Endogenous Viral Element-Derived Piwi-Interacting RNAs (piRNAs) Are Not Required for Production of Ping-Pong-Dependent piRNAs from Diaphorina citri Densovirus

ABSTRACT Piwi-interacting RNAs (piRNAs) are a class of small RNAs primarily responsible for silencing transposons in the animal germ line. The ping-pong cycle, the posttranscriptional silencing branch of the piRNA pathway, relies on piRNAs produced from endogenous transposon remnants to direct cleavage of transposon RNA via association with Piwi-family Argonaute proteins. In some mosquito species and mosquito-derived cell lines expressing a functionally expanded group of Piwi-family Argonaute proteins, both RNA and DNA viruses are targeted by piRNAs in a manner thought to involve direct processing of exogenous viral RNA into piRNAs. Whether viruses are targeted by piRNAs in nonmosquito species is unknown. Partial integrations of DNA and nonretroviral RNA virus genomes, termed endogenous viral elements (EVEs), are abundant in arthropod genomes and often produce piRNAs that are speculated to target cognate viruses through the ping-pong cycle. Here, we describe a Diaphorina citri densovirus (DcDV)-derived EVE in the genome of Diaphorina citri. We found that this EVE gives rise to DcDV-specific primary piRNAs and is unevenly distributed among D. citri populations. Unexpectedly, we found that DcDV is targeted by ping-pong-dependent virus-derived piRNAs (vpiRNAs) in D. citri lacking the DcDV-derived EVE, while four naturally infecting RNA viruses of D. citri are not targeted by vpiRNAs. Furthermore, a recombinant Cricket paralysis virus containing a portion of the DcDV genome corresponding to the DcDV-derived EVE was not targeted by vpiRNAs during infection in D. citri harboring the EVE. These results demonstrate that viruses can be targeted by piRNAs in a nonmosquito species independently of endogenous piRNAs. IMPORTANCE Small RNAs serve as specificity determinants of antiviral responses in insects. Piwi-interacting RNAs (piRNAs) are a class of small RNAs found in animals, and their primary role is to direct antitransposon responses. These responses require endogenous piRNAs complementary to transposon RNA. Additionally, piRNAs have been shown to target RNA and DNA viruses in some mosquito species. In contrast to transposons, targeting of viruses by the piRNA pathway in these mosquito species does not require endogenous piRNAs. Here, we show that piRNAs target a DNA virus, but not RNA viruses, in an agricultural insect pest. We found that targeting of this DNA virus did not require endogenous piRNAs and that endogenous piRNAs did not mediate targeting of an RNA virus with which they shared complementary sequence. Our results highlight differences between mosquitoes and our experimental system and raise the possibility that DNA viruses may be targeted by piRNAs in other species.

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Temporal landscape of human gut RNA and DNA virome in SARS-CoV-2 infection and severity

10.21203/rs.3.rs-66879/v2 ◽

2020 ◽

Author(s):

Tao Zuo ◽

Qin Liu ◽

Fen Zhang ◽

Yun Kit Yeoh ◽

Yating Wan ◽

...

Keyword(s):

