scholarly journals Use of Flow Cytometry for Rapid, Quantitative Detection of Poliovirus-Infected Cells via TAT Peptide-Delivered Molecular Beacons

2012 ◽  
Vol 79 (2) ◽  
pp. 696-700 ◽  
Author(s):  
Divya Sivaraman ◽  
Hsiao-Yun Yeh ◽  
Ashok Mulchandani ◽  
Marylynn V. Yates ◽  
Wilfred Chen
Keyword(s):  
Flow Cytometry ◽  
Viral Infections ◽  
Molecular Beacons ◽  
Disease Spread ◽  
Quantitative Detection ◽  
Tat Peptide ◽  
Detection Scheme ◽  
Efficient Detection ◽  
Order Of Magnitude ◽  
Infected Cells

ABSTRACTRapid and efficient detection of viral infection is crucial for the prevention of disease spread during an outbreak and for timely clinical management. In this paper, the utility of Tat peptide-modified molecular beacons (MBs) as a rapid diagnostic tool for the detection of virus-infected cells was demonstrated. The rapid intracellular delivery mediated by the Tat peptide enabled the detection of infected cells within 30 s, reaching saturation in signal in 30 min. This rapid detection scheme was coupled with flow cytometry (FC), resulting in an automated, high-throughput method for the identification of virus-infected cells. Because of the 2-order-of-magnitude difference in fluorescence intensity between infected and uninfected cells, as few as 1% infected cells could be detected. Because of its speed and sensitivity, this approach may be adapted for the practical diagnosis of multiple viral infections.

scholarly journals Quantitative Detection of Hepadnavirus-Infected Lymphoid Cells by In Situ PCR Combined with Flow Cytometry: Implications for the Study of Occult Virus Persistence

2003 ◽  
Vol 77 (2) ◽  
pp. 970-979 ◽  
Author(s):  
Patricia M. Mulrooney ◽  
Tomasz I. Michalak
Keyword(s):  
Flow Cytometry ◽  
Nucleic Acid ◽  
Standard Error ◽  
Persistent Infection ◽  
Lymphoid Cells ◽  
Quantitative Detection ◽  
In Situ Pcr ◽  
Infected Cells ◽  
The Mean

ABSTRACT The detection of small amounts of viral pathogens in infected cells by classical PCR is hampered by a partial loss of virus nucleic acid due to extraction and by difficulties in discrimination between truly intracellular virus genome material and that possibly adhered to the cell surface. These impediments limit reliable identification of virus traces within infected cells, which are typically encountered in latent and persistent occult infections. In this study, hepadnavirus-specific in situ PCR combined with the enzymatic elimination of extracellular virus and flow cytometry permitted detection of viral genomes in lymphoid cells without nucleic acid isolation and allowed quantification of infected cells during the course of persistent infection with woodchuck hepatitis virus (WHV). The validity of the procedure was confirmed by hybridization analysis of the in situ-amplified viral sequences. The results showed that hepadnavirus can be directly detected within lymphoid cells not only in serologically accountable infection, but also years after recovery from viral hepatitis and in the course of primary occult virus carriage. Percentages of infected peripheral lymphoid cells in symptomatic WHV hepatitis fluctuate between 3.4 and 20.4% (mean ± standard error of the mean, 9.6% ± 1.7%), whereas those in persistent, serologically mute WHV infection range from 1.1 to 14.6% (mean ± standard error of the mean, 4.8% ± 0.8%) (P = 0.005). The data obtained provide further evidence that WHV infection continues indefinitely in the lymphatic system independently of whether it is symptomatic or concealed. They document that hepadnavirus can be detected in a significant proportion of circulating lymphoid cells in both immunovirologically apparent as well as occult persistent infection.

scholarly journals Detection of Infective Poliovirus by a Simple, Rapid, and Sensitive Flow Cytometry Method Based on Fluorescence Resonance Energy Transfer Technology

2009 ◽  
Vol 76 (2) ◽  
pp. 584-588 ◽  
Author(s):  
Jason L. Cantera ◽  
Wilfred Chen ◽  
Marylynn V. Yates
Keyword(s):  
Flow Cytometry ◽  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Infectious Virus ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Disease Spread ◽  
Infected Cells ◽  
Fluorescence Resonance

