scholarly journals Single-stranded positive-sense RNA viruses generated in days using infectious subgenomic amplicons

2014 ◽  
Vol 95 (11) ◽  
pp. 2462-2467 ◽  
Author(s):  
Fabien Aubry ◽  
Antoine Nougairède ◽  
Lauriane de Fabritus ◽  
Gilles Querat ◽  
Ernest A. Gould ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Rna Viruses ◽  
Simple Procedure ◽  
Genetically Modified ◽  
Genetic Material ◽  
Biological Properties ◽  
Wild Type ◽  
Viral Genomes ◽  
Positive Sense

Reverse genetics is a key methodology for producing genetically modified RNA viruses and deciphering cellular and viral biological properties, but methods based on the preparation of plasmid-based complete viral genomes are laborious and unpredictable. Here, both wild-type and genetically modified infectious RNA viruses were generated in days using the newly described ISA (infectious-subgenomic-amplicons) method. This new versatile and simple procedure may enhance our capacity to obtain infectious RNA viruses from PCR-amplified genetic material.

scholarly journals An inability to maintain the ribonucleoprotein genomic structure is responsible for host detection of negative-sense RNA viruses

2020 ◽  
Author(s):  
Daniel Blanco-Melo ◽  
Benjamin E. Nilsson-Payant ◽  
Skyler Uhl ◽  
Beatriz Escudero-Pèrez ◽  
Silke Olschewski ◽  
...  
Keyword(s):  
Rna Viruses ◽  
Genomic Structure ◽  
Genetic Material ◽  
List Type ◽  
Human Pathogens ◽  
Viral Genomes ◽  
Optimal Drug ◽  
Host Detection ◽  
Increasing Demand ◽  
Negative Sense

SUMMARYCellular biology has a uniformity not shared amongst viruses. This is perhaps best exemplified by negative-sense RNA viruses that encode their genetic material as a ribonucleoprotein complex composed of genome, RNA-dependent RNA polymerase, and the nucleoprotein. Here we demonstrate that limiting nucleoprotein availability not only universally culminates in a replicative catastrophe for negative-sense RNA viruses, but it results in the production of aberrant genomic material and induction of the interferon-based host defenses. This dynamic illustrates the tremendous stress imposed on negative-sense RNA viruses during replication as genomic products accumulate in an environment that requires an increasing demand on nucleoprotein availability. We show that limiting NP by RNA interference or drug targeting blocks replication and primes neighboring cells through the production of interferon. Together, these results demonstrate that the nucleoprotein represents the Achilles heel of the entire phylum of negative-sense RNA viruses. Here we establish this principle for a diverse collection of human pathogens and propose that the nucleoprotein should be a primary target for the development of future antiviral drugs.HIGHLIGHTSLimited levels of NP result in production of defective viral genomesDefective viral genomes and viral antagonists are key determinants of the host antiviral responseThe host response and defective viral genome generation further exasperate NP availabilityNP is an optimal drug target for the whole phylum of negative-sense RNA viruses

scholarly journals Bottleneck, Isolate, Amplify, Select (BIAS) as a Mechanistic Framework for Intracellular Population Dynamics of Positive Sense RNA Viruses

2020 ◽  
Author(s):  
Feng Qu ◽  
Limin Zheng ◽  
Shaoyan Zhang ◽  
Rong Sun ◽  
Jason Slot ◽  
...  
Keyword(s):  
Rna Viruses ◽  
Virus Disease ◽  
Dependent Manner ◽  
Population Bottlenecks ◽  
Concentration Dependent Manner ◽  
Beneficial Mutations ◽  
Phenotypic Differences ◽  
Viral Genomes ◽  
Positive Sense ◽  
Infected Cells