Disease Severity ◽

Rna Virus ◽

Host Immunity ◽

Virus Species ◽

Human Gut ◽

Dna Virus ◽

Dna Viruses ◽

Cell Counts ◽

Faecal Samples ◽

Rna And Dna

Abstract Background: Coronavirus Disease 2019 (COVID-19) caused by the enveloped RNA virus SARS-CoV-2 primarily affects the respiratory and gastrointestinal tracts. SARS-CoV-2 was isolated from faecal samples and active viral replication was reported in human intestinal cells. The human gut also harbors an enormous amount of resident viruses (collectively known as the virome) that play a role in regulating host immunity and disease pathophysiology. Understanding gut virome perturbation that underlies SARS-CoV-2 infection and severity is an unmet need.Methods: We enrolled 98 COVID-19 patients with varying disease severity (3 asymptomatic, 53 mild, 34 moderate, 5 severe, 3 critical) and 78 non-COVID-19 controls matched for gender and co-morbidities. All subjects had faecal specimens sampled at inclusion. Blood specimens were collected for COVID-19 patients at admission to test for inflammatory markers and white cell counts. Among COVID-19 cases, 37 (38%) patients had serial faecal samples collected 2 to 3 times per week from time of hospitalization until after discharge. Using shotgun metagenomics sequencing, we sequenced and profiled the faecal RNA and DNA virome. We investigated alterations and longitudinal dynamics of the gut virome in association with disease severity and blood parameters.Results: Patients with COVID-19 showed underrepresentation of Pepper mild mottle virus (RNA virus) and multiple bacteriophage lineages (DNA viruses) and enrichment of environment-derived eukaryotic DNA viruses in faecal samples, compared to non-COVID-19 subjects. Such gut virome alterations persisted up to 30 days after disease resolution. Faecal virome in SARS-CoV-2 infection harboured more stress-, inflammation- and virulence-associated gene encoding capacities including those pertaining to bacteriophage integration, DNA repair, and metabolism and virulence associated with their bacterial host. Baseline fecal abundance of 10 virus species (1 RNA virus, Pepper chlorotic spot virus, and 9 DNA virus species) inversely correlated with disease COVID-19 severity. These viruses inversely correlated with blood levels of pro-inflammatory proteins, white cells and neutrophils. Among the 10 COVID-19 severity-associated DNA virus species, 4 showed inverse correlation with age; 5 showed persistent lower abundance both during disease course and after disease resolution relative to non-COVID-19 subjects.Conclusions: Both enteric RNA and DNA virome in COVID-19 patients were different from non-COVID-19 subjects, which persisted after disease resolution of COVID-19. Gut virome may calibrate host immunity and regulate severity to SARS-CoV-2 infection. Our observation that gut viruses inversely correlated with both severity of COVID-19 and host age may partly explain that older subjects are prone to severe and worse COVID-19 outcomes. Altogether our data highlight the importance of human gut virome in severity and potentially therapeutics of COVID-19.

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Temporal Landscape of Human Gut RNA and DNA Viromes in SARS-CoV-2 Infection and Severity

10.21203/rs.3.rs-66879/v1 ◽

2020 ◽

Author(s):

Tao Zuo ◽

Qin Liu ◽

Fen Zhang ◽

Yun Kit Yeoh ◽

Yating Wan ◽

...

Keyword(s):

Disease Severity ◽

Rna Virus ◽

Host Immunity ◽

Virus Species ◽

Disease Course ◽

Human Gut ◽

Dna Virus ◽

Dna Viruses ◽

Faecal Samples ◽

Rna And Dna

Abstract Background: Coronavirus Disease 2019 (COVID-19) caused by the enveloped RNA virus SARS-CoV-2 primarily affects the respiratory and gastrointestinal tracts. SARS-CoV-2 was isolated from faecal samples and active viral replication was reported in human intestinal cells. The human gut also harbors an enormous amount of resident viruses (collectively known as virome) that play a role in regulating host immunity and pathophysiology. Understanding gut virome perturbation that underlies SARS-CoV-2 infection and severity is an unmet need.Methods:We enrolled 98 COVID-19 patients with varying disease severity (3 asymptomatic, 53 mild, 34 moderate, 5 severe, 3 critical) and 78 non-COVID-19 controls matched for gender and co-morbidities. All study subjects had faecal specimens sampled at inclusion. Blood specimens were sampled for COVID-19 patients at admission to test for inflammatory markers and white cell counts. Among COVID-19 cases, 37 (38%) patients had serially faecal samples collected 2 to 3 times per week from time of hospitalization until after discharge. Using shotgun metagenomics sequencing, we sequenced and profiled the faecal RNA and DNA virome respectively. We investigated alterations and longitudinal dynamics of the gut virome in association with disease severity and blood parameters.Results: Patients with COVID-19 showed underrepresentation of Pepper mild mottle virus (RNA virus) and multiple bacteriophage lineages (DNA viruses) and enrichment of environment-derived eukaryotic DNA viruses in faecal samples, compared to non-COVID-19 subjects. Such gut virome dysbiosis persisted up to 30 days after disease resolution. Faecal virome in SARS-CoV-2 infection harboured more stress-, inflammation- and virulence-associated gene encoding capacities including those pertaining to bacteriophage integration, DNA repair, and metabolism and virulence associated with their bacterial host. Human faecal baseline abundance of 10 virus species (1 RNA virus, Pepper chlorotic spot virus, and 9 DNA virus species) inversely correlated with disease severity of COVID-19. These viruses were also inversely associated with blood levels of pro-inflammatory proteins, white cells and neutrophils. Among the 10 COVID-19 severity-associated DNA virus species, 4 showed inverse correlation with age; 5 showed persistent lower abundance both during disease course and after disease resolution relative to non-COVID-19 subjects.Conclusions: Both enteric RNA and DNA viromes were perturbed in COVID-19, which prolonged even after disease resolution. Gut virome may calibrate host immunity and regulate severity to SARS-CoV-2 infection. Our observation that gut viruses inversely correlated with both severity of COVID-19 and host age partly explains that older subjects are prone to severe and unfavorable COVID-19 outcomes. Our data altogether highlight the significance of human gut virome in COVID-19 disease course and potentially therapeutics.

