scholarly journals Cell Cultures for Virology: Usability, Advantages, and Prospects

2020 ◽  
Vol 21 (21) ◽  
pp. 7978
Author(s):  
Alexander A. Dolskiy ◽  
Irina V. Grishchenko ◽  
Dmitry V. Yudkin
Keyword(s):  
Cell Lines ◽  
Cell Cultures ◽  
Virus Isolation ◽  
Virus Detection ◽  
Clinical Samples ◽  
Laboratory Setting ◽  
Reporter Cell ◽  
Isolation And Identification ◽  
Reporter Systems ◽  
Reporter Proteins

Virus detection in natural and clinical samples is a complicated problem in research and diagnostics. There are different approaches for virus isolation and identification, including PCR, CRISPR/Cas technology, NGS, immunoassays, and cell-based assays. Following the development of genetic engineering methods, approaches that utilize cell cultures have become useful and informative. Molecular biology methods allow increases in the sensitivity and specificity of cell cultures for certain viruses and can be used to generate reporter cell lines. These cell lines express specific reporter proteins (e.g., GFP, luciferase, and CAT) in response to virus infection that can be detected in a laboratory setting. The development of genome editing and synthetic biology methods has given rise to new perspectives regarding the design of virus reporter systems in cell cultures. This review is aimed at describing both virology methods in general and examples of the development of cell-based methods that exist today.

scholarly journals Comparative performance of fetal goat tongue cell line ZZ-R 127 and fetal porcine kidney cell line LFBK-αvβ6 for Foot-and-mouth disease virus isolation

2015 ◽  
Vol 27 (4) ◽  
pp. 516-521 ◽  
Author(s):  
Katsuhiko Fukai ◽  
Kazuki Morioka ◽  
Manabu Yamada ◽  
Tatsuya Nishi ◽  
Kazuo Yoshida ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Kidney Cell ◽  
Virus Isolation ◽  
Foot And Mouth Disease ◽  
Clinical Samples ◽  
Porcine Kidney ◽  
Kidney Cell Line ◽  
Mouth Disease Virus ◽  
Porcine Kidney Cell

The fetal goat tongue cell line ZZ-R 127 and the fetal porcine kidney cell line LFBK-αvβ6 have been reported to have high sensitivity to various Foot-and-mouth disease virus (FMDV) strains. The suitability of ZZ-R 127 cells for FMDV isolation not only from epithelial suspensions but also from other clinical samples has already been confirmed in a previous study. However, to our knowledge, the suitability of LFBK-αvβ6 cells has not been evaluated using clinical samples other than epithelial materials. In addition, both cell lines have never been compared, in terms of use for FMDV isolation, under the same conditions. Therefore, in the current study, the virus isolation rates of both cell lines were compared using clinical samples collected from animals infected experimentally with FMDV. Viruses were successfully isolated from clinical samples other than epithelial suspensions for both cell lines. The virus isolation rates for the 2 cell lines were not significantly different. The Cohen kappa coefficients between the virus isolation results for both cell lines were significantly high. Taken together, these results confirmed the suitability of LFBK-αvβ6 cells for FMDV isolation from clinical samples other than epithelial suspensions. The levels of susceptibility of both cell lines to FMDV isolation were also confirmed to be almost the same.

scholarly journals An epidemic of acute diarrhoea in rural southern India associated with echovirus type 11 infection

1985 ◽  
Vol 95 (2) ◽  
pp. 483-492 ◽  
Author(s):  
J. R. Patel ◽  
J. Daniel ◽  
V. I. Mathan
Keyword(s):  
Cell Lines ◽  
Shigella Flexneri ◽  
Serum Antibody ◽  
Acute Diarrhoea ◽  
Southern India ◽  
Clinical Samples ◽  
Isolation And Identification ◽  
Epithelial Cell Lines ◽  
Second Wave ◽  
Echovirus Type

SUMMARYAn epidemic of diarrhoea with two distinct waves affected a village of 1375 people in southern India in 1983. The first wave of the epidemic, from the last week of December 1982, had a sharp peak in January 1983 and was over by March. Echovirus type 11 was isolated from patients, who also had a serum antibody response to the virus. During the second wave of the epidemic, from May to September 1983, the clinical features were different andShigella flexneriwas isolated without significant viral isolates. Infection during the first wave did not protect from the second wave. Virus isolation was in human intestinal tumourderived differentiated epithelial cell lines; such cell lines may be useful for the isolation and identification of entcroviruses in clinical samples.

scholarly journals Development of Cellular Models to Study Efficiency and Safety of Gene Edition by Homologous Directed Recombination Using the CRISPR/Cas9 System

