scholarly journals Aedes anphevirus (AeAV): an insect-specific virus distributed worldwide inAedes aegyptimosquitoes that has complex interplays withWolbachiaand dengue virus infection in cells

2018 ◽  
Author(s):  
Rhys Parry ◽  
Sassan Asgari
Keyword(s):  
Dengue Virus ◽  
Cell Lines ◽  
Rna Viruses ◽  
Rna Virus ◽  
Asia Pacific ◽  
Mammalian Cell Lines ◽  
Persistent Virus ◽  
Specific Virus ◽  
Laboratory Colonies

AbstractInsect specific viruses (ISVs) of the yellow fever mosquitoAedes aegyptihave been demonstrated to modulate transmission of arboviruses such as dengue virus (DENV) and West Nile virus by the mosquito. The diversity and composition of the virome ofAe. aegypti, however, remains poorly understood. In this study, we characterised Aedes anphevirus (AeAV), a negative-sense RNA virus from the orderMononegavirales. AeAV identified fromAedescell lines were infectious to bothAe. aegyptiandAedes albopictuscells, but not to three mammalian cell lines. To understand the incidence and genetic diversity of AeAV, we assembled 17 coding-complete and two partial genomes of AeAV from available RNA-Seq data. AeAV appears to transmit vertically and be present in laboratory colonies, wild-caught mosquitoes and cell lines worldwide. Phylogenetic analysis of AeAV strains indicates that as theAe. aegyptimosquito has expanded into the Americas and Asia-Pacific, AeAV has evolved into monophyletic African, American and Asia-Pacific lineages. The endosymbiotic bacteriumWolbachia pipientisrestricts positive-sense RNA viruses inAe. aegypti. Re-analysis of a small RNA library ofAe. aegypticells co-infected with AeAV andWolbachiaproduces an abundant RNAi response consistent with persistent virus replication. We foundWolbachiaenhances replication of AeAV when compared to a tetracycline cleared cell line, and AeAV modestly reduces DENV replicationin vitro. The results from our study improve understanding of the diversity and evolution of the virome ofAe. aegyptiand adds to previous evidence that showsWolbachiadoes not restrict a range of negative strand RNA viruses.

scholarly journals Aedes Anphevirus: an Insect-Specific Virus Distributed Worldwide inAedes aegyptiMosquitoes That Has Complex Interplays withWolbachiaand Dengue Virus Infection in Cells

2018 ◽  
Vol 92 (17) ◽  
Author(s):  
Rhys Parry ◽  
Sassan Asgari
Keyword(s):  
Aedes Aegypti ◽  
Dengue Virus ◽  
Cell Line ◽  
Cell Lines ◽  
Virus Replication ◽  
Rna Viruses ◽  
Asia Pacific ◽  
Content Type ◽  
Negative Strand ◽  
Positive Sense

ABSTRACTInsect-specific viruses (ISVs) of the yellow fever mosquitoAedes aegyptihave been demonstrated to modulate transmission of arboviruses such as dengue virus (DENV) and West Nile virus by the mosquito. The diversity and composition of the virome ofA. aegypti, however, remains poorly understood. In this study, we characterized Aedes anphevirus (AeAV), a negative-sense RNA virus from the orderMononegavirales. AeAV identified fromAedescell lines was infectious to bothA. aegyptiandAedes albopictuscells but not to three mammalian cell lines. To understand the incidence and genetic diversity of AeAV, we assembled 17 coding-complete and two partial genomes of AeAV from available transcriptome sequencing (RNA-Seq) data. AeAV appears to transmit vertically and be present in laboratory colonies, wild-caught mosquitoes, and cell lines worldwide. Phylogenetic analysis of AeAV strains indicates that as theA. aegyptimosquito has expanded into the Americas and Asia-Pacific, AeAV has evolved into monophyletic African, American, and Asia-Pacific lineages. The endosymbiotic bacteriumWolbachia pipientisrestricts positive-sense RNA viruses inA. aegypti. Reanalysis of a small RNA library ofA. aegypticells coinfected with AeAV andWolbachiaproduces an abundant RNA interference (RNAi) response consistent with persistent virus replication. We foundWolbachiaenhances replication of AeAV compared to a tetracycline-cleared cell line, and AeAV modestly reduces DENV replicationin vitro. The results from our study improve understanding of the diversity and evolution of the virome ofA. aegyptiand adds to previous evidence that showsWolbachiadoes not restrict a range of negative-strand RNA viruses.IMPORTANCEThe mosquitoAedes aegyptitransmits a number of arthropod-borne viruses (arboviruses), such as dengue virus and Zika virus. Mosquitoes also harbor insect-specific viruses that may affect replication of pathogenic arboviruses in their body. Currently, however, there are only a few insect-specific viruses described fromA. aegyptiin the literature. Here, we characterize a novel negative-strand virus, AeAV. Meta-analysis ofA. aegyptisamples showed that it is present inA. aegyptimosquitoes worldwide and is vertically transmitted.Wolbachia-transinfected mosquitoes are currently being used in biocontrol, as they effectively block transmission of several positive-sense RNA viruses in mosquitoes. Our results demonstrate thatWolbachiaenhances the replication of AeAV and modestly reduces dengue virus replication in a cell line model. This study expands our understanding of the virome inA. aegyptias well as providing insight into the complexity of theWolbachiavirus restriction phenotype.

