scholarly journals A simple reverse genetics method to generate recombinant coronaviruses 

2021 ◽  
Author(s):  
Julien Mélade ◽  
Géraldine Piorkowski ◽  
Franck Touret ◽  
Toscane Fourié ◽  
Jean-Sélim Driouich ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Biological Properties ◽  
Viral Pathogens ◽  
Simple Method ◽  
Fluorescent Reporter ◽  
Recombinant Viruses ◽  
Large Size ◽  
Therapeutic Measures ◽  
Reporter Strains

Abstract Engineering recombinant viruses is capital for deciphering the biology of emerging viral pathogens such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the large size of coronaviruses genome makes reverse genetics methods challenging.Here we describe a simple method based on “infectious subgenomic amplicons” (ISA) technology to generate recombinant infectious coronaviruses with no need for reconstructing a full genomic cDNA. The method was applied to the SARS-CoV-2 and the feline enteric coronavirus, and allowed to rescue wild-type viruses with biological characteristics closely similar to original strains. Mutations and fluorescent red reporter gene were rapidly incorporated into the SARS-CoV-2 genome allowing the generation of a genomic variant and a fluorescent reporter strains which were studied during in vivo experiments, serological diagnosis and antiviral assays. The swiftness and simplicity of the ISA method has the potential to facilitate the advance of coronavirus reverse genetics studies and to explore biological properties of SARS-CoV-2 variants or accelerating the development of therapeutic measures.

scholarly journals Expression of Separate Foreign Proteins from the Rotavirus NSP3 Genome Segment Using a Translational 2A Stop-Restart Element

2020 ◽  
Author(s):  
Asha A. Philip ◽  
John T. Patton
Keyword(s):  
Reverse Genetics ◽  
Fluorescent Protein ◽  
Viral Proteins ◽  
Live Cell ◽  
Nuclear Accumulation ◽  
Foreign Protein ◽  
Fluorescent Reporter ◽  
Recombinant Viruses ◽  
Full Complement ◽  
Foreign Proteins

AbstractThe segmented 18.5-kB dsRNA genome of rotavirus expresses 6 structural and 6 nonstructural proteins. We investigated the possibility of using the recently-developed plasmid-based rotavirus reverse genetics (RG) system to generate recombinant viruses that express a separate foreign protein, in addition to the 12 viral proteins. To address this, we replaced the NSP3 open-reading-frame (ORF) of the segment 7 (pT7/NSP3) transcription vector used in the RG system with an ORF encoding NSP3 fused to a fluorescent reporter protein (i.e., UnaG, mRuby, mKate, or TagBFP). Inserted at the fusion junction was a teschovirus 2A-like self-cleaving element designed to direct the separate expression of NSP3 and the fluorescent protein. Recombinant rotaviruses made with the modified pT7/NSP3 vectors were well growing, generally genetically stable, and expressed NSP3 and a separate fluorescent protein detectable by live cell imaging. NSP3 made by the recombinant viruses was functional, inducing nuclear accumulation of cellular poly(A)-binding protein. Further modification of the NSP3 ORF showed that it was possible to generate recombinant viruses encoding 2 foreign proteins (mRuby and UnaG) in addition to NSP3. Our results demonstrate that, through modification of segment 7, the rotavirus genome can be increased in size to at least 19.8 kB and can be used to produce recombinant rotaviruses expressing a full complement of viral proteins and multiple foreign proteins. The generation of recombinant rotaviruses expressing fluorescent proteins will be valuable for the study of rotavirus replication and pathogenesis by live cell imagining and suggest that rotaviruses may prove useful as expression vectors.ImportanceRotaviruses are a major cause of severe gastroenteritis in infants and young children. Recently, a highly efficient reverse genetics system was developed that allows genetic manipulation of the rotavirus segmented double-stranded RNA genome. Using the reverse genetics system, we show that it is possible to modify one of the rotavirus genome segments (segment 7) such that virus gains the capacity to express a separate foreign protein, in addition to the full complement of viral proteins. Through this approach, we have generated wildtype-like rotaviruses that express various fluorescent reporter proteins, including UnaG (green), mRuby (far red), mKate (red), and TagBFP (blue). Such strains will be of value in probing rotavirus biology and pathogenesis by live-cell imagining techniques. Notably, our work indicates that the rotavirus genome is remarkably flexible and able to accommodate significant amounts of foreign RNA sequence, raising the possibility of using the virus as vaccine expression vector.