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Temporal landscape of human gut RNA and DNA virome in SARS-CoV-2 infection and severity

Microbiome ◽

10.1186/s40168-021-01008-x ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

Tao Zuo ◽

Qin Liu ◽

Fen Zhang ◽

Yun Kit Yeoh ◽

Yating Wan ◽

...

Keyword(s):

Disease Severity ◽

Rna Virus ◽

Host Immunity ◽

Virus Species ◽

Human Gut ◽

Dna Virus ◽

Dna Viruses ◽

Fecal Samples ◽

Cell Counts ◽

Rna And Dna

Abstract Background Coronavirus disease 2019 (COVID-19) caused by the enveloped RNA virus SARS-CoV-2 primarily affects the respiratory and gastrointestinal tracts. SARS-CoV-2 was isolated from fecal samples, and active viral replication was reported in human intestinal cells. The human gut also harbors an enormous amount of resident viruses (collectively known as the virome) that play a role in regulating host immunity and disease pathophysiology. Understanding gut virome perturbation that underlies SARS-CoV-2 infection and severity is an unmet need. Methods We enrolled 98 COVID-19 patients with varying disease severity (3 asymptomatic, 53 mild, 34 moderate, 5 severe, 3 critical) and 78 non-COVID-19 controls matched for gender and co-morbidities. All subjects had fecal specimens sampled at inclusion. Blood specimens were collected for COVID-19 patients at admission to test for inflammatory markers and white cell counts. Among COVID-19 cases, 37 (38%) patients had serial fecal samples collected 2 to 3 times per week from time of hospitalization until after discharge. Using shotgun metagenomics sequencing, we sequenced and profiled the fecal RNA and DNA virome. We investigated alterations and longitudinal dynamics of the gut virome in association with disease severity and blood parameters. Results Patients with COVID-19 showed underrepresentation of Pepper mild mottle virus (RNA virus) and multiple bacteriophage lineages (DNA viruses) and enrichment of environment-derived eukaryotic DNA viruses in fecal samples, compared to non-COVID-19 subjects. Such gut virome alterations persisted up to 30 days after disease resolution. Fecal virome in SARS-CoV-2 infection harbored more stress-, inflammation-, and virulence-associated gene encoding capacities including those pertaining to bacteriophage integration, DNA repair, and metabolism and virulence associated with their bacterial host. Baseline fecal abundance of 10 virus species (1 RNA virus, pepper chlorotic spot virus, and 9 DNA virus species) inversely correlated with disease COVID-19 severity. These viruses inversely correlated with blood levels of pro-inflammatory proteins, white cells, and neutrophils. Among the 10 COVID-19 severity-associated DNA virus species, 4 showed inverse correlation with age; 5 showed persistent lower abundance both during disease course and after disease resolution relative to non-COVID-19 subjects. Conclusions Both enteric RNA and DNA virome in COVID-19 patients were different from non-COVID-19 subjects, which persisted after disease resolution of COVID-19. Gut virome may calibrate host immunity and regulate severity to SARS-CoV-2 infection. Our observation that gut viruses inversely correlated with both severity of COVID-19 and host age may partly explain that older subjects are prone to severe and worse COVID-19 outcomes. Altogether, our data highlight the importance of human gut virome in severity and potentially therapeutics of COVID-19.