Many positive sense RNA viruses, especially those infecting plants, are known to experience stringent, stochastic population bottlenecks inside the cells they invade, but exactly how and why these populations become bottlenecked are unclear. A model proposed ten years ago advocates that such bottlenecks are evolutionarily favored because they cause the isolation of individual viral variants in separate cells. Such isolation in turn allows the viral variants to manifest the phenotypic differences they encode. Recently published observations lend mechanistic support to this model, and prompt us to refine the model with novel molecular details. The refined model, designated Bottleneck, Isolate, Amplify, Select (BIAS), postulates that these viruses impose population bottlenecks on themselves by encoding bottleneck-enforcing proteins (BNEPs) that function in a concentration-dependent manner. In cells simultaneously invaded by numerous virions of the same virus, BNEPs reach the bottleneck-ready concentration sufficiently early to arrest nearly all internalized viral genomes. As a result, very few (as few as one) viral genomes stochastically escape to initiate reproduction. Repetition of this process in successively infected cells isolate viral genomes with different mutations in separate cells. This isolation prevents mutant viruses encoding defective viral proteins from hitchhiking on sister genome-encoded products, leading to the swift purging of such mutants. Importantly, genome isolation also ensures viral genomes harboring beneficial mutations accrue the cognate benefit exclusively to themselves, leading to the fixation of such beneficial mutations. Further interrogation of the BIAS hypothesis promises to deepen our understanding of virus evolution, and inspire new solutions to virus disease mitigation.

scholarly journals Infectious Subgenomic Amplicons method to expedite reverse genetics of SARS-CoV-2 and other coronaviruses

2020 ◽  
Author(s):  
Julien Mélade ◽  
Géraldine Piorkowski ◽  
Franck Touret ◽  
Toscane Fourié ◽  
Bruno Coutard ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Rna Viruses ◽  
Virus Genome ◽  
Biological Properties ◽  
Entire Genome ◽  
Wild Fauna ◽  
Viral Particles ◽  
Human Coronaviruses ◽  
Genotypic Characteristics ◽  
Cdna Fragments

Abstract There is a need for simple reverse genetics methods to decipher the biological properties of animal and human coronaviruses. Here, we attempted to rescue the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the Feline enteric coronavirus (FeCoV) using the rapid “Infectious-Subgenomic Amplicons” (ISA) method. For each virus, transfection into permissive cells of eight overlapping subgenomic cDNA fragments covering the entire genome allowed reconstruction of the complete virus genome and generated infectious viral particles. Rescued viruses replicated the phenotypic and genotypic characteristics of the original isolates. In conclusion, the ISA method, which had been previously used for RNA viruses with shorter genomes (e.g., flaviviruses, alphaviruses and enteroviruses) can be used to rescue viruses with substantially longer genomes and usefully complements pre-existing methods for reverse genetics of coronaviruses. Its extreme simplicity and versatility makes it a strong option to decipher the biological properties of coronaviruses circulating in human, domestic or wild fauna populations.

scholarly journals Application of Reverse Genetics in Functional Genomics of Potyvirus

Viruses ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 803
Author(s):  
Maathavi Kannan ◽  
Zamri Zainal ◽  
Ismanizan Ismail ◽  
Syarul Nataqain Baharum ◽  
Hamidun Bunawan
Keyword(s):  
Protein Function ◽  
Reverse Genetics ◽  
Agronomic Traits ◽  
Reverse Genetic ◽  
Genetic Technology ◽  
Wild Type ◽  
Reporter System ◽  
Viral Genomes ◽  
Host Interaction ◽  
Genetic Techniques

Numerous potyvirus studies, including virus biology, transmission, viral protein function, as well as virus–host interaction, have greatly benefited from the utilization of reverse genetic techniques. Reverse genetics of RNA viruses refers to the manipulation of viral genomes, transfection of the modified cDNAs into cells, and the production of live infectious progenies, either wild-type or mutated. Reverse genetic technology provides an opportunity of developing potyviruses into vectors for improving agronomic traits in plants, as a reporter system for tracking virus infection in hosts or a production system for target proteins. Therefore, this review provides an overview on the breakthroughs achieved in potyvirus research through the implementation of reverse genetic systems.

scholarly journals Influenza A Virus Defective Viral Genomes Are Inefficiently Packaged into Virions Relative to Wild-Type Genomic RNAs

mBio ◽  
2021 ◽  
Author(s):  
Fadi G. Alnaji ◽  
William K. Reiser ◽  
Joel Rivera-Cardona ◽  
Aartjan J. W. te Velthuis ◽  
Christopher B. Brooke
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Rna Viruses ◽  
Influenza Viruses ◽  
Clinical Infection ◽  
Wild Type ◽  
Genomic Rnas ◽  
Defective Interfering Particles ◽  
Viral Genomes

