scholarly journals Attenuation of Bluetongue Virus (BTV) in an in ovo Model Is Related to the Changes of Viral Genetic Diversity of Cell-Culture Passaged BTV

Viruses ◽  
2019 ◽  
Vol 11 (5) ◽  
pp. 481 ◽  
Author(s):  
Fabian Z. X. Lean ◽  
Matthew J. Neave ◽  
John R. White ◽  
Jean Payne ◽  
Teresa Eastwood ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Cell Culture ◽  
Bluetongue Virus ◽  
Genomic Analysis ◽  
Sequencing Analysis ◽  
Structural Protein ◽  
In Ovo ◽  
Novel Approach ◽  
Amino Acid Mutations

The embryonated chicken egg (ECE) is routinely used for the laboratory isolation and adaptation of Bluetongue virus (BTV) in vitro. However, its utility as an alternate animal model has not been fully explored. In this paper, we evaluated the pathogenesis of BTV in ovo using a pathogenic isolate of South African BTV serotype 3 (BTV-3) derived from the blood of an infected sheep. Endothelio- and neurotropism of BTV-3 were observed by immunohistochemistry of non-structural protein 1 (NS1), NS3, NS3/3a, and viral protein 7 (VP7) antigens. In comparing the pathogenicity of BTV from infectious sheep blood with cell-culture-passaged BTV, including virus propagated through a Culicoides-derived cell line (KC) or ECE, we found virus attenuation in ECE following cell-culture passage. Genomic analysis of the consensus sequences of segments (Seg)-2, -5, -6, -7, -8, -9, and -10 identified several nucleotide and amino-acid mutations among the cell-culture-propagated BTV-3. Deep sequencing analysis revealed changes in BTV-3 genetic diversity in various genome segments, notably a reduction of Seg-7 diversity following passage in cell culture. Using this novel approach to investigate BTV pathogenicity in ovo, our findings support the notion that pathogenic BTV becomes attenuated in cell culture and that this change is associated with virus quasispecies evolution.

scholarly journals Global Genomic Analysis of Newly Diagnosed t(4 ;14) Multiple Myeloma Reveals a Specific Mutational Spectrum and Identifies PKD2 As a Potential Therapeutic Target

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4462-4462
Author(s):  
Xiu Ly Song ◽  
Raphaël Szalat ◽  
Alexis Talbot ◽  
HaiVu Nguyen ◽  
Mehmet K. Samur ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Plasma Cells ◽  
Genomic Analysis ◽  
Sequencing Analysis ◽  
Newly Diagnosed ◽  
Potential Therapeutic Target ◽  
Mutational Landscape

Abstract In Multiple Myeloma (MM), the t(4;14) translocation is associated with a poor outcome. However, beside this translocation, the genetic events which determine the adverse evolution of the disease and the resistance to treatments remain elusive. In this study we performed whole exome or RNA sequencing analysis of samples from 65 newly diagnosed t(4;14) MM. We found that NRAS, KRAS, MAPK and FGFR3 are frequently mutated (12%, 9%, 13.8%, and 20% respectively). Overall, the FGFR3/RAS/BRAF/MAPK genes were mutated in 36 cases (54%). There was a negative correlation between mutations in FGFR3 and those occurring in NRAS, KRAS and BRAF as expected from the mutually exclusive occurrence of mutations in these genes. In addition to alterations in TP53 and DIS3, we found marked elevated frequency of mutations in PRKD2 (10.7%), ATM/ATR (10.7%) and MYCBP2 (7.6%), reduced frequency in FAM46C (1.5%) and no mutation in TRAF3 and CCND1. Mutations in ATM/ATR were strongly associated with the MB4-2 breakpoint (Bp) (p = 1.62 10-4) and significantly correlated with mutations affecting genes coding for members of the MAPK family. We observed a positive correlation between non-silent mutations in PRKD2 and the MB4-1 or MB4-3 Bp (p = 1.3 10-2). Of note, PRKD2 mutations are exclusively found in 3 t(4;14) MM cell lines and among the 84 MM sequenced by Bolli et al. (1), none of the non t(4;14) patient were mutated in PRKD2, indicating that this genetic lesion is associated with t(4;14) MM. In the NCI-H929 t(4;14) MM cell line, which is mutated for PRKD2, encoding the PKD2 serine/threonine kinase, we observed elevated levels of phosphorylated PKD2. Furthermore, inhibition of PKD, decreased PKD2 phosphorylation and triggered reduced proliferation and apoptosis of MM cell lines and fresh plasma cells from patients in vitro. These results define a specific mutational landscape for t(4;14) MM and identify PKD2 as a potential therapeutic target in MM patients. Altogether, these results define a specific mutational landscape for t(4;14) MM and identify PKD2 as a potential therapeutic target in MM patients. Reference 1. Bolli, N., Avet-Loiseau, H., Wedge, D.C., Van Loo, P., Alexandrov, L.B., Martincorena, I., Dawson, K.J., Iorio, F., Nik-Zainal, S., Bignell, G.R., et al. (2014). Heterogeneity of genomic evolution and mutational profiles in multiple myeloma. Nat Commun 5, 2997. Disclosures Munshi: Janssen: Consultancy; Takeda: Consultancy; Celgene: Consultancy; Amgen: Consultancy; Merck: Consultancy; Pfizer: Consultancy; Oncopep: Patents & Royalties.