scholarly journals Zinc finger antiviral protein (ZAP) activity in mammalian and avian hosts in CpG and UpA-mediated restriction of RNA viruses and investigation of ZAP-mediated shaping of host transcriptome compositions

2021 ◽  
Author(s):  
Valerie Odon ◽  
Steven fiddaman ◽  
Adrian Smith ◽  
Peter Simmonds
Keyword(s):  
Cell Lines ◽  
Zinc Finger ◽  
Influenza A ◽  
Rna Viruses ◽  
Rna Virus ◽  
Virus Genome ◽  
Antiviral Protein ◽  
Cpg Dinucleotides ◽  
Antiviral State ◽  
Mammalian Cell Lines

The ability of zinc finger antiviral protein (ZAP) to recognise and respond to RNA virus sequences with elevated frequencies of CpG dinucleotides has been proposed as a functional part of the vertebrate innate immune antiviral response. It has been further proposed that ZAP activity shapes compositions of cytoplasmic mRNA sequences to avoid self-recognition, particularly mRNAs for interferons (IFNs) and IFN-stimulated genes highly expressed when ZAP is upregulated during the antiviral state. We investigated the ZAP functional activity in different species of mammals and birds, and potential downstream effects of differences in CpG and UpA dinucleotide representations in host transcriptomes and in RNA viruses that infect them. Cell lines from different bird orders showed variability in restriction of influenza A virus and echovirus 7 replicons with elevated CpG frequencies and none restricted UpA-high mutants, in marked contrast to mammalian cell lines. Given this variability, we compared CpG and UpA representation in coding regions of ISGs and IFNs with the total cellular transcriptome to determine whether differences in ZAP activity shaped dinucleotide compositions of highly expressed genes during the antiviral state. While type 1 IFN genes typically showed often profound suppression of CpG and UpA frequencies, there was no over-suppression of CpGs or UpAs in ISGs in any species, irrespective of underlying ZAP activity. Similarly, mammalian and avian RNA virus genome sequences were compositionally equivalent as were IAV serotypes recovered from ducks, chickens and humans. Overall, we found no evidence for host variability in ZAP function impacting compositions of antiviral genes.

Viral RNA structural equilibria and their interactions with host proteins

2019 ◽  
Author(s):  
◽  
Samantha Elizabeth Brady
Keyword(s):  
Reverse Transcription ◽  
Rna Structure ◽  
Drug Targets ◽  
Rna Viruses ◽  
Rna Virus ◽  
Protein Translation ◽  
Viral Rna ◽  
Primer Binding Site ◽  
In Vitro Techniques

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] Understanding viral RNA structure and how it functions is crucial in elucidating new drug targets. There are many kinds of viruses that utilize RNA as a critical component of their life cycle, such as retroviruses, single-stranded plus or minus sense RNA viruses, and double-stranded RNA viruses. Two viruses that are studied in this thesis are human immunodeficiency virus (HIV), which is a retrovirus, and hepatitis C virus (HCV), which is a single-stranded plus sense RNA virus. It has been previously reported that a human host factor, RNA helicase A (RHA), is packaged into HIV virions by binding to the primer binding site (PBS) segment of the 5'untranslated region in the HIV genomic RNA. We determined RHA is required for efficient reverse transcription prior to capsid uncoating by utilizing cell based and in vitro techniques. It has also been suggested that RHA plays other roles during HIV infection besides reverse transcription. Utilizing NMR, we demonstrated that RHA binds to the monomeric 5'UTR at the bottom of the TAR hairpin, which is different from how it binds during viral packaging. Next, we employed NMR techniques to probe the 3'end of the HCV genome called 3'X. We determined that the 3'X is in structural equilibrium between two states: an open conformation and a closed conformation. These two conformations have been suggested to play a role in minus sense synthesis and viral protein translation, respectively. Taken together, my thesis work has elucidated how many viruses manipulate and utilize their RNA structure to modulate their outcome.

