scholarly journals CRISPR-Csy4-mediated editing of rotavirus double-stranded RNA genome

2020 ◽  
Author(s):  
Guido Papa ◽  
Luca Venditti ◽  
Luca Braga ◽  
Edoardo Schneider ◽  
Mauro Giacca ◽  
...  
Keyword(s):  
Genome Editing ◽  
Double Stranded Rna ◽  
Viral Genomes ◽  
Viral Particles ◽  
Dsrna Genome ◽  
Dsrna Viruses ◽  
Level Of Understanding ◽  
Virus Genomes ◽  
First Time ◽  
Replication Intermediates

ABSTRACTCRISPR-nucleases have been widely applied for editing cellular and viral genomes, but nuclease-mediated genome editing of double-stranded RNA (dsRNA) viruses has not yet been reported. Here, by engineering CRISPR-Csy4 nuclease to localise to rotavirus viral factories, we achieved the first nuclease-mediated genome editing of rotavirus, an important human and livestock pathogen with a multi-segmented dsRNA genome. Rotavirus replication intermediates cleaved by Csy4 were repaired through the formation of defined deletions in the targeted genome segments in a single replication cycle. Using CRISPR-Csy4-mediated editing of rotavirus genome, we labelled for the first time the products of rotavirus secondary transcription made by newly assembled viral particles during rotavirus replication, demonstrating that this step largely contributes to the overall production of viral proteins. We anticipate that the nuclease-mediated cleavage of dsRNA virus genomes will promote a new level of understanding of viral replication and host-pathogen interactions, offering the opportunity to develop new therapeutics.

scholarly journals The First Detection of Co-Infection of Double-Stranded RNA Virus 1, 2 and 3 in Iranian Isolates of Trichomonas vaginalis

2020 ◽  
Author(s):  
Farah BOKHARAEI-SALIM ◽  
Abdoulreza ESTEGHAMATI ◽  
Khadijeh KHANALIHA ◽  
Maryam ESGHAEI ◽  
Tahereh DONYAVI ◽  
...  
Keyword(s):  
Trichomonas Vaginalis ◽  
Rna Virus ◽  
Rna Extraction ◽  
Multiple Infections ◽  
Rt Pcr ◽  
Double Stranded Rna ◽  
Pcr Products ◽  
Dsrna Viruses ◽  
Different Strains ◽  
First Time

Background: The Totiviridae family includes a number of double-stranded RNA viruses that can infect Trichomonas vaginalis. Some T. vaginalis isolates are infected with one or more double-stranded RNA (dsRNA) viruses. In this study, different strains of double-stranded RNA virus in Iranian isolates of T. vaginalis were evaluated for the first time in Iran.  Methods: Vaginal swabs were collected from 1550 participants who were referred to hospitals associated with Iran University of Medical Sciences, Tehran, Iran from June to November 2018.    T. vaginalis isolates were cultured in Diamond's modified medium. After the extraction of nucleic acids using a DNA/RNA extraction kit, RT-PCR was performed and PCR products were purified and sequenced. Results: In general 9 (0.6%) isolates were confirmed as T. vaginalis among 1550 collected vaginal samples. Among 9 isolates of T. vaginalis, three of them were infected with TVV1. One isolate has multiple infections with T. vaginalis virus (TVV1, TVV2 and TVV3) as coinfection. The nucleotide BLAST indicated that the T. vaginalis virus 1(TVV1) isolates were most closely related to TVV1-OC5, TVV1-UR1-1.The T. vaginalis virus 2 (TVV2) sequence had also a similarity with TVV2-UR1-1, TVV2-UR1 and TVV2-OC3. The sequence of T. vaginalis virus 3(TVV3) had similarity with TVV3-OC5, TVV3-UR1-1 and TVV3-UR1. Conclusion: Three dsRNA viruses T. vaginalis virus (TVV1, TVV2 and TVV3) were detected using RT-PCR in T. vaginalis Iranian isolates. The coinfection of TVV1, TVV2 and TVV3 in one isolate of T.vaginalis was observed for the first time in Iran.

