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1
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Amono R, Markussen T, Singh VK, Lund M, Manji F, Mor SK, Evensen Ø, Mikalsen AB. Unraveling the genomic landscape of piscine myocarditis virus: mutation frequencies, viral diversity and evolutionary dynamics in Atlantic salmon. Virus Evol 2024; 10:veae097. [PMID: 39717704 PMCID: PMC11665822 DOI: 10.1093/ve/veae097] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2024] [Revised: 11/06/2024] [Accepted: 11/15/2024] [Indexed: 12/25/2024] Open
Abstract
Over a decade since its discovery, piscine myocarditis virus (PMCV) remains a significant pathogen in Atlantic salmon aquaculture. Despite this significant impact, the genomic landscape, evolutionary dynamics, and virulence factors of PMCV are poorly understood. This study enhances the existing PMCV sequence dataset by adding 34 genome sequences and 202 new ORF3 sequences from clinical cardiomyopathy syndrome (CMS) cases in Norwegian aquaculture. Phylogenetic analyses, also including sequences from the Faroe Islands and Ireland revealed that PMCV sequences are highly conserved with distinct clustering by country of origin. Still, single CMS outbreaks display multiple PMCV variants, and although some clustering was seen by case origin, occasional grouping of sequences from different cases was also apparent. Temporal data from selected cases indicated increased sequence diversity in the population. We hypothesize that multiple bottlenecks and changing infection dynamics in the host population, with transfer to naïve individuals over time, represent a continuous selection pressure on the virus populations. No clear relation was found between PMCV variants and the severity of heart pathology. However, specific non-synonymous and synonymous mutations that might impact protein function and gene expression efficiency were identified. An additional factor that may impact PMCV replication is the presence of defective viral genomes, a novel finding for viruses of the order Ghabrivirales. This study provides new insights into PMCV genomic characteristics and evolutionary dynamics, highlighting the complex interplay of genetic diversity, virulence markers, and host-pathogen interactions, underscoring the epidemiological complexity of the virus. Keywords: piscine myocarditis virus; evolutionary dynamics; diversity; phylogeny; genomic sequencing; defective viral genomes.
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Affiliation(s)
- Racheal Amono
- Department of Paraclinical Sciences, Norwegian University of Life Sciences, Post box 5003, Ås 1432, Norway
| | - Turhan Markussen
- Department of Paraclinical Sciences, Norwegian University of Life Sciences, Post box 5003, Ås 1432, Norway
| | - Vikash K Singh
- Department of Veterinary Population Medicine and Veterinary Diagnostic Laboratory, University of Minnesota, 1333 Gortner Avenue, St. Paul, MN 55108, United States
| | - Morten Lund
- PatoGen AS, Rasmus Rønnebergs Gate 21, Ålesund 6002, Norway
| | - Farah Manji
- Mowi ASA, Post box 4102, Bergen 5835, Norway
| | - Sunil K Mor
- Department of Veterinary Population Medicine and Veterinary Diagnostic Laboratory, University of Minnesota, 1333 Gortner Avenue, St. Paul, MN 55108, United States
- Department of Veterinary and Biomedical Sciences and Animal Disease Research & Diagnostic Laboratory, South Dakota State University, Post box 2175 University Station, Brookings, SD 57007, USA
| | - Øystein Evensen
- Department of Paraclinical Sciences, Norwegian University of Life Sciences, Post box 5003, Ås 1432, Norway
| | - Aase B Mikalsen
- Department of Paraclinical Sciences, Norwegian University of Life Sciences, Post box 5003, Ås 1432, Norway
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2
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Henke D, Piedra FA, Avadhanula V, Doddapaneni H, Muzny DM, Menon VK, Hoffman KL, Ross MC, Javornik Cregeen SJ, Metcalf G, Gibbs RA, Petrosino JF, Piedra PA. Examining intra-host genetic variation of RSV by short read high-throughput sequencing. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2023.05.17.541198. [PMID: 39282457 PMCID: PMC11398394 DOI: 10.1101/2023.05.17.541198] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 09/20/2024]
Abstract
Every viral infection entails an evolving population of viral genomes. High-throughput sequencing technologies can be used to characterize such populations, but to date there are few published examples of such work. In addition, mixed sequencing data are sometimes used to infer properties of infecting genomes without discriminating between genome-derived reads and reads from the much more abundant, in the case of a typical active viral infection, transcripts. Here we apply capture probe-based short read high-throughput sequencing to nasal wash samples taken from a previously described group of adult hematopoietic cell transplant (HCT) recipients naturally infected with respiratory syncytial virus (RSV). We separately analyzed reads from genomes and transcripts for the levels and distribution of genetic variation by calculating per position Shannon entropies. Our analysis reveals a low level of genetic variation within the RSV infections analyzed here, but with interesting differences between genomes and transcripts in 1) average per sample Shannon entropies; 2) the genomic distribution of variation 'hotspots'; and 3) the genomic distribution of hotspots encoding alternative amino acids. In all, our results suggest the importance of separately analyzing reads from genomes and transcripts when interpreting high-throughput sequencing data for insight into intra-host viral genome replication, expression, and evolution.
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Affiliation(s)
- David Henke
- Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA
| | - Felipe-Andrés Piedra
- Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA
| | - Vasanthi Avadhanula
- Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA
| | - Harsha Doddapaneni
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA
| | - Donna M. Muzny
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA
| | - Vipin K. Menon
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA
| | - Kristi L. Hoffman
- Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA
| | - Matthew C. Ross
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA
| | | | - Ginger Metcalf
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA
| | - Richard A. Gibbs
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA
| | - Joseph F. Petrosino
- Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA
| | - Pedro A. Piedra
- Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA
- Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA
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3
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Ferreira Sa Antunes T, Huguet-Tapia JC, Elena SF, Folimonova SY. Intra-Host Citrus Tristeza Virus Populations during Prolonged Infection Initiated by a Well-Defined Sequence Variant in Nicotiana benthamiana. Viruses 2024; 16:1385. [PMID: 39339861 PMCID: PMC11437405 DOI: 10.3390/v16091385] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2024] [Revised: 08/24/2024] [Accepted: 08/27/2024] [Indexed: 09/30/2024] Open
Abstract
Due to the error-prone nature of viral RNA-dependent RNA polymerases, the replication of RNA viruses results in a diversity of viral genomes harboring point mutations, deletions, insertions, and genome rearrangements. Citrus tristeza virus (CTV), a causal agent of diseases of economically important citrus species, shows intrinsic genetic stability. While the virus appears to have some mechanism that limits the accumulation of single-nucleotide variants, the production of defective viral genomes (DVGs) during virus infection has been reported for certain variants of CTV. The intra-host diversity generated during plant infection with variant T36 (CTV-T36) remains unclear. To address this, we analyzed the RNA species accumulated in the initially infected and systemic leaves of Nicotiana benthamiana plants inoculated with an infectious cDNA clone of CTV-T36, which warranted that infection was initiated by a known, well-defined sequence variant of the virus. CTV-T36 limited the accumulation of single-nucleotide mutants during infection. With that, four types of DVGs-deletions, insertions, and copy- and snap-backs-were found in all the samples, with deletions and insertions being the most common types. Hot-spots across the genome for DVG recombination and short direct sequence repeats suggest that sequence complementarity could mediate DVG formation. In conclusion, our study illustrates the formation of diverse DVGs during CTV-T36 infection. To the best of our knowledge, this is the first study that has analyzed the genetic variability and recombination of a well-defined sequence variant of CTV in an herbaceous host.
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Affiliation(s)
| | - José C. Huguet-Tapia
- Department of Plant Pathology, University of Florida, Gainesville, FL 32611, USA; (T.F.S.A.); (J.C.H.-T.)
| | - Santiago F. Elena
- Instituto de Biología Integrativa de Sistemas (I2SysBio), CSIC-Universitat de València, 46980 Valencia, Spain;
- Santa Fe Institute, Santa Fe, NM 87501, USA
| | - Svetlana Y. Folimonova
- Department of Plant Pathology, University of Florida, Gainesville, FL 32611, USA; (T.F.S.A.); (J.C.H.-T.)
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4
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Zhang S, Yang C, Qiu Y, Liao R, Xuan Z, Ren F, Dong Y, Xie X, Han Y, Wu D, Ramos-González PL, Freitas-Astúa J, Yang H, Zhou C, Cao M. Conserved untranslated regions of multipartite viruses: Natural markers of novel viral genomic components and tags of viral evolution. Virus Evol 2024; 10:veae004. [PMID: 38361819 PMCID: PMC10868557 DOI: 10.1093/ve/veae004] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2023] [Revised: 12/20/2023] [Accepted: 01/09/2024] [Indexed: 02/17/2024] Open
Abstract
Viruses with split genomes are classified as being either segmented or multipartite based on whether their genomic segments occur within a single virion or across different virions. Despite variations in number and sequence during evolution, the genomic segments of many viruses are conserved within the untranslated regions (UTRs). In this study, we present a methodology that combines RNA sequencing with iterative BLASTn of UTRs (https://github.com/qq371260/Iterative-blast-v.1.0) to identify new viral genomic segments. Some novel multipartite-like viruses related to the phylum Kitrinoviricota were annotated using sequencing data from field plant samples and public databases. We identified potentially plant-infecting jingmen-related viruses (order Amarillovirales) and jivi-related viruses (order Martellivirales) with at least six genomic components. The number of RNA molecules associated with a genome of the novel viruses in the families Closteroviridae, Kitaviridae, and Virgaviridae within the order Martellivirales reached five. Several of these viruses seem to represent new taxa at the subgenus, genus, and family levels. The diversity of novel genomic components and the multiple duplication of proteins or protein domains within single or multiple genomic components emphasize the evolutionary roles of genetic recombination (horizontal gene transfer), reassortment, and deletion. The relatively conserved UTRs at the genome level might explain the relationships between monopartite and multipartite viruses, as well as how subviral agents such as defective RNAs and satellite viruses can coexist with their helper viruses.
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Affiliation(s)
| | - Caixia Yang
- Liaoning Key Laboratory of Urban Integrated Pest Management and Ecological Security, College of Life Science and Engineering, Shenyang University, 21 Huanan Street, Shenyang, Liaoning 110044, China
| | - Yuanjian Qiu
- National Citrus Engineering and Technology Research Center, Integrative Science Center of Germplasm Creation in Western China (CHONGQING) Science City, Citrus Research Institute, Southwest University, 2 Tiansheng Road, Beibei, Chongqing 400712, China
| | - Ruiling Liao
- National Citrus Engineering and Technology Research Center, Integrative Science Center of Germplasm Creation in Western China (CHONGQING) Science City, Citrus Research Institute, Southwest University, 2 Tiansheng Road, Beibei, Chongqing 400712, China
| | - Zhiyou Xuan
- National Citrus Engineering and Technology Research Center, Integrative Science Center of Germplasm Creation in Western China (CHONGQING) Science City, Citrus Research Institute, Southwest University, 2 Tiansheng Road, Beibei, Chongqing 400712, China
| | - Fang Ren
- Research Institute of Pomology, Chinese Academy of Agricultural Sciences, 98 Xinghainan Street, Xingcheng, Liaoning 125100, China
| | - Yafeng Dong
- Research Institute of Pomology, Chinese Academy of Agricultural Sciences, 98 Xinghainan Street, Xingcheng, Liaoning 125100, China
| | - Xiaoying Xie
- Vector-borne Virus Research Center, College of Plant Protection, Fujian Agriculture and Forestry University, 15 Shangxiadian Road, Fuzhou, Fujian 350002, China
| | - Yanhong Han
- Vector-borne Virus Research Center, College of Plant Protection, Fujian Agriculture and Forestry University, 15 Shangxiadian Road, Fuzhou, Fujian 350002, China
| | - Di Wu
- College of Horticulture and Landscape Architecture, Southwest University, 2 Tiansheng Road, Beibei, Chongqing 400712, China
| | - Pedro Luis Ramos-González
- Laboratório de Biologia Molecular Aplicada, Instituto Biológico, Av. Cons. Rodrigues Alves 1252, São Paulo SP, 04014-002, Brazil
| | - Juliana Freitas-Astúa
- Laboratório de Biologia Molecular Aplicada, Instituto Biológico, Av. Cons. Rodrigues Alves 1252, São Paulo SP, 04014-002, Brazil
- Embrapa Mandioca e Fruticultura, Rua da Embrapa, Caixa Postal 007, CEP, Cruz das Almas BA, 44380-000, Brazil
| | - Huadong Yang
- Hunan Agricultural University, 1 Nongda Road, Changsha, Hunan 410125, China
| | - Changyong Zhou
- National Citrus Engineering and Technology Research Center, Integrative Science Center of Germplasm Creation in Western China (CHONGQING) Science City, Citrus Research Institute, Southwest University, 2 Tiansheng Road, Beibei, Chongqing 400712, China
- Guangxi Citrus Breeding and Cultivation Technology Innovation Center, Guangxi Academy of Specialty Crops, 40 Putuo Road, Guilin, Guangxi 541010, China
- Guangxi Key Laboratory of Germplasm Innovation and Utilization of Specialty Commercial Crops in North Guangxi, Guangxi Academy of Specialty Crops, 40 Putuo Road, Guilin, Guangxi 541010, China
| | - Mengji Cao
- National Citrus Engineering and Technology Research Center, Integrative Science Center of Germplasm Creation in Western China (CHONGQING) Science City, Citrus Research Institute, Southwest University, 2 Tiansheng Road, Beibei, Chongqing 400712, China
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5
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Nemes K, Gil JF, Liebe S, Mansi M, Poimenopoulou E, Lennefors BL, Varrelmann M, Savenkov EI. Intermolecular base-pairing interactions, a unique topology and exoribonuclease-resistant noncoding RNAs drive formation of viral chimeric RNAs in plants. THE NEW PHYTOLOGIST 2024; 241:861-877. [PMID: 37897070 DOI: 10.1111/nph.19346] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/04/2023] [Accepted: 10/02/2023] [Indexed: 10/29/2023]
Abstract
In plants, exoribonuclease-resistant RNAs (xrRNAs) are produced by many viruses. Whereas xrRNAs contribute to the pathogenicity of these viruses, the role of xrRNAs in the virus infectious cycle remains elusive. Here, we show that xrRNAs produced by a benyvirus (a multipartite RNA virus with four genomic segments) in plants are involved in the formation of monocistronic coat protein (CP)-encoding chimeric RNAs. Naturally occurring chimeric RNAs, we discovered, are composed of 5'-end of RNA 2 and 3'-end of either RNA 3 or RNA 4 bearing conservative exoribonuclease-resistant 'coremin' region. Using computational tools and site-directed mutagenesis, we show that de novo formation of chimeric RNAs requires intermolecular base-pairing interaction between 'coremin' and 3'-proximal part of the CP gene of RNA 2 as well as a stem-loop structure immediately adjacent to the CP gene. Moreover, knockdown of the expression of the XRN4 gene, encoding 5'→3' exoribonuclease, inhibits biogenesis of both xrRNAs and chimeric RNAs. Our findings suggest a novel mechanism involving a unique tropology of the intermolecular base-pairing complex between xrRNAs and RNA2 to promote formation of chimeric RNAs in plants. XrRNAs, essential for chimeric RNA biogenesis, are generated through the action of cytoplasmic Xrn 4 5'→3' exoribonuclease conserved in all plant species.