scholarly journals Sequence-Specific Modifications Enhance the Broad-Spectrum Antiviral Response Activated by RIG-I Agonists

2015 ◽  
Vol 89 (15) ◽  
pp. 8011-8025 ◽  
Author(s):  
Cindy Chiang ◽  
Vladimir Beljanski ◽  
Kevin Yin ◽  
David Olagnier ◽  
Fethia Ben Yebdri ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Virus Infection ◽  
Inflammatory Responses ◽  
Rna Viruses ◽  
Rna Virus ◽  
A549 Cells ◽  
Antiviral Response ◽  
Interferon Response

ABSTRACTThe cytosolic RIG-I (retinoic acid-inducible gene I) receptor plays a pivotal role in the initiation of the immune response against RNA virus infection by recognizing short 5′-triphosphate (5′ppp)-containing viral RNA and activating the host antiviral innate response. In the present study, we generated novel 5′ppp RIG-I agonists of varieous lengths, structures, and sequences and evaluated the generation of the antiviral and inflammatory responses in human epithelial A549 cells, human innate immune primary cells, and murine models of influenza and chikungunya viral pathogenesis. A 99-nucleotide, uridine-rich hairpin 5′pppRNA termed M8 stimulated an extensive and robust interferon response compared to other modified 5′pppRNA structures, RIG-I aptamers, or poly(I·C). Interestingly, manipulation of the primary RNA sequence alone was sufficient to modulate antiviral activity and inflammatory response, in a manner dependent exclusively on RIG-I and independent of MDA5 and TLR3. Both prophylactic and therapeutic administration of M8 effectively inhibited influenza virus and dengue virus replicationin vitro. Furthermore, multiple strains of influenza virus that were resistant to oseltamivir, an FDA-approved therapeutic treatment for influenza, were highly sensitive to inhibition by M8. Finally, prophylactic M8 treatmentin vivoprolonged survival and reduced lung viral titers of mice challenged with influenza virus, as well as reducing chikungunya virus-associated foot swelling and viral load. Altogether, these results demonstrate that 5′pppRNA can be rationally designed to achieve a maximal RIG-I-mediated protective antiviral response against human-pathogenic RNA viruses.IMPORTANCEThe development of novel therapeutics to treat human-pathogenic RNA viral infections is an important goal to reduce spread of infection and to improve human health and safety. This study investigated the design of an RNA agonist with enhanced antiviral and inflammatory properties against influenza, dengue, and chikungunya viruses. A novel, sequence-dependent, uridine-rich RIG-I agonist generated a protective antiviral responsein vitroandin vivoand was effective at concentrations 100-fold lower than prototype sequences or other RNA agonists, highlighting the robust activity and potential clinical use of the 5′pppRNA against RNA virus infection. Altogether, the results identify a novel, sequence-specific RIG-I agonist as an attractive therapeutic candidate for the treatment of a broad range of RNA viruses, a pressing issue in which a need for new and more effective options persists.

scholarly journals In VitroandIn VivoAntimalarial Evaluations of Myrtle Extract, a Plant Traditionally Used for Treatment of Parasitic Disorders

2013 ◽  
Vol 2013 ◽  
pp. 1-5 ◽  
Author(s):  
Farzaneh Naghibi ◽  
Somayeh Esmaeili ◽  
Noor Rain Abdullah ◽  
Mehdi Nateghpour ◽  
Mahdieh Taghvai ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mammalian Cell ◽  
Methanolic Extract ◽  
Cytotoxic Activities ◽  
Traditional Use ◽  
Aerial Parts ◽  
Mammalian Cell Lines ◽  
Phytochemical Investigation

Based on the collected ethnobotanical data from the Traditional Medicine and Materia Medica Research Center (TMRC), Iran,Myrtus communisL. (myrtle) was selected for the assessment ofin vitroandin vivoantimalarial and cytotoxic activities. Methanolic extract of myrtle was prepared from the aerial parts and assessed for antiplasmodial activity, using the parasite lactate dehydrogenase (pLDH) assay against chloroquine-resistant (K1) and chloroquine-sensitive (3D7) strains ofPlasmodium falciparum. The 4-day suppressive test was employed to determine the parasitemia suppression of the myrtle extract againstP. berghei  in vivo. The IC50values of myrtle extract were 35.44 µg/ml against K1 and 0.87 µg/ml against 3D7. Myrtle extract showed a significant suppression of parasitaemia (84.8 ± 1.1% at 10 mg/kg/day) in mice infected withP. bergheiafter 4 days of treatment. Cytotoxic activity was carried out against mammalian cell lines using methyl thiazol tetrazolium (MTT) assay. No cytotoxic effect on mammalian cell lines up to 100 µg/mL was shown. The results support the traditional use of myrtle in malaria. Phytochemical investigation and understanding the mechanism of action would be in our upcoming project.