scholarly journals Novel Mycoviruses Discovered in the Mycovirome of a Necrotrophic Fungus

mBio ◽  
2021 ◽  
Vol 12 (3) ◽  
Author(s):  
Ana Ruiz-Padilla ◽  
Julio Rodríguez-Romero ◽  
Irene Gómez-Cid ◽  
Davide Pacifico ◽  
María A. Ayllón
Keyword(s):  
Chemical Control ◽  
Pathogenic Fungus ◽  
Pathogenic Fungi ◽  
General Knowledge ◽  
Double Stranded Rna ◽  
Positive Sense ◽  
Dsrna Genome ◽  
Horizontal Transfers ◽  
First Time ◽  
Negative Sense

ABSTRACT Botrytis cinerea is one of the most important plant-pathogenic fungus. Products based on microorganisms can be used in biocontrol strategies alternative to chemical control, and mycoviruses have been explored as putative biological agents in such approaches. Here, we have explored the mycovirome of B. cinerea isolates from grapevine of Italy and Spain to increase the knowledge about mycoviral diversity and evolution, and to search for new widely distributed mycoviruses that could be active ingredients in biological products to control this hazardous fungus. A total of 248 B. cinerea field isolates were used for our metatranscriptomic study. Ninety-two mycoviruses were identified: 62 new mycoviral species constituting putative novel viral genera and families. Of these mycoviruses, 57 had a positive-sense single-stranded RNA (ssRNA) genome, 19 contained a double-stranded RNA (dsRNA) genome, 15 had a negative-sense ssRNA genome, and 1 contained a single-stranded DNA (ssDNA) genome. In general, ssRNA mycoviruses were widely distributed in all sampled regions, the ssDNA mycovirus was more frequently found in Spain, and dsRNA mycoviruses were scattered in some pools of both countries. Some of the identified mycoviruses belong to clades that have never been found associated with Botrytis species: Botrytis-infecting narnaviruses; alpha-like, umbra-like, and tymo-like ssRNA+ mycoviruses; trisegmented ssRNA− mycovirus; bisegmented and tetrasegmented dsRNA mycoviruses; and finally, an ssDNA mycovirus. Among the results obtained in this massive mycovirus screening, the discovery of novel bisegmented viruses, phylogenetically related to narnaviruses, is remarkable. IMPORTANCE The results obtained here have expanded our knowledge of mycoviral diversity, horizontal transfers, and putative cross-kingdom events. To date, this study presents the most extensive and wide diversity collection of mycoviruses infecting the necrotrophic fungus B. cinerea. The collection included all types of mycoviruses, with dsRNA, ssRNA+, ssRNA–, and ssDNA genomes, most of which were discovered here, and some of which were previously reported as infecting B. cinerea or other plant-pathogenic fungi. Some of these mycoviruses are reported for the first time here associated with B. cinerea, as a trisegmented ssRNA– mycovirus and as an ssDNA mycovirus, but even more remarkablly, we also describe here four novel bisegmented viruses (binarnaviruses) not previously described in nature. The present findings significantly contribute to general knowledge in virology and more particularly in the field of mycovirology.

scholarly journals Induction of Promoter DNA Methylation Upon High-Pressure Spraying of Double-Stranded RNA in Plants

Agronomy ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 789
Author(s):  
Athanasios Dalakouras ◽  
Ioannis Ganopoulos
Keyword(s):  
High Pressure ◽  
De Novo ◽  
Epigenetic Changes ◽  
Double Stranded Rna ◽  
Rna Molecules ◽  
Promoter Sequences ◽  
Promoter Dna Methylation ◽  
Promoter Dna ◽  
First Time ◽  
De Novo Methylation

Exogenous application of RNA molecules is a potent method to trigger RNA interference (RNAi) in plants in a transgene-free manner. So far, all exogenous RNAi (exo-RNAi) applications have aimed to trigger mRNA degradation of a given target. However, the issue of concomitant epigenetic changes was never addressed. Here, we report for the first time that high-pressure spraying of dsRNAs can trigger de novo methylation of promoter sequences in plants.

scholarly journals Reovirus Low-Density Particles Package Cellular RNA

Viruses ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1096
Author(s):  
Timothy W. Thoner ◽  
Xiang Ye ◽  
John Karijolich ◽  
Kristen M. Ogden
Keyword(s):  
Rna Polymerase ◽  
Viral Proteins ◽  
Viral Rna ◽  
Low Density ◽  
Rna Packaging ◽  
Genome Packaging ◽  
Double Stranded Rna ◽  
Viral Genomes ◽  
Mammalian Orthoreovirus ◽  
Insight Into