ABSTRACT The rapid and effective detection of virus infection is critical for clinical management and prevention of disease spread during an outbreak. Several methods have been developed for this purpose, of which classical serological and viral nucleic acid detection are the most common. We describe an alternative approach that utilizes engineered cells expressing fluorescent proteins undergoing fluorescence resonance energy transfer (FRET) upon cleavage by the viral 2A protease (2Apro) as an indication of infection. Quantification of the infectious-virus titers was resolved by using flow cytometry, and utility was demonstrated for the detection of poliovirus 1 (PV1) infection. Engineered buffalo green monkey kidney (BGMK) cells expressing the cyan fluorescent protein (CFP)-yellow fluorescent protein (YFP) substrate linked by a cleavage recognition site for PV1 2Apro were infected with different titers of PV1. After incubation at various time points, cells were harvested, washed, and subjected to flow cytometry analysis. The number of infected cells was determined by counting the number of cells with an increased CFP-to-YFP ratio. As early as 5 h postinfection, a significant number of infected cells (3%) was detected by flow cytometry, and cells infected with only 1 PFU were detected after 12 h postinfection. When applied to an environmental water sample spiked with PV1, the flow cytometry-based assay provided a level of sensitivity similar to that of the plaque assay for detecting and quantifying infectious virus particles. This approach, therefore, is more rapid than plaque assays and can be used to detect other viruses that frequently do not form clear plaques on cell cultures.

scholarly journals Immunosurveillance of Cancer and Viral Infections with Regard to Alterations of Human NK Cells Originating from Lifestyle and Aging

Biomedicines ◽  
2021 ◽  
Vol 9 (5) ◽  
pp. 557
Author(s):  
Xuewen Deng ◽  
Hiroshi Terunuma ◽  
Mie Nieda
Keyword(s):  
Nk Cells ◽  
Viral Infections ◽  
Nk Cell ◽  
Healthy Lifestyle ◽  
Cell Activity ◽  
Nk Cell Activity ◽  
Effector Cells ◽  
Cell Dysfunction ◽  
Forest Bathing ◽  
Infected Cells

Natural killer (NK) cells are cytotoxic immune cells with an innate capacity for eliminating cancer cells and virus- infected cells. NK cells are critical effector cells in the immunosurveillance of cancer and viral infections. Patients with low NK cell activity or NK cell deficiencies are predisposed to increased risks of cancer and severe viral infections. However, functional alterations of human NK cells are associated with lifestyles and aging. Personal lifestyles, such as cigarette smoking, alcohol consumption, stress, obesity, and aging are correlated with NK cell dysfunction, whereas adequate sleep, moderate exercise, forest bathing, and listening to music are associated with functional healthy NK cells. Therefore, adherence to a healthy lifestyle is essential and will be favorable for immunosurveillance of cancer and viral infections with healthy NK cells.

scholarly journals Poxviral Strategies to Overcome Host Cell Apoptosis

Pathogens ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 6
Author(s):  
Chathura D. Suraweera ◽  
Mark G. Hinds ◽  
Marc Kvansakul
Keyword(s):  
Viral Infections ◽  
B Cell Lymphoma ◽  
Intrinsic Apoptosis ◽  
Host Cell Apoptosis ◽  
Successful Infection ◽  
Infected Cells ◽  
Host Virus Interactions ◽  
Almost All ◽  
Virus Interactions ◽  
Multicellular Organisms

Apoptosis is a form of cellular suicide initiated either via extracellular (extrinsic apoptosis) or intracellular (intrinsic apoptosis) cues. This form of programmed cell death plays a crucial role in development and tissue homeostasis in multicellular organisms and its dysregulation is an underlying cause for many diseases. Intrinsic apoptosis is regulated by members of the evolutionarily conserved B-cell lymphoma-2 (Bcl-2) family, a family that consists of pro- and anti-apoptotic members. Bcl-2 genes have also been assimilated by numerous viruses including pox viruses, in particular the sub-family of chordopoxviridae, a group of viruses known to infect almost all vertebrates. The viral Bcl-2 proteins are virulence factors and aid the evasion of host immune defenses by mimicking the activity of their cellular counterparts. Viral Bcl-2 genes have proved essential for the survival of virus infected cells and structural studies have shown that though they often share very little sequence identity with their cellular counterparts, they have near-identical 3D structures. However, their mechanisms of action are varied. In this review, we examine the structural biology, molecular interactions, and detailed mechanism of action of poxvirus encoded apoptosis inhibitors and how they impact on host–virus interactions to ultimately enable successful infection and propagation of viral infections.

scholarly journals Mass Spectrometry versus Conventional Techniques of Protein Detection: Zika Virus NS3 Protease Activity towards Cellular Proteins

Molecules ◽  
2021 ◽  
Vol 26 (12) ◽  
pp. 3732
Author(s):  
Agnieszka Dabrowska ◽  
Aleksandra Milewska ◽  
Joanna Ner-Kluza ◽  
Piotr Suder ◽  
Krzysztof Pyrc
Keyword(s):  
Mass Spectrometry ◽  
Zika Virus ◽  
Viral Infections ◽  
Protein Detection ◽  
Extensive Discussion ◽  
Protein Targets ◽  
Highly Sensitive ◽  
Infected Cells ◽  
Ns3 Protease ◽  
To Receive