Cells ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1492
Author(s):  
Sabina Sánchez-Hernández ◽  
Araceli Aguilar-González ◽  
Beatriz Guijarro-Albaladejo ◽  
Noelia Maldonado-Pérez ◽  
Iris Ramos-Hernández ◽  
...  
Keyword(s):  
Cell Lines ◽  
Main Concern ◽  
Target Sequence ◽  
Gene Repair ◽  
Reporter Cell ◽  
Delivery Method ◽  
Cellular Models ◽  
Target Sites ◽  
Reporter Systems ◽  
Or Gene

In spite of the enormous potential of CRISPR/Cas in basic and applied science, the levels of undesired genomic modifications cells still remain mostly unknown and controversial. Nowadays, the efficiency and specificity of the cuts generated by CRISPR/Cas is the main concern. However, there are also other potential drawbacks when DNA donors are used for gene repair or gene knock-ins. These GE strategies should take into account not only the specificity of the nucleases, but also the fidelity of the DNA donor to carry out their function. The current methods to quantify the fidelity of DNA donor are costly and lack sensitivity to detect illegitimate DNA donor integrations. In this work, we have engineered two reporter cell lines (K562_SEWAS84 and K562GWP) that efficiently quantify both the on-target and the illegitimate DNA donor integrations in a WAS-locus targeting setting. K562_SEWAS84 cells allow the detection of both HDR-and HITI-based donor integration, while K562GWP cells only report HDR-based GE. To the best of our knowledge, these are the first reporter systems that allow the use of gRNAs targeting a relevant locus to measure efficacy and specificity of DNA donor-based GE strategies. By using these models, we have found that the specificity of HDR is independent of the delivery method and that the insertion of the target sequence into the DNA donor enhances efficiency but do not affect specificity. Finally, we have also shown that the higher the number of the target sites is, the higher the specificity and efficacy of GE will be.

scholarly journals Assessment of the infectious threshold of SARS-CoV-2 in primary airway epithelial cells

2021 ◽  
Author(s):  
Manel ESSAIDI-LAZIOSI ◽  
Francisco Javier Perez Rodriguez ◽  
Pascale Sattonnet Roche ◽  
Nicolas Hulo ◽  
Frederique Jacquerioz ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cell Lines ◽  
Virus Isolation ◽  
Airway Epithelial Cells ◽  
Clinical Samples ◽  
Airway Epithelial ◽  
Airway Epithelia ◽  
Viral Loads ◽  
Human Airway ◽  
Human Airway Epithelia

Comparison of virus isolation success from clinical samples across a range of viral loads inoculated in parallel on Vero E6 and human airway epithelia (HAE) showed lower success of virus isolation in HAE, suggesting an overestimation of actual infectiousness in humans using Vero E6 cell lines, commonly considered as reference.

scholarly journals HT-29 and Caco-2 Reporter Cell Lines for Functional Studies of Nuclear Factor Kappa B Activation

2015 ◽  
Vol 2015 ◽  
pp. 1-13 ◽  
Author(s):  
Giuliana Mastropietro ◽  
Inés Tiscornia ◽  
Karen Perelmuter ◽  
Soledad Astrada ◽  
Mariela Bollati-Fogolín
Keyword(s):  
Cell Lines ◽  
Nuclear Factor Kappa B ◽  
Biological Processes ◽  
Bacterial Strains ◽  
Reporter Cell ◽  
Functional Studies ◽  
Primary Screening ◽  
Screening And Identification ◽  
Reporter Systems ◽  
Ht 29

The NF-κB is a transcription factor which plays a key role in regulating biological processes. In response to signals, NF-κB activation occurs via phosphorylation of its inhibitor, which dissociates from the NF-κB dimer allowing the translocation to the nucleus, inducing gene expression. NF-κB activation has direct screening applications for drug discovery for several therapeutic indications. Thus, pathway-specific reporter cell systems appear as useful tools to screen and unravel the mode of action of probiotics and natural and synthetic compounds. Here, we describe the generation, characterization, and validation of human epithelial reporter cell lines for functional studies of NF-κB activation by different pro- and anti-inflammatory agents. Caco-2 and HT-29 cells were transfected with a pNF-κB-hrGFP plasmid which contains the GFP gene under the control of NF-κB binding elements. Three proinflammatory cytokines (TNF-α, IL-1β, and LPS) were able to activate the reporter systems in a dose-response manner, which corresponds to the activation of the NF-κB signaling pathway. Finally, the reporter cell lines were validated using lactic acid bacteria and a natural compound. We have established robust Caco-2-NF-κB-hrGFP and HT-29-NF-κB-hrGFP reporter cell lines which represent a valuable tool for primary screening and identification of bacterial strains and compounds with a potential therapeutic interest.

