scholarly journals Establishment of a reverse genetics system for Schmallenberg virus, a newly emerged orthobunyavirus in Europe

2013 ◽  
Vol 94 (4) ◽  
pp. 851-859 ◽  
Author(s):  
Richard M. Elliott ◽  
Gjon Blakqori ◽  
Ingeborg C. van Knippenberg ◽  
Elina Koudriakova ◽  
Ping Li ◽  
...  
Keyword(s):  
Host Cell ◽  
Recombinant Virus ◽  
Reverse Genetics ◽  
Cell Metabolism ◽  
Schmallenberg Virus ◽  
Wild Type Virus ◽  
Cell Protein ◽  
Virus Recombinant ◽  
Rescue System

Schmallenberg virus (SBV) is a newly emerged orthobunyavirus that has caused widespread disease in cattle, sheep and goats in Europe. Like other orthobunyaviruses, SBV is characterized by a tripartite negative-sense RNA genome that encodes four structural and two non-structural proteins. This study showed that SBV has a wide in vitro host range, and that BHK-21 cells are a convenient host for both SBV propagation and assay by plaque titration. The SBV genome segments were cloned as cDNA and a three-plasmid rescue system was established to recover infectious virus. Recombinant virus behaved similarly in cell culture to authentic virus. The ORF for the non-structural NSs protein, encoded on the smallest genome segment, was disrupted by introduction of translation stop codons in the appropriate cDNA, and when this plasmid was used in reverse genetics, a recombinant virus that lacked NSs expression was recovered. This virus had reduced capacity to shut-off host-cell protein synthesis compared with the wild-type virus. In addition, the NSs-deleted virus induced interferon (IFN) in cells, indicating that, like other orthobunyaviruses, NSs functions as an IFN antagonist, most probably by globally inhibiting host-cell metabolism. The development of a robust reverse genetics system for SBV will facilitate investigation of its pathogenic mechanisms as well as the creation of attenuated strains that could be candidate vaccines.

scholarly journals Revisiting Ehrlichia ruminantium Replication Cycle Using Proteomics: The Host and the Bacterium Perspectives

2021 ◽  
Vol 9 (6) ◽  
pp. 1144
Author(s):  
Isabel Marcelino ◽  
Philippe Holzmuller ◽  
Ana Coelho ◽  
Gabriel Mazzucchelli ◽  
Bernard Fernandez ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Host Cell ◽  
Cell Metabolism ◽  
Replication Cycle ◽  
Host Cells ◽  
Ehrlichia Ruminantium ◽  
Host Interaction ◽  
Infected Cells ◽  
Related Proteins

The Rickettsiales Ehrlichia ruminantium, the causal agent of the fatal tick-borne disease Heartwater, induces severe damage to the vascular endothelium in ruminants. Nevertheless, E. ruminantium-induced pathobiology remains largely unknown. Our work paves the way for understanding this phenomenon by using quantitative proteomic analyses (2D-DIGE-MS/MS, 1DE-nanoLC-MS/MS and biotin-nanoUPLC-MS/MS) of host bovine aorta endothelial cells (BAE) during the in vitro bacterium intracellular replication cycle. We detect 265 bacterial proteins (including virulence factors), at all time-points of the E. ruminantium replication cycle, highlighting a dynamic bacterium–host interaction. We show that E. ruminantium infection modulates the expression of 433 host proteins: 98 being over-expressed, 161 under-expressed, 140 detected only in infected BAE cells and 34 exclusively detected in non-infected cells. Cystoscape integrated data analysis shows that these proteins lead to major changes in host cell immune responses, host cell metabolism and vesicle trafficking, with a clear involvement of inflammation-related proteins in this process. Our findings led to the first model of E. ruminantium infection in host cells in vitro, and we highlight potential biomarkers of E. ruminantium infection in endothelial cells (such as ROCK1, TMEM16K, Albumin and PTPN1), which may be important to further combat Heartwater, namely by developing non-antibiotic-based strategies.

scholarly journals Brucella abortusinduces a Warburg shift in host metabolism that is linked to enhanced intracellular survival of the pathogen

2017 ◽  
Author(s):  
Daniel M. Czyż ◽  
Jonathan Willett ◽  
Sean Crosson
Keyword(s):  
Lactic Acid ◽  
Host Cell ◽  
Brucella Abortus ◽  
Cell Metabolism ◽  
Lactate Production ◽  
Human Macrophage ◽  
Metabolic Shift ◽  
Intracellular Infections ◽  
Host Metabolism