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Affiliation(s)
- Katalin Nemes
- Department of Plant Biology, Linnean Center for Plant Biology, Swedish University of Agricultural Sciences (SLU), Uppsala, 75007, Sweden
| | - Jose F Gil
- Department of Plant Biology, Linnean Center for Plant Biology, Swedish University of Agricultural Sciences (SLU), Uppsala, 75007, Sweden
- VEDAS Corporación de Investigación e Innovación (VEDAS CII), Medellín, 050024, Colombia
| | - Sebastian Liebe
- Department of Phytopathology, Institute of Sugar Beet Research, Göttingen, 37079, Germany
| | - Mansi Mansi
- Department of Plant Biology, Linnean Center for Plant Biology, Swedish University of Agricultural Sciences (SLU), Uppsala, 75007, Sweden
| | - Efstratia Poimenopoulou
- Department of Plant Biology, Linnean Center for Plant Biology, Swedish University of Agricultural Sciences (SLU), Uppsala, 75007, Sweden
| | | | - Mark Varrelmann
- Department of Phytopathology, Institute of Sugar Beet Research, Göttingen, 37079, Germany
| | - Eugene I Savenkov
- Department of Plant Biology, Linnean Center for Plant Biology, Swedish University of Agricultural Sciences (SLU), Uppsala, 75007, Sweden
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6
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Tercero B, Makino S. Reverse Genetics System for Rift Valley Fever Virus. Methods Mol Biol 2024; 2733:101-113. [PMID: 38064029 DOI: 10.1007/978-1-0716-3533-9_7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2023]
Abstract
Rift Valley fever virus (RVFV) is an important mosquito-borne virus that can cause severe disease manifestations in humans including ocular damage, vision loss, late-onset encephalitis, and hemorrhagic fever. In ruminants, RVFV can cause high mortality rates in young animals and high rates of abortion in pregnant animals resulting in an enormous negative impact on the economy of affected regions. To date, no licensed vaccines in humans or anti-RVFV therapeutics for animal or human use are available. The development of reverse genetics has facilitated the generation of recombinant infectious viruses that serve as powerful tools for investigating the molecular biology and pathogenesis of RVFV. Infectious recombinant RVFV can be rescued entirely from cDNAs containing predetermined mutations in their genomes to investigate virus-host interactions and mechanisms of pathogenesis and generate live-attenuated vaccines. In this chapter, we will describe the experimental procedures for the implementation of RVFV reverse genetics.
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Affiliation(s)
- Breanna Tercero
- Departments of Microbiology and Immunology, Galveston, TX, USA
| | - Shinji Makino
- Departments of Microbiology and Immunology, Galveston, TX, USA.
- Institute of Human Infection and Immunity, Galveston, TX, USA.
- Center for Biodefense and Emerging Infectious Diseases, Galveston, TX, USA.
- UTMB Center for Tropical Diseases, Galveston, TX, USA.
- The Sealy Institute for Vaccine Sciences, Galveston, TX, USA.
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7
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Abstract
Robust plant immune systems are fine-tuned by both protein-coding genes and non-coding RNAs. Long non-coding RNAs (lncRNAs) refer to RNAs with a length of more than 200 nt and usually do not have protein-coding function and do not belong to any other well-known non-coding RNA types. The non-protein-coding, low expression, and non-conservative characteristics of lncRNAs restrict their recognition. Although studies of lncRNAs in plants are in the early stage, emerging studies have shown that plants employ lncRNAs to regulate plant immunity. Moreover, in response to stresses, numerous lncRNAs are differentially expressed, which manifests the actions of low-expressed lncRNAs and makes plant-microbe/insect interactions a convenient system to study the functions of lncRNAs. Here, we summarize the current advances in plant lncRNAs, discuss their regulatory effects in different stages of plant immunity, and highlight their roles in diverse plant-microbe/insect interactions. These insights will not only strengthen our understanding of the roles and actions of lncRNAs in plant-microbe/insect interactions but also provide novel insight into plant immune responses and a basis for further research in this field.
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Affiliation(s)
- Juan Huang
- State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
- CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of Sciences, Beijing, China
| | - Wenling Zhou
- State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
- CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of Sciences, Beijing, China
| | - Xiaoming Zhang
- State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
- CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of Sciences, Beijing, China
- HainanYazhou Bay Seed Lab, Sanya, China
| | - Yi Li
- State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, China
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8
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Leeks A, Young PG, Turner PE, Wild G, West SA. Cheating leads to the evolution of multipartite viruses. PLoS Biol 2023; 21:e3002092. [PMID: 37093882 PMCID: PMC10159356 DOI: 10.1371/journal.pbio.3002092] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2022] [Revised: 05/04/2023] [Accepted: 03/22/2023] [Indexed: 04/25/2023] Open
Abstract
In multipartite viruses, the genome is split into multiple segments, each of which is transmitted via a separate capsid. The existence of multipartite viruses poses a problem, because replication is only possible when all segments are present within the same host. Given this clear cost, why is multipartitism so common in viruses? Most previous hypotheses try to explain how multipartitism could provide an advantage. In so doing, they require scenarios that are unrealistic and that cannot explain viruses with more than 2 multipartite segments. We show theoretically that selection for cheats, which avoid producing a shared gene product, but still benefit from gene products produced by other genomes, can drive the evolution of both multipartite and segmented viruses. We find that multipartitism can evolve via cheating under realistic conditions and does not require unreasonably high coinfection rates or any group-level benefit. Furthermore, the cheating hypothesis is consistent with empirical patterns of cheating and multipartitism across viruses. More broadly, our results show how evolutionary conflict can drive new patterns of genome organisation in viruses and elsewhere.
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Affiliation(s)
- Asher Leeks
- Department of Ecology and Evolutionary Biology, Yale University, New Haven, Connecticut, United States of America
- Department of Biology, University of Oxford, Oxford, United Kingdom
| | | | - Paul Eugene Turner
- Department of Ecology and Evolutionary Biology, Yale University, New Haven, Connecticut, United States of America
- Quantitative Biology Institute, Yale University, New Haven, Connecticut, United States of America
| | - Geoff Wild
- Department of Mathematics, The University of Western Ontario, London, Canada
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9
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Atari N, Erster O, Shteinberg YH, Asraf H, Giat E, Mandelboim M, Goldstein I. Proof-of-concept for effective antiviral activity of an in silico designed decoy synthetic mRNA against SARS-CoV-2 in the Vero E6 cell-based infection model. Front Microbiol 2023; 14:1113697. [PMID: 37152730 PMCID: PMC10157240 DOI: 10.3389/fmicb.2023.1113697] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2022] [Accepted: 04/03/2023] [Indexed: 05/09/2023] Open
Abstract
The positive-sense single-stranded (ss) RNA viruses of the Betacoronavirus (beta-CoV) genus can spillover from mammals to humans and are an ongoing threat to global health and commerce, as demonstrated by the current zoonotic pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Current anti-viral strategies focus on vaccination or targeting key viral proteins with antibodies and drugs. However, the ongoing evolution of new variants that evade vaccination or may become drug-resistant is a major challenge. Thus, antiviral compounds that circumvent these obstacles are needed. Here we describe an innovative antiviral modality based on in silico designed fully synthetic mRNA that is replication incompetent in uninfected cells (termed herein PSCT: parasitic anti-SARS-CoV-2 transcript). The PSCT sequence was engineered to include key untranslated cis-acting regulatory RNA elements of the SARS-CoV-2 genome, so as to effectively compete for replication and packaging with the standard viral genome. Using the Vero E6 cell-culture based SARS-CoV-2 infection model, we determined that the intracellular delivery of liposome-encapsulated PSCT at 1 hour post infection significantly reduced intercellular SARS-CoV-2 replication and release into the extracellular milieu as compared to mock treatment. In summary, our findings are a proof-of-concept for the therapeutic feasibility of in silico designed mRNA compounds formulated to hinder the replication and packaging of ssRNA viruses sharing a comparable genomic-structure with beta-CoVs.
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Affiliation(s)
- Nofar Atari
- Central Virology Laboratory, Public Health Services, Ministry of Health, Sheba Medical Center, Tel HaShomer, Israel
- Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel
| | - Oran Erster
- Central Virology Laboratory, Public Health Services, Ministry of Health, Sheba Medical Center, Tel HaShomer, Israel
| | | | - Hadar Asraf
- Central Virology Laboratory, Public Health Services, Ministry of Health, Sheba Medical Center, Tel HaShomer, Israel
| | - Eitan Giat
- Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel
- The Department of Medicine, Sheba Medical Center, Ramat Gan, Israel
| | - Michal Mandelboim
- Central Virology Laboratory, Public Health Services, Ministry of Health, Sheba Medical Center, Tel HaShomer, Israel
- The Department of Medicine, Sheba Medical Center, Ramat Gan, Israel
| | - Itamar Goldstein
- Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel
- The Department of Medicine, Sheba Medical Center, Ramat Gan, Israel
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10
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Budzyńska D, Zwart MP, Hasiów-Jaroszewska B. Defective RNA Particles of Plant Viruses-Origin, Structure and Role in Pathogenesis. Viruses 2022; 14:2814. [PMID: 36560818 PMCID: PMC9786237 DOI: 10.3390/v14122814] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2022] [Revised: 12/14/2022] [Accepted: 12/15/2022] [Indexed: 12/23/2022] Open
Abstract
The genomes of RNA viruses may be monopartite or multipartite, and sub-genomic particles such as defective RNAs (D RNAs) or satellite RNAs (satRNAs) can be associated with some of them. D RNAs are small, deletion mutants of a virus that have lost essential functions for independent replication, encapsidation and/or movement. D RNAs are common elements associated with human and animal viruses, and they have been described for numerous plant viruses so far. Over 30 years of studies on D RNAs allow for some general conclusions to be drawn. First, the essential condition for D RNA formation is prolonged passaging of the virus at a high cellular multiplicity of infection (MOI) in one host. Second, recombination plays crucial roles in D RNA formation. Moreover, during virus propagation, D RNAs evolve, and the composition of the particle depends on, e.g., host plant, virus isolate or number of passages. Defective RNAs are often engaged in transient interactions with full-length viruses-they can modulate accumulation, infection dynamics and virulence, and are widely used, i.e., as a tool for research on cis-acting elements crucial for viral replication. Nevertheless, many questions regarding the generation and role of D RNAs in pathogenesis remain open. In this review, we summarise the knowledge about D RNAs of plant viruses obtained so far.
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Affiliation(s)
- Daria Budzyńska
- Department of Virology and Bacteriology, Institute of Plant Protection-National Research Institute, Wl Wegorka 20, 60-318 Poznan, Poland
| | - Mark P. Zwart
- Department of Microbial Ecology, Netherlands Institute of Ecology (NIOO-KNAW), Droevendaalsesteeg 10, 6708 PB Wageningen, The Netherlands
| | - Beata Hasiów-Jaroszewska
- Department of Virology and Bacteriology, Institute of Plant Protection-National Research Institute, Wl Wegorka 20, 60-318 Poznan, Poland
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11
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Genetic Diversity of Tomato Black Ring Virus Satellite RNAs and Their Impact on Virus Replication. Int J Mol Sci 2022; 23:ijms23169393. [PMID: 36012656 PMCID: PMC9409425 DOI: 10.3390/ijms23169393] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2022] [Revised: 08/17/2022] [Accepted: 08/19/2022] [Indexed: 11/16/2022] Open
Abstract
Viral satellite RNAs (satRNAs) are small subviral particles that are associated with the genomic RNA of a helper virus (HV). Their replication, encapsidation, and movement depend on the HV. In this paper, we performed a global analysis of the satRNAs associated with different isolates of tomato black ring virus (TBRV). We checked the presence of satRNAs in 42 samples infected with TBRV, performed recombination and genetic diversity analyses, and examined the selective pressure affecting the satRNAs population. We identified 18 satRNAs in total that differed in length and the presence of point mutations. Moreover, we observed a strong effect of selection operating upon the satRNA population. We also constructed infectious cDNA clones of satRNA and examined the viral load of different TBRV isolates in the presence and absence of satRNAs, as well as the accumulation of satRNA molecules on infected plants. Our data provide evidence that the presence of satRNAs significantly affects viral load; however, the magnitude of this effect differs among viral isolates and plant hosts. We also showed a positive correlation between the number of viral genomic RNAs (gRNAs) and satRNAs for two analysed TBRV isolates.
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12
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Lessons Learned and Yet-to-Be Learned on the Importance of RNA Structure in SARS-CoV-2 Replication. Microbiol Mol Biol Rev 2022; 86:e0005721. [PMID: 35862724 PMCID: PMC9491204 DOI: 10.1128/mmbr.00057-21] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022] Open
Abstract
SARS-CoV-2, the etiological agent responsible for the COVID-19 pandemic, is a member of the virus family Coronaviridae, known for relatively extensive (~30-kb) RNA genomes that not only encode for numerous proteins but are also capable of forming elaborate structures. As highlighted in this review, these structures perform critical functions in various steps of the viral life cycle, ultimately impacting pathogenesis and transmissibility. We examine these elements in the context of coronavirus evolutionary history and future directions for curbing the spread of SARS-CoV-2 and other potential human coronaviruses. While we focus on structures supported by a variety of biochemical, biophysical, and/or computational methods, we also touch here on recent evidence for novel structures in both protein-coding and noncoding regions of the genome, including an assessment of the potential role for RNA structure in the controversial finding of SARS-CoV-2 integration in “long COVID” patients. This review aims to serve as a consolidation of previous works on coronavirus and more recent investigation of SARS-CoV-2, emphasizing the need for improved understanding of the role of RNA structure in the evolution and adaptation of these human viruses.