Packaging of segmented, double-stranded RNA viral genomes requires coordination of viral proteins and RNA segments. For mammalian orthoreovirus (reovirus), evidence suggests either all ten or zero viral RNA segments are simultaneously packaged in a highly coordinated process hypothesized to exclude host RNA. Accordingly, reovirus generates genome-containing virions and “genomeless” top component particles. Whether reovirus virions or top component particles package host RNA is unknown. To gain insight into reovirus packaging potential and mechanisms, we employed next-generation RNA-sequencing to define the RNA content of enriched reovirus particles. Reovirus virions exclusively packaged viral double-stranded RNA. In contrast, reovirus top component particles contained similar proportions but reduced amounts of viral double-stranded RNA and were selectively enriched for numerous host RNA species, especially short, non-polyadenylated transcripts. Host RNA selection was not dependent on RNA abundance in the cell, and specifically enriched host RNAs varied for two reovirus strains and were not selected solely by the viral RNA polymerase. Collectively, these findings indicate that genome packaging into reovirus virions is exquisitely selective, while incorporation of host RNAs into top component particles is differentially selective and may contribute to or result from inefficient viral RNA packaging.

Improved detection of flaviviruses in Australian mosquito populations via replicative intermediates

2021 ◽  
Vol 102 (7) ◽  
Author(s):  
Caitlin A. O'Brien ◽  
Jessica J. Harrison ◽  
Agathe M. G. Colmant ◽  
Renee J. Traves ◽  
Devina Paramitha ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Untranslated Regions ◽  
Fixation Method ◽  
Enveloped Viruses ◽  
Double Stranded Rna ◽  
Vertebrate Cell ◽  
Infected Cells ◽  
Replicative Intermediates ◽  
Dsrna Viruses ◽  
Arboviral Disease

Mosquito-borne flaviviruses are significant contributors to the arboviral disease burdens both in Australia and globally. While routine arbovirus surveillance remains a vital exercise to identify known flaviviruses in mosquito populations, novel or divergent and emerging species can be missed by these traditional methods. The MAVRIC (monoclonal antibodies to viral RNA intermediates in cells) system is an ELISA-based method for broad-spectrum isolation of positive-sense and double-stranded RNA (dsRNA) viruses based on detection of dsRNA in infected cells. While the MAVRIC ELISA has successfully been used to detect known and novel flaviviruses in Australian mosquitoes, we previously reported that dsRNA could not be detected in dengue virus-infected cells using this method. In this study we identified additional flaviviruses which evade detection of dsRNA by the MAVRIC ELISA. Utilising chimeric flaviviruses we demonstrated that this outcome may be dictated by the non-structural proteins and/or untranslated regions of the flaviviral genome. In addition, we report a modified fixation method that enables improved detection of flavivirus dsRNA and inactivation of non-enveloped viruses from mosquito populations using the MAVRIC system. This study demonstrates the utility of anti-dsRNA monoclonal antibodies for identifying viral replication in insect and vertebrate cell systems and highlights a unique characteristic of flavivirus replication.

scholarly journals Virus-Like Microfossils in the 1.64 Ga Siliceous Rocks From Hogland Island, Russia

2021 ◽  
Author(s):  
Anatoly Belyaev
Keyword(s):  
Systematic Position ◽  
Base Pairs ◽  
Molecular Weights ◽  
Siliceous Rocks ◽  
Viral Particles ◽  
Unicellular Organisms ◽  
Giant Viruses ◽  
First Time ◽  
The Gulf Of Finland ◽  
Size Of Nuclei