ABSTRACTIntracellular bacterial pathogens exploit host cell resources to replicate and survive inside the host. Targeting these host systems is one promising approach to developing novel antimicrobials to treat intracellular infections. We show that human macrophage-like cells infected withBrucella abortusundergo a metabolic shift characterized by attenuated tricarboxylic acid cycle metabolism, reduced amino acid consumption, altered mitochondrial localization, and increased lactate production. This shift to an aerobic glycolytic state resembles the Warburg effect, a change in energy production that is well-described in cancer cells, and also occurs in activated inflammatory cells.B. abortusefficiently uses lactic acid as its sole carbon and energy source and requires the ability to metabolize lactate for normal survival in human macrophage-like cells. We demonstrate that chemical inhibitors of host glycolysis and lactate production do not affectin vitrogrowth ofB. abortusin axenic culture, but decrease its survival in the intracellular niche. Our data support a model in which infection shifts host metabolism to a Warburg-like state, andB. abortususes this change in metabolism to promote intracellular survival. Pharmacological perturbation of these features of host cell metabolism may be a useful strategy to inhibit infection by intracellular pathogens.IMPORTANCEBrucellaspp. are intracellular bacterial pathogens that cause disease in a range of mammals, including livestock. Transmission from livestock to humans is common and can lead to chronic human disease. Human macrophage-like cells infected withBrucella abortusundergo a Warburg-like metabolic shift to an aerobic glycolytic state where the host cells produce lactic acid and have reduced amino acid catabolism. We provide evidence that the pathogen can exploit this change in host metabolism to support growth and survival in the intracellular niche. Drugs that inhibit this shift in host cell metabolism inhibit intracellular replication and decrease the survival ofB. abortusin anin vitroinfection model; these drugs may be broadly useful therapeutics for intracellular infections.

scholarly journals Recovery of Infectious Virus by Transfection of In Vitro-Generated RNA from Tulane Calicivirus cDNA

2008 ◽  
Vol 82 (22) ◽  
pp. 11429-11436 ◽  
Author(s):  
Chao Wei ◽  
Tibor Farkas ◽  
Karol Sestak ◽  
Xi Jiang
Keyword(s):  
Reverse Genetics ◽  
Genomic Sequence ◽  
Rhesus Macaques ◽  
Wild Type Virus ◽  
Primate Research ◽  
Complete Genomic Sequence ◽  
Subgenomic Rna ◽  
Reading Frame ◽  
Complete Genomic

ABSTRACT Tulane virus (TV) is a newly reported calicivirus that was isolated from stool samples of captive rhesus macaques from the Tulane National Primate Research Center (TNPRC). The virus has been cultivated successfully in LLC-MK2 rhesus monkey kidney cells. Its complete genomic sequence suggests that TV represents a new genus and is evolutionarily more closely related to Norovirus than to any other genus of Caliciviridae. In this study, we demonstrated that RNA transcripts made in vitro from the full-length genomic cDNA of TV were infectious upon transfection into permissive LLC-MK2 cells. The recombinant virus exhibited plaque morphologies and growth kinetics similar to those of the wild-type virus in this cell line. Capping was required for TV RNA infectivity. Although a subgenomic RNA has been detected in TV-transfected cells, a separate subgenomic RNA transcript was not required for the initial transfection to establish the replication. Transfection of truncated RNA lacking open reading frame 2 (ORF2) and ORF3 or TV-norovirus chimeric RNA resulted in abortive replication without the production of infectious progeny viruses, indicating that both ORFs are essential for the replication of TV. A heterologous insertion at the 5′ end of the genome also hampered viral replication, suggesting that an authentic 5′ end of the genome is critical for replication. The availability of the complete genomic sequence and the reverse genetics system described herein make TV a valuable model for studying calicivirus pathogenesis and replication.

scholarly journals In Vitro: Natural Compounds (Thymol, Carvacrol, Hesperidine, And Thymoquinone) Against Sars-Cov2 Strain Isolated From Egyptian Patients

2020 ◽  
Author(s):  
Mohamed G Seadawy ◽  
Ahmed F. Gad ◽  
Mohamed Shamel ◽  
Bassem Elharty ◽  
Mostfa F. Mohamed ◽  
...  
Keyword(s):  
Host Cell ◽  
Natural Compounds ◽  
Cell Protein ◽  
Protein Targets ◽  
Plaque Reduction Assay ◽  
Main Protease ◽  
Immunodeficiency Virus ◽  
Egyptian Patients ◽  
Clinical Trail