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13
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Perdoncini Carvalho C, Ren R, Han J, Qu F. Natural Selection, Intracellular Bottlenecks of Virus Populations, and Viral Superinfection Exclusion. Annu Rev Virol 2022; 9:121-137. [PMID: 35567296 DOI: 10.1146/annurev-virology-100520-114758] [Citation(s) in RCA: 15] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Natural selection acts on cellular organisms by ensuring the genes responsible for an advantageous phenotype consistently reap the phenotypic advantage. This is possible because reproductive cells of these organisms are almost always haploid, separating the beneficial gene from its rival allele at every generation. How natural selection acts on plus-strand RNA viruses is unclear because these viruses frequently load host cells with numerous genome copies and replicate thousands of progeny genomes in each cell. Recent studies suggest that these viruses encode the Bottleneck, Isolate, Amplify, Select (BIAS) mechanism that blocks all but a few viral genome copies from replication, thus creating the environment in which the bottleneck-escaping viral genome copies are isolated from each other, allowing natural selection to reward beneficial mutations and purge lethal errors. This BIAS mechanism also blocks the genomes of highly homologous superinfecting viruses, thus explaining cellular-level superinfection exclusion. Expected final online publication date for the Annual Review of Virology, Volume 9 is September 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
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Affiliation(s)
| | - Ruifan Ren
- Department of Plant Pathology, The Ohio State University, Wooster, Ohio, USA;
| | - Junping Han
- Department of Plant Pathology, The Ohio State University, Wooster, Ohio, USA;
| | - Feng Qu
- Department of Plant Pathology, The Ohio State University, Wooster, Ohio, USA;
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14
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Krishnamoorthy P, Raj AS, Kumar P, Das N, Kumar H. Host and viral non-coding RNAs in dengue pathogenesis. Rev Med Virol 2022; 32:e2360. [PMID: 35510480 DOI: 10.1002/rmv.2360] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2021] [Revised: 04/15/2022] [Accepted: 04/20/2022] [Indexed: 11/10/2022]
Abstract
Dengue virus (DENV) is a mosquito-borne flavivirus that causes frequent outbreaks in tropical countries. Due to the four different serotypes and ever-mutating RNA genome, it is challenging to develop efficient therapeutics. Early diagnosis is crucial to prevent the severe form of dengue, leading to mortality. In the past decade, rapid advancement in the high throughput sequencing technologies has shed light on the crucial regulating role of non-coding RNAs (ncRNAs), also known as the "dark matter" of the genome, in various pathological processes. In addition to the human host ncRNAs like microRNAs and circular RNAs, DENV also produces ncRNAs such as subgenomic flaviviral RNAs that can modulate the virus life cycle and regulate disease outcomes. This review outlines the advances in understanding the interplay between the human host and DENV ncRNAs, their regulation of the innate immune system of the host, and the prospects of the ncRNAs in clinical applications such as dengue diagnosis and promising therapeutics.
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Affiliation(s)
- Pandikannan Krishnamoorthy
- Department of Biological Sciences, Laboratory of Immunology and Infectious Disease Biology, Indian Institute of Science Education and Research (IISER) Bhopal, Bhopal, Madhya Pradesh, India
| | - Athira S Raj
- Department of Biological Sciences, Laboratory of Immunology and Infectious Disease Biology, Indian Institute of Science Education and Research (IISER) Bhopal, Bhopal, Madhya Pradesh, India
| | - Pramod Kumar
- Department of Biological Sciences, Laboratory of Immunology and Infectious Disease Biology, Indian Institute of Science Education and Research (IISER) Bhopal, Bhopal, Madhya Pradesh, India
| | - Nilanjana Das
- Department of Biological Sciences, Laboratory of Immunology and Infectious Disease Biology, Indian Institute of Science Education and Research (IISER) Bhopal, Bhopal, Madhya Pradesh, India
| | - Himanshu Kumar
- Department of Biological Sciences, Laboratory of Immunology and Infectious Disease Biology, Indian Institute of Science Education and Research (IISER) Bhopal, Bhopal, Madhya Pradesh, India.,Laboratory of Host Defense, WPI Immunology, Frontier Research Centre, Osaka University, Osaka, Japan
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15
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Telwatte S, Martin HA, Marczak R, Fozouni P, Vallejo-Gracia A, Kumar GR, Murray V, Lee S, Ott M, Wong JK, Yukl SA. Novel RT-ddPCR assays for measuring the levels of subgenomic and genomic SARS-CoV-2 transcripts. Methods 2022; 201:15-25. [PMID: 33882362 PMCID: PMC8105137 DOI: 10.1016/j.ymeth.2021.04.011] [Citation(s) in RCA: 21] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2021] [Revised: 04/12/2021] [Accepted: 04/14/2021] [Indexed: 11/20/2022] Open
Abstract
The replication of SARS-CoV-2 and other coronaviruses depends on transcription of negative-sense RNA intermediates that serve as the templates for the synthesis of positive-sense genomic RNA (gRNA) and multiple different subgenomic mRNAs (sgRNAs) encompassing fragments arising from discontinuous transcription. Recent studies have aimed to characterize the expression of subgenomic SARS-CoV-2 transcripts in order to investigate their clinical significance. Here, we describe a novel panel of reverse transcription droplet digital PCR (RT-ddPCR) assays designed to specifically quantify multiple different subgenomic SARS-CoV-2 transcripts and distinguish them from transcripts that do not arise from discontinuous transcription at each locus. These assays can be applied to samples from SARS-CoV-2 infected patients to better understand the regulation of SARS-CoV-2 transcription and how different sgRNAs may contribute to viral pathogenesis and clinical disease severity.
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Affiliation(s)
- Sushama Telwatte
- Department of Medicine, University of California, San Francisco (UCSF), San Francisco, CA, United States; Department of Medicine, San Francisco VA Health Care System, San Francisco, CA, United States
| | - Holly Anne Martin
- Department of Medicine, University of California, San Francisco (UCSF), San Francisco, CA, United States; Department of Medicine, San Francisco VA Health Care System, San Francisco, CA, United States
| | - Ryan Marczak
- University of California, Santa Barbara, CA, United States
| | - Parinaz Fozouni
- Gladstone Institute of Virology, Gladstone Institutes, San Francisco, CA, United States
| | - Albert Vallejo-Gracia
- Gladstone Institute of Virology, Gladstone Institutes, San Francisco, CA, United States
| | - G Renuka Kumar
- Gladstone Institute of Virology, Gladstone Institutes, San Francisco, CA, United States
| | - Victoria Murray
- Department of Medicine, University of California, San Francisco (UCSF), San Francisco, CA, United States
| | - Sulggi Lee
- Department of Medicine, University of California, San Francisco (UCSF), San Francisco, CA, United States
| | - Melanie Ott
- Department of Medicine, University of California, San Francisco (UCSF), San Francisco, CA, United States; Gladstone Institute of Virology, Gladstone Institutes, San Francisco, CA, United States
| | - Joseph K Wong
- Department of Medicine, University of California, San Francisco (UCSF), San Francisco, CA, United States; Department of Medicine, San Francisco VA Health Care System, San Francisco, CA, United States
| | - Steven A Yukl
- Department of Medicine, University of California, San Francisco (UCSF), San Francisco, CA, United States; Department of Medicine, San Francisco VA Health Care System, San Francisco, CA, United States.
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16
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Di Gioacchino A, Legendre R, Rahou Y, Najburg V, Charneau P, Greenbaum BD, Tangy F, van der Werf S, Cocco S, Komarova AV. sgDI-tector: defective interfering viral genome bioinformatics for detection of coronavirus subgenomic RNAs. RNA (NEW YORK, N.Y.) 2022; 28:277-289. [PMID: 34937774 PMCID: PMC8848934 DOI: 10.1261/rna.078969.121] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/30/2021] [Accepted: 12/07/2021] [Indexed: 06/14/2023]
Abstract
Coronavirus RNA-dependent RNA polymerases produce subgenomic RNAs (sgRNAs) that encode viral structural and accessory proteins. User-friendly bioinformatic tools to detect and quantify sgRNA production are urgently needed to study the growing number of next-generation sequencing (NGS) data of SARS-CoV-2. We introduced sgDI-tector to identify and quantify sgRNA in SARS-CoV-2 NGS data. sgDI-tector allowed detection of sgRNA without initial knowledge of the transcription-regulatory sequences. We produced NGS data and successfully detected the nested set of sgRNAs with the ranking M > ORF3a > N>ORF6 > ORF7a > ORF8 > S > E>ORF7b. We also compared the level of sgRNA production with other types of viral RNA products such as defective interfering viral genomes.
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Affiliation(s)
- Andrea Di Gioacchino
- Sorbonne Université, Université de Paris, Laboratoire de Physique de l'Ecole Normale Supérieure, PSL & CNRS UMR8063, 75005, Paris, France
| | - Rachel Legendre
- Institut Pasteur, Université de Paris, Hub de Bioinformatique et Biostatistique - Département Biologie Computationnelle, 75015, Paris, France
| | - Yannis Rahou
- Institut Pasteur, Université de Paris, CNRS UMR3569, Génétique Moléculaire des Virus à ARN, F-75015 Paris, France
| | - Valérie Najburg
- Institut Pasteur, Université de Paris, Laboratory of Innovative Vaccines, 75015, Paris, France
| | - Pierre Charneau
- Institut Pasteur, Pasteur-TheraVectys joined unit, 75015, Paris, France
| | - Benjamin D Greenbaum
- Computational Oncology, Department of Epidemiology and Biostatistics, Memorial Sloan Kettering Cancer Center, New York 10065, USA
- Physiology, Biophysics and Systems Biology, Weill Cornell Medicine, Weill Cornell Medical College, New York 10065, USA
| | - Frédéric Tangy
- Institut Pasteur, Université de Paris, Laboratory of Innovative Vaccines, 75015, Paris, France
| | - Sylvie van der Werf
- Institut Pasteur, Université de Paris, CNRS UMR3569, Génétique Moléculaire des Virus à ARN, F-75015 Paris, France
| | - Simona Cocco
- Sorbonne Université, Université de Paris, Laboratoire de Physique de l'Ecole Normale Supérieure, PSL & CNRS UMR8063, 75005, Paris, France
| | - Anastassia V Komarova
- Institut Pasteur, Université de Paris, CNRS UMR3569, Génétique Moléculaire des Virus à ARN, F-75015 Paris, France
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17
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Takahashi H, Tabara M, Miyashita S, Ando S, Kawano S, Kanayama Y, Fukuhara T, Kormelink R. Cucumber Mosaic Virus Infection in Arabidopsis: A Conditional Mutualistic Symbiont? Front Microbiol 2022; 12:770925. [PMID: 35069476 PMCID: PMC8776717 DOI: 10.3389/fmicb.2021.770925] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2021] [Accepted: 12/02/2021] [Indexed: 11/13/2022] Open
Abstract
A cucumber mosaic virus isolate, named Ho [CMV(Ho)], was isolated from a symptomless Arabidopsis halleri field sample containing low virus titers. An analysis of CMV(Ho) RNA molecules indicated that the virus isolate, besides the usual cucumovirus tripartite RNA genome, additionally contained defective RNA3 molecules and a satellite RNA. To study the underlying mechanism of the persistent CMV(Ho) infection in perennial A. halleri, infectious cDNA clones were generated for all its genetic elements. CMV, which consists of synthetic transcripts from the infectious tripartite RNA genomes, and designated CMV(Ho)tr, multiplied in A. halleri and annual Arabidopsis thaliana Col-0 to a similar level as the virulent strain CMV(Y), but did not induce any symptoms in them. The response of Col-0 to a series of reassortant CMVs between CMV(Ho)tr and CMV(Y) suggested that the establishment of an asymptomatic phenotype of CMV(Ho) infection was due to the 2b gene of CMV RNA2, but not due to the presence of the defective RNA3 and satellite RNA. The accumulation of CMV(Ho) 2b protein tagged with the FLAG epitope (2b.Ho-FLAG) in 2b.Ho-FLAG-transformed Col-0 did not induce any symptoms, suggesting a 2b-dependent persistency of CMV(Ho)tr infection in Arabidopsis. The 2b protein interacted with Argonaute 4, which is known to regulate the cytosine methylation levels of host genomic DNA. Whole genomic bisulfite sequencing analysis of CMV(Ho)tr- and mock-inoculated Col-0 revealed that cytosine hypomethylation in the promoter regions of 82 genes, including two genes encoding transcriptional regulators (DOF1.7 and CBP1), was induced in response to CMV(Ho)tr infection. Moreover, the increased levels of hypomethylation in the promoter region of both genes, during CMV(Ho)tr infection, were correlated with the up- or down-regulation of their expression. Taken altogether, the results indicate that during persistent CMV(Ho) infection in Arabidopsis, host gene expression may be epigenetically modulated resulting from a 2b-mediated cytosine hypomethylation of host genomic DNA.
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Affiliation(s)
- Hideki Takahashi
- Graduate School of Agricultural Science, Tohoku University, Sendai, Japan
| | - Midori Tabara
- Department of Applied Biological Sciences, Tokyo University of Agriculture and Technology, Fuchu, Japan
- Ritsumeikan Global Innovation Research Organization, Ritsumeikan University, Kusatsu, Japan
| | - Shuhei Miyashita
- Graduate School of Agricultural Science, Tohoku University, Sendai, Japan
| | - Sugihiro Ando
- Graduate School of Agricultural Science, Tohoku University, Sendai, Japan
| | - Shuichi Kawano
- Graduate School of Informatics and Engineering, The University of Electro-Communications, Chofu, Japan
| | - Yoshinori Kanayama
- Graduate School of Agricultural Science, Tohoku University, Sendai, Japan
| | - Toshiyuki Fukuhara
- Department of Applied Biological Sciences, Tokyo University of Agriculture and Technology, Fuchu, Japan
| | - Richard Kormelink
- Laboratory of Virology, Department of Plant Sciences, Wageningen University and Research, Wageningen, Netherlands
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18
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Abstract
The success of many viruses depends upon cooperative interactions between viral genomes. However, whenever cooperation occurs, there is the potential for 'cheats' to exploit that cooperation. We suggest that: (1) the biology of viruses makes viral cooperation particularly susceptible to cheating; (2) cheats are common across a wide range of viruses, including viral entities that are already well studied, such as defective interfering genomes, and satellite viruses. Consequently, the evolutionary theory of cheating could help us understand and manipulate viral dynamics, while viruses also offer new opportunities to study the evolution of cheating.