scholarly journals Treatment with Exogenous Trypsin Expands In Vitro Cellular Tropism of the Avian Coronavirus Infectious Bronchitis Virus

Viruses ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 1102
Author(s):  
Phoebe Stevenson-Leggett ◽  
Sarah Keep ◽  
Erica Bickerton
Keyword(s):  
Cell Culture ◽  
Infectious Bronchitis Virus ◽  
Ex Vivo ◽  
Serial Passage ◽  
In Ovo ◽  
Infectious Bronchitis ◽  
Protease Treatment ◽  
Cellular Tropism ◽  
Passage Cell

The Gammacoronavirus infectious bronchitis virus (IBV) causes a highly contagious and economically important respiratory disease in poultry. In the laboratory, most IBV strains are restricted to replication in ex vivo organ cultures or in ovo and do not replicate in cell culture, making the study of their basic virology difficult. Entry of IBV into cells is facilitated by the large glycoprotein on the surface of the virion, the spike (S) protein, comprised of S1 and S2 subunits. Previous research showed that the S2′ cleavage site is responsible for the extended tropism of the IBV Beaudette strain. This study aims to investigate whether protease treatment can extend the tropism of other IBV strains. Here we demonstrate that the addition of exogenous trypsin during IBV propagation in cell culture results in significantly increased viral titres. Using a panel of IBV strains, exhibiting varied tropisms, the effects of spike cleavage on entry and replication were assessed by serial passage cell culture in the presence of trypsin. Replication could be maintained over serial passages, indicating that the addition of exogenous protease is sufficient to overcome the barrier to infection. Mutations were identified in both S1 and S2 subunits following serial passage in cell culture. This work provides a proof of concept that exogenous proteases can remove the barrier to IBV replication in otherwise non-permissive cells, providing a platform for further study of elusive field strains and enabling sustainable vaccine production in vitro.

scholarly journals In vitro evolution of herpes simplex virus 1 (HSV-1) reveals selection for syncytia and other minor variants in Vero cell culture

2019 ◽  
Author(s):  
Chad V. Kuny ◽  
Christopher D. Bowen ◽  
Daniel W. Renner ◽  
Christine M. Johnston ◽  
Moriah L. Szpara
Keyword(s):  
Genetic Diversity ◽  
Cell Culture ◽  
Herpes Simplex ◽  
Genetic Variants ◽  
Viral Population ◽  
Selective Pressures ◽  
Minor Variant ◽  
Simplex Virus ◽  
Hsv 1