scholarly journals Expression of Separate Heterologous Proteins from the Rotavirus NSP3 Genome Segment Using a Translational 2A Stop-Restart Element

2020 ◽  
Vol 94 (18) ◽  
Author(s):  
Asha A. Philip ◽  
John T. Patton
Keyword(s):  
Reverse Genetics ◽  
Heterologous Protein ◽  
Fluorescent Protein ◽  
Viral Proteins ◽  
Live Cell ◽  
Nuclear Accumulation ◽  
Heterologous Proteins ◽  
Fluorescent Reporter ◽  
Recombinant Viruses ◽  
Full Complement

ABSTRACT The segmented 18.5-kbp dsRNA genome of rotavirus expresses 6 structural and 6 nonstructural proteins. We investigated the possibility of using the recently developed plasmid-based rotavirus reverse genetics (RG) system to generate recombinant viruses that express a separate heterologous protein in addition to the 12 viral proteins. To address this, we replaced the NSP3 open reading frame (ORF) of the segment 7 (pT7/NSP3) transcription vector used in the RG system with an ORF encoding NSP3 fused to a fluorescent reporter protein (i.e., UnaG, mRuby, mKate, or TagBFP). Inserted at the fusion junction was a teschovirus translational 2A stop-restart element designed to direct the separate expression of NSP3 and the fluorescent protein. Recombinant rotaviruses made with the modified pT7/NSP3 vectors were well growing and generally genetically stable, and they expressed NSP3 and a separate fluorescent protein detectable by live cell imaging. NSP3 made by the recombinant viruses was functional, inducing nuclear accumulation of cellular poly(A)-binding protein. Further modification of the NSP3 ORF showed that it was possible to generate recombinant viruses encoding 2 heterologous proteins (mRuby and UnaG) in addition to NSP3. Our results demonstrate that, through modification of segment 7, the rotavirus genome can be increased in size to at least 19.8 kbp and can be used to produce recombinant rotaviruses expressing a full complement of viral proteins and multiple heterologous proteins. The generation of recombinant rotaviruses expressing fluorescent proteins will be valuable for the study of rotavirus replication and pathogenesis by live cell imagining and suggest that rotaviruses will prove useful as expression vectors. IMPORTANCE Rotaviruses are a major cause of severe gastroenteritis in infants and young children. Recently, a highly efficient reverse genetics system was developed that allows genetic manipulation of the rotavirus segmented double-stranded RNA genome. Using the reverse genetics system, we show that it is possible to modify one of the rotavirus genome segments (segment 7) such that virus gains the capacity to express a separate heterologous protein in addition to the full complement of viral proteins. Through this approach, we have generated wild-type-like rotaviruses that express various fluorescent reporter proteins, including UnaG (green), mRuby (far red), mKate (red), and TagBFP (blue). Such strains will be of value in probing rotavirus biology and pathogenesis by live cell imagining techniques. Notably, our work indicates that the rotavirus genome is remarkably flexible and able to accommodate significant amounts of heterologous RNA sequence, raising the possibility of using the virus as a vaccine expression vector.

scholarly journals In vivo fate mapping identifies pre-TCRα expression as an intra- and extrathymic, but not prethymic, marker of T lymphopoiesis

2013 ◽  
Vol 210 (4) ◽  
pp. 699-714 ◽  
Author(s):  
Hervé Luche ◽  
Tata Nageswara Rao ◽  
Suresh Kumar ◽  
Alpaslan Tasdogan ◽  
Franziska Beckel ◽  
...  
Keyword(s):  
T Cell ◽  
Molecular Marker ◽  
Γδ T Cells ◽  
Cell Receptor ◽  
Lineage Tracing ◽  
Fluorescent Reporter ◽  
History Of ◽  
Reporter Strains ◽  
Consistent Labeling

Expression of the pre–T cell receptor α (pTα) gene has been exploited in previous studies as a molecular marker to identify tiny cell populations in bone marrow (BM) and blood that were suggested to contain physiologically relevant thymus settling progenitors (TSPs). But to what extent these cells genuinely contribute to thymopoiesis has remained obscure. We have generated a novel pTαiCre knockin mouse line and performed lineage-tracing experiments to precisely quantitate the contribution of pTα-expressing progenitors to distinct differentiation pathways and to the genealogy of mature hematopoietic cells under physiological in vivo conditions. Using these mice in combination with fluorescent reporter strains, we observe highly consistent labeling patterns that identify pTα expression as a faithful molecular marker of T lineage commitment. Specifically, the fate of pTα-expressing progenitors was found to include all αβ and most γδ T cells but, in contrast to previous assumptions, to exclude B, NK, and thymic dendritic cells. Although we could detect small numbers of T cell progenitors with a history of pTα expression in BM and blood, our data clearly exclude these populations as physiologically important precursors of thymopoiesis and indicate that they instead belong to a pathway of T cell maturation previously defined as extrathymic.

