scholarly journals A STUDY OF THE ULTRASTRUCTURE OF THE SURFACE OF THE TRANSPLANTABLE LINE VERO CELLS INFECTED WITH THE RABIES VIRUS (RABV, LISSAVIRUS, RHABDOVIRIDAE)

2017 ◽  
Vol 62 (5) ◽  
pp. 227-232 ◽  
Author(s):  
S. V. Generalov ◽  
P. S. Erokhin ◽  
T. Yu. Krasovskaya ◽  
N. A. Osina ◽  
E. G. Abramova ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Rabies Virus ◽  
Membrane Surface ◽  
Methodological Approach ◽  
Vero Cells ◽  
Force Microscopy ◽  
Length Width ◽  
Infected Cells ◽  
Membrane Roughness

Characteristics of the effect of attenuated rabies virus strain «Moscow 3253» on morphological parameters of transplantable line Vero cells were studied by atomic force microscopy (AFM). Methods based on phase contrast microscopy and immunofluorescence were used to confirm the specificity of interaction and to identify the infectious activity of the rabies virus. Images of intact Vero cells and Vero cells infected with rabies virus were obtained at different periods of cultivation. The character of changes in the cell dimensions (length, width, height) and the cell membrane roughness depending on the rabies virus cultivation time was determined. During the observation period both increases and decreases in the size of the cells were recorded. The size of the infected cells exceeded that of the intact. An increase in the membrane roughness in cells exposed to rabies occurred during the entire period of observation, since the first hours of the interaction of the virus with the cell, while the intact Vero cells exhibited only minor changes in the membrane surface roughness, which were not dependent on the age of the culture. The dependence of the increase in the cell membrane roughness on the infecting dose of the rabies virus was determined. The obtained results open up the prospect of developing a methodological approach to the quantitative in vitro evaluation of the rabies virus using AFM. Changes in the cell membrane roughness appear to be the most indicative parameter for such evaluation.

scholarly journals Biological Atomic Force Microscopy for Imaging Gold-Labeled Liposomes on Human Coronary Artery Endothelial Cells

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Ana-María Zaske ◽  
Delia Danila ◽  
Michael C. Queen ◽  
Eva Golunski ◽  
Jodie L. Conyers
Keyword(s):  
Atomic Force Microscopy ◽  
Endothelial Cells ◽  
Coronary Artery ◽  
Cell Membrane ◽  
Fluorescent Microscopy ◽  
Human Coronary Artery ◽  
Force Microscopy ◽  
Cell Internalization ◽  
Atomic Force

Although atomic force microscopy (AFM) has been used extensively to characterize cell membrane structure and cellular processes such as endocytosis and exocytosis, the corrugated surface of the cell membrane hinders the visualization of extracellular entities, such as liposomes, that may interact with the cell. To overcome this barrier, we used 90 nm nanogold particles to label FITC liposomes and monitor their endocytosis on human coronary artery endothelial cells (HCAECs) in vitro. We were able to study the internalization process of gold-coupled liposomes on endothelial cells, by using AFM. We found that the gold-liposomes attached to the HCAEC cell membrane during the first 15–30 min of incubation, liposome cell internalization occurred from 30 to 60 min, and most of the gold-labeled liposomes had invaginated after 2 hr of incubation. Liposomal uptake took place most commonly at the periphery of the nuclear zone. Dynasore monohydrate, an inhibitor of endocytosis, obstructed the internalization of the gold-liposomes. This study showed the versatility of the AFM technique, combined with fluorescent microscopy, for investigating liposome uptake by endothelial cells. The 90 nm colloidal gold nanoparticles proved to be a noninvasive contrast agent that efficiently improves AFM imaging during the investigation of biological nanoprocesses.

scholarly journals The Major Phosphorylation Sites of the Respiratory Syncytial Virus Phosphoprotein Are Dispensable for Virus Replication In Vitro

2002 ◽  
Vol 76 (21) ◽  
pp. 10776-10784 ◽  
Author(s):  
Bin Lu ◽  
Chien-Hui Ma ◽  
Robert Brazas ◽  
Hong Jin
Keyword(s):  
Respiratory Syncytial Virus ◽  
Virus Replication ◽  
Vero Cells ◽  
Phosphorylation Sites ◽  
N Protein ◽  
P Protein ◽  
Infected Cells ◽  
Syncytial Virus