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Affiliation(s)
- Asher Leeks
- Department of Zoology, University of Oxford, Oxford, OX1 3PS, UK.
| | - Stuart A West
- Department of Zoology, University of Oxford, Oxford, OX1 3PS, UK
| | - Melanie Ghoul
- Department of Zoology, University of Oxford, Oxford, OX1 3PS, UK
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19
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Long S. SARS-CoV-2 Subgenomic RNAs: Characterization, Utility, and Perspectives. Viruses 2021; 13:1923. [PMID: 34696353 PMCID: PMC8539008 DOI: 10.3390/v13101923] [Citation(s) in RCA: 45] [Impact Index Per Article: 11.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2021] [Revised: 09/12/2021] [Accepted: 09/16/2021] [Indexed: 12/11/2022] Open
Abstract
SARS-CoV-2, the etiologic agent at the root of the ongoing COVID-19 pandemic, harbors a large RNA genome from which a tiered ensemble of subgenomic RNAs (sgRNAs) is generated. Comprehensive definition and investigation of these RNA products are important for understanding SARS-CoV-2 pathogenesis. This review summarizes the recent progress on SARS-CoV-2 sgRNA identification, characterization, and application as a viral replication marker. The significance of these findings and potential future research areas of interest are discussed.
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Affiliation(s)
- Samuel Long
- Independent Researcher, Clarksburg, MD 20871, USA
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20
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Wong CH, Ngan CY, Goldfeder RL, Idol J, Kuhlberg C, Maurya R, Kelly K, Omerza G, Renzette N, De Abreu F, Li L, Browne FA, Liu ET, Wei CL. Reduced subgenomic RNA expression is a molecular indicator of asymptomatic SARS-CoV-2 infection. COMMUNICATIONS MEDICINE 2021; 1:33. [PMID: 35602196 PMCID: PMC9053197 DOI: 10.1038/s43856-021-00034-y] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2021] [Accepted: 09/02/2021] [Indexed: 01/12/2023] Open
Abstract
Background It is estimated that up to 80% of infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are asymptomatic and asymptomatic patients can still effectively transmit the virus and cause disease. While much of the effort has been placed on decoding single nucleotide variation in SARS-CoV-2 genomes, considerably less is known about their transcript variation and any correlation with clinical severity in human hosts, as defined here by the presence or absence of symptoms. Methods To assess viral genomic signatures of disease severity, we conducted a systematic characterization of SARS-CoV-2 transcripts and genetic variants in 81 clinical specimens collected from symptomatic and asymptomatic individuals using multi-scale transcriptomic analyses including amplicon-seq, short-read metatranscriptome and long-read Iso-seq. Results Here we show a highly coordinated and consistent pattern of sgRNA expression from individuals with robust SARS-CoV-2 symptomatic infection and their expression is significantly repressed in the asymptomatic infections. We also observe widespread inter- and intra-patient variants in viral RNAs, known as quasispecies frequently found in many RNA viruses. We identify unique sets of deletions preferentially found primarily in symptomatic individuals, with many likely to confer changes in SARS-CoV-2 virulence and host responses. Moreover, these frequently occurring structural variants in SARS-CoV-2 genomes serve as a mechanism to further induce SARS-CoV-2 proteome complexity. Conclusions Our results indicate that differential sgRNA expression and structural mutational burden are highly correlated with the clinical severity of SARS-CoV-2 infection. Longitudinally monitoring sgRNA expression and structural diversity could further guide treatment responses, testing strategies, and vaccine development.
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Affiliation(s)
- Chee Hong Wong
- The Jackson Laboratory for Genomic Medicine, Farmington, CT 06032 USA
| | - Chew Yee Ngan
- The Jackson Laboratory for Genomic Medicine, Farmington, CT 06032 USA
| | | | - Jennifer Idol
- The Jackson Laboratory for Genomic Medicine, Farmington, CT 06032 USA
| | - Chris Kuhlberg
- The Jackson Laboratory for Genomic Medicine, Farmington, CT 06032 USA
| | - Rahul Maurya
- The Jackson Laboratory for Genomic Medicine, Farmington, CT 06032 USA
| | - Kevin Kelly
- The Jackson Laboratory for Genomic Medicine, Farmington, CT 06032 USA
| | - Gregory Omerza
- The Jackson Laboratory for Genomic Medicine, Farmington, CT 06032 USA
| | - Nicholas Renzette
- The Jackson Laboratory for Genomic Medicine, Farmington, CT 06032 USA
| | - Francine De Abreu
- The Jackson Laboratory for Genomic Medicine, Farmington, CT 06032 USA
| | - Lei Li
- The Jackson Laboratory for Genomic Medicine, Farmington, CT 06032 USA
| | | | - Edison T. Liu
- The Jackson Laboratory for Genomic Medicine, Farmington, CT 06032 USA
| | - Chia-Lin Wei
- The Jackson Laboratory for Genomic Medicine, Farmington, CT 06032 USA
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21
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Zhao Y, Sun J, Li Y, Li Z, Xie Y, Feng R, Zhao J, Hu Y. The strand-biased transcription of SARS-CoV-2 and unbalanced inhibition by remdesivir. iScience 2021; 24:102857. [PMID: 34278249 PMCID: PMC8277956 DOI: 10.1016/j.isci.2021.102857] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2020] [Revised: 03/13/2021] [Accepted: 07/12/2021] [Indexed: 01/18/2023] Open
Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a positive single-stranded RNA virus, causes the coronavirus disease 19 pandemic. During the viral replication and transcription, the RNA-dependent RNA polymerase "jumps" along the genome template, resulting in discontinuous negative-stranded transcripts. Although the sense-mRNA architectures of SARS-CoV-2 were reported, its negative strand was unexplored. Here, we deeply sequenced both strands of RNA and found SARS-CoV-2 transcription is strongly biased to form the sense strand with variable transcription efficiency for different genes. During negative strand synthesis, numerous non-canonical fusion transcripts are also formed, driven by 3-15 nt sequence homology scattered along the genome but more prone to be inhibited by SARS-CoV-2 RNA polymerase inhibitor remdesivir. The drug also represses more of the negative than the positive strand synthesis as supported by a mathematic simulation model and experimental quantifications. Overall, this study opens new sights into SARS-CoV-2 biogenesis and may facilitate the antiviral vaccine development and drug design.
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Affiliation(s)
- Yan Zhao
- Shenzhen Key Laboratory of Gene Regulation and Systems Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen 518005, China.,Department of Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen 518005, China.,Department of Computational Molecular Biology, Max-Planck-Institute for Molecular Genetics, Berlin 14195, Germany.,Department of Mathematics and Computer Science, Free University Berlin, Berlin 14195, Germany
| | - Jing Sun
- State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou 510182, Guangdong, China
| | - Yunfei Li
- Shenzhen Key Laboratory of Gene Regulation and Systems Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen 518005, China.,Department of Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen 518005, China
| | - Zhengxuan Li
- Shenzhen Key Laboratory of Gene Regulation and Systems Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen 518005, China.,Department of Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen 518005, China
| | - Yu Xie
- Shenzhen Key Laboratory of Gene Regulation and Systems Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen 518005, China.,Department of Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen 518005, China
| | - Ruoqing Feng
- Shenzhen Key Laboratory of Gene Regulation and Systems Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen 518005, China.,Department of Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen 518005, China
| | - Jincun Zhao
- State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou 510182, Guangdong, China
| | - Yuhui Hu
- Shenzhen Key Laboratory of Gene Regulation and Systems Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen 518005, China.,Department of Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen 518005, China.,Academy for Advanced Interdisciplinary Studies, Southern University of Science and Technology, Shenzhen 518055, Guangdong, China
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22
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Wang D, Jiang A, Feng J, Li G, Guo D, Sajid M, Wu K, Zhang Q, Ponty Y, Will S, Liu F, Yu X, Li S, Liu Q, Yang XL, Guo M, Li X, Chen M, Shi ZL, Lan K, Chen Y, Zhou Y. The SARS-CoV-2 subgenome landscape and its novel regulatory features. Mol Cell 2021; 81:2135-2147.e5. [PMID: 33713597 PMCID: PMC7927579 DOI: 10.1016/j.molcel.2021.02.036] [Citation(s) in RCA: 73] [Impact Index Per Article: 18.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2020] [Revised: 10/28/2020] [Accepted: 02/24/2021] [Indexed: 12/31/2022]
Abstract
Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is currently a global pandemic. CoVs are known to generate negative subgenomes (subgenomic RNAs [sgRNAs]) through transcription-regulating sequence (TRS)-dependent template switching, but the global dynamic landscapes of coronaviral subgenomes and regulatory rules remain unclear. Here, using next-generation sequencing (NGS) short-read and Nanopore long-read poly(A) RNA sequencing in two cell types at multiple time points after infection with SARS-CoV-2, we identified hundreds of template switches and constructed the dynamic landscapes of SARS-CoV-2 subgenomes. Interestingly, template switching could occur in a bidirectional manner, with diverse SARS-CoV-2 subgenomes generated from successive template-switching events. The majority of template switches result from RNA-RNA interactions, including seed and compensatory modes, with terminal pairing status as a key determinant. Two TRS-independent template switch modes are also responsible for subgenome biogenesis. Our findings reveal the subgenome landscape of SARS-CoV-2 and its regulatory features, providing a molecular basis for understanding subgenome biogenesis and developing novel anti-viral strategies.
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Affiliation(s)
- Dehe Wang
- State Key Laboratory of Virology, Modern Virology Research Center, College of Life Sciences, Wuhan University, Wuhan, China; Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, China
| | - Ao Jiang
- State Key Laboratory of Virology, Modern Virology Research Center, College of Life Sciences, Wuhan University, Wuhan, China
| | - Jiangpeng Feng
- State Key Laboratory of Virology, Modern Virology Research Center, College of Life Sciences, Wuhan University, Wuhan, China
| | - Guangnan Li
- State Key Laboratory of Virology, Modern Virology Research Center, College of Life Sciences, Wuhan University, Wuhan, China; Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, China
| | - Dong Guo
- State Key Laboratory of Virology, Modern Virology Research Center, College of Life Sciences, Wuhan University, Wuhan, China
| | - Muhammad Sajid
- State Key Laboratory of Virology, Modern Virology Research Center, College of Life Sciences, Wuhan University, Wuhan, China
| | - Kai Wu
- State Key Laboratory of Virology, Modern Virology Research Center, College of Life Sciences, Wuhan University, Wuhan, China; Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, China
| | - Qiuhan Zhang
- State Key Laboratory of Virology, Modern Virology Research Center, College of Life Sciences, Wuhan University, Wuhan, China
| | - Yann Ponty
- CNRS UMR 7161 LIX, Ecole Polytechnique, Institut Polytechnique de Paris, Paris, France
| | - Sebastian Will
- CNRS UMR 7161 LIX, Ecole Polytechnique, Institut Polytechnique de Paris, Paris, France
| | - Feiyan Liu
- State Key Laboratory of Virology, Modern Virology Research Center, College of Life Sciences, Wuhan University, Wuhan, China; Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, China
| | - Xinghai Yu
- State Key Laboratory of Virology, Modern Virology Research Center, College of Life Sciences, Wuhan University, Wuhan, China; Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, China
| | - Shaopeng Li
- State Key Laboratory of Virology, Modern Virology Research Center, College of Life Sciences, Wuhan University, Wuhan, China; Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, China
| | - Qianyun Liu
- State Key Laboratory of Virology, Modern Virology Research Center, College of Life Sciences, Wuhan University, Wuhan, China
| | - Xing-Lou Yang
- CAS Key Laboratory of Special Pathogens, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, China
| | - Ming Guo
- State Key Laboratory of Virology, Modern Virology Research Center, College of Life Sciences, Wuhan University, Wuhan, China
| | - Xingqiao Li
- State Key Laboratory of Virology, Modern Virology Research Center, College of Life Sciences, Wuhan University, Wuhan, China; Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, China
| | - Mingzhou Chen
- State Key Laboratory of Virology, Modern Virology Research Center, College of Life Sciences, Wuhan University, Wuhan, China
| | - Zheng-Li Shi
- CAS Key Laboratory of Special Pathogens, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, China
| | - Ke Lan
- State Key Laboratory of Virology, Modern Virology Research Center, College of Life Sciences, Wuhan University, Wuhan, China; Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, China.
| | - Yu Chen
- State Key Laboratory of Virology, Modern Virology Research Center, College of Life Sciences, Wuhan University, Wuhan, China.
| | - Yu Zhou
- State Key Laboratory of Virology, Modern Virology Research Center, College of Life Sciences, Wuhan University, Wuhan, China; Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, China.
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Ludwig-Begall LF, Di Felice E, Toffoli B, Ceci C, Di Martino B, Marsilio F, Mauroy A, Thiry E. Analysis of Synchronous and Asynchronous In Vitro Infections with Homologous Murine Norovirus Strains Reveals Time-Dependent Viral Interference Effects. Viruses 2021; 13:823. [PMID: 34063220 PMCID: PMC8147416 DOI: 10.3390/v13050823] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2021] [Revised: 04/26/2021] [Accepted: 04/29/2021] [Indexed: 11/16/2022] Open
Abstract
Viral recombination is a key mechanism in the evolution and diversity of noroviruses. In vivo, synchronous single-cell coinfection by multiple viruses, the ultimate prerequisite to viral recombination, is likely to be a rare event and delayed secondary infections are a more probable occurrence. Here, we determine the effect of a temporal separation of in vitro infections with the two homologous murine norovirus strains MNV-1 WU20 and CW1 on the composition of nascent viral populations. WU20 and CW1 were either synchronously inoculated onto murine macrophage cell monolayers (coinfection) or asynchronously applied (superinfection with varying titres of CW1 at half-hour to 24-h delays). Then, 24 h after initial co-or superinfection, quantification of genomic copy numbers and discriminative screening of plaque picked infectious progeny viruses demonstrated a time-dependent predominance of primary infecting WU20 in the majority of viral progenies. Our results indicate that a time interval from one to two hours onwards between two consecutive norovirus infections allows for the establishment of a barrier that reduces or prevents superinfection.