scholarly journals Development and Characterization of a Reverse Genetics System for a Human-Derived Severe Fever With Thrombocytopenia Syndrome Virus Isolate From South Korea

2021 ◽  
Vol 12 ◽  
Author(s):  
Seok-Min Yun ◽  
Tae-Young Lee ◽  
Hee-Young Lim ◽  
Jungsang Ryou ◽  
Joo-Yeon Lee ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Reverse Genetics ◽  
Biological Properties ◽  
Encephalomyocarditis Virus ◽  
Entry Site ◽  
T7 Promoter ◽  
Helper Plasmids ◽  
Severe Fever

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging, tick-borne Bandavirus that causes lethal disease in humans. As there are no licensed vaccines and therapeutics for SFTSV, there is an urgent need to develop countermeasures against it. In this respect, a reverse genetics (RG) system is a powerful tool to help achieve this goal. Herein, we established a T7 RNA polymerase-driven RG system to rescue infectious clones of a Korean SFTSV human isolate entirely from complementary DNA (cDNA). To establish this system, we cloned cDNAs encoding the three antigenomic segments into transcription vectors, with each segment transcribed under the control of the T7 promoter and the hepatitis delta virus ribozyme (HdvRz) sequences. We also constructed two helper plasmids expressing the nucleoprotein (NP) or viral RNA-dependent RNA polymerase (RdRp) under the control of the T7 promoter and the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES). After co-transfection into BHK/T7-9 cells with three transcription and two helper plasmids, then passaging in Vero E6 or Huh-7 cells, we confirmed efficient rescue of the recombinant SFTSV. By evaluating the in vitro and in vivo virological properties of the parental and rescued SFTSVs, we show that the rescued virus exhibited biological properties similar to those of the parental virus. This system will be useful for identifying molecular viral determinants of SFTSV infection and pathogenesis and for facilitating the development of vaccine and antiviral approaches.

scholarly journals Mapping nonapoptotic caspase activity with a transgenic reporter in mice

2017 ◽  
Author(s):  
P. J. Nicholls ◽  
Thomas F. Pack ◽  
Nikhil M. Urs ◽  
Sunil Kumar ◽  
Gabor Turu ◽  
...  
Keyword(s):  
Cell Death ◽  
Neural Plasticity ◽  
Structural Changes ◽  
Caspase 3 ◽  
Neuronal Firing ◽  
Caspase Activity ◽  
Live Animal ◽  
Simple Method ◽  
Fluorescent Reporter

AbstractThe protease caspase-3 is a key mediator of apoptotic programmed cell death. But weak or transient caspase activity can contribute to neuronal differentiation, axonal pathfinding, and synaptic long-term depression. Despite the importance of sublethal, or nonapoptotic, caspase activity in neurodevelopment and neural plasticity, there has been no simple method for mapping and quantifying nonapoptotic caspase activity in rodent brains. We therefore generated a transgenic mouse expressing a highly sensitive and specific fluorescent reporter of caspase activity, with peak signal localized to the nucleus. Surprisingly, nonapoptotic caspase activity was prevalent in healthy adult brains and influenced neuronal functional connectivity. We quantified the relationship between caspase activity and cell firing and morphology. We also notably observed a sex-specific persistent elevation in amygdalar caspase activity in females after restraint stress. This simple in vivo caspase activity reporter will facilitate systems-level studies of apoptotic and nonapoptotic phenomena in behavioral and pathological models.Significance StatementCaspase-3 is an enzyme that can cause cell death when highly active but can also perform important cellular functions, such as maturation and structural changes, when only weakly or transiently active. Despite the importance of this nonlethal type of caspase activity, there is no straightforward method to measure it in live rodents. We therefore developed mice that have a fluorescent reporter that is sensitive enough to detect nonlethal caspase activity. Surprisingly, we found that weak caspase activity is widespread in healthy brains and influences the synchrony of neuronal firing across different brain regions. We also observed increased caspase activity in female mice after severe stress. This simple, live-animal caspase activity reporter can subserve multiple applications in behavior and pathology research.