Defective interfering particles (DIPs) are commonly produced by RNA viruses and have been implicated in modulating clinical infection outcomes; hence, there is increasing interest in the potential of DIPs as antiviral therapeutics. For influenza viruses, DIPs are formed by the packaging of genomic RNAs harboring internal deletions.

scholarly journals Infectious Subgenomic Amplicons method to expedite reverse genetics of SARS-CoV-2 and other coronaviruses

2021 ◽  
Author(s):  
Julien Mélade ◽  
Géraldine Piorkowski ◽  
Franck Touret ◽  
Toscane Fourié ◽  
Bruno Coutard ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Rna Viruses ◽  
Virus Genome ◽  
Biological Properties ◽  
Entire Genome ◽  
Wild Fauna ◽  
Viral Particles ◽  
Human Coronaviruses ◽  
Genotypic Characteristics ◽  
Cdna Fragments

Abstract There is a need for simple reverse genetics methods to decipher the biological properties of animal and human coronaviruses. Here, we attempted to rescue the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the Feline enteric coronavirus (FeCoV) using the rapid “Infectious-Subgenomic Amplicons” (ISA) method. For each virus, transfection into permissive cells of eight overlapping subgenomic cDNA fragments covering the entire genome allowed reconstruction of the complete virus genome and generated infectious viral particles. Rescued viruses replicated the phenotypic and genotypic characteristics of the original isolates. In conclusion, the ISA method, which had been previously used for RNA viruses with shorter genomes (e.g., flaviviruses, alphaviruses and enteroviruses) can be used to rescue viruses with substantially longer genomes and usefully complements pre-existing methods for reverse genetics of coronaviruses. Its extreme simplicity and versatility makes it a strong option to decipher the biological properties of coronaviruses circulating in human, domestic or wild fauna populations.

scholarly journals Bottleneck, Isolate, Amplify, Select (BIAS) as a mechanistic framework for intracellular population dynamics of positive-sense RNA viruses

2020 ◽  
Vol 6 (2) ◽  
Author(s):  
Feng Qu ◽  
Limin Zheng ◽  
Shaoyan Zhang ◽  
Rong Sun ◽  
Jason Slot ◽  
...  
Keyword(s):  
Rna Viruses ◽  
Virus Disease ◽  
Dependent Manner ◽  
Population Bottlenecks ◽  
Concentration Dependent Manner ◽  
Beneficial Mutations ◽  
Phenotypic Differences ◽  
Viral Genomes ◽  
Positive Sense ◽  
Infected Cells

Abstract Many positive-sense RNA viruses, especially those infecting plants, are known to experience stringent, stochastic population bottlenecks inside the cells they invade, but exactly how and why these populations become bottlenecked are unclear. A model proposed ten years ago advocates that such bottlenecks are evolutionarily favored because they cause the isolation of individual viral variants in separate cells. Such isolation in turn allows the viral variants to manifest the phenotypic differences they encode. Recently published observations lend mechanistic support to this model and prompt us to refine the model with novel molecular details. The refined model, designated Bottleneck, Isolate, Amplify, Select (BIAS), postulates that these viruses impose population bottlenecks on themselves by encoding bottleneck-enforcing proteins (BNEPs) that function in a concentration-dependent manner. In cells simultaneously invaded by numerous virions of the same virus, BNEPs reach the bottleneck-ready concentration sufficiently early to arrest nearly all internalized viral genomes. As a result, very few (as few as one) viral genomes stochastically escape to initiate reproduction. Repetition of this process in successively infected cells isolates viral genomes with different mutations in separate cells. This isolation prevents mutant viruses encoding defective viral proteins from hitchhiking on sister genome-encoded products, leading to the swift purging of such mutants. Importantly, genome isolation also ensures viral genomes harboring beneficial mutations accrue the cognate benefit exclusively to themselves, leading to the fixation of such beneficial mutations. Further interrogation of the BIAS hypothesis promises to deepen our understanding of virus evolution and inspire new solutions to virus disease mitigation.