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Affiliation(s)
- Louisa F. Ludwig-Begall
- FARAH Research Centre, Faculty of Veterinary Medicine, Veterinary Virology and Animal Viral Diseases, Department of Infectious and Parasitic Diseases, Liège University, 4000 Liège, Belgium; (L.F.L.-B.); (B.T.); (A.M.)
| | - Elisabetta Di Felice
- Department of Diagnosis and Surveillance of Exotic Disease, IZS Istituto Zooprofilattico Sperimentale A&M G. Caporale, 64100 Teramo, Italy;
| | - Barbara Toffoli
- FARAH Research Centre, Faculty of Veterinary Medicine, Veterinary Virology and Animal Viral Diseases, Department of Infectious and Parasitic Diseases, Liège University, 4000 Liège, Belgium; (L.F.L.-B.); (B.T.); (A.M.)
| | - Chiara Ceci
- Faculty of Veterinary Medicine, Università degli Studi di Teramo, 64100 Teramo, Italy; (C.C.); (B.D.M.); (F.M.)
| | - Barbara Di Martino
- Faculty of Veterinary Medicine, Università degli Studi di Teramo, 64100 Teramo, Italy; (C.C.); (B.D.M.); (F.M.)
| | - Fulvio Marsilio
- Faculty of Veterinary Medicine, Università degli Studi di Teramo, 64100 Teramo, Italy; (C.C.); (B.D.M.); (F.M.)
| | - Axel Mauroy
- FARAH Research Centre, Faculty of Veterinary Medicine, Veterinary Virology and Animal Viral Diseases, Department of Infectious and Parasitic Diseases, Liège University, 4000 Liège, Belgium; (L.F.L.-B.); (B.T.); (A.M.)
- Staff Direction for Risk Assessment, Control Policy, FASFC, 1000 Brussels, Belgium
| | - Etienne Thiry
- FARAH Research Centre, Faculty of Veterinary Medicine, Veterinary Virology and Animal Viral Diseases, Department of Infectious and Parasitic Diseases, Liège University, 4000 Liège, Belgium; (L.F.L.-B.); (B.T.); (A.M.)
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24
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Host-virus interactions mediated by long non-coding RNAs. Virus Res 2021; 298:198402. [PMID: 33771610 DOI: 10.1016/j.virusres.2021.198402] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2021] [Revised: 03/17/2021] [Accepted: 03/18/2021] [Indexed: 11/21/2022]
Abstract
Viruses are obligate pathogens that cause a wide range of diseases across all kingdoms of life. They have a colossal impact on the economy and healthcare infrastructure world-wide. Plants and animals have developed sophisticated molecular mechanisms to defend themselves against viruses and viruses in turn hijack host mechanisms to ensure their survival inside their hosts. Long non-coding (lnc) RNAs have emerged as important macromolecules that regulate plant-virus and animal-virus interactions. Both pro-viral and anti-viral lncRNAs have been reported and they show immense potential to be used as markers and in therapeutics. The current review is focussed on the recent developments that have been made in viral interactions of animals and plants.
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25
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O’Leary VB, Dolly OJ, Höschl C, Černa M, Ovsepian SV. Unpacking Pandora From Its Box: Deciphering the Molecular Basis of the SARS-CoV-2 Coronavirus. Int J Mol Sci 2020; 22:ijms22010386. [PMID: 33396557 PMCID: PMC7795774 DOI: 10.3390/ijms22010386] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2020] [Revised: 12/25/2020] [Accepted: 12/29/2020] [Indexed: 02/07/2023] Open
Abstract
An enigmatic localized pneumonia escalated into a worldwide COVID-19 pandemic from Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). This review aims to consolidate the extensive biological minutiae of SARS-CoV-2 which requires decipherment. Having one of the largest RNA viral genomes, the single strand contains the genes ORF1ab, S, E, M, N and ten open reading frames. Highlighting unique features such as stem-loop formation, slippery frameshifting sequences and ribosomal mimicry, SARS-CoV-2 represents a formidable cellular invader. Hijacking the hosts translational engine, it produces two polyprotein repositories (pp1a and pp1ab), armed with self-cleavage capacity for production of sixteen non-structural proteins. Novel glycosylation sites on the spike trimer reveal unique SARS-CoV-2 features for shielding and cellular internalization. Affording complexity for superior fitness and camouflage, SARS-CoV-2 challenges diagnosis and vaccine vigilance. This review serves the scientific community seeking in-depth molecular details when designing drugs to curb transmission of this biological armament.
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Affiliation(s)
- Valerie Bríd O’Leary
- Department of Medical Genetics, Third Faculty of Medicine, Charles University, Ruska 87, Vinohrady, 10000 Prague, Czech Republic;
- Department of Experimental Neurobiology, National Institute of Mental Health, Research Programme 1, Topolova 748, 25067 Klecany, Czech Republic; (C.H.); (S.V.O.)
- Correspondence:
| | - Oliver James Dolly
- International Centre for Neurotherapeutics, Dublin City University, Collins Avenue, Dublin 9, Ireland;
| | - Cyril Höschl
- Department of Experimental Neurobiology, National Institute of Mental Health, Research Programme 1, Topolova 748, 25067 Klecany, Czech Republic; (C.H.); (S.V.O.)
- Department of Psychiatry and Medical Psychology, Third Faculty of Medicine, Charles University, Ruska 87, Vinohrady, 10000 Prague, Czech Republic
| | - Marie Černa
- Department of Medical Genetics, Third Faculty of Medicine, Charles University, Ruska 87, Vinohrady, 10000 Prague, Czech Republic;
| | - Saak Victor Ovsepian
- Department of Experimental Neurobiology, National Institute of Mental Health, Research Programme 1, Topolova 748, 25067 Klecany, Czech Republic; (C.H.); (S.V.O.)
- International Centre for Neurotherapeutics, Dublin City University, Collins Avenue, Dublin 9, Ireland;
- Department of Psychiatry and Medical Psychology, Third Faculty of Medicine, Charles University, Ruska 87, Vinohrady, 10000 Prague, Czech Republic
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26
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Vandelli A, Monti M, Milanetti E, Armaos A, Rupert J, Zacco E, Bechara E, Delli Ponti R, Tartaglia GG. Structural analysis of SARS-CoV-2 genome and predictions of the human interactome. Nucleic Acids Res 2020. [PMID: 33068416 DOI: 10.1101/2020.03.28.013789] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/07/2023] Open
Abstract
Specific elements of viral genomes regulate interactions within host cells. Here, we calculated the secondary structure content of >2000 coronaviruses and computed >100 000 human protein interactions with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The genomic regions display different degrees of conservation. SARS-CoV-2 domain encompassing nucleotides 22 500-23 000 is conserved both at the sequence and structural level. The regions upstream and downstream, however, vary significantly. This part of the viral sequence codes for the Spike S protein that interacts with the human receptor angiotensin-converting enzyme 2 (ACE2). Thus, variability of Spike S is connected to different levels of viral entry in human cells within the population. Our predictions indicate that the 5' end of SARS-CoV-2 is highly structured and interacts with several human proteins. The binding proteins are involved in viral RNA processing, include double-stranded RNA specific editases and ATP-dependent RNA-helicases and have strong propensity to form stress granules and phase-separated assemblies. We propose that these proteins, also implicated in viral infections such as HIV, are selectively recruited by SARS-CoV-2 genome to alter transcriptional and post-transcriptional regulation of host cells and to promote viral replication.
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Affiliation(s)
- Andrea Vandelli
- Centre for Genomic Regulation (CRG), The Barcelona Institute for Science and Technology, Dr. Aiguader 88, 08003 Barcelona, Spain and Universitat Pompeu Fabra (UPF), 08003 Barcelona, Spain
- Systems Biology of Infection Lab, Department of Biochemistry and Molecular Biology, Biosciences Faculty, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain
| | - Michele Monti
- Centre for Genomic Regulation (CRG), The Barcelona Institute for Science and Technology, Dr. Aiguader 88, 08003 Barcelona, Spain and Universitat Pompeu Fabra (UPF), 08003 Barcelona, Spain
- Center for Human Technologies, Istituto Italiano di Tecnologia, Via Enrico Melen 83, 16152 Genoa, Italy
| | - Edoardo Milanetti
- Department of Physics, Sapienza University, Piazzale Aldo Moro 5, 00185 Rome, Italy
- Center for Life Nanoscience, Istituto Italiano di Tecnologia, Viale Regina Elena 291, 00161 Rome, Italy
| | - Alexandros Armaos
- Centre for Genomic Regulation (CRG), The Barcelona Institute for Science and Technology, Dr. Aiguader 88, 08003 Barcelona, Spain and Universitat Pompeu Fabra (UPF), 08003 Barcelona, Spain
- Center for Human Technologies, Istituto Italiano di Tecnologia, Via Enrico Melen 83, 16152 Genoa, Italy
| | - Jakob Rupert
- Center for Human Technologies, Istituto Italiano di Tecnologia, Via Enrico Melen 83, 16152 Genoa, Italy
- Department of Biology 'Charles Darwin', Sapienza University of Rome, P.le A. Moro 5, Rome 00185, Italy
| | - Elsa Zacco
- Center for Human Technologies, Istituto Italiano di Tecnologia, Via Enrico Melen 83, 16152 Genoa, Italy
| | - Elias Bechara
- Centre for Genomic Regulation (CRG), The Barcelona Institute for Science and Technology, Dr. Aiguader 88, 08003 Barcelona, Spain and Universitat Pompeu Fabra (UPF), 08003 Barcelona, Spain
- Center for Human Technologies, Istituto Italiano di Tecnologia, Via Enrico Melen 83, 16152 Genoa, Italy
| | - Riccardo Delli Ponti
- School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore
| | - Gian Gaetano Tartaglia
- Centre for Genomic Regulation (CRG), The Barcelona Institute for Science and Technology, Dr. Aiguader 88, 08003 Barcelona, Spain and Universitat Pompeu Fabra (UPF), 08003 Barcelona, Spain
- Center for Human Technologies, Istituto Italiano di Tecnologia, Via Enrico Melen 83, 16152 Genoa, Italy
- Department of Biology 'Charles Darwin', Sapienza University of Rome, P.le A. Moro 5, Rome 00185, Italy
- Institucio Catalana de Recerca i Estudis Avançats (ICREA), 23 Passeig Lluis Companys, 08010 Barcelona, Spain
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27
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Vandelli A, Monti M, Milanetti E, Armaos A, Rupert J, Zacco E, Bechara E, Delli Ponti R, Tartaglia G. Structural analysis of SARS-CoV-2 genome and predictions of the human interactome. Nucleic Acids Res 2020; 48:11270-11283. [PMID: 33068416 PMCID: PMC7672441 DOI: 10.1093/nar/gkaa864] [Citation(s) in RCA: 67] [Impact Index Per Article: 13.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2020] [Revised: 09/15/2020] [Accepted: 09/25/2020] [Indexed: 12/17/2022] Open
Abstract
Specific elements of viral genomes regulate interactions within host cells. Here, we calculated the secondary structure content of >2000 coronaviruses and computed >100 000 human protein interactions with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The genomic regions display different degrees of conservation. SARS-CoV-2 domain encompassing nucleotides 22 500-23 000 is conserved both at the sequence and structural level. The regions upstream and downstream, however, vary significantly. This part of the viral sequence codes for the Spike S protein that interacts with the human receptor angiotensin-converting enzyme 2 (ACE2). Thus, variability of Spike S is connected to different levels of viral entry in human cells within the population. Our predictions indicate that the 5' end of SARS-CoV-2 is highly structured and interacts with several human proteins. The binding proteins are involved in viral RNA processing, include double-stranded RNA specific editases and ATP-dependent RNA-helicases and have strong propensity to form stress granules and phase-separated assemblies. We propose that these proteins, also implicated in viral infections such as HIV, are selectively recruited by SARS-CoV-2 genome to alter transcriptional and post-transcriptional regulation of host cells and to promote viral replication.