Abstract Background: The current pandemic of the coronavirus disease-2019 (COVID-19) has badly affected our life during the year 2020. SARS-CoV-2 is the primary causative agent of the newly emerged pandemic. Natural flavonoids, Terpenoid and Thymoquinone are tested against different viral and host-cell protein targets. These natural compounds have a good history in treating Hepatitis C Virus (HCV) and Human Immunodeficiency Virus (HIV). Methods: Molecular docking combined with cytotoxicity and plaque reduction assay is used to test the natural compounds against different viral (Spike, RdRp, and Mpro) and host-cell (TMPRSS II, keap 1, and ACE2) targets. Results: The results demonstrate the binding possibility of the natural compounds (Thymol, Carvacrol, Hesperidine, and Thymoquinone) to the viral main protease (Mpro). Some of these natural compounds were approved to start clinical trail from Egypt Center for Research and Regenerative Medicine ECRRM IRB (Certificate No.IRB00012517)Conclusion: Development of an effective anti-viral for SARS-CoV-2 could help to limit the viral load. Benchmarking testing of those natural compounds against other potential antivirals for SARS-CoV-2 with alternative mechanisms of action would thus be important as soon as practicable.

scholarly journals Induced Expression of the Legionella pneumophila Gene Encoding a 20-Kilodalton Protein during Intracellular Infection

1998 ◽  
Vol 66 (1) ◽  
pp. 203-212 ◽  
Author(s):  
Yousef Abu Kwaik
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Host Cell ◽  
Legionella Pneumophila ◽  
Cell Protein ◽  
Intracellular Bacteria ◽  
Host Cell Protein ◽  
Bacterial Gene ◽  
Intracellular Infection

ABSTRACT The eukaryotic protein synthesis inhibitor cycloheximid has been used by many investigators to selectively radiolabel intracellular bacteria. Although cycloheximide has no direct effect on bacterial gene expression, there are concerns that long-term inhibition of the host cell protein synthesis may have secondary effects on bacterial gene expression. Therefore, prior to further identification and cloning of the macrophage-induced (MI) genes of Legionella pneumophila, the effects of cycloheximide on L. pneumophila-infected U937 cells were evaluated by transmission electron microscopy. Inhibition of protein synthesis of the host cell for 6 h had no major effect on the ultrastructure of the host cell, on the formation of rough endoplasmic reticulum-surrounded replicative phagosome, or on initiation of intracellular bacterial replication. In contrast, by 15 h of cycloheximide treatment, there was profound deterioration in the host cell as well as in the phagosome. To examine protein synthesis by L. pneumophila during the intracellular infection, U937 macrophage-like cells were infected with L. pneumophila, and intracellular bacteria were radiolabeled during a 2-h cycloheximide treatment or following 12 h of cycloheximide treatment. Comparison by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the protein profile of radiolabeled in vitro-grown L. pneumophila to that of intracellularly radiolabeled bacteria showed that 23 proteins were induced in response to the intracellular environment during 2 h of inhibition of host cell protein biosynthesis. Twelve MI proteins ofL. pneumophila were artifactually induced due to prolonged inhibition of the host cell protein synthesis. The gene encoding a 20-kDa MI protein was cloned by a reverse genetics technique. Sequence analysis showed that the cloned gene encoded a protein that was 80% similar to the enzyme inorganic pyrophosphatase. Studies of promoter fusion to a promoterless lacZ gene showed that compared to in vitro-grown bacteria, expression of the pyrophosphatase gene (ppa) was induced fourfold throughout the intracellular infection. There was no detectable induction in transcription of the ppa promoter during exposure to stress stimuli in vitro. The ppa gene of L. pneumophila is the first example of a regulated ppagene which is selectively induced during intracellular infection and which may reflect enhanced capabilities of macromolecular biosynthesis by intracellular L. pneumophila. The data indicate caution in the long-term use of inhibition of host cell protein synthesis to selectively examine gene expression by intracellular bacteria.

scholarly journals Switching Species Tropism: an Effective Way To Manipulate the Feline Coronavirus Genome

2003 ◽  
Vol 77 (8) ◽  
pp. 4528-4538 ◽  
Author(s):  
Bert Jan Haijema ◽  
Haukeliene Volders ◽  
Peter J. M. Rottier
Keyword(s):  
Recombinant Virus ◽  
Reverse Genetics ◽  
Hepatitis Virus ◽  
Rna Recombination ◽  
Recombination System ◽  
Lethal Infection ◽  
Positive Strand Rna ◽  
Targeted Recombination ◽  
Selection Of