Mass spectrometry (MS) used in proteomic approaches is able to detect hundreds of proteins in a single assay. Although undeniable high analytical power of MS, data acquired sometimes lead to confusing results, especially during a search of very selective, unique interactions in complex biological matrices. Here, we would like to show an example of such confusing data, providing an extensive discussion on the observed phenomenon. Our investigations focus on the interaction between the Zika virus NS3 protease, which is essential for virus replication. This enzyme is known for helping to remodel the microenvironment of the infected cells. Several reports show that this protease can process cellular substrates and thereby modify cellular pathways that are important for the virus. Herein, we explored some of the targets of NS3, clearly shown by proteomic techniques, as processed during infection. Unfortunately, we could not confirm the biological relevance of protein targets for viral infections detected by MS. Thus, although mass spectrometry is highly sensitive and useful in many instances, also being able to show directions where cell/virus interaction occurs, we believe that deep recognition of their biological role is essential to receive complete insight into the investigated process.

scholarly journals Kinetic aspects of virus targeting by nanoparticles in vivo

2021 ◽  
Author(s):  
Vladimir P. Zhdanov
Keyword(s):  
Immune System ◽  
Kinetic Model ◽  
Order Of Magnitude ◽  
Eradication Of Infection ◽  
Infected Cells

AbstractOne of the suggested ways of the use of nanoparticles in virology implies their association with and subsequent deactivation of virions. The conditions determining the efficiency of this approach in vivo are now not clear. Herein, I propose the first kinetic model describing the corresponding processes and clarifying these conditions. My analysis indicates that nanoparticles can decrease concentration of infected cells by a factor of one order of magnitude, but this decrease itself (without feedback of the immune system) is insufficient for full eradication of infection. It can, however, induce delay in the progress of infection, and this delay can help to form sufficient feedback of the immune system.

scholarly journals Enhanced replica detection scheme for efficient analysis of intrusion detection in MANET

2017 ◽  
Vol 7 (1.1) ◽  
pp. 565
Author(s):  
P. Bakeyalakshmi ◽  
S. K. Mahendran
Keyword(s):  
Intrusion Detection ◽  
Secure Communication ◽  
Mobile Ad Hoc Network ◽  
Delivery Ratio ◽  
Ratio Test ◽  
Network Simulator ◽  
Efficient Manner ◽  
Detection Scheme ◽  
Efficient Detection ◽  
Replica Detection

Nowadays, detection scheme of intrusion is placing a major role for efficient access and analysis in Mobile Ad-hoc network (MANET). In the past, the detection scheme of Intrusion was used to identify the efficiency of the network and in maximum systems it performs with huge rate of false alarm. In this paper, an Effective approach of the Enhanced Replica Detection scheme (ERDS) based on Sequential Probability Ratio Test (SPRT) is proposed to detect the malicious actions and to have a secure path without claim in an efficient manner. Also, provides strategies to avoid attacker and to provide secure communication. In order to have an efficient analysis of intrusion detection the proposed approach is implemented based on the anomaly. To achieve this, the detection scheme is established based on SPRT and demonstrated the performances of detection with less claim. The simulation results of control overhead, packet delivery ratio, efficient detection, energy consumption and average claims are carried out for the analysis of performance to show the improvement than the existing by using the network simulator tool. Also, the performance of the proposed system illustrated the detection of intrusion in the normal and attacker states of the network.

scholarly journals Ex vivo analysis of SIV-infected cells by flow cytometry

2010 ◽  
Vol 77A (11) ◽  
pp. 1059-1066 ◽  
Author(s):  
Matthew R. Reynolds ◽  
Shari M. Piaskowski ◽  
Kimberly L Weisgrau ◽  
Andrea M. Weiler ◽  
Thomas C. Friedrich ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Ex Vivo ◽  
Infected Cells

scholarly journals Three Severe Cases of Viral Infections with Post-Kidney Transplantation Successfully Confirmed by Polymerase Chain Reaction and Flow Cytometry

2018 ◽  
Vol 8 (3) ◽  
pp. 198-206
Author(s):  
Keita Nakanishi ◽  
Hiroshi Kaito ◽  
Miki Ogi ◽  
Denshi Takai ◽  
Junya Fujimura ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Polymerase Chain Reaction ◽  
Kidney Transplantation ◽  
Viral Infections ◽  
Specific Treatment ◽  
Epstein Barr Virus Infection ◽  
Chain Reaction ◽  
Barr Virus ◽  
Polymerase Chain ◽  
Epstein Barr

Viral infections in patients with post-kidney transplantation are often difficult to diagnose as well as treat. We herein report three cases with severe viral infections after kidney transplantation. All their causative pathogens could be detected promptly by polymerase chain reaction and flow cytometry during the early stages of infection. These examinations would also be of great use to monitor therapeutic responses and disease activity. It is indeed true that no specific treatment is available for most of the viral infections, but we should be aware that some infections, such as Epstein-Barr virus infection, can be treatable with prompt and specific treatment, such as rituximab.

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