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 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 Locked Nucleic Acid and Flow Cytometry-FluorescenceIn SituHybridization for the Detection of Bacterial Small Noncoding RNAs

2011 ◽  
Vol 78 (1) ◽  
pp. 14-20 ◽  
Author(s):  
Kelly L. Robertson ◽  
Gary J. Vora
Keyword(s):  
Flow Cytometry ◽  
Nucleic Acid ◽  
Stationary Phase ◽  
Noncoding Rnas ◽  
Locked Nucleic Acid ◽  
Lag Phase ◽  
Bacterial Cells ◽  
Small Noncoding Rnas ◽  
Qrt Pcr

ABSTRACTWe describe the development and testing of a high-throughput method that enables the detection of small noncoding RNAs (ncRNAs) from single bacterial cells using locked nucleic acid probes (LNA) and flow cytometry-fluorescencein situhybridization (flow-FISH). The LNA flow-FISH method and quantitative reverse transcription-PCR (qRT-PCR) were used to monitor the expression of three ncRNAs (6S, CsrB, and TPP-2) inVibrio campbelliiATCC BAA-1116 cultures during lag phase, mid-log phase, and stationary phase. Both LNA flow-FISH and qRT-PCR revealed that CsrB and TPP-2 were highly expressed during lag phase but markedly reduced in mid-log phase and stationary phase, whereas 6S demonstrated no to little expression during lag phase but increased thereafter. Importantly, while LNA flow-FISH and qRT-PCR demonstrated similar overall expression trends, only LNA flow-FISH, which enabled the detection of ncRNAs in individual cells as opposed to the lysate-based ensemble measurements generated by qRT-PCR, was able to capture the cell-to-cell heterogeneity in ncRNA expression. As such, this study demonstrates a new method that simultaneously enables thein situdetection of ncRNAs and the determination of gene expression heterogeneity within an isogenic bacterial population.

scholarly journals Discrimination of bacteriophage infected cells using locked nucleic acid fluorescent in situ hybridization (LNA-FISH)

Biofouling ◽  
2016 ◽  
Vol 32 (2) ◽  
pp. 179-190 ◽  
Author(s):  
Diana Vilas Boas ◽  
Carina Almeida ◽  
Sanna Sillankorva ◽  
Ana Nicolau ◽  
Joana Azeredo ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Nucleic Acid ◽  
Fluorescent In Situ Hybridization ◽  
Locked Nucleic Acid ◽  
Infected Cells

Could an ocean climate atlas generated by a model compete with an observational one?

2020 ◽  
Author(s):  
Georgy I. Shapiro ◽  
Jose M. Gonzalez-Ondina ◽  
Xavier Francis ◽  
Hyee S. Lim ◽  
Ali Almehrezi
Keyword(s):  
Standard Error ◽  
Sea Surface ◽  
Vertical Profiles ◽  
Individual Data ◽  
Model Simulations ◽  
Ocean Climate ◽  
Data Points ◽  
In Situ Observations ◽  
The Mean

<p>Modern numerical ocean models have matured over the last decades and are able to provide accurate fore- and hind-cast of the ocean state. The most accurate data could be obtained from the reanalysis where the model run in a hindcast mode with assimilation of available observational data. An obvious benefit of model simulation is that it provides the spatial density and temporal resolution which cannot be achieved by in-situ observations or satellite derived measurements. It is not unusual that even a relatively small area of the ocean model can have in access of 100,000 nodes in the horizontal, each containing vertical profiles of temperature, salinity, velocity and other ocean parameters with a temporal resolution theoretically as high as a few minutes. Remotely sensed (satellite) observations of sea surface temperature can compete with the models in terms of spatial resolution, however they only produce data at the sea surface not the vertical profiles. On the other hand, in-situ observations have a benefit of being much more precise than model simulations. For instance a widely used CTD profiler SBE 911plus has accuracy of about 0.001 °C, which is not achievable by models.</p><p>In the creation of a climatic atlas the higher accuracy of individual profiles provided by in-situ measurements may become less beneficial. Assuming the normal distribution of data at each location, the standard error of the mean (SEM) is calculated as SE=S/SQRT(N), where S is the standard deviation of individual data points around the mean, and N is the number of data points. The climatic data are obtained by averaging a large number of individual data points, and here the benefit of having more data points may become a greater advantage than the accuracy of a single observation.  </p><p>In this study we have created an ocean climate atlas for the northern part of the Indian Ocean including the Red Sea and the Arabian Gulf using model generated data. The data were taken from Copernicus Marine Environment Monitoring Service (CMEMS) reanalysis product GLOBAL_REANALYSIS_PHY_001_030 with 1/12° horizontal resolution and 50 vertical levels for the period 1998 to 2017. The model component is the NEMO platform driven at the surface by ECMWF ERA-Interim reanalysis. The model assimilates along track altimeter data, satellite Sea Surface Temperature, as well as in-situ temperature and salinity vertical profiles where available. The monthly data from CMEMS were then averaged over 20 years to produce an atlas at the surface, 10, 20, 30, 75, 100, 125, 150, 200, 250, 300, 400, and 500 m depths.  The standard error of the mean has been calculated for each point and each depth level on the native grid (1/12 degree).</p><p>The atlas based on model simulations was compared with the latest version of the World Ocean Atlas (WOA)  2018 published by the NCEI.  WOA has objectively analysed climatological mean fields on a ¼  degree grid. The differences between the mean values and SEMs from observational and simulated atlases are analysed, and the potential causes of mismatch are discussed.</p>

scholarly journals In Situ Localization of Barley Yellow Dwarf Virus-PAV 17-kDa Protein and Nucleic Acids in Oats

1998 ◽  
Vol 88 (10) ◽  
pp. 1031-1039 ◽  
Author(s):  
Petra H. Nass ◽  
Leslie L. Domier ◽  
Birute P. Jakstys ◽  
Cleora J. D'Arcy
Keyword(s):  
In Situ Hybridization ◽  
Nucleic Acid ◽  
Barley Yellow Dwarf Virus ◽  
Yellow Dwarf ◽  
Viral Rna ◽  
Barley Yellow Dwarf ◽  
Infected Cells ◽  
Yellow Dwarf Virus ◽  
Filamentous Material

Barley yellow dwarf virus strain PAV (BYDV-PAV) RNA and the 17-kDa protein were localized in BYDV-PAV-infected oat cells using in situ hybridization and in situ immunolocalization assays, respectively. The in situ hybridization assay showed labeling of filamentous material in the nucleus, cytoplasm, and virus-induced vesicles with both sense and antisense nucleic acid probes, suggesting that the filamentous material found in BYDV-PAV-infected cells contains viral RNA. BYDV-PAV negative-strand RNA was detected before virus particles were observed, which indicates that RNA replication is initiated before synthesis of viral coat protein in the cytoplasm. The 17-kDa protein was associated with filamentous material in the cytoplasm, nucleus, and virus-induced vesicles. The labeling densities observed using antibodies against the 17-kDa protein were similar in the nucleus and cytoplasm. No labeling of the 17-kDa protein was observed in plasmodesmata, but filaments in the nuclear pores occasionally were labeled. Since BYDV-PAV RNA and 17-kDa protein colocalized within infected cells, it is possible that single-stranded viral RNA is always associated with the 17-kDa protein in vivo. The 17-kDa protein may be required for viral nucleic acid filaments to traverse the nuclear membrane or other membrane systems.

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