AbstractThe large dsDNA virus HSV-1 is often considered to be genetically stable, however it is known to rapidly evolve in response to strong selective pressures such as antiviral drug treatment. Deep sequencing analysis has revealed that clinical and laboratory isolates of this virus exist as populations that contain a mixture of minor alleles or variants, similar to many RNA viruses. Classical virology methods often used plaque-purified virus populations to demonstrate consistent genetic inheritance of viral traits. Plaque purification represents a severe genetic bottleneck which may or may not be representative of natural transmission of HSV-1. Since HSV-1 has a low error rate polymerase but exhibits substantial genetic diversity, the virus likely uses other mechanisms to generate genetic diversity, including recombination, contraction and expansion of tandem repeats, and imprecise DNA repair mechanisms. We sought to study the evolution of HSV-1 in vitro, to examine the impact of this genetic diversity in evolution, in the setting of standard laboratory conditions for viral cell culture, and in the absence of strong selective pressures. We found that a mixed population of HSV-1 was more able to evolve and adapt in culture than a plaque-purified population, though this adaptation generally occurred in a minority of the viral population. We found that certain genetic variants appeared to be positively selected for rapid growth and spread in Vero cell culture, a phenotype which was also observed in clinical samples during their first passages in culture. In the case of a minor variant that induces a visually observable syncytial phenotype, we found that changes in minor variant frequency can have a large effect on the overall phenotype of a viral population.Author SummaryHerpes simplex virus type 1 (HSV-1) is a common virus, affecting over half of the adult human population, although it presents variable levels of disease burden and frequency of symptomatic recurrence. Antiviral treatments for HSV-1 infections are available, but thus far attempts at vaccine development have been foiled by insufficient immunity and/or viral escape. As a virus with a double-stranded DNA genome, HSV-1 is generally considered to be genetically stable and to have limited evolutionary potential. As these two statements are in conflict, we examined the ability of HSV-1 to evolve in a standardized cell culture setting. We utilized two HSV-1 isolates in this experiment, one with multiple viral genotypes present, which is similar to the viral populations seen in clinical settings, and one with a highly clonal viral population, which is similar to those often used in laboratory settings. After multiple rounds of replication, we analyzed the sequences of each passaged population. We found that the mixed viral population changed substantially over passage, and we were able to track specific genetic variants to phenotypic traits. By comparison, evolution in the clonal virus population was more limited. These data indicate that HSV-1 is capable of evolving rapidly, and that this evolution is facilitated by diversity in the viral population.

scholarly journals Organoids: An Emerging Tool To Study Aging Signature Across Human Tissues. Modeling Aging With Patient-derived Organoids

2021 ◽  
Author(s):  
Margalida Torrens-Mas ◽  
Catalina Perello-Reus ◽  
Cayetano Navas-Enamorado ◽  
Lesly Ibargüen ◽  
Andres Sanchez -Polo ◽  
...  
Keyword(s):  
Cell Culture ◽  
3D Cell Culture ◽  
Adverse Outcomes ◽  
Biological Aging ◽  
Preclinical Model ◽  
Biomarkers Of Aging ◽  
Novel Approach ◽  
New Biomarkers ◽  
Biology Of Aging

The biology of aging is focused on the identification of novel pathways that regulate the underlying processes of aging to develop interventions aimed at delaying the onset and progression of chronic diseases to extend lifespan. However, the research on the aging field has been conducted mainly in animal models, yeast, Caenorhabditis elegans and cell culture. Thus, it is unclear to what extent this knowledge is transferable to humans since they might not reflect the complexity of aging in people. Organoid culture is an in vitro 3D cell-culture technology that reproduces the physiological and cellular composition of the tissues and/or organs. This technology is being used in the cancer field to predict the response of a patient-derived tumor to a certain drug or treatment serving as patient stratification and drug-guidance approaches. Modeling aging with patient-derived organoids has a tremendous potential as a preclinical model tool to discover new biomarkers of aging, to predict adverse outcomes during aging and to design personalized approaches for prevention and treatment of aging-related diseases and geriatric syndromes. This could represent a novel approach to study chronological and/or biological aging paving the way to personalized interventions targeting the biology of aging.

scholarly journals A natural polymorphism in Zika virus NS2A protein responsible of virulence in mice

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Gines Ávila-Pérez ◽  
Aitor Nogales ◽  
Jun-Gyu Park ◽  
Silvia Márquez-Jurado ◽  
Francisco J. Iborra ◽  
...  
Keyword(s):  
Amino Acid ◽  
Zika Virus ◽  
Lethal Dose ◽  
Single Amino Acid ◽  
Sequencing Analysis ◽  
Structural Protein ◽  
Genetic Risk Assessment ◽  
Zikv Infection ◽  
Natural Polymorphism