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Artificial Small RNA-Based Silencing Tools for Antiviral Resistance in Plants

Plants ◽

10.3390/plants9060669 ◽

2020 ◽

Vol 9 (6) ◽

pp. 669 ◽

Cited By ~ 1

Author(s):

Adriana E. Cisneros ◽

Alberto Carbonell

Keyword(s):

Small Rnas ◽

Rational Design ◽

High Specificity ◽

Plant Viruses ◽

Antiviral Resistance ◽

Small Interfering Rnas ◽

Crop Species ◽

Dna Viruses ◽

Induce Resistance ◽

Rna And Dna

Artificial small RNAs (art-sRNAs), such as artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs), are highly specific 21-nucleotide small RNAs designed to recognize and silence complementary target RNAs. Art-sRNAs are extensively used in gene function studies or for improving crops, particularly to protect plants against viruses. Typically, antiviral art-sRNAs are computationally designed to target one or multiple sites in viral RNAs with high specificity, and art-sRNA constructs are generated and introduced into plants that are subsequently challenged with the target virus(es). Numerous studies have reported the successful application of art-sRNAs to induce resistance against a large number of RNA and DNA viruses in model and crop species. However, the application of art-sRNAs as an antiviral tool has limitations, such as the difficulty to predict the efficacy of a particular art-sRNA or the emergence of virus variants with mutated target sites escaping to art-sRNA-mediated degradation. Here, we review the different classes, features, and uses of art-sRNA-based tools to induce antiviral resistance in plants. We also provide strategies for the rational design of antiviral art-sRNAs and discuss the latest advances in developing art-sRNA-based methodologies for enhanced resistance to plant viruses.

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Flavonoids as Antiviral Agents for Enterovirus A71 (EV-A71)

Viruses ◽

10.3390/v12020184 ◽

2020 ◽

Vol 12 (2) ◽

pp. 184 ◽

Cited By ~ 15

Author(s):

Salima Lalani ◽

Chit Laa Poh

Keyword(s):

Molecular Mechanisms ◽

Molecular Level ◽

Rna Virus ◽

Antiviral Treatment ◽

Antiviral Agents ◽

Foot And Mouth Disease ◽

Dna Viruses ◽

Enterovirus A71 ◽

Causative Agents ◽

Rna And Dna

Flavonoids are natural biomolecules that are known to be effective antivirals. These biomolecules can act at different stages of viral infection, particularly at the molecular level to inhibit viral growth. Enterovirus A71 (EV-A71), a non-enveloped RNA virus, is one of the causative agents of hand, foot and mouth disease (HFMD), which is prevalent in Asia. Despite much effort, no clinically approved antiviral treatment is available for children suffering from HFMD. Flavonoids from plants serve as a vast reservoir of therapeutically active constituents that have been explored as potential antiviral candidates against RNA and DNA viruses. Here, we reviewed flavonoids as evidence-based natural sources of antivirals against non-picornaviruses and picornaviruses. The detailed molecular mechanisms involved in the inhibition of EV-A71 infections are discussed.

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Engineering RNA Virus Interference via the CRISPR/Cas13 Machinery in Arabidopsis

Viruses ◽

10.3390/v10120732 ◽

2018 ◽

Vol 10 (12) ◽

pp. 732 ◽

Cited By ~ 22

Author(s):

Rashid Aman ◽

Ahmed Mahas ◽

Haroon Butt ◽

Fatimah Aljedaani ◽

Magdy Mahfouz

Keyword(s):