scholarly journals Comparison of ZMAC and MARC-145 Cell Lines for Improving Porcine Reproductive and Respiratory Syndrome Virus Isolation from Clinical Samples

2020 ◽  
Author(s):  
Wannarat Yim-im ◽  
Haiyan Huang ◽  
Jie Park ◽  
Chong Wang ◽  
Gabriela Calzada ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Virus Isolation ◽  
Clinical Samples ◽  
Respiratory Syndrome Virus ◽  
Genetic Lineage ◽  
Success Rates ◽  
Serum Samples ◽  
Genetic Lineages ◽  
Wide Range

MARC-145 cell line is commonly used to isolate porcine reproductive and respiratory syndrome virus (PRRSV) for diagnostics, research and vaccine production, but it yields frustratingly low success rates of virus isolation (VI). ZMAC cell line, derived from porcine alveolar macrophages, has become available but its utilization for PRRSV VI from clinical samples has not been evaluated. This study compared PRRSV VI in ZMAC and MARC-145 cells from 375 clinical samples (including 104 lung, 140 serum, 90 oral fluid, and 41 processing fluid samples). PRRSV VI success rate was very low in oral fluids and processing fluids regardless of using ZMAC or MARC-145 cells. PRRSV VI success rates from lung and serum samples were significantly higher in ZMAC than in MARC-145 cells. Lung and serum samples with CT<30 had better VI success. PRRSV-2 in genetic lineages-1 and -8 were isolated more successfully in ZMAC than in MARC-145 whereas PRRSV-2 in genetic lineage-5 were isolated in two cell lines with similar success rates. For samples with VI positive in both ZMAC and MARC-145, 14 of 23 PRRSV-2 isolates had similar titers in two cell lines. Fifty-one of 95 (53.7%) ZMAC-obtained PRRSV-2 or PRRSV-1 isolates grew in MARC-145 and all 46 (100%) MARC-145-obtained isolates grew in ZMAC. In summary, ZMAC allows better isolation of a wide range of PRRSV field strains; however, not all of the ZMAC-obtained PRRSV isolates grow in MARC-145 cells. This study provides important guidelines to improve isolating PRRSV from clinical samples for further characterization and/or for producing autogenous vaccines.

scholarly journals Practical aspects on identification, cultivation and characteristics of varicella-zoster virus isolates

2020 ◽  
Vol 10 (2) ◽  
pp. 387-396
Author(s):  
F. G. Nagieva ◽  
E. P. Barkova ◽  
A. N. Lisakov ◽  
A. V. Sidorov ◽  
V. V. Zverev ◽  
...  
Keyword(s):  
Herpes Zoster ◽  
Varicella Zoster Virus ◽  
Cell Lines ◽  
Cell Cultures ◽  
Culture Medium ◽  
Clinical Isolates ◽  
Clinical Samples ◽  
Target Cells ◽  
Varicella Zoster ◽  
Extracellular Virus

Until now, it has been considered that infectivity of the varicella-zoster virus (VZV) is closely related to target cell, and newly formed virus is not released into the culture medium. It is also known that it is hard to grow VZV in cell cultures, due to its slow replication rate and a limited range of sensitive cell cultures. In addition, VZV isolation depends on type of cell culture used, nature of clinical material, presence of viable virus and transport time. Objectives. To study production of infectious extracellular VZV in various cell cultures. Materials and methods. Eight cell cultures were used, including human embryonic diploid lung cells and human embryonic dermomuscular tissue (KM-27), as well as continuous human and monkey cell lines. Crusts detached from vesicular lesions were used as clinical isolates, which were placed into cryo-vials added with transport medium and transferred in liquid nitrogen. VZV infectivity was assessed in cell cultures by using hemo-adsorption assay with erythrocyte suspension isolated from guinea pig or human zero group blood and confirmed by indirect immunofluorescence with polyclonal sera from varicella or herpes zoster convalescents. Results. There were examined 27 clinical samples consisting of crusts from vesicular lesions isolated from patients with chickenpox, as well as one sample from 63-year old patient with exacerbated recurrent herpes zoster. Primary infection with clinical isolates was performed on diploid human lung embryo cells (HLEC) at low temperature. It was found that clinical samples collected within day 1–18 inclusive after the onset of skin eruption were able to induce cytopathic effects in HLEC cell monolayer such as cytolysis around dermal crusts. Specificity of cytopathic effect was confirmed by using real-time polymerase chain reaction (RT-PCR). Viral antigens were prepared on 7 cell lines infected with the laboratory strain Ellen VZV (USA) to assess the immune sera. A high anti-VZV specificity of mouse sera was detected by ELISA while all the lysates of infected cell lines were used as the solid-phase sorbent. In experiments on VZV reproduction demonstrated that extracellular virus was released into the culture medium starting from day 1 after infection of target cells, and infectivity of the virus-containing fluid ascends during further cultivation.