ABSTRACT The phosphoprotein (P protein) of respiratory syncytial virus (RSV) is a key component of the viral RNA-dependent RNA polymerase complex. The protein is constitutively phosphorylated at the two clusters of serine residues (116, 117, and 119 [116/117/119] and 232 and 237 [232/237]). To examine the role of phosphorylation of the RSV P protein in virus replication, these five serine residues were altered to eliminate their phosphorylation potential, and the mutant proteins were analyzed for their functions with a minigenome assay. The reporter gene expression was reduced by 20% when all five phosphorylation sites were eliminated. Mutants with knockout mutations at two phosphorylation sites (S232A/S237A [PP2]) and at five phosphorylation sites (S116L/S117R/S119L/S232A/S237A [PP5]) were introduced into the infectious RSV A2 strain. Immunoprecipitation of 33Pi-labeled infected cells showed that P protein phosphorylation was reduced by 80% for rA2-PP2 and 95% for rA2-PP5. The interaction between the nucleocapsid (N) protein and P protein was reduced in rA2-PP2- and rA2-PP5-infected cells by 30 and 60%, respectively. Although the two recombinant viruses replicated well in Vero cells, rA2-PP2 and, to a greater extent, rA2-PP5, replicated poorly in HEp-2 cells. Virus budding from the infected HEp-2 cells was affected by dephosphorylation of P protein, because the majority of rA2-PP5 remained cell associated. In addition, rA2-PP5 was also more attenuated than rA2-PP2 in replication in the respiratory tracts of mice and cotton rats. Thus, our data suggest that although the major phosphorylation sites of RSV P protein are dispensable for virus replication in vitro, phosphorylation of P protein is required for efficient virus replication in vitro and in vivo.

DISEASED RED BLOOD CELLS STUDIED BY ATOMIC FORCE MICROSCOPY

2002 ◽  
Vol 01 (05n06) ◽  
pp. 683-688 ◽  
Author(s):  
YONG CHEN ◽  
JIYE CAI ◽  
JINGXIAN ZHAO
Keyword(s):  
Lung Cancer ◽  
Atomic Force Microscopy ◽  
Cell Membrane ◽  
Erythrocyte Membrane ◽  
Mammalian Cells ◽  
Membrane Surface ◽  
Force Microscopy ◽  
Membrane Surfaces ◽  
Atomic Force ◽  
Mean Width

In recent years, many mammalian cells, especially erythrocytes because of simpleness of their membrane surfaces, were widely studied by atomic force microscopy. In our study, diseased erythrocytes were taken from patients of lung cancer, myelodisplastic syndrome (MDS), and so on. We obtained many clear topographical images of numerous erythrocytes, single erythrocyte, and ultramicrostructure of erythrocyte membrane surfaces from normal persons and patients. By studying the red cells of lung cancer patients, we found that many erythrocytes of lung cancer patient have changed into echinocytes. One erythrocyte has 10–20 short projections, most of which, with a mean width of 589.0 nm and a length of 646.7 nm, are on the edge of cell. The projections in the center of echinocytes are lodged and embedded, but in conventional model of echinocytes, the projections in the center stretch outside cell membrane, so a novel model of erythrocytes was designed in our paper. After observation of microstructure of MDS patient's erythrocyte membrane surface, we found that many apertures with different diameters of tens to hundreds nanometers appeared on the surface of cell membrane. It can be concluded that AFM may be widely applied in clinic pathological inspection.

Aptamers targeting rabies virus-infected cells inhibit viral replication both in vitro and in vivo

2013 ◽  
Vol 173 (2) ◽  
pp. 398-403 ◽  
Author(s):  
Hong-Ru Liang ◽  
Quan Liu ◽  
Xue-Xing Zheng ◽  
Wei-Wei Gai ◽  
Xiang-hong Xue ◽  
...  
Keyword(s):  
Viral Replication ◽  
Rabies Virus ◽  
Infected Cells

scholarly journals Evidences for lipid involvement in SARS-CoV-2 cytopathogenesis

2021 ◽  
Vol 12 (3) ◽  
Author(s):  
Roberta Nardacci ◽  
Francesca Colavita ◽  
Concetta Castilletti ◽  
Daniele Lapa ◽  
Giulia Matusali ◽  
...  
Keyword(s):  
Lipid Droplets ◽  
Vero Cells ◽  
Blood Cholesterol ◽  
Ultrastructural Changes ◽  
Type Ii ◽  
Cytopathic Effects ◽  
Type Ii Pneumocytes ◽  
Infected Cells ◽  
Accumulation Of Lipids