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Affiliation(s)
- Andrea Vandelli
- Centre for Genomic Regulation (CRG), The Barcelona Institute for Science and Technology, Dr. Aiguader 88, 08003 Barcelona, Spain and Universitat Pompeu Fabra (UPF), 08003 Barcelona, Spain
- Systems Biology of Infection Lab, Department of Biochemistry and Molecular Biology, Biosciences Faculty, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain
| | - Michele Monti
- Centre for Genomic Regulation (CRG), The Barcelona Institute for Science and Technology, Dr. Aiguader 88, 08003 Barcelona, Spain and Universitat Pompeu Fabra (UPF), 08003 Barcelona, Spain
- Center for Human Technologies, Istituto Italiano di Tecnologia, Via Enrico Melen 83, 16152 Genoa, Italy
| | - Edoardo Milanetti
- Department of Physics, Sapienza University, Piazzale Aldo Moro 5, 00185 Rome, Italy
- Center for Life Nanoscience, Istituto Italiano di Tecnologia, Viale Regina Elena 291, 00161 Rome, Italy
| | - Alexandros Armaos
- Centre for Genomic Regulation (CRG), The Barcelona Institute for Science and Technology, Dr. Aiguader 88, 08003 Barcelona, Spain and Universitat Pompeu Fabra (UPF), 08003 Barcelona, Spain
- Center for Human Technologies, Istituto Italiano di Tecnologia, Via Enrico Melen 83, 16152 Genoa, Italy
| | - Jakob Rupert
- Center for Human Technologies, Istituto Italiano di Tecnologia, Via Enrico Melen 83, 16152 Genoa, Italy
- Department of Biology ‘Charles Darwin’, Sapienza University of Rome, P.le A. Moro 5, Rome 00185, Italy
| | - Elsa Zacco
- Center for Human Technologies, Istituto Italiano di Tecnologia, Via Enrico Melen 83, 16152 Genoa, Italy
| | - Elias Bechara
- Centre for Genomic Regulation (CRG), The Barcelona Institute for Science and Technology, Dr. Aiguader 88, 08003 Barcelona, Spain and Universitat Pompeu Fabra (UPF), 08003 Barcelona, Spain
- Center for Human Technologies, Istituto Italiano di Tecnologia, Via Enrico Melen 83, 16152 Genoa, Italy
| | - Riccardo Delli Ponti
- School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore
| | - Gian Gaetano Tartaglia
- Centre for Genomic Regulation (CRG), The Barcelona Institute for Science and Technology, Dr. Aiguader 88, 08003 Barcelona, Spain and Universitat Pompeu Fabra (UPF), 08003 Barcelona, Spain
- Center for Human Technologies, Istituto Italiano di Tecnologia, Via Enrico Melen 83, 16152 Genoa, Italy
- Department of Biology ‘Charles Darwin’, Sapienza University of Rome, P.le A. Moro 5, Rome 00185, Italy
- Institucio Catalana de Recerca i Estudis Avançats (ICREA), 23 Passeig Lluis Companys, 08010 Barcelona, Spain
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28
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Shrestha N, Bujarski JJ. Long Noncoding RNAs in Plant Viroids and Viruses: A Review. Pathogens 2020; 9:E765. [PMID: 32961969 PMCID: PMC7559573 DOI: 10.3390/pathogens9090765] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2020] [Revised: 09/15/2020] [Accepted: 09/16/2020] [Indexed: 12/11/2022] Open
Abstract
Infectious long-noncoding (lnc) RNAs related to plants can be of both viral and non-viral origin. Viroids are infectious plant lncRNAs that are not related to viruses and carry the circular, single-stranded, non-coding RNAs that replicate with host enzymatic activities via a rolling circle mechanism. Viroids interact with host processes in complex ways, emerging as one of the most productive tools for studying the functions of lncRNAs. Defective (D) RNAs, another category of lnc RNAs, are found in a variety of plant RNA viruses, most of which are noncoding. These are derived from and are replicated by the helper virus. D RNA-virus interactions evolve into mutually beneficial combinations, enhancing virus fitness via competitive advantages of moderated symptoms. Yet the satellite RNAs are single-stranded and include either large linear protein-coding ss RNAs, small linear ss RNAs, or small circular ss RNAs (virusoids). The satellite RNAs lack sequence homology to the helper virus, but unlike viroids need a helper virus to replicate and encapsidate. They can attenuate symptoms via RNA silencing and enhancement of host defense, but some can be lethal as RNA silencing suppressor antagonists. Moreover, selected viruses produce lncRNAs by incomplete degradation of genomic RNAs. They do not replicate but may impact viral infection, gene regulation, and cellular functions. Finally, the host plant lncRNAs can also contribute during plant-virus interactions, inducing plant defense and the regulation of gene expression, often in conjunction with micro and/or circRNAs.
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Affiliation(s)
- Nipin Shrestha
- Department of Biological Sciences and Plant Molecular and Bioinformatics Center, Northern Illinois University, DeKalb, IL 60115, USA
| | - Józef J. Bujarski
- Department of Biological Sciences and Plant Molecular and Bioinformatics Center, Northern Illinois University, DeKalb, IL 60115, USA
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Sanfaçon H. Modulation of disease severity by plant positive-strand RNA viruses: The complex interplay of multifunctional viral proteins, subviral RNAs and virus-associated RNAs with plant signaling pathways and defense responses. Adv Virus Res 2020; 107:87-131. [PMID: 32711736 DOI: 10.1016/bs.aivir.2020.04.003] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Plant viruses induce a range of symptoms of varying intensity, ranging from severe systemic necrosis to mild or asymptomatic infection. Several evolutionary constraints drive virus virulence, including the dependence of viruses on host factors to complete their infection cycle, the requirement to counteract or evade plant antiviral defense responses and the mode of virus transmission. Viruses have developed an array of strategies to modulate disease severity. Accumulating evidence has highlighted not only the multifunctional role that viral proteins play in disrupting or highjacking plant factors, hormone signaling pathways and intracellular organelles, but also the interaction networks between viral proteins, subviral RNAs and/or other viral-associated RNAs that regulate disease severity. This review focusses on positive-strand RNA viruses, which constitute the majority of characterized plant viruses. Using well-characterized viruses with different genome types as examples, recent advances are discussed as well as knowledge gaps and opportunities for further research.
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Affiliation(s)
- Hélène Sanfaçon
- Summerland Research and Development Centre, Agriculture and Agri-Food Canada, Summerland, BC, Canada.
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30
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Kim D, Lee JY, Yang JS, Kim JW, Kim VN, Chang H. The Architecture of SARS-CoV-2 Transcriptome. Cell 2020; 181:914-921.e10. [PMID: 32330414 PMCID: PMC7179501 DOI: 10.1016/j.cell.2020.04.011] [Citation(s) in RCA: 1526] [Impact Index Per Article: 305.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/13/2020] [Revised: 03/25/2020] [Accepted: 04/07/2020] [Indexed: 12/31/2022]
Abstract
SARS-CoV-2 is a betacoronavirus responsible for the COVID-19 pandemic. Although the SARS-CoV-2 genome was reported recently, its transcriptomic architecture is unknown. Utilizing two complementary sequencing techniques, we present a high-resolution map of the SARS-CoV-2 transcriptome and epitranscriptome. DNA nanoball sequencing shows that the transcriptome is highly complex owing to numerous discontinuous transcription events. In addition to the canonical genomic and 9 subgenomic RNAs, SARS-CoV-2 produces transcripts encoding unknown ORFs with fusion, deletion, and/or frameshift. Using nanopore direct RNA sequencing, we further find at least 41 RNA modification sites on viral transcripts, with the most frequent motif, AAGAA. Modified RNAs have shorter poly(A) tails than unmodified RNAs, suggesting a link between the modification and the 3' tail. Functional investigation of the unknown transcripts and RNA modifications discovered in this study will open new directions to our understanding of the life cycle and pathogenicity of SARS-CoV-2.
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Affiliation(s)
- Dongwan Kim
- Center for RNA Research, Institute for Basic Science (IBS), Seoul 08826, Republic of Korea; School of Biological Sciences, Seoul National University, Seoul 08826, Republic of Korea
| | - Joo-Yeon Lee
- Korea National Institute of Health, Korea Centers for Disease Control and Prevention, Osong 28159, Republic of Korea
| | - Jeong-Sun Yang
- Korea National Institute of Health, Korea Centers for Disease Control and Prevention, Osong 28159, Republic of Korea
| | - Jun Won Kim
- Korea National Institute of Health, Korea Centers for Disease Control and Prevention, Osong 28159, Republic of Korea
| | - V Narry Kim
- Center for RNA Research, Institute for Basic Science (IBS), Seoul 08826, Republic of Korea; School of Biological Sciences, Seoul National University, Seoul 08826, Republic of Korea.
| | - Hyeshik Chang
- Center for RNA Research, Institute for Basic Science (IBS), Seoul 08826, Republic of Korea; School of Biological Sciences, Seoul National University, Seoul 08826, Republic of Korea.
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You J, Zhou K, Liu X, Wu M, Yang L, Zhang J, Chen W, Li G. Defective RNA of a Novel Mycovirus with High Transmissibility Detrimental to Biocontrol Properties of Trichoderma spp. Microorganisms 2019; 7:microorganisms7110507. [PMID: 31671828 PMCID: PMC6920978 DOI: 10.3390/microorganisms7110507] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2019] [Revised: 10/20/2019] [Accepted: 10/25/2019] [Indexed: 01/07/2023] Open
Abstract
Trichoderma species are a group of fungi which is widely distributed in major terrestrial ecosystems; they are also commonly used as biocontrol agents for many plant diseases. A virus, namely Trichoderma harzianum hypovirus 1 (ThHV1), was identified in T. harzianum isolate T-70, and also infected isolate T-70D, together with its defective RNA (ThHV1-S). The ThHV1 genome possessed two Open Reading Frames (ORFs), namely ORF1 and ORF2. The start codon of ORF2 overlapped with the stop codon of ORF1 in a 43 nt long region. The polypeptide encoded by ORF2 of ThHV1 shared sequence similarities with those of betahypoviruses, indicating that ThHV1 is a novel member of Hypoviridea. Isolate T-70D, carrying both ThHV1 and ThHV1-S, showed abnormal biological properties, notably a decreased mycoparasitism ability when compared with isolate T-70. Both ThHV1 and ThHV1-S could be vertically transmitted to conidia and horizontally transmitted to T. harzianum isolate T-68 and T. koningiopsis T-51. The derivative strains carrying both ThHV1 and ThHV1-S showed decreased mycoparasitism ability, whereas strains carrying ThHV1 alone were normal, indicating that ThHV1-S is closely associated with the decreased mycoparasitism ability of T. harzianum isolate T-70D. ThHV1 was widely detected in isolates of T. harzianum, T. koningiopsis and T. atroviride originating from soil of China. Therefore, viruses in fungal biocontrol agents may also be a factor associated with the stability of their application.
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Affiliation(s)
- Jiaqi You
- State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China.
- The Key Laboratory of Plant Pathology of Hubei Province, Huazhong Agricultural University, Wuhan 430070, China.
- Horticultural Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai 201106, China.
| | - Kang Zhou
- State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China.
- The Key Laboratory of Plant Pathology of Hubei Province, Huazhong Agricultural University, Wuhan 430070, China.
| | - Xiaolin Liu
- State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China.
- The Key Laboratory of Plant Pathology of Hubei Province, Huazhong Agricultural University, Wuhan 430070, China.
| | - Mingde Wu
- State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China.
- The Key Laboratory of Plant Pathology of Hubei Province, Huazhong Agricultural University, Wuhan 430070, China.
| | - Long Yang
- State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China.
- The Key Laboratory of Plant Pathology of Hubei Province, Huazhong Agricultural University, Wuhan 430070, China.
| | - Jing Zhang
- State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China.
- The Key Laboratory of Plant Pathology of Hubei Province, Huazhong Agricultural University, Wuhan 430070, China.
| | - Weidong Chen
- U.S. Department of Agriculture, Agricultural Research Service, Washington State University, Pullman, WA 99164, USA.
| | - Guoqing Li
- State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China.
- The Key Laboratory of Plant Pathology of Hubei Province, Huazhong Agricultural University, Wuhan 430070, China.
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Bora M, Yousuf RW, Dhar P, Manu M, Zafir I, Mishra B, Rajak KK, Singh RP. Characterization of defective interfering (DI) particles of Pestedes petitsruminants vaccine virus Sungri/96 strain-implications in vaccine upscaling. Biologicals 2019; 62:57-64. [PMID: 31588012 DOI: 10.1016/j.biologicals.2019.09.008] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2019] [Revised: 08/03/2019] [Accepted: 09/24/2019] [Indexed: 10/25/2022] Open
Abstract
The present investigation deals with the characterization of defective interfering (DI) particles of Peste-des-petits ruminants (PPR) vaccine Sungri/96 strain generated as a result of high MOI in Vero cells. During the serial 10 passages, infectivity titres drastically reduced from 6.5 to 2.25 log10TCID50/ml at high MOI. Further, attenuation of CPE with high MOI indicated generation of DI particles that resulted in no/slow progression of CPE during the late passages. Monoclonal antibody based cell ELISA indicated normal protein (N & H) packaging in samples with DI activity. At genomic level, inconsistency in amplicon intensity of H gene was observed in RT-PCR, indicating a possible defect of H gene. Further analysis of copy number of PPRV by RT-qPCR indicated intermittent fluctuations of viral genomic RNA copies. The significant decline of viral RNA copies with MOI 3 (314 copies) compared to low MOI (512804 copies), proved that higher DI multiplicities cause more interference with the replication process of the standard virus. Therefore, MOI is critical for manufacturing of vaccines. These investigations will help in upscaling of PPR vaccines in view of ongoing National and Global PPR control and eradication programme.
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Affiliation(s)
- Mousumi Bora
- Division of Biological Products, ICAR-Indian Veterinary Research Institute, Uttar Pradesh, India
| | - Raja Wasim Yousuf
- Division of Biological Products, ICAR-Indian Veterinary Research Institute, Uttar Pradesh, India
| | - Pronab Dhar
- Division of Biological Standardization, ICAR-Indian Veterinary Research Institute, Uttar Pradesh, India
| | - M Manu
- Division of Biological Standardization, ICAR-Indian Veterinary Research Institute, Uttar Pradesh, India
| | - Insha Zafir
- Division of Biological Products, ICAR-Indian Veterinary Research Institute, Uttar Pradesh, India
| | - Bina Mishra
- Division of Biological Products, ICAR-Indian Veterinary Research Institute, Uttar Pradesh, India
| | - Kaushal Kishor Rajak
- Division of Biological Products, ICAR-Indian Veterinary Research Institute, Uttar Pradesh, India
| | - Rabindra Prasad Singh
- Division of Biological Products, ICAR-Indian Veterinary Research Institute, Uttar Pradesh, India.
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Viehweger A, Krautwurst S, Lamkiewicz K, Madhugiri R, Ziebuhr J, Hölzer M, Marz M. Direct RNA nanopore sequencing of full-length coronavirus genomes provides novel insights into structural variants and enables modification analysis. Genome Res 2019; 29:1545-1554. [PMID: 31439691 DOI: 10.1101/483693] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2018] [Accepted: 08/05/2019] [Indexed: 05/24/2023]
Abstract
Sequence analyses of RNA virus genomes remain challenging owing to the exceptional genetic plasticity of these viruses. Because of high mutation and recombination rates, genome replication by viral RNA-dependent RNA polymerases leads to populations of closely related viruses, so-called "quasispecies." Standard (short-read) sequencing technologies are ill-suited to reconstruct large numbers of full-length haplotypes of (1) RNA virus genomes and (2) subgenome-length (sg) RNAs composed of noncontiguous genome regions. Here, we used a full-length, direct RNA sequencing (DRS) approach based on nanopores to characterize viral RNAs produced in cells infected with a human coronavirus. By using DRS, we were able to map the longest (∼26-kb) contiguous read to the viral reference genome. By combining Illumina and Oxford Nanopore sequencing, we reconstructed a highly accurate consensus sequence of the human coronavirus (HCoV)-229E genome (27.3 kb). Furthermore, by using long reads that did not require an assembly step, we were able to identify, in infected cells, diverse and novel HCoV-229E sg RNAs that remain to be characterized. Also, the DRS approach, which circumvents reverse transcription and amplification of RNA, allowed us to detect methylation sites in viral RNAs. Our work paves the way for haplotype-based analyses of viral quasispecies by showing the feasibility of intra-sample haplotype separation. Even though several technical challenges remain to be addressed to exploit the potential of the nanopore technology fully, our work illustrates that DRS may significantly advance genomic studies of complex virus populations, including predictions on long-range interactions in individual full-length viral RNA haplotypes.