Arabidopsis 4-COUMAROYL-COA LIGASE 8 contributes to the biosynthesis of the benzenoid ring of coenzyme Q in peroxisomes

2019 ◽  
Vol 476 (22) ◽  
pp. 3521-3532
Author(s):  
Eric Soubeyrand ◽  
Megan Kelly ◽  
Shea A. Keene ◽  
Ann C. Bernert ◽  
Scott Latimer ◽  
...  
Keyword(s):  
Oxidative Metabolism ◽  
Time Course ◽  
Reverse Genetics ◽  
De Novo ◽  
Coenzyme Q ◽  
Control Level ◽  
Wild Type ◽  
Fluorescent Reporters ◽  
Coa Ligase ◽  
Peroxisomal Targeting

Plants have evolved the ability to derive the benzenoid moiety of the respiratory cofactor and antioxidant, ubiquinone (coenzyme Q), either from the β-oxidative metabolism of p-coumarate or from the peroxidative cleavage of kaempferol. Here, isotopic feeding assays, gene co-expression analysis and reverse genetics identified Arabidopsis 4-COUMARATE-COA LIGASE 8 (4-CL8; At5g38120) as a contributor to the β-oxidation of p-coumarate for ubiquinone biosynthesis. The enzyme is part of the same clade (V) of acyl-activating enzymes than At4g19010, a p-coumarate CoA ligase known to play a central role in the conversion of p-coumarate into 4-hydroxybenzoate. A 4-cl8 T-DNA knockout displayed a 20% decrease in ubiquinone content compared with wild-type plants, while 4-CL8 overexpression boosted ubiquinone content up to 150% of the control level. Similarly, the isotopic enrichment of ubiquinone's ring was decreased by 28% in the 4-cl8 knockout as compared with wild-type controls when Phe-[Ring-13C6] was fed to the plants. This metabolic blockage could be bypassed via the exogenous supply of 4-hydroxybenzoate, the product of p-coumarate β-oxidation. Arabidopsis 4-CL8 displays a canonical peroxisomal targeting sequence type 1, and confocal microscopy experiments using fused fluorescent reporters demonstrated that this enzyme is imported into peroxisomes. Time course feeding assays using Phe-[Ring-13C6] in a series of Arabidopsis single and double knockouts blocked in the β-oxidative metabolism of p-coumarate (4-cl8; at4g19010; at4g19010 × 4-cl8), flavonol biosynthesis (flavanone-3-hydroxylase), or both (at4g19010 × flavanone-3-hydroxylase) indicated that continuous high light treatments (500 µE m−2 s−1; 24 h) markedly stimulated the de novo biosynthesis of ubiquinone independently of kaempferol catabolism.

COVID 19, All a Radiologist Needs to Know: A Narrative Review

2020 ◽  
Vol 16 ◽  
Author(s):  
Farzaneh Shobeirian
Keyword(s):  
Computed Tomography ◽  
Rna Viruses ◽  
Imaging Findings ◽  
Protective Measures ◽  
Infection Transmission ◽  
The World ◽  
Positive Sense ◽  
Global Concern ◽  
Interlobular Septal Thickening ◽  
Radiologic Characteristics

Background: Coronaviruses are non-segmented enveloped positive-sense single-strand RNA viruses, and COVID-19 is the seventh known coronavirus, infecting humans. Objective: As the COVID-19 continued to spread the world wildly, every radiologist or clinician needs to be familiar with its imaging findings. Methods: In this study, we reviewed available studies to provide a comprehensive statement on COVID-19 imaging findings. Results: Ground-glass opacities, linear opacities, interlobular septal thickening, consolidation, and Crazy-paving patterns are the most frequent findings in computed tomography (CT) of lungs in patients with COVID-19 pneumonia, which are mostly bilateral, multifocal, and peripheral. Staff needs to follow some rules to reduce infection transmission. Conclusion: COVID-19 pneumonia is a new global concern which has many unknown features. In this article, the radiologic characteristics of COVID-19 pneumonia are discussed. We also discussed appropriate protective measures that the radiology team should be aware of.

Inhibitions of Positive-Sense (ss) RNA Viruses RNA-Dependent RNA Polymerases

2009 ◽  
Vol 5 (4) ◽  
pp. 234-244 ◽  
Author(s):  
Kajohn Boonrod ◽  
Gabriele Krczal
Keyword(s):  
Rna Viruses ◽  
Rna Polymerases ◽  
Positive Sense
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