ABSTRACT Feline infectious peritonitis virus (FIPV), a coronavirus, is the causative agent of an invariably lethal infection in cats. Like other coronaviruses, FIPV contains an extremely large positive-strand RNA genome of ca. 30 kb. We describe here the development and use of a reverse genetics strategy for FIPV based on targeted RNA recombination that is analogous to what has been described for the mouse hepatitis virus (MHV) (L. Kuo et al., J. Virol. 74:1393-1406, 2000). In this two-step process, we first constructed by targeted recombination a mutant of FIPV, designated mFIPV, in which the ectodomain of the spike glycoprotein was replaced by that of MHV. This switch allowed for the selection of the recombinant virus in murine cells: mFIPV grows to high titers in these cells but has lost the ability to grow in feline cells. In a second, reverse process, mFIPV was used as the recipient, and the reintroduction of the FIPV spike now allowed for selection of candidate recombinants by their regained ability to grow in feline cells. In this fashion, we reconstructed a wild-type recombinant virus (r-wtFIPV) and generated a directed mutant FIPV in which the initiation codon of the nonstructural gene 7b had been disrupted (FIPVΔ7b). The r-wtFIPV was indistinguishable from its parental virus FIPV 79-1146 not only for its growth characteristics in tissue culture but also in cats, exhibiting a highly lethal phenotype. FIPVΔ7b had lost the expression of its 7b gene but grew unimpaired in cell culture, confirming that the 7b glycoprotein is not required in vitro. We establish the second targeted RNA recombination system for coronaviruses and provide a powerful tool for the genetic engineering of the FIPV genome.

scholarly journals Inhibition of host cell RNA polymerase III-mediated transcription by poliovirus: inactivation of specific transcription factors.

1987 ◽  
Vol 7 (11) ◽  
pp. 3880-3887 ◽  
Author(s):  
L G Fradkin ◽  
S K Yoshinaga ◽  
A J Berk ◽  
A Dasgupta
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Dna Binding ◽  
Rna Polymerase ◽  
Host Cell ◽  
Rna Polymerase Iii ◽  
Cell Protein ◽  
Polymerase Iii ◽  
Infected Cells

The inhibition of transcription by RNA polymerase III in poliovirus-infected cells was studied. Experiments utilizing two different cell lines showed that the initiation step of transcription by RNA polymerase III was impaired by infection of these cells with the virus. The observed inhibition of transcription was not due to shut-off of host cell protein synthesis by poliovirus. Among four distinct components required for accurate transcription in vitro from cloned DNA templates, activities of RNA polymerase III and transcription factor TFIIIA were not significantly affected by virus infection. The activity of transcription factor TFIIIC, the limiting component required for transcription of RNA polymerase III genes, was severely inhibited in infected cells, whereas that of transcription factor TFIIIB was inhibited to a lesser extent. The sequence-specific DNA-binding of TFIIIC to the adenovirus VA1 gene internal promoter, however, was not altered by infection of cells with the virus. We conclude that (i) at least two transcription factors, TFIIIB and TFIIIC, are inhibited by infection of cells with poliovirus, (ii) inactivation of TFIIIC does not involve destruction of its DNA-binding domain, and (iii) sequence-specific DNA binding by TFIIIC may be necessary but is not sufficient for the formation of productive transcription complexes.

scholarly journals Synergistic PA and HA mutations confer mouse adaptation of a contemporary A/H3N2 influenza virus

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Mariana Baz ◽  
Zeineb M’hamdi ◽  
Julie Carbonneau ◽  
Sophie Lavigne ◽  
Christian Couture ◽  
...  
Keyword(s):  
Animal Model ◽  
Influenza Virus ◽  
Reverse Genetics ◽  
Polymerase Activity ◽  
Influenza Viruses ◽  
H3n2 Virus ◽  
Wild Type Virus ◽  
Virus Research ◽  
H3n2 Influenza

Abstract The mouse is the most widely used animal model for influenza virus research. However, the susceptibility of mice to seasonal influenza virus depends on the strain of mouse and on the strain of the influenza virus. Seasonal A/H3N2 influenza viruses do not replicate well in mice and therefore they need to be adapted to this animal model. In this study, we generated a mouse-adapted A/H3N2 virus (A/Switzerland/9715293/2013 [MA-H3N2]) by serial passaging in mouse lungs that exhibited greater virulence compared to the wild-type virus (P0-H3N2). Seven mutations were found in the genome of MA-H3N2: PA(K615E), NP(G384R), NA(G320E) and HA(N122D, N144E, N246K, and A304T). Using reverse genetics, two synergistically acting genes were found as determinants of the pathogenicity in mice. First, the HA substitutions were shown to enhanced viral replication in vitro and, second, the PA-K615E substitution increased polymerase activity, although did not alter virus replication in vitro or in mice. Notably, single mutations had only limited effects on virulence in vitro. In conclusion, a co-contribution of HA and PA mutations resulted in a lethal mouse model of seasonal A/H3N2 virus. Such adapted virus is an excellent tool for evaluation of novel drugs or vaccines and for study of influenza pathogenesis.

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