽

pp. 185-194 ◽

Cited By ~ 2

Author(s):

T. MAGUIRE ◽

O. B. USTA ◽

M. L. YARMUSH

Keyword(s):

Clinical Trials ◽

Convective Transport ◽

In Vitro Assays ◽

Phase I Clinical Trials ◽

Clearance Rates ◽

Culture Systems ◽

Functional Efficiency ◽

Gene And Protein Expression

About 50% of all candidate drugs that make it past Phase-I clinical trials ultimately fail to receive U.S. Food and Drug Administration (FDA) marketing approval due to human toxicity or bioavailability problems that the drug develops. To remedy this state of affairs, in recent years, researchers have attempted to develop improved in vitro assays which can be applied pre-clinically to produce data with a higher degree of correlation to in vivo responses, and which circumvent the allometric limitations inherent in animal models. Such in vitro assays are intended to enable preclinical research and development groups to better predict the pharmacokinetic and pharmacodynamic action of candidate molecules prior to clinical trials, thereby reducing high late-stage failure rates. While most such in vitro systems comprise a static cell culture environment, microfluidic systems have been shown to provide better results. For example, experiments performed with the HμREL® microfluidics system demonstrated increased clearance rates and better in vitro–in vivo correlation (IVIVC) than that observed with static culture systems. Several hypotheses were suggested to account for these improvements: (1) enhanced mass transport resulting in better functional efficiency; (2) a flow-induced transduction of gene and protein expression and function; and (3) a flow-induced effect resulting in increased cellular uptake. In this paper, we describe a framework, utilizing computational fluid dynamics (CFD), that can be used to study culture systems with a level of scrutiny and spatial precision not offered using standard in vitro assays. Using this approach, we were able to successfully model experimental observations of increases in the clearance rates of high and medium clearance compounds in the microfluidics system. Based on these results we posit that the increase in clearance is most likely due to the addition of convective transport, and a thinning of the boundary layer present in static and mixed plate cultures systems.

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SARS-CoV-2 growth, furin-cleavage-site adaptation and neutralization using serum from acutely infected, hospitalized COVID-19 patients

10.1101/2020.06.19.154930 ◽

2020 ◽

Cited By ~ 3

Author(s):

William B. Klimstra ◽

Natasha L. Tilston-Lunel ◽

Sham Nambulli ◽

James Boslett ◽

Cynthia M. McMillen ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Serum Samples ◽

Furin Cleavage ◽

Furin Cleavage Site ◽

Similar Disease ◽

Antibody Titers ◽

Low Passage

AbstractSARS-CoV-2, the causative agent of COVID-19, emerged at the end of 2019 and by mid-June 2020, the virus has spread to at least 215 countries, caused more than 8,000,000 confirmed infections and over 450,000 deaths, and overwhelmed healthcare systems worldwide. Like SARS-CoV, which emerged in 2002 and caused a similar disease, SARS-CoV-2 is a betacoronavirus. Both viruses use human angiotensin-converting enzyme 2 (hACE2) as a receptor to enter cells. However, the SARS-CoV-2 spike (S) glycoprotein has a novel insertion that generates a putative furin cleavage signal and this has been postulated to expand the host range. Two low passage (P) strains of SARS-CoV-2 (Wash1: P4 and Munich: P1) were cultured twice in Vero-E6 cells and characterized virologically. Sanger and MinION sequencing demonstrated significant deletions in the furin cleavage signal of Wash1: P6 and minor variants in the Munich: P3 strain. Cleavage of the S glycoprotein in SARS-CoV-2-infected Vero-E6 cell lysates was inefficient even when an intact furin cleavage signal was present. Indirect immunofluorescence demonstrated the S glycoprotein reached the cell surface. Since the S protein is a major antigenic target for the development of neutralizing antibodies we investigated the development of neutralizing antibody titers in serial serum samples obtained from COVID-19 human patients. These were comparable regardless of the presence of an intact or deleted furin cleavage signal. These studies illustrate the need to characterize virus stocks meticulously prior to performing either in vitro or in vivo pathogenesis studies.

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Enhanced Virulence of Aeromonas hydrophila Is Induced by Stress and Serial Passaging in Mice

Animals ◽

10.3390/ani11020508 ◽

2021 ◽

Vol 11 (2) ◽

pp. 508

Author(s):

Kyoo-Tae Kim ◽

Seung-Hun Lee ◽

Kyoung-Ki Lee ◽

Jee Eun Han ◽

Dongmi Kwak

Keyword(s):

Aeromonas Hydrophila ◽

Virulence Genes ◽

Stress Hormones ◽

Vero Cells ◽

In Vitro Assays ◽

Stress Effect ◽

African Black ◽

Spheniscus Demersus

Aeromonas hydrophila was isolated from an African black-footed penguin (Spheniscus demersus) that died while in zoo captivity. At necropsy, the virulence of A. hydrophila appeared to be enhanced by stress, so was assessed in the presence of in vitro and in vivo stressors and serial passaging in mice. Virulence genes from the isolate were amplified by PCR. In vitro assays were conducted to test the hemolytic activity, cytotoxicity, and effect of stress hormones on A. hydrophila virulence. In vivo assays were conducted to test the stress effect on mortality of A. hydrophila-infected mice and virulence in mice. Two virulence genes coding for hemolysin (ahh1) and aerolysin (aerA) were detected, and the cytotoxic potential of the isolate was demonstrated in baby hamster kidney and Vero cells. Some or all mice inoculated with A. hydrophila and exposed to stress hormones (epinephrine and norepinephrine) or low temperature died, while mice inoculated with A. hydrophila and exposed to fasting or agitation stressors or no stressors survived. We concluded that stress can be fatal in mice experimentally infected with A. hydrophila and that serial passaging in mice dramatically enhances the virulence of A. hydrophila.