Several varieties of virus-like microfossils, morphologically similar to modern giant viruses of the Mimiviridae family, have been identified in microquartzites in the 1.64 Ga volcanogenic-sedimentary strata in Hogland Island in the Gulf of Finland, Russia. Microquartzites contain graphite enriched in a light carbon isotope 12С, as is typical for the rocks forming with participation of living matter. Abundant remains of silificated and ferruginizated microfossils of planktonic microorganisms and virus-like structures were found in fragments of silificated biofilms. However, virus-like microfossils exceed modern giant viruses in linear dimensions by a factor of a thousand or more (Belyaev, 2018; 2019; Belyaev, Yukhalin, 2021) and contain structures similar to eukaryotic nuclei. In addition, data were obtained that can be interpreted as a fact of parasitic relationships of virus-like formations with microfossils of amoeba-like microorganisms. Inside, and in the immediate surroundings of some virus-like structures, small oval zonal formations occur, which, possibly, represent silificated viral particles, the most ancient obligate super parasites similar to "satellite" virophages in mimiviruses (La Scola, et al., 2008). Apatite grains found in the mineralized cytoplasm and nuclei of virus-like microfossils, most likely, crystallized from phosphoric acid residues of decayed nucleotides. This allowed for the first time to roughly estimate the size of the genomes of the most ancient virus-like structures, which exceeded the genomes of modern giant viruses and unicellular organisms by a factor of thousands (Belyaev, Yukhalin, 2021). The genome masses of eukaryotic microfossils and virus-like structures were also estimated following the principle of genomic-nuclear proportionality, according to which the molecular weights of genomes are directly proportional to the size of the nuclei. In this case, the size of genomes of virus-like structures estimated both form the enclosed apatite grains and the size of nuclei, averaged tens of thousands of picograms and, thus, could contain tens of thousands billions of base pairs. It is assumed that microfossils of virus-like structures from the group of unclear systematic position Dinoviridae Incertae sedis were representatives of the extinct family of unicellular facultative parasites or were the ancestors of giant viruses of the Mimiviridae family.

scholarly journals Saccharomyces paradoxus Transcriptional Alterations in Cells of Distinct Phenotype and Viral dsRNA Content

2020 ◽  
Vol 8 (12) ◽  
pp. 1902
Author(s):  
Bazilė Ravoitytė ◽  
Juliana Lukša ◽  
Vyacheslav Yurchenko ◽  
Saulius Serva ◽  
Elena Servienė
Keyword(s):  
Food Industry ◽  
Antifungal Agents ◽  
Acid Biosynthesis ◽  
Viral Dsrna ◽  
Saccharomyces Paradoxus ◽  
Distinct Phenotype ◽  
Transcriptional Alterations ◽  
Potential Applications ◽  
Dsrna Viruses ◽  
First Time

Killer yeasts are attractive antifungal agents with great potential applications in the food industry. Natural Saccharomyces paradoxus isolates provide new dsRNA-based killer systems available for investigation. The presence of viral dsRNA may alter transcriptional profile of S. paradoxus. To test this possibility, a high-throughput RNA sequencing was employed to compare the transcriptomes of S. paradoxus AML 15-66 K66 killer strains after curing them of either M-66 alone or both M-66 and L-A-66 dsRNA viruses. The S. paradoxus cells cured of viral dsRNA(s) showed respiration deficient or altered sporulation patterns. We have identified numerous changes in the transcription profile of genes including those linked to ribosomes and amino acid biosynthesis, as well as mitochondrial function. Our work advance studies of transcriptional adaptations of Saccharomyces spp. induced by changes in phenotype and set of dsRNA viruses, reported for the first time.

scholarly journals High-Throughput Sequencing Reveals Further Diversity of Little Cherry Virus 1 with Implications for Diagnostics

Viruses ◽  
2018 ◽  
Vol 10 (7) ◽  
pp. 385 ◽  
Author(s):  
Asimina Katsiani ◽  
Varvara Maliogka ◽  
Nikolaos Katis ◽  
Laurence Svanella-Dumas ◽  
Antonio Olmos ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Sour Cherry ◽  
Prunus Species ◽  
Cherry Tree ◽  
High Genetic Diversity ◽  
Viral Genomes ◽  
Virus Diversity ◽  
First Time

Little cherry virus 1 (LChV1, Velarivirus, Closteroviridae) is a widespread pathogen of sweet or sour cherry and other Prunus species, which exhibits high genetic diversity and lacks a putative efficient transmission vector. Thus far, four distinct phylogenetic clusters of LChV1 have been described, including isolates from different Prunus species. The recent application of high throughput sequencing (HTS) technologies in fruit tree virology has facilitated the acquisition of new viral genomes and the study of virus diversity. In the present work, several new LChV1 isolates from different countries were fully sequenced using different HTS approaches. Our results reveal the presence of further genetic diversity within the LChV1 species. Interestingly, mixed infections of the same sweet cherry tree with different LChV1 variants were identified for the first time. Taken together, the high intra-host and intra-species diversities of LChV1 might affect its pathogenicity and have clear implications for its accurate diagnostics.