scholarly journals Single cell analysis of M. tuberculosis phenotype and macrophage lineages in the infected lung

2021 ◽  
Vol 218 (9) ◽  
Author(s):  
Davide Pisu ◽  
Lu Huang ◽  
Vipin Narang ◽  
Monique Theriault ◽  
Gabrielle Lê-Bury ◽  
...  
Keyword(s):  
Single Cell Analysis ◽  
Conceptual Approach ◽  
Fluorescent Reporter ◽  
Surface Marker Expression ◽  
Novel Approach ◽  
Bacterial Fitness ◽  
Reporter Strains ◽  
Different Populations ◽  
Macrophage Populations

In this study, we detail a novel approach that combines bacterial fitness fluorescent reporter strains with scRNA-seq to simultaneously acquire the host transcriptome, surface marker expression, and bacterial phenotype for each infected cell. This approach facilitates the dissection of the functional heterogeneity of M. tuberculosis–infected alveolar (AMs) and interstitial macrophages (IMs) in vivo. We identify clusters of pro-inflammatory AMs associated with stressed bacteria, in addition to three different populations of IMs with heterogeneous bacterial phenotypes. Finally, we show that the main macrophage populations in the lung are epigenetically constrained in their response to infection, while inter-species comparison reveals that most AMs subsets are conserved between mice and humans. This conceptual approach is readily transferable to other infectious disease agents with the potential for an increased understanding of the roles that different host cell populations play during the course of an infection.

scholarly journals Gene 5 of the Avian Coronavirus Infectious Bronchitis Virus Is Not Essential for Replication

2005 ◽  
Vol 79 (13) ◽  
pp. 8065-8078 ◽  
Author(s):  
Rosa Casais ◽  
Marc Davies ◽  
David Cavanagh ◽  
Paul Britton
Keyword(s):  
Infectious Bronchitis Virus ◽  
Reverse Genetics ◽  
Ex Vivo ◽  
Wild Type Virus ◽  
In Ovo ◽  
Amino Acid Similarity ◽  
Recombinant Viruses ◽  
Infectious Bronchitis ◽  
Ns Gene

ABSTRACT The avian coronavirus Infectious bronchitis virus (IBV), like other coronaviruses, expresses several small nonstructural (ns) proteins in addition to those from gene 1 (replicase) and the structural proteins. These coronavirus ns genes differ both in number and in amino acid similarity between the coronavirus groups but show some concordance within a group or subgroup. The functions and requirements of the small ns gene products remain to be elucidated. With the advent of reverse genetics for coronaviruses, the first steps in elucidating their role can be investigated. We have used our reverse genetics system for IBV (R. Casais, V. Thiel, S. G. Siddell, D. Cavanagh, and P. Britton, J. Virol. 75:12359-12369, 2001) to investigate the requirement of IBV gene 5 for replication in vivo, in ovo, and ex vivo. We produced a series of recombinant viruses, with an isogenic background, in which complete expression of gene 5 products was prevented by the inactivation of gene 5 following scrambling of the transcription-associated sequence, thereby preventing the expression of IBV subgenomic mRNA 5, or scrambling either separately or together of the translation initiation codons for the two gene 5 products. As all of the recombinant viruses replicated very similarly to the wild-type virus, Beau-R, we conclude that the IBV gene 5 products are not essential for IBV replication per se and that they are accessory proteins.

The Filter Loop Technique as a Method of Measuring Platelet Aggregation in the Flowing Blood of the Rat; the Inhibitory Activity of 5-oxo-l-cyclopentene-l-heptanoic Acid (AY-16,804) on Platelet Aggregation

1973 ◽  
Vol 30 (01) ◽  
pp. 138-147 ◽  
Author(s):  
Christopher R. Muirhead
Keyword(s):  
Platelet Aggregation ◽  
Prostaglandin E1 ◽  
Suitable Model ◽  
Heptanoic Acid ◽  
Simple Method ◽  
Loop Technique ◽  
Flowing Blood ◽  
Filter Loop

SummaryThe filter loop technique which measures platelet aggregation in vivo in the flowing-blood of the rat was compared to the optical density technique of Born which is carried out in vitro with platelet rich plasma. Using these two experimental models the effect on platelet aggregation of three known inhibitors sulfinpyrazone, dipyridamole and prostaglandin E1, and a novel compound 5-oxo-l-cyclopentene-l-heptanoic acid (AY-16, 804) was determined.The effects on platelet aggregation of the known inhibitors were consistent with information in the literature. Prostaglandin E1 was the most potent inhibitor in both techniques; sulfinpyrazone inhibited aggregation in both models but was less potent than prostaglandin E1. AY-16, 804 exhibited activity in vitro and in vivo similar to that of sulfinpyrazone. Dipyridamole did not inhibit platelet aggregation in vivo and did not inhibit aggregation in vitro in concentrations at which it remained soluble.The filter loop technique is a suitable model for measuring platelet aggregation in the flowing blood of the rat. It is a relatively simple method of determining aggregation and easily adapted to other species.