Genome Engineering ◽

Rna Viruses ◽

Rna Virus ◽

Basic Research ◽

Dna Viruses ◽

Eukaryotic Species ◽

Exciting Potential ◽

Rna Genome ◽

Target Rna ◽

Rna And Dna

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems are key immune mechanisms helping prokaryotic species fend off RNA and DNA viruses. CRISPR/Cas9 has broad applications in basic research and biotechnology and has been widely used across eukaryotic species for genome engineering and functional analysis of genes. The recently developed CRISPR/Cas13 systems target RNA rather than DNA and thus offer new potential for transcriptome engineering and combatting RNA viruses. Here, we used CRISPR/LshCas13a to stably engineer Arabidopsis thaliana for interference against the RNA genome of Turnip mosaic virus (TuMV). Our data demonstrate that CRISPR RNAs (crRNAs) guiding Cas13a to the sequences encoding helper component proteinase silencing suppressor (HC-Pro) or GFP target 2 (GFP-T2) provide better interference compared to crRNAs targeting other regions of the TuMV RNA genome. This work demonstrates the exciting potential of CRISPR/Cas13 to be used as an antiviral strategy to obstruct RNA viruses, and encourages the search for more robust and effective Cas13 variants or CRISPR systems that can target RNA.

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Evaluation of Ultraviolet-C Light for Rapid Decontamination of Airport Security Bins in the Era of SARS-CoV-2

Pathogens and Immunity ◽

10.20411/pai.v5i1.373 ◽

2020 ◽

Vol 5 (1) ◽

pp. 133 ◽

Cited By ~ 5

Author(s):

Jennifer Cadnum ◽

Daniel F. Li ◽

Lucas D. Jones ◽

Sarah N. Redmond ◽

Basya Pearlmutter ◽

...

Keyword(s):

Rna Virus ◽

Test Method ◽

Airport Security ◽

Dna Viruses ◽

Bacteriophage Ms2 ◽

Test Organisms ◽

Ultraviolet C ◽

American Society ◽

Uv C ◽

Rna And Dna

Background: Contaminated surfaces are a potential source for spread of respiratory viruses including SARS-CoV-2. Ultraviolet-C (UV-C) light is effective against RNA and DNA viruses and could be useful for decontamination of high-touch fomites that are shared by multiple users. Methods: A modification of the American Society for Testing and Materials standard quantitative carrier disk test method (ASTM E-2197-11) was used to examine the effectiveness of ultraviolet-C (UV-C) light for rapid decontamination of plastic airport security bins inoculated at 3 sites with methicillin-resistant Staphylococcus aureus (MRSA) and bacteriophages MS2, PhiX174, and Phi6, an enveloped RNA virus used as a surrogate for coronaviruses. Three log10 reductions on inoculated plastic bins were considered effective for decontamination. Results: UV-C light administered as 10-, 20-, or 30-second cycles in proximity to a plastic bin reduced contamination on each of the test sites, including vertical and horizontal surfaces. The 30-second cycle met criteria for decontamination of all 3 test sites for all the test organisms except bacteriophage MS2 which was reduced by greater than 2 log10 PFU at each site. Conclusions: UV-C light is an attractive technology for rapid decontamination of airport security bins. Further work is needed to evaluate the utility of UV-C light in real-world settings and to develop methods to provide automated movement of bins through a UV-C decontamination process.

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Density dependent enhancement effect ofWolbachiaand the host RNAi response to a densovirus inAedescells

10.1101/441584 ◽

2018 ◽

Author(s):

Rhys Parry ◽

Cameron Bishop ◽

Lachlan de Hayr ◽

Sassan Asgari

Keyword(s):

Enhancement Effect ◽

Dependent Manner ◽

Dna Virus ◽

Dna Viruses ◽

Density Dependent ◽

Host Interaction ◽

Ping Pong ◽

Infected Cells ◽

First Time ◽

Rnai Response

AbstractThe endosymbiotic bacteriumWolbachia pipientishas been shown to restrict a range of RNA viruses inDrosophila melanogasterand transinfected dengue mosquito,Aedes aegypti. Here, we show thatWolbachiainfection enhances replication of Aedes albopictus densovirus (AalDNV-1), a single stranded DNA virus, inAedescell lines in a density-dependent manner. Analysis of previously produced small RNAs of Aag2 cells showed thatWolbachia-infected cells produced greater proportions of viral derived short interfering RNAs as compared to uninfected cells. Additionally, we found production of viral derived PIWI-like RNAs (vpiRNA) produced in response to AalDNV-1 infection. Nuclear fractions of Aag2 cells produced a primary vpiRNA signature U1bias whereas the typical “ping-pong” signature (U1- A10) was evident in the cytoplasmic fraction. This is the first report of the density-dependent enhancement of DNA viruses byWolbachia. Further, we report the generation of vpiRNAs in a DNA virus-host interaction for the first time.