AbstractZika virus (ZIKV) infection is currently one of the major concerns in human public health due to its association with neurological disorders. Intensive effort has been implemented for the treatment of ZIKV, however there are not currently approved vaccines or antivirals available to combat ZIKV infection. In this sense, the identification of virulence factors associated with changes in ZIKV virulence could help to develop safe and effective countermeasures to treat ZIKV or to prevent future outbreaks. Here, we have compared the virulence of two related ZIKV strains from the recent outbreak in Brazil (2015), Rio Grande do Norte Natal (RGN) and Paraiba. In spite of both viruses being identified in the same period of time and region, significant differences in virulence and replication were observed using a validated mouse model of ZIKV infection. While ZIKV-RGN has a 50% mouse lethal dose (MLD50) of ~105 focus forming units (FFUs), ZIKV-Paraiba infection resulted in 100% of lethality with less than 10 FFUs. Combining deep-sequencing analysis and our previously described infectious ZIKV-RGN cDNA clone, we identified a natural polymorphism in the non-structural protein 2 A (NS2A) that increase the virulence of ZIKV. Moreover, results demonstrate that the single amino acid alanine to valine substitution at position 117 (A117V) in the NS2A was sufficient to convert the attenuated rZIKV-RGN in a virulent Paraiba-like virus (MLD50 < 10 FFU). The mechanism of action was also evaluated and data indicate that substitution A117V in ZIKV NS2A protein reduces host innate immune responses and viral-induced apoptosis in vitro. Therefore, amino acid substitution A117V in ZIKV NS2A could be used as a genetic risk-assessment marker for future ZIKV outbreaks.

High-throughput sequencing analysis reveals the genetic diversity of different regions of the murine norovirus genome during in vitro replication

2016 ◽  
Vol 162 (4) ◽  
pp. 1019-1023 ◽  
Author(s):  
Axel Mauroy ◽  
Bernard Taminiau ◽  
Carine Nezer ◽  
Elsa Ghurburrun ◽  
Denis Baurain ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Sequencing Analysis ◽  
Murine Norovirus ◽  
In Vitro Replication

scholarly journals DIPG-22. DISSECTING THE ONCOGENIC ROLE OF FOXR2 IN DIFFUSE INTRINSIC PONTINE GLIOMA

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii291-iii291
Author(s):  
Jessica W Tsai ◽  
Smruti K Patel ◽  
Heather Bear ◽  
Frank Dubois ◽  
Prasidda Khadka ◽  
...  
Keyword(s):  
Cell Lines ◽  
Therapeutic Target ◽  
Transcriptional Profiling ◽  
Genomic Analysis ◽  
In Utero ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Sequencing Analysis ◽  
Integrative Genomics

Abstract BACKGROUND Diffuse intrinsic pontine gliomas (DIPGs) pose particular challenges for treatment. We recently completed a genomic analysis of close to 200 DIPGs and high-grade gliomas. We identified that nearly 10% of all DIPGs have increased expression of the fork head domain transcription factor FOXR2. We hypothesize that FOXR2 accelerates gliomagenesis in histone mutant DIPGs and represents a previously unexplored therapeutic target. METHODS To determine whether FOXR2 is sufficient to mediate gliomagenesis, we applied an integrative genomics approach using both in vitro and in vivo DIPG models: mouse neural stem cell models expressing FOXR2, in vivo mouse models using in utero brainstem electroporation, patient-derived DIPG cell lines, and RNA sequencing analysis of human and mouse tumors expressing FOXR2. RESULTS Our data shows that FOXR2 indeed is an oncogene that rapidly accelerates gliomagenesis using an in vivo brainstem in utero electroporation model of DIPG. In human tumors, increased FOXR2 expression is mutually exclusive with MYC amplification suggesting functional redundancy. In vivo, FOXR2 results in large brainstem gliomas and rapid neurologic decline of animals. Transcriptional profiling of these tumors demonstrates activation of MYC signaling pathways. In vitro, we have further identified patient-derived cell lines with increased expression of FOXR2. CONCLUSION FOXR2 is sufficient to enhance gliomagenesis and represents a previously understudied therapeutic target for patients with the devastating disease DIPG.

scholarly journals Growing neuronal islands on multi-electrode arrays using an Accurate Positioning-μCP device

2015 ◽  
Author(s):  
Robert Samhaber ◽  
Manuel Schottdorf ◽  
Ahmed El Hady ◽  
Kai Broeking ◽  
Andreas Daus ◽  
...  
Keyword(s):  
Cell Culture ◽  
Neuronal Networks ◽  
Standard Cell ◽  
Electrode Arrays ◽  
Unit Recording ◽  
Contact Printing ◽  
Novel Approach ◽  
Custom Made ◽  
Typical Cell