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Virulence parameters in the characterization of strains of Entamoeba histolytica

Revista do Instituto de Medicina Tropical de São Paulo ◽

10.1590/s0036-46651997000200001 ◽

1997 ◽

Vol 39 (2) ◽

pp. 65-70 ◽

Cited By ~ 2

Author(s):

Maria A GOMES ◽

Maria N. MELO ◽

Gil P.M. PENA ◽

Edward F. SILVA

Keyword(s):

Entamoeba Histolytica ◽

Cytopathic Effect ◽

Axenic Culture ◽

Vero Cells ◽

In Vitro Assays ◽

Biological Assays ◽

Restriction Fragment Pattern

Differences in virulence of strains of Entamoeba histolytica have long been detected by various experimental assays, both in vivo and in vitro. Discrepancies in the strains characterization have been arisen when different biological assays are compared. In order to evaluate different parameters of virulence in the strains characterization, five strains of E. histolytica, kept under axenic culture, were characterized in respect to their, capability to induce hamster liver abscess, erythrophagocytosis rate and cytopathic effect upon VERO cells. It was found significant correlation between in vitro biological assays, but not between in vivo and in vitro assays. Good correlation was found between cytopathic effect and the mean number of uptaken erythrocytes, but not with percentage of phagocytic amoebae, showing that great variability can be observed in the same assay, according to the variable chosen. It was not possible to correlate isoenzyme and restriction fragment pattern with virulence indexes since all studied strains presented pathogenic patterns. The discordant results observed in different virulence assays suggests that virulence itself may not the directly assessed. What is in fact assessed are different biological characteristics or functions of the parasite more than virulence itself. These characteristics or functions may be related or not with pathogenic mechanisms occurring in the development of invasive amoebic disease

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Furin Cleavage Site Is Key to SARS-CoV-2 Pathogenesis

10.1101/2020.08.26.268854 ◽

2020 ◽

Cited By ~ 11

Author(s):

Bryan A. Johnson ◽

Xuping Xie ◽

Birte Kalveram ◽

Kumari G. Lokugamage ◽

Antonio Muruato ◽

...

Keyword(s):

Cleavage Site ◽

Critical Role ◽

Vero Cells ◽

Mutant Virus ◽

Spike Protein ◽

Furin Cleavage ◽

Furin Cleavage Site ◽

Wide Range ◽

The World

AbstractSARS-CoV-2 has resulted in a global pandemic and shutdown economies around the world. Sequence analysis indicates that the novel coronavirus (CoV) has an insertion of a furin cleavage site (PRRAR) in its spike protein. Absent in other group 2B CoVs, the insertion may be a key factor in the replication and virulence of SARS-CoV-2. To explore this question, we generated a SARS-CoV-2 mutant lacking the furin cleavage site (ΔPRRA) in the spike protein. This mutant virus replicated with faster kinetics and improved fitness in Vero E6 cells. The mutant virus also had reduced spike protein processing as compared to wild-type SARS-CoV-2. In contrast, the ΔPRRA had reduced replication in Calu3 cells, a human respiratory cell line, and had attenuated disease in a hamster pathogenesis model. Despite the reduced disease, the ΔPRRA mutant offered robust protection from SARS-CoV-2 rechallenge. Importantly, plaque reduction neutralization tests (PRNT50) with COVID-19 patient sera and monoclonal antibodies against the receptor-binding domain found a shift, with the mutant virus resulting in consistently reduced PRNT50 titers. Together, these results demonstrate a critical role for the furin cleavage site insertion in SARS-CoV-2 replication and pathogenesis. In addition, these findings illustrate the importance of this insertion in evaluating neutralization and other downstream SARS-CoV-2 assays.ImportanceAs COVID-19 has impacted the world, understanding how SARS-CoV-2 replicates and causes virulence offers potential pathways to disrupt its disease. By removing the furin cleavage site, we demonstrate the importance of this insertion to SARS-CoV-2 replication and pathogenesis. In addition, the findings with Vero cells indicate the likelihood of cell culture adaptations in virus stocks that can influence reagent generation and interpretation of a wide range of data including neutralization and drug efficacy. Overall, our work highlights the importance of this key motif in SARS-CoV-2 infection and pathogenesis.Article SummaryA deletion of the furin cleavage site in SARS-CoV-2 amplifies replication in Vero cells, but attenuates replication in respiratory cells and pathogenesis in vivo. Loss of the furin site also reduces susceptibility to neutralization in vitro.

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