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The discovery, distribution and diversity of DNA viruses associated with Drosophila melanogaster in Europe

10.1101/2020.10.16.342956 ◽

2020 ◽

Cited By ~ 1

Author(s):

Megan A. Wallace ◽

Kelsey A. Coffman ◽

Clément Gilbert ◽

Sanjana Ravindran ◽

Gregory F. Albery ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Rna Virus ◽

Large Population ◽

Experimental Studies ◽

Low Frequency ◽

Dna Virus ◽

Dna Viruses ◽

Virus Prevalence ◽

Salivary Gland Hypertrophy ◽

Salivary Gland Hypertrophy Virus

AbstractDrosophila melanogaster is an important model for antiviral immunity in arthropods, but very few DNA viruses have been described in association with the Drosophilidae. This has limited the opportunity to use natural host-pathogen combinations in experimental studies, and may have biased our understanding of the Drosophila virome. Here we describe fourteen DNA viruses detectable by metagenomic analysis of 6.5 thousand pool-sequenced Drosophila, sampled from 47 European locations between 2014 and 2016. These include three new Nudiviruses, a new and divergent Entomopox virus, a virus related to Leptopilina boulardi filamentous virus, and a virus related to Musca domestica salivary gland hypertrophy virus. We also find an endogenous genomic copy of Galbut virus, an RNA Partitivirus, segregating at very low frequency. Remarkably, we show that Vesanto virus, a small DNA virus previously described as a Bidnavirus, may be composed of up to 12 segments and represents a new lineage of segmented DNA viruses. Only two of the DNA viruses, Kallithea virus (Nudiviridae) and Vesanto virus (Bidna-virus like) are common, being found in 2% or more of wild flies. The other viruses are rare, with many likely to be represented by a single infected fly in the collection. We find that virus prevalence in Europe reflects that seen in publicly-available datasets, with Kallithea virus and Vesanto virus being commonly detectable in data from wild-caught flies and large population cages, and the others being rare or absent. These analyses suggest that DNA viruses are generally rarer than RNA viruses in D. melanogaster, and may be less likely to persist in laboratory cultures. Our findings go some way to redress the earlier bias toward RNA virus studies in Drosophila, and lay the foundation needed to harness the power of Drosophila as a model system for the study of DNA viruses.

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piRNA-Guided Genome Defense: From Biogenesis to Silencing

Annual Review of Genetics ◽

10.1146/annurev-genet-120417-031441 ◽

2018 ◽

Vol 52 (1) ◽

pp. 131-157 ◽

Cited By ~ 89

Author(s):

Benjamin Czech ◽

Marzia Munafò ◽

Filippo Ciabrelli ◽

Evelyn L. Eastwood ◽

Martin H. Fabry ◽

...

Keyword(s):

Transcriptional Gene Silencing ◽

Cytoplasmic Process ◽

Main Function ◽

Argonaute Proteins ◽

Repressive Chromatin ◽

Genome Defense ◽

Ping Pong ◽

Sequence Complementarity ◽

And Function ◽

Pirna Pathway

PIWI-interacting RNAs (piRNAs) and their associated PIWI clade Argonaute proteins constitute the core of the piRNA pathway. In gonadal cells, this conserved pathway is crucial for genome defense, and its main function is to silence transposable elements. This is achieved through posttranscriptional and transcriptional gene silencing. Precursors that give rise to piRNAs require specialized transcription and transport machineries because piRNA biogenesis is a cytoplasmic process. The ping-pong cycle, a posttranscriptional silencing mechanism, combines the cleavage-dependent silencing of transposon RNAs with piRNA production. PIWI proteins also function in the nucleus, where they scan for nascent target transcripts with sequence complementarity, instructing transcriptional silencing and deposition of repressive chromatin marks at transposon loci. Although studies have revealed numerous factors that participate in each branch of the piRNA pathway, the precise molecular roles of these factors often remain unclear. In this review, we summarize our current understanding of the mechanisms involved in piRNA biogenesis and function.

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