AbstractThe pathogenesis of SARS-CoV-2 remains to be completely understood, and detailed SARS-CoV-2 cellular cytopathic effects requires definition. We performed a comparative ultrastructural study of SARS-CoV-1 and SARS-CoV-2 infection in Vero E6 cells and in lungs from deceased COVID-19 patients. SARS-CoV-2 induces rapid death associated with profound ultrastructural changes in Vero cells. Type II pneumocytes in lung tissue showed prominent altered features with numerous vacuoles and swollen mitochondria with presence of abundant lipid droplets. The accumulation of lipids was the most striking finding we observed in SARS-CoV-2 infected cells, both in vitro and in the lungs of patients, suggesting that lipids can be involved in SARS-CoV-2 pathogenesis. Considering that in most cases, COVID-19 patients show alteration of blood cholesterol and lipoprotein homeostasis, our findings highlight a peculiar important topic that can suggest new approaches for pharmacological treatment to contrast the pathogenicity of SARS-CoV-2.

scholarly journals Antiviral Activity of Vacuolar ATPase Blocker Diphyllin against SARS-CoV-2

2021 ◽  
Vol 9 (3) ◽  
pp. 471
Author(s):  
Michal Stefanik ◽  
Petra Strakova ◽  
Jan Haviernik ◽  
Andrew D. Miller ◽  
Daniel Ruzek ◽  
...  
Keyword(s):  
Colorectal Adenocarcinoma ◽  
Vero Cells ◽  
Early Step ◽  
Endosomal Membranes ◽  
Infected Cells ◽  
Ph Dependent ◽  
Viral Envelopes ◽  
Vacuolar Atpases ◽  
Related Compounds

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a causative agent of the pandemic coronavirus disease 2019 (COVID-19), which has resulted in over two million deaths worldwide to date. Diphyllin and diphyllinosides are known as natural blockers of cellular vacuolar ATPases, and so can act as inhibitors of the pH-dependent fusion of viral envelopes with host cell endosomal membranes. Such pH-dependent fusion is a critical early step during the SARS-CoV-2 replication cycle. Accordingly, the anti-SARS-CoV-2 profiles and cytotoxicities of diphyllin, diphyllinoside cleistanthin B, and two structurally related compounds, helioxanthin 8-1 and helioxanthin 5-4-2, are evaluated here using in vitro cell-based assay systems. Neither helioxanthin exhibits any obvious anti-SARS-CoV-2 effects in vitro. By contrast diphyllin and cleistanthin B do exhibit anti-SARS-CoV-2 effects in Vero cells, with respective 50% effective concentrations (EC50) values of 1.92 and 6.51 µM. Diphyllin displays anti-SARS-CoV-2 effect also in colorectal adenocarcinoma (CaCo-2) cells. Moreover, when diphyllin is added at various times post infection, a significant decrease in viral titer is observed in SARS-CoV-2-infected Vero cells, even at high viral multiplicities of infection. Importantly, neither diphyllin nor cleistanthin B are found cytotoxic to Vero cells in concentrations up to 100 µM. However, the cytotoxic effect of diphyllin is more pronounced in Vero E6 and CaCo-2 cells. Overall, our data demonstrate that diphyllin and diphyllin analogues might be perfected as anti-SARS-CoV-2 agents in future preclinical studies, most especially if nanomedicine approaches may be invoked to optimize functional drug delivery to virus infected cells.

scholarly journals Dengue Virus Non-structural (NS) 1 Gene as A Molecular Marker for Early Detection of in vitro Dengue Virus Infection

2021 ◽  
Vol 50 (10) ◽  
pp. 3035-3043
Author(s):  
Nur Azizah A Rahman ◽  
Fadhilah Moh Djamil ◽  
Vinod RMT Balasubramaniam ◽  
Sharifah Syed Hassan ◽  
Wei Boon Yap
Keyword(s):  
Early Detection ◽  
Dengue Virus ◽  
Early Phase ◽  
Disease Onset ◽  
Vero Cells ◽  
Specific Primers ◽  
Cytopathic Effects ◽  
Ns1 Gene ◽  
Infected Cells