scholarly journals The Molecular Pharmacology of Pateamine A

2021 ◽  
Author(s):  
◽  
James Henry Matthews
Keyword(s):  
Protein Synthesis ◽  
Cell Lines ◽  
Mammalian Cell ◽  
Mode Of Action ◽  
Protein Synthesis Inhibitors ◽  
Primary Target ◽  
Dead Box ◽  
Mammalian Cell Lines ◽  
Pateamine A

<p>Pateamine A is a cytotoxic terpenoid isolated from the marine sponge Mycale hentscheli that induces apoptosis in mammalian cell lines and is growth inhibitory to yeasts and fungi, yet shows no inhibitory action in prokaryotes. The targets of pateamine in mammalian cell lines were isolated and identified using a combination of affinity chromatography and mass spectrometry, putative targets included the DEAD-Box helicase eIF4A family of proteins, β-tubulin and actin. In vitro assessment of tubulin and actin polymerization showed pateamine was able to affect them only at high micromolar concentrations, whereas the effect on eIF4A in vitro was shown by others to occur at nanomolar concentrations. Additionally, pateamine was shown to inhibit cap-dependent protein synthesis in vivo, suggesting eIF4A as a primary target. The generation of a pateamine resistance-conferring mutation in the yeast eIF4A encoding gene TIF1, suggested further that eIF4A is a primary target in both mammalian and yeast cells, and allows the speculation of the position of the binding site for pateamine on the N-terminal lobe of eIF4A and the proposal of potential covalent interaction between this drug and its target. Given the size of the DEAD-Box helicase family, all of which share considerable homology with the eIF4As, FAL1 especially which is essential for rRNA maturation, a chemogenomic screen was performed in an attempt to establish the breadth of functional interactions of pateamine. The results of hierarchical clustering of these screen results suggest that pateamine has a mode-of-action distinct from other compounds screened previously, despite its effect on protein synthesis it failed to cluster with any other protein synthesis inhibitors regardless of their separate mechanisms, though, as a class, protein synthesis inhibitors were not found to form a discrete cluster in any of the variations of cluster analysis performed. Functional analysis, by GO term enrichment, of the genes whose deletions are hypersensitive to pateamine indicates that deletions of genes involved in numerous aspects of RNA metabolism affect pateamine sensitivity, however clear results regarding the involvement of FAL1 or any other non-eIF4A target in pateamine’s mode-of-action were not found.</p>

scholarly journals High-Yield Expression and Purification of Recombinant Influenza Virus Proteins from Stably-Transfected Mammalian Cell Lines

Vaccines ◽  
2020 ◽  
Vol 8 (3) ◽  
pp. 462
Author(s):  
Jeffrey W. Ecker ◽  
Greg A. Kirchenbaum ◽  
Spencer R. Pierce ◽  
Amanda L. Skarlupka ◽  
Rodrigo B. Abreu ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Cell Lines ◽  
Mammalian Cell ◽  
Recombinant Proteins ◽  
Influenza Viruses ◽  
Scale Production ◽  
Vaccine Platform ◽  
Mammalian Cell Lines

Influenza viruses infect millions of people each year, resulting in significant morbidity and mortality in the human population. Therefore, generation of a universal influenza virus vaccine is an urgent need and would greatly benefit public health. Recombinant protein technology is an established vaccine platform and has resulted in several commercially available vaccines. Herein, we describe the approach for developing stable transfected human cell lines for the expression of recombinant influenza virus hemagglutinin (HA) and recombinant influenza virus neuraminidase (NA) proteins for the purpose of in vitro and in vivo vaccine development. HA and NA are the main surface glycoproteins on influenza virions and the major antibody targets. The benefits for using recombinant proteins for in vitro and in vivo assays include the ease of use, high level of purity and the ability to scale-up production. This work provides guidelines on how to produce and purify recombinant proteins produced in mammalian cell lines through either transient transfection or generation of stable cell lines from plasmid creation through the isolation step via Immobilized Metal Affinity Chromatography (IMAC). Collectively, the establishment of this pipeline has facilitated large-scale production of recombinant HA and NA proteins to high purity and with consistent yields, including glycosylation patterns that are very similar to proteins produced in a human host.

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