Multi-electrode arrays (MEAs) allow non-invasive multi-unit recording in-vitro from cultured neuronal networks. For sufficient neuronal growth and adhesion on such MEAs, substrate preparation is required. Plating of dissociated neurons on a uniformly prepared MEA's surface results in the formation of spatially extended random networks with substantial inter-sample variability. Such cultures are not optimally suited to study the relationship between defined structure and dynamics in neuronal networks. To overcome these shortcomings, neurons can be cultured with pre-defined topology by spatially structured surface modification. Spatially structuring a MEA surface accurately and reproducibly with the equipment of a typical cell-culture laboratory is challenging. In this paper, we present a novel approach utilizing micro-contact printing (μCP) combined with a custom-made device to accurately position patterns on MEAs with high precision. We call this technique AP-μCP (accurate positioning micro-contact printing). Other approaches presented in the literature using μCP for patterning either relied on facilities or techniques not readily available in a standard cell culture laboratory, or they did not specify means of precise pattern positioning. Here we present a relatively simple device for reproducible and precise patterning in a standard cell-culture laboratory setting. The patterned neuronal islands on MEAs provide a basis for high throughput electrophysiology to study the dynamics of single neurons and neuronal networks.

scholarly journals Using Next Generation Sequencing to Study the Genetic Diversity of Candidate Live Attenuated Zika Vaccines

Vaccines ◽  
2020 ◽  
Vol 8 (2) ◽  
pp. 161
Author(s):  
Natalie D. Collins ◽  
Chao Shan ◽  
Bruno T.D. Nunes ◽  
Steven G. Widen ◽  
Pei-Yong Shi ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Next Generation Sequencing ◽  
Rna Virus ◽  
Nonstructural Protein ◽  
Sequencing Analysis ◽  
Next Generation ◽  
Single Nucleotide Variants ◽  
Live Attenuated Vaccine ◽  
Generation Sequencing

Zika virus (ZIKV) is a mosquito-transmitted positive-sense RNA virus in the family Flaviviridae. Candidate live-attenuated vaccine (LAV) viruses with engineered deletions in the 3’ untranslated region (UTR) provide immunity and protection in animal models of ZIKV infection, and phenotypic studies show that LAVs retain protective abilities following in vitro passage. The present study investigated the genetic diversity of wild-type (WT) parent ZIKV and its candidate LAVs using next generation sequencing analysis of five sequential in vitro passages. The results show that genomic entropy of WT ZIKV steadily increases during in vitro passage, whereas that of LAVs also increased by passage number five but was variable throughout passaging. Additionally, clusters of single nucleotide variants (SNVs) were found to be present in the pre-membrane/membrane (prM), envelope (E), nonstructural protein NS1 (NS1), and other nonstructural protein genes, depending on the specific deletion, whereas in the parent WT ZIKV, they are more abundant in prM and NS1. Ultimately, both the parental WT and LAV derivatives increase in genetic diversity, with evidence of adaptation following passage.

In vitro cytocompatibility testing of oxidative degradation products

2021 ◽  
pp. 088391152110031
Author(s):  
Scott M Herting ◽  
Mary Beth B Monroe ◽  
Andrew C Weems ◽  
Sam T Briggs ◽  
Grace K Fletcher ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Cell Culture ◽  
Culture Media ◽  
Degradation Products ◽  
Oxidative Degradation ◽  
Cell Culture Media ◽  
Novel Approach ◽  
Gene And Protein Expression

Implantable medical devices must undergo thorough evaluation to ensure safety and efficacy before use in humans. If a device is designed to degrade, it is critical to understand the rate of degradation and the degradation products that will be released. Oxidative degradation is typically modeled in vitro by immersing materials or devices in hydrogen peroxide, which can limit further analysis of degradation products in many cases. Here we demonstrate a novel approach for testing the cytocompatibility of degradation products for oxidatively-degradable biomaterials where the materials are exposed to hydrogen peroxide, and then catalase enzyme is used to convert the hydrogen peroxide to water and oxygen so that the resulting aqueous solution can be added to cell culture media. To validate our results, expected degradation products are also synthesized then added to cell culture media. We used these methods to evaluate the cytocompatibility of degradation products from an oxidatively-degradable shape memory polyurethane designed in our lab and found that the degradation of these polymers is unlikely to cause a cytotoxic response in vivo based on the guidance provided by ISO 10993-5. These methods may also be applicable to other biocompatibility tests such as tests for mutagenicity or systemic toxicity, and evaluations of cell proliferation, migration, or gene and protein expression.

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