Serology-based dengue assays at times produce inaccurate results especially in the early phase of disease onset. A more precise diagnostic approach detecting dengue infections in the early phase enables better management of the disease. This helps reduce dengue-associated morbidity and mortality. Besides, an early diagnosis of dengue is also very beneficial in a dengue outbreak and in endemic regions. In this light, this study aimed to determine the potential of the dengue virus (DENV) non-structural 1 (NS1) gene as an early detection biomarker. The cytopathic effects (CPE) were monitored and the cell death of DENV serotype-2 (DENV2)-infected Vero cells was evaluated for fourteen consecutive days. Only Lemos and in-house NS1-specific primer pairs showed positive amplifications in the preliminary primer validation. Thus, both of the primer pairs were then used to amplify the NS1 gene from the infected cells. The NS1 gene was detected as early as day-2 post-infection using the in-house primers. There was no amplicon produced using the Lemos primers. This is speculated to be attributable to the relatively lower complementarity of the primer sequences with that of the template and low amount of viral mRNA in the DENV2-infected cells. Conclusively, the DENV NS1 gene is a potential early detection marker, however, the NS1-specific primers should be pre-validated to ensure a reliable dengue diagnosis.

scholarly journals DETERMINATION OF THE RABIES VIRUS-INFECTED VERO LINE CELL PORTION BY FLOW CYTOMETRY

2019 ◽  
Vol 1 (3) ◽  
pp. 18-25 ◽  
Author(s):  
A. L. Kravtsov ◽  
S. V. Generalov ◽  
V. A. Kozhevnikov ◽  
Yu. K. Gavrilova ◽  
E. G. Abramova ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Rabies Virus ◽  
Relative Number ◽  
Vero Cells ◽  
Time Interval ◽  
Vero Cell Line ◽  
Rabies Virus Strain ◽  
Infected Cells ◽  
Effective Use ◽  
And Control

Aim. Experimental substantiation of possibility to determine the rabies virus-infected Vero cell line portion in culture by flow cytometry (FC) and FITC labeled diagnostic anti-rabies immunoglobulin (DAI), manufactured in Russia. Materials and methods. Fixation and permeabilization of Vero cells, infected by rabies virus strain «Moscow 3253», was carried out by means of Суtofix/Cytoperm reagent (BD Biosciences, USA) according the Vengatesan D. et al. method (2006) and then intracellular rabies antigen was stained by DAI. Percentage of infected cells was determined by FC in 24, 48 and 72 h and as well in 48 h when the cell cultures were infected with tenfold dilutions of virus-containing fluid from 10-1 to 10-8. Results. There was a significant increase in the percentage of infected cells on average from 30 to 70% in time interval from 24 to 48 h. With 1000-fold dilution of viral-containing fluid the FC method detected the 6,9±0,21% of infected cells in Vero cultures (P<0,001, n=3). Conclusion. FC has proved to be a fast, sensitive and reliable method for determining the relative number of virus- infected Vero cells in cultures. The drug DAI had a sufficient activity for its effective use in the automated version of MFA based on the FC method. The use of FC is possible at various stages of anti-rabies drug production and control, and is also promising in terms of further improving of the rabies diagnosis.

New permeability pathways induced by the malarial parasite in the membrane of its host erythrocyte: Potential routes for targeting of drugs into infected cells

1987 ◽  
Vol 7 (6) ◽  
pp. 455-463 ◽  
Author(s):  
Hagai Ginsburg ◽  
Wilfred D. Stein
Keyword(s):  
Cell Membrane ◽  
Host Cell ◽  
Malarial Parasite ◽  
Structural Defects ◽  
Transport Systems ◽  
Host Cell Membrane ◽  
Transport Pathways ◽  
Infected Cells ◽  
New Permeability Pathways

Malarial parasites propagate asexually inside the erythrocytes of their vertebrate host. Six hours after invasion, the permeability of the host cell membrane to anions and small nonelectrolytes starts to increase and reaches its peak as the parasite matures. This increased permeability differs from the native transport systems of the normal erythrocyte in its solute selectivity pattern, its enthalpy of activation and its susceptibility to inhibitors, suggesting the appearance of new transport pathways. A biophysical analysis of the permeability data indicates that the selectivity barrier discriminates between permeants according to their hydrogen bonding capacity and has solubilization properties compared to those of iso-butanol. The new permeability pathways could result from structural defects caused in the host cell membrane by the insertion of parasite-derived polypeptides. It is suggested that the unique transport properties of the new pathways be used to target drugs into infected cells, to affect the parasite either directly or through the modulation of the intraerythrocytic environment. The feasibility of drug targeting is demonstrated in in vitro cultures of the human malarial parasite Plasmodium falciparum.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

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