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Affiliation(s)
- Adrian Viehweger
- RNA Bioinformatics and High-Throughput Analysis, Friedrich Schiller University Jena, 07743 Jena, Germany
- European Virus Bioinformatics Center, Friedrich Schiller University Jena, 07743 Jena, Germany
| | - Sebastian Krautwurst
- RNA Bioinformatics and High-Throughput Analysis, Friedrich Schiller University Jena, 07743 Jena, Germany
- European Virus Bioinformatics Center, Friedrich Schiller University Jena, 07743 Jena, Germany
| | - Kevin Lamkiewicz
- RNA Bioinformatics and High-Throughput Analysis, Friedrich Schiller University Jena, 07743 Jena, Germany
- European Virus Bioinformatics Center, Friedrich Schiller University Jena, 07743 Jena, Germany
| | - Ramakanth Madhugiri
- Institute of Medical Virology, Justus Liebig University Gießen, 35390 Gießen, Germany
| | - John Ziebuhr
- European Virus Bioinformatics Center, Friedrich Schiller University Jena, 07743 Jena, Germany
- Institute of Medical Virology, Justus Liebig University Gießen, 35390 Gießen, Germany
| | - Martin Hölzer
- RNA Bioinformatics and High-Throughput Analysis, Friedrich Schiller University Jena, 07743 Jena, Germany
- European Virus Bioinformatics Center, Friedrich Schiller University Jena, 07743 Jena, Germany
| | - Manja Marz
- RNA Bioinformatics and High-Throughput Analysis, Friedrich Schiller University Jena, 07743 Jena, Germany
- European Virus Bioinformatics Center, Friedrich Schiller University Jena, 07743 Jena, Germany
- Leibniz Institute on Aging-Fritz Lipmann Institute, 07743 Jena, Germany
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34
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Viehweger A, Krautwurst S, Lamkiewicz K, Madhugiri R, Ziebuhr J, Hölzer M, Marz M. Direct RNA nanopore sequencing of full-length coronavirus genomes provides novel insights into structural variants and enables modification analysis. Genome Res 2019; 29:1545-1554. [PMID: 31439691 PMCID: PMC6724671 DOI: 10.1101/gr.247064.118] [Citation(s) in RCA: 147] [Impact Index Per Article: 24.5] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2018] [Accepted: 08/05/2019] [Indexed: 01/09/2023]
Abstract
Sequence analyses of RNA virus genomes remain challenging owing to the exceptional genetic plasticity of these viruses. Because of high mutation and recombination rates, genome replication by viral RNA-dependent RNA polymerases leads to populations of closely related viruses, so-called “quasispecies.” Standard (short-read) sequencing technologies are ill-suited to reconstruct large numbers of full-length haplotypes of (1) RNA virus genomes and (2) subgenome-length (sg) RNAs composed of noncontiguous genome regions. Here, we used a full-length, direct RNA sequencing (DRS) approach based on nanopores to characterize viral RNAs produced in cells infected with a human coronavirus. By using DRS, we were able to map the longest (∼26-kb) contiguous read to the viral reference genome. By combining Illumina and Oxford Nanopore sequencing, we reconstructed a highly accurate consensus sequence of the human coronavirus (HCoV)-229E genome (27.3 kb). Furthermore, by using long reads that did not require an assembly step, we were able to identify, in infected cells, diverse and novel HCoV-229E sg RNAs that remain to be characterized. Also, the DRS approach, which circumvents reverse transcription and amplification of RNA, allowed us to detect methylation sites in viral RNAs. Our work paves the way for haplotype-based analyses of viral quasispecies by showing the feasibility of intra-sample haplotype separation. Even though several technical challenges remain to be addressed to exploit the potential of the nanopore technology fully, our work illustrates that DRS may significantly advance genomic studies of complex virus populations, including predictions on long-range interactions in individual full-length viral RNA haplotypes.
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Affiliation(s)
- Adrian Viehweger
- RNA Bioinformatics and High-Throughput Analysis, Friedrich Schiller University Jena, 07743 Jena, Germany.,European Virus Bioinformatics Center, Friedrich Schiller University Jena, 07743 Jena, Germany
| | - Sebastian Krautwurst
- RNA Bioinformatics and High-Throughput Analysis, Friedrich Schiller University Jena, 07743 Jena, Germany.,European Virus Bioinformatics Center, Friedrich Schiller University Jena, 07743 Jena, Germany
| | - Kevin Lamkiewicz
- RNA Bioinformatics and High-Throughput Analysis, Friedrich Schiller University Jena, 07743 Jena, Germany.,European Virus Bioinformatics Center, Friedrich Schiller University Jena, 07743 Jena, Germany
| | - Ramakanth Madhugiri
- Institute of Medical Virology, Justus Liebig University Gießen, 35390 Gießen, Germany
| | - John Ziebuhr
- European Virus Bioinformatics Center, Friedrich Schiller University Jena, 07743 Jena, Germany.,Institute of Medical Virology, Justus Liebig University Gießen, 35390 Gießen, Germany
| | - Martin Hölzer
- RNA Bioinformatics and High-Throughput Analysis, Friedrich Schiller University Jena, 07743 Jena, Germany.,European Virus Bioinformatics Center, Friedrich Schiller University Jena, 07743 Jena, Germany
| | - Manja Marz
- RNA Bioinformatics and High-Throughput Analysis, Friedrich Schiller University Jena, 07743 Jena, Germany.,European Virus Bioinformatics Center, Friedrich Schiller University Jena, 07743 Jena, Germany.,Leibniz Institute on Aging-Fritz Lipmann Institute, 07743 Jena, Germany
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35
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Abstract
Defective viral genomes (DVGs) are generated during viral replication and are unable to carry out a full replication cycle unless coinfected with a full-length virus. DVGs are produced by many viruses, and their presence correlates with alterations in infection outcomes. Historically, DVGs were studied for their ability to interfere with standard virus replication as well as for their association with viral persistence. More recently, a critical role for DVGs in inducing the innate immune response during infection was appreciated. Here we review the role of DVGs of RNA viruses in shaping outcomes of experimental as well as natural infections and explore the mechanisms by which DVGs impact infection outcome.
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Affiliation(s)
- Emmanuelle Genoyer
- Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA;
| | - Carolina B López
- Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA;
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36
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Sun Y, Kim EJ, Felt SA, Taylor LJ, Agarwal D, Grant GR, López CB. A specific sequence in the genome of respiratory syncytial virus regulates the generation of copy-back defective viral genomes. PLoS Pathog 2019; 15:e1007707. [PMID: 30995283 PMCID: PMC6504078 DOI: 10.1371/journal.ppat.1007707] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2019] [Revised: 05/07/2019] [Accepted: 03/15/2019] [Indexed: 01/12/2023] Open
Abstract
Defective viral genomes of the copy-back type (cbDVGs) are the primary initiators of the antiviral immune response during infection with respiratory syncytial virus (RSV) both in vitro and in vivo. However, the mechanism governing cbDVG generation remains unknown, thereby limiting our ability to manipulate cbDVG content in order to modulate the host response to infection. Here we report a specific genomic signal that mediates the generation of a subset of RSV cbDVG species. Using a customized bioinformatics tool, we identified regions in the RSV genome frequently used to generate cbDVGs during infection. We then created a minigenome system to validate the function of one of these sequences and to determine if specific nucleotides were essential for cbDVG generation at that position. Further, we created a recombinant virus unable to produce a subset of cbDVGs due to mutations introduced in this sequence. The identified sequence was also found as a site for cbDVG generation during natural RSV infections, and common cbDVGs originated at this sequence were found among samples from various infected patients. These data demonstrate that sequences encoded in the viral genome determine the location of cbDVG formation and, therefore, the generation of cbDVGs is not a stochastic process. These findings open the possibility of genetically manipulating cbDVG formation to modulate infection outcome.
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Affiliation(s)
- Yan Sun
- Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, United States
| | - Eun Ji Kim
- Institute of Translational Medicine and Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States
| | - Sébastien A. Felt
- Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, United States
| | - Louis J. Taylor
- Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States
| | - Divyansh Agarwal
- Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, United States
| | - Gregory R. Grant
- Institute of Translational Medicine and Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States
- Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States
| | - Carolina B. López
- Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, United States
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Defective Viral Genomes Alter How Sendai Virus Interacts with Cellular Trafficking Machinery, Leading to Heterogeneity in the Production of Viral Particles among Infected Cells. J Virol 2019; 93:JVI.01579-18. [PMID: 30463965 DOI: 10.1128/jvi.01579-18] [Citation(s) in RCA: 20] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2018] [Accepted: 11/10/2018] [Indexed: 12/12/2022] Open
Abstract
Defective viral genomes (DVGs) generated during RNA virus replication determine infection outcome by triggering innate immunity, diminishing virulence, and, in many cases, facilitating the establishment of persistent infections. Despite their critical role during virus-host interactions, the mechanisms regulating the production and propagation of DVGs are poorly understood. Visualization of viral genomes using RNA fluorescent in situ hybridization revealed a striking difference in the intracellular localization of DVGs and full-length viral genomes during infections with the paramyxovirus Sendai virus. In cells enriched in full-length virus, viral genomes clustered in a perinuclear region and associated with cellular trafficking machinery, including microtubules and the GTPase Rab11a. However, in cells enriched in DVGs, defective genomes distributed diffusely throughout the cytoplasm and failed to interact with this cellular machinery. Consequently, cells enriched in full-length genomes produced both DVG- and full-length-genome-containing viral particles, while DVG-high cells poorly produced viral particles yet strongly stimulated antiviral immunity. These findings reveal the selective production of both standard and DVG-containing particles by a subpopulation of infected cells that can be differentiated by the intracellular localization of DVGs. This study highlights the importance of considering this functional heterogeneity in analyses of virus-host interactions during infection.IMPORTANCE Defective viral genomes (DVGs) generated during Sendai virus infections accumulate in the cytoplasm of some infected cells and stimulate antiviral immunity and cell survival. DVGs are packaged and released as defective particles and have a significant impact on infection outcome. We show that the subpopulation of DVG-high cells poorly engages the virus packaging and budding machinery and do not effectively produce viral particles. In contrast, cells enriched in full-length genomes are the primary producers of both standard and defective viral particles during infection. This study demonstrates heterogeneity in the molecular interactions occurring within infected cells and highlights distinct functional roles for cells as either initiators of immunity or producers and perpetuators of viral particles depending on their content of viral genomes and their intracellular localization.
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38
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Sato Y, Castón JR, Suzuki N. The biological attributes, genome architecture and packaging of diverse multi-component fungal viruses. Curr Opin Virol 2018; 33:55-65. [DOI: 10.1016/j.coviro.2018.07.009] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2018] [Revised: 06/29/2018] [Accepted: 07/05/2018] [Indexed: 12/19/2022]
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Paudel DB, Sanfaçon H. Exploring the Diversity of Mechanisms Associated With Plant Tolerance to Virus Infection. FRONTIERS IN PLANT SCIENCE 2018; 9:1575. [PMID: 30450108 PMCID: PMC6224807 DOI: 10.3389/fpls.2018.01575] [Citation(s) in RCA: 61] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/28/2018] [Accepted: 10/09/2018] [Indexed: 05/17/2023]
Abstract
Tolerance is defined as an interaction in which viruses accumulate to some degree without causing significant loss of vigor or fitness to their hosts. Tolerance can be described as a stable equilibrium between the virus and its host, an interaction in which each partner not only accommodate trade-offs for survival but also receive some benefits (e.g., protection of the plant against super-infection by virulent viruses; virus invasion of meristem tissues allowing vertical transmission). This equilibrium, which would be associated with little selective pressure for the emergence of severe viral strains, is common in wild ecosystems and has important implications for the management of viral diseases in the field. Plant viruses are obligatory intracellular parasites that divert the host cellular machinery to complete their infection cycle. Highjacking/modification of plant factors can affect plant vigor and fitness. In addition, the toxic effects of viral proteins and the deployment of plant defense responses contribute to the induction of symptoms ranging in severity from tissue discoloration to malformation or tissue necrosis. The impact of viral infection is also influenced by the virulence of the specific virus strain (or strains for mixed infections), the host genotype and environmental conditions. Although plant resistance mechanisms that restrict virus accumulation or movement have received much attention, molecular mechanisms associated with tolerance are less well-understood. We review the experimental evidence that supports the concept that tolerance can be achieved by reaching the proper balance between plant defense responses and virus counter-defenses. We also discuss plant translation repression mechanisms, plant protein degradation or modification pathways and viral self-attenuation strategies that regulate the accumulation or activity of viral proteins to mitigate their impact on the host. Finally, we discuss current progress and future opportunities toward the application of various tolerance mechanisms in the field.