scholarly journals Evidence ofIn VivoProphage Induction during Clostridium difficile Infection

2012 ◽  
Vol 78 (21) ◽  
pp. 7662-7670 ◽  
Author(s):  
Mathieu Meessen-Pinard ◽  
Ognjen Sekulovic ◽  
Louis-Charles Fortier
Keyword(s):  
Clostridium Difficile ◽  
Bioinformatics Analyses ◽  
Content Type ◽  
Unique Character ◽  
Viral Particles ◽  
Potential Impact ◽  
First Time ◽  
Culture Supernatants

ABSTRACTProphages contribute to the evolution and virulence of most bacterial pathogens, but their role inClostridium difficileis unclear. Here we describe the isolation of fourMyoviridaephages, ϕMMP01, ϕMMP02, ϕMMP03, and ϕMMP04, that were recovered as free viral particles in the filter-sterilized stool supernatants of patients suffering fromC. difficileinfection (CDI). Furthermore, identical prophages were found in the chromosomes ofC. difficileisolated from the corresponding fecal samples. We therefore provide, for the first time, evidence ofin vivoprophage induction during CDI. We completely sequenced the genomes of ϕMMP02 and ϕMMP04, and bioinformatics analyses did not reveal the presence of virulence factors but underlined the unique character of ϕMMP04. We also studied the mobility of ϕMMP02 and ϕMMP04 prophagesin vitro. Both prophages were spontaneously induced, with 4 to 5 log PFU/ml detected in the culture supernatants of the corresponding lysogens. When lysogens were grown in the presence of subinhibitory concentrations of ciprofloxacin, moxifloxacin, levofloxacin, or mitomycin C, the phage titers further increased, reaching 8 to 9 log PFU/ml in the case of ϕMMP04. In summary, our study highlights the extensive genetic diversity and mobility ofC. difficileprophages. Moreover, antibiotics known to represent risk factors for CDI, such as quinolones, can stimulate prophage mobilityin vitroand probablyin vivoas well, which underscores their potential impact on phage-mediated horizontal gene transfer events and the evolution ofC. difficile.

scholarly journals Probing the Transcription Mechanisms of Reovirus Cores with Molecules That Alter RNA Duplex Stability

2009 ◽  
Vol 83 (11) ◽  
pp. 5659-5670 ◽  
Author(s):  
Alexander A. Demidenko ◽  
Max L. Nibert
Keyword(s):  
Small Molecules ◽  
Dimethyl Sulfoxide ◽  
Active Fraction ◽  
Core Particle ◽  
Double Stranded Rna ◽  
Promoter Escape ◽  
Duplex Stability ◽  
Rna Duplexes ◽  
Dsrna Viruses ◽  
Transcript Initiation

ABSTRACT The mammalian reovirus (MRV) genome comprises 10 double-stranded RNA (dsRNA) segments, packaged along with transcriptase complexes inside each core particle. Effects of four small molecules on transcription by MRV cores were studied for this report, chosen for their known capacities to alter RNA duplex stability. Spermidine and spermine, which enhance duplex stability, inhibited transcription, whereas dimethyl sulfoxide and trimethylglycine, which attenuate duplex stability, stimulated transcription. Different mechanisms were identified for inhibition or activation by these molecules. With spermidine, one round of transcription occurred normally, but subsequent rounds were inhibited. Thus, inhibition occurred at the transition between the end of elongation in one round and initiation in the next round of transcription. Dimethyl sulfoxide or trimethylglycine, on the other hand, had no effect on transcription by a constitutively active fraction of cores in each preparation but activated transcription in another fraction that was otherwise silent for the production of elongated transcripts. Activation of this other fraction occurred at the transition between transcript initiation and elongation, i.e., at promoter escape. These results suggest that the relative stability of RNA duplexes is most important for certain steps in the particle-associated transcription cycles of dsRNA viruses and that small molecules are useful tools for probing these and probably other steps.

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