Investigation of duck plague virus in hoar areas of Bangladesh

2020 ◽  
Vol 26 (1-2) ◽  
pp. 73-78
Author(s):  
A Hossen ◽  
MH Rahman ◽  
MZ Ali ◽  
MA Yousuf ◽  
MZ Hassan ◽  
...  
Keyword(s):  
Cytopathic Effect ◽  
Clinical Sample ◽  
Chorioallantoic Membrane ◽  
Clinical Samples ◽  
Isolation And Identification ◽  
Duck Plague Virus ◽  
Pcr Method ◽  
Polymerase Chain ◽  
The Individual ◽  
Free Ranging

Duck plague (DP) is the most important infectious disease of geese, ducks and free-ranging water birds. The present study was conducted to determine the prevalence of duck plague virus followed by isolation and identification. For these purposes, a total of 155 cloacal swabs samples were collected randomly from duck of different haor areas of Bangladesh including 45 (41 surveillance and 4 clinical) samples from Netrokona; 42 (40 surveillance and 2 clinical) samples from Kishoregonj; 30 samples from Brahmanbaria and 38 samples from Sunamganj. The samples were processed and pooled (1:5 ratio) for initial screening of target polymerase gene of duck plague virus by polymerase chain reaction (PCR) method. All the samples of a positive pool were then tested individually for identifying the individual positive samples. The result showed that out of 155 samples, 41 (26.45%) were found positive in which 17 were from Netrokona, where 15 (36.58%) were from surveillance samples and 2 (50%) were from clinical sample; 16 were from Kishoregonj, where 14 (35%) were from surveillance samples and 2 (100%) were from clinical sample; 2 (6.6%) were from Brahmanbaria and 5 (13.15%) were from Sunamganj. These positive samples were inoculated into 9-10 days embryonated duck eggs (EDE) through chorioallantoic membrane (CAM) route for the isolation of virus. The EDE died earlier was also chilled, and in a similar way, the CAMs were collected and again performed PCR for id entification of virus. Out of 41 PCR positive samples, 26 samples were isolated and reconfirmed by PCR. Subsequently, DPV was isolated in primary duck embryo fibroblasts cell culture and confirmed by observing cytopathic effect (CPE). Bang. J. Livs. Res. Vol. 26 (1&2), 2019: P. 73-78

Hepatoma Cell Cultures as In Vitro Models for the Hepatotoxicity of Xenobiotics

1988 ◽  
Vol 16 (1) ◽  
pp. 32-37
Author(s):  
Margherita Ferro ◽  
Anna Maria Bassi ◽  
Giorgio Nanni
Keyword(s):  
Cell Lines ◽  
Cell Cultures ◽  
Membrane Lipids ◽  
Hepatoma Cell ◽  
Positive Control ◽  
In Vitro Models ◽  
Low Concentrations ◽  
Formation Efficiency ◽  
Dose Dependent

Two hepatoma cell cultures were examined as in vitro models to be used in genotoxicity and cytotoxicity tests without the addition of bioactivating enzymes. The MH1C1, and HTC hepatoma lines were used in this study to establish their sensitivity to a number of xenobiotics, namely, cyclophosphamide (CP), the classical positive control in bioactivation tests; benzaldehyde (BA), a short-chain aldehyde; and 4-hydroxynonenal (HNE), a major toxic end-product of the peroxidative degradation of cell membrane lipids. As a first approach, we compared the following cytotoxicity tests: release of lactate dehydrogenase (LDH), and colony formation efficiency (CF). Colony-forming cells were exposed to the drugs according to different procedures, before or after the anchorage phase. The leakage of LDH into the medium following exposure of both cell lines to HNE, CP and BA for up to 24 hours was found not to be a good index of cytotoxicity. A better indicator of cytotoxicity was CF, as evaluated by exposure of the cells 24 hours after seeding. The effects were detectable at very low concentrations, corresponding to 10, 90 and 100μM for HNE, CP and BA, respectively. The impairment of CF efficiency was dose-dependent and time-dependent, and several differences between the two cell lines were observed.

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