Nanodrugs as a new approach in therapy of cardiovascular diseases and cancer with tumor-associated angiogenesis

2020 ◽  
Vol 28 ◽  
Author(s):  
Justyna Hajtuch ◽  
Karolina Niska ◽  
Iwona Inkielewicz-Stepniak
Keyword(s):  
Cardiovascular Disease ◽  
Cardiovascular Diseases ◽  
Controlled Drug Release ◽  
Biological Properties ◽  
Comprehensive Information ◽  
Conventional Drug ◽  
Key Factor ◽  
Functionalized Materials

Background: Cancer along with cardiovascular diseases are globally defined as leading causes of death. Importantly, some risk factors are common to these diseases. The process of angiogenesis and platelets aggregation are observed in cancer development and progression. In recent years, studies have been conducted on nanodrugs in these diseases that have provided important information on the biological and physicochemical properties of nanoparticles. Their attractive features are that they are made of biocompatible, well-characterized and easily functionalized materials. Unlike conventional drug delivery, sustained and controlled drug release can be obtained by using nanomaterials. Methods: In this article, we review the latest research to provide comprehensive information on nanoparticle-based drugs for the treatment of cancer, cardiovascular disease associated with abnormal haemostasis, and the inhibition of tumorassociated angiogenesis. Results: The results of the analysis of data based on nanoparticles with drugs confirm their improved pharmaceutical and biological properties, which gives promising antiplatelet, anticoagulant and antiangiogenic effects. Moreover, the review included in vitro, in vivo research and presented nanodrugs with chemotherapeutics approved by Food and Drug Administration. Conclusion: By the optimization of nanoparticles size and surface properties, nanotechnology are able to deliver drugs with enhanced bioavailability in treatment of cardiovascular disease, cancer and inhibition of cancer-related angiogenesis. Thus, nanotechnology can improve the therapeutic efficacy of the drug, but there is a need for a better understanding of the nanodrugs interaction in the human body, because this is a key factor in the success of potential nanotherapeutics.

Tamarix articulata (T. articulata) - An Important Halophytic Medicinal Plant with Potential Pharmacological Properties

2019 ◽  
Vol 20 (4) ◽  
pp. 285-292 ◽  
Author(s):  
Abdullah M. Alnuqaydan ◽  
Bilal Rah
Keyword(s):  
Medicinal Plant ◽  
Biological Activities ◽  
Wide Spectrum ◽  
Biological Properties ◽  
In Vivo Model ◽  
Drug Candidate ◽  
Phytochemical Analysis ◽  
Pharmacological Properties

Background:Tamarix Articulata (T. articulata), commonly known as Tamarisk or Athal in Arabic region, belongs to the Tamaricaece species. It is an important halophytic medicinal plant and a good source of polyphenolic phytochemical(s). In traditional medicines, T. articulata extract is commonly used, either singly or in combination with other plant extracts against different ailments since ancient times.Methods:Electronic database survey via Pubmed, Google Scholar, Researchgate, Scopus and Science Direct were used to review the scientific inputs until October 2018, by searching appropriate keywords. Literature related to pharmacological activities of T. articulata, Tamarix species, phytochemical analysis of T. articulata, biological activities of T. articulata extracts. All of these terms were used to search the scientific literature associated with T. articulata; the dosage of extract, route of administration, extract type, and in-vitro and in-vivo model.Results:Numerous reports revealed that T. articulata contains a wide spectrum of phytochemical(s), which enables it to have a wide window of biological properties. Owing to the presence of high content of phytochemical compounds like polyphenolics and flavonoids, T. articulata is a potential source of antioxidant, anti-inflammatory and antiproliferative properties. In view of these pharmacological properties, T. articulata could be a potential drug candidate to treat various clinical conditions including cancer in the near future.Conclusion:In this review, the spectrum of phytochemical(s) has been summarized for their pharmacological properties and the mechanisms of action, and the possible potential therapeutic applications of this plant against various diseases discussed.

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