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Affiliation(s)
- Dinesh Babu Paudel
- Department of Botany, The University of British Columbia, Vancouver, BC, Canada
| | - Hélène Sanfaçon
- Summerland Research and Development Centre, Agriculture and Agri-Food Canada, Summerland, BC, Canada
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40
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Jo Y, Cho WK. RNA viromes of the oriental hybrid lily cultivar "Sorbonne". BMC Genomics 2018; 19:748. [PMID: 30316297 PMCID: PMC6186116 DOI: 10.1186/s12864-018-5138-3] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2018] [Accepted: 10/02/2018] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND The lily is a perennial flowering plant belonging to the genus Lilium in the family Liliaceae. Most cultivated lily plants are propagated by bulbs. Therefore, numerous lily bulbs are frequently infected by diverse viruses causing viral diseases. To date, no study has examined the viromes of plants of one type with identical genetic backgrounds collected from different geographical regions. RESULTS Here, we examined different viromes of the lily cultivar "Sorbonne" using 172 gigabytes of transcriptome data composed of 23 libraries from four different projects for the cultivar "Sorbonne." We identified 396 virus-associated contigs from all but one library. We identified six different viruses, including Plantago asiatica mosaic virus (PlAMV), Cucumber mosaic virus (CMV), Lily symptomless virus (LSV), Tulip virus X (TVX), Lily mottle virus (LMoV), and Tobacco rattle virus (TRV). Of them, PlAMV was the most common virus infecting the lily. Scale and flower samples possessed a high number of virus-associated reads. We assembled 32 nearly complete genomes for the six identified viruses possessing the polyadenylate tails. Genomes of all six viruses were highly conserved in the lily cultivar "Sorbonne" based on mutation analysis. We identified defective RNAs from LSV, TVX, and PlAMV localized in the triple gene block region. Phylogenetic analyses showed that virus genomes are highly correlated with geographical regions and host plants. CONCLUSIONS We conducted comprehensive virome analyses of a single lily cultivar, "Sorbonne," using transcriptome data. Our results shed light on an array of lily virome-associated topics, including virus identification, the dominant virus, virus accumulation in different plant tissues, virus genome assembly, virus mutation, identification of defective RNAs, and phylogenetic relationships of identified viruses. Taken together, we provide very useful methods and valuable results that can be applied in other virome-associated studies.
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Affiliation(s)
- Yeonhwa Jo
- Research Institute of Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University, Seoul, 08826 Republic of Korea
| | - Won Kyong Cho
- Research Institute of Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University, Seoul, 08826 Republic of Korea
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Leeks A, Segredo-Otero EA, Sanjuán R, West SA. Beneficial coinfection can promote within-host viral diversity. Virus Evol 2018; 4:vey028. [PMID: 30288300 PMCID: PMC6166523 DOI: 10.1093/ve/vey028] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
In many viral infections, a large number of different genetic variants can coexist within a host, leading to more virulent infections that are better able to evolve antiviral resistance and adapt to new hosts. But how is this diversity maintained? Why do faster-growing variants not outcompete slower-growing variants, and erode this diversity? One hypothesis is if there are mutually beneficial interactions between variants, with host cells infected by multiple different viral genomes producing more, or more effective, virions. We modelled this hypothesis with both mathematical models and simulations, and found that moderate levels of beneficial coinfection can maintain high levels of coexistence, even when coinfection is relatively rare, and when there are significant fitness differences between competing variants. Rare variants are more likely to be coinfecting with a different variant, and hence beneficial coinfection increases the relative fitness of rare variants through negative frequency dependence, and maintains diversity. We further find that coexisting variants sometimes reach unequal frequencies, depending on the extent to which different variants benefit from coinfection, and the ratio of variants which leads to the most productive infected cells. These factors could help drive the evolution of defective interfering particles, and help to explain why the different segments of multipartite viruses persist at different equilibrium frequencies.
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Affiliation(s)
- Asher Leeks
- Department of Zoology, University of Oxford, Oxford, UK
| | - Ernesto A Segredo-Otero
- Institute for Integrative Systems Biology (I2SysBio), Universitat de València, València, Spain
| | - Rafael Sanjuán
- Institute for Integrative Systems Biology (I2SysBio), Universitat de València, València, Spain
| | - Stuart A West
- Department of Zoology, University of Oxford, Oxford, UK
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An overview of process intensification and thermo stabilization for upscaling of Peste des petits ruminants vaccines in view of global control and eradication. Virusdisease 2018; 29:285-296. [PMID: 30159362 DOI: 10.1007/s13337-018-0455-3] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2018] [Accepted: 05/03/2018] [Indexed: 12/22/2022] Open
Abstract
Peste des petits ruminants (PPR) has been recognized as a globally distributed disease affecting the small ruminant population. The disease results in severe economic losses mainly to small land holders and low input farming systems. The control of PPR is mainly achieved through vaccination with available live attenuated vaccines. The thermo labile nature of PPR virus poses a major constraint in production of quality vaccines which often results in vaccine failures. The lack of quality vaccine production jeopardize the wide vaccination coverage especially in countries with poor infrastructure due to which PPR persists endemically. The vaccine production system may require augmentation to attain consistent and quality vaccines through efforts of process intensification integrated with suitable stabilizer formulations with appropriate freeze drying cycles for improved thermo tolerance. Manufacturing of live attenuated PPR vaccines during batch cultures might introduce defective interfering particles (DIPs) as a result of high multiplicity of infection (MOI) of inoculums, which has a huge impact on virus dynamics and yield. Accumulation of DIPs adversely affects the quality of the manufactured vaccines which can be avoided through use of appropriate MOI of virus inoculums and quality control of working seed viruses. Therefore, adherence to critical manufacturing standard operating procedures in vaccine production and ongoing efforts on development of thermo tolerant vaccine will help a long way in PPR control and eradication programme globally. The present review focuses on the way forward to achieve the objectives of quality vaccine production and easy upscaling to help the global PPR control and eradication by mass vaccination as an important tool.
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43
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Hasiów-Jaroszewska B, Minicka J, Zarzyńska-Nowak A, Budzyńska D, Elena SF. Defective RNA particles derived from Tomato black ring virus genome interfere with the replication of parental virus. Virus Res 2018; 250:87-94. [PMID: 29665369 DOI: 10.1016/j.virusres.2018.04.010] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2018] [Revised: 04/05/2018] [Accepted: 04/10/2018] [Indexed: 12/18/2022]
Abstract
Tomato black ring virus (TBRV) is the only member of the Nepovirus genus that is known to form defective RNA particles (D RNAs) during replication. Here, de novo generation of D RNAs was observed during prolonged passages of TBRV isolates originated from Solanum lycopersicum and Lactuca sativa in Chenopodium quinoa plants. D RNAs of about 500 nt derived by a single deletion in the RNA1 molecule and contained a portion of the 5' untranslated region and viral replicase, and almost the entire 3' non-coding region. Short regions of sequence complementarity were found at the 5' and 3' junction borders, which can facilitate formation of the D RNAs. Moreover, in this study we analyzed the effects of D RNAs on TBRV replication and symptoms development of infected plants. C. quinoa, S. lycopersicum, Nicotiana tabacum, and L. sativa were infected with the original TBRV isolates (TBRV-D RNA) and those containing additional D RNA particles (TBRV + D RNA). The viral accumulation in particular hosts was measured up to 28 days post inoculation by RT-qPCR. Statistical analyses revealed that D RNAs interfere with TBRV replication and thus should be referred to as defective interfering particles. The magnitude of the interference effect depends on the interplay between TBRV isolate and host species.
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Affiliation(s)
- Beata Hasiów-Jaroszewska
- Institute of Plant Protection-National Research Institute, Department of Virology and Bacteriology, ul. Wł. Węgorka 20, 60-318 Poznań, Poland.
| | - Julia Minicka
- Institute of Plant Protection-National Research Institute, Department of Virology and Bacteriology, ul. Wł. Węgorka 20, 60-318 Poznań, Poland
| | - Aleksandra Zarzyńska-Nowak
- Institute of Plant Protection-National Research Institute, Department of Virology and Bacteriology, ul. Wł. Węgorka 20, 60-318 Poznań, Poland
| | - Daria Budzyńska
- Institute of Plant Protection-National Research Institute, Department of Virology and Bacteriology, ul. Wł. Węgorka 20, 60-318 Poznań, Poland
| | - Santiago F Elena
- Instituto de Biología Molecular y Celular de Plantas, Consejo Superior de Investigaciones Científicas-Universitat Politècnica de València, València, Spain; Instituto de Biología Integrativa de Sistemas, Consejo Superior de Investigaciones Científicas-Universitat de València, València, Spain; The Santa Fe Institute, Santa Fe, New Mexico, USA
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44
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Abstract
Reproduction of RNA viruses is typically error-prone due to the infidelity of their replicative machinery and the usual lack of proofreading mechanisms. The error rates may be close to those that kill the virus. Consequently, populations of RNA viruses are represented by heterogeneous sets of genomes with various levels of fitness. This is especially consequential when viruses encounter various bottlenecks and new infections are initiated by a single or few deviating genomes. Nevertheless, RNA viruses are able to maintain their identity by conservation of major functional elements. This conservatism stems from genetic robustness or mutational tolerance, which is largely due to the functional degeneracy of many protein and RNA elements as well as to negative selection. Another relevant mechanism is the capacity to restore fitness after genetic damages, also based on replicative infidelity. Conversely, error-prone replication is a major tool that ensures viral evolvability. The potential for changes in debilitated genomes is much higher in small populations, because in the absence of stronger competitors low-fit genomes have a choice of various trajectories to wander along fitness landscapes. Thus, low-fit populations are inherently unstable, and it may be said that to run ahead it is useful to stumble. In this report, focusing on picornaviruses and also considering data from other RNA viruses, we review the biological relevance and mechanisms of various alterations of viral RNA genomes as well as pathways and mechanisms of rehabilitation after loss of fitness. The relationships among mutational robustness, resilience, and evolvability of viral RNA genomes are discussed.
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45
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Pyle JD, Scholthof KBG. De novo generation of helper virus-satellite chimera RNAs results in disease attenuation and satellite sequence acquisition in a host-dependent manner. Virology 2018; 514:182-191. [PMID: 29197268 DOI: 10.1016/j.virol.2017.11.006] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2017] [Revised: 11/07/2017] [Accepted: 11/08/2017] [Indexed: 12/27/2022]
Abstract
Panicum mosaic virus (PMV) is a helper RNA virus for satellite RNAs (satRNAs) and a satellite virus (SPMV). Here, we describe modifications that occur at the 3'-end of a satRNA of PMV, satS. Co-infections of PMV+satS result in attenuation of the disease symptoms induced by PMV alone in Brachypodium distachyon and proso millet. The 375 nt satS acquires ~100-200 nts from the 3'-end of PMV during infection and is associated with decreased abundance of the PMV RNA and capsid protein in millet. PMV-satS chimera RNAs were isolated from native infections of St. Augustinegrass and switchgrass. Phylogenetic analyses revealed that the chimeric RNAs clustered according to the host species from which they were isolated. Additionally, the chimera satRNAs acquired non-viral "linker" sequences in a host-specific manner. These results highlight the dynamic regulation of viral pathogenicity by satellites, and the selective host-dependent, sequence-based pressures for driving satRNA generation and genome compositions.
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Affiliation(s)
- Jesse D Pyle
- Department of Plant Pathology & Microbiology, Texas A&M University, College Station, TX 77843, United States.
| | - Karen-Beth G Scholthof
- Department of Plant Pathology & Microbiology, Texas A&M University, College Station, TX 77843, United States.
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Nonencapsidated 5' Copy-Back Defective Interfering Genomes Produced by Recombinant Measles Viruses Are Recognized by RIG-I and LGP2 but Not MDA5. J Virol 2017; 91:JVI.00643-17. [PMID: 28768856 DOI: 10.1128/jvi.00643-17] [Citation(s) in RCA: 37] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2017] [Accepted: 07/24/2017] [Indexed: 12/20/2022] Open
Abstract
Attenuated measles virus (MV) is one of the most effective and safe vaccines available, making it an attractive candidate vector for preventing other infectious diseases. Yet the great capacity of this vaccine still needs to be understood at the molecular level. MV vaccine strains have different type I interferon (IFN)-inducing abilities that partially depend on the presence of 5' copy-back defective interfering genomes (DI-RNAs). DI-RNAs are pathogen-associated molecular patterns recognized by RIG-I-like receptors (RLRs) (RIG-I, MDA5, and LGP2) that activate innate immune signaling and shape the adaptive immune response. In this study, we characterized the DI-RNAs produced by various modified recombinant MVs (rMVs), including vaccine candidates, as well as wild-type MV. All tested rMVs produced 5' copy-back DI-RNAs that were different in length and nucleotide sequence but still respected the so-called "rule of six." We correlated the presence of DI-RNAs with a larger stimulation of the IFN-β pathway and compared their immunostimulatory potentials. Importantly, we revealed that encapsidation of DI-RNA molecules within the MV nucleocapsid abolished their immunoactive properties. Furthermore, we identified specific interactions of DI-RNAs with both RIG-I and LGP2 but not MDA5. Our results suggest that DI-RNAs produced by rMV vaccine candidates may indeed strengthen their efficiency by triggering RLR signaling.IMPORTANCE Having been administered to hundreds of millions of children, the live attenuated measles virus (MV) vaccine is the safest and most widely used human vaccine, providing high protection with long-term memory. Additionally, recombinant MVs carrying heterologous antigens are promising vectors for new vaccines. The great capacity of this vaccine still needs to be elucidated at the molecular level. Here we document that recombinant MVs produce defective interfering genomes that have high immunostimulatory properties via their binding to RIG-I and LGP2 proteins, both of which are cytosolic nonself RNA sensors of innate immunity. Defective interfering genome production during viral replication should be considered of great importance due to the immunostimulatory properties of these genomes as intrinsic adjuvants produced by the vector that increase recognition by the innate immune system.
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Vasou A, Sultanoglu N, Goodbourn S, Randall RE, Kostrikis LG. Targeting Pattern Recognition Receptors (PRR) for Vaccine Adjuvantation: From Synthetic PRR Agonists to the Potential of Defective Interfering Particles of Viruses. Viruses 2017; 9:v9070186. [PMID: 28703784 PMCID: PMC5537678 DOI: 10.3390/v9070186] [Citation(s) in RCA: 47] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2017] [Revised: 07/11/2017] [Accepted: 07/11/2017] [Indexed: 12/13/2022] Open
Abstract
Modern vaccinology has increasingly focused on non-living vaccines, which are more stable than live-attenuated vaccines but often show limited immunogenicity. Immunostimulatory substances, known as adjuvants, are traditionally used to increase the magnitude of protective adaptive immunity in response to a pathogen-associated antigen. Recently developed adjuvants often include substances that stimulate pattern recognition receptors (PRRs), essential components of innate immunity required for the activation of antigen-presenting cells (APCs), which serve as a bridge between innate and adaptive immunity. Nearly all PRRs are potential targets for adjuvants. Given the recent success of toll-like receptor (TLR) agonists in vaccine development, molecules with similar, but additional, immunostimulatory activity, such as defective interfering particles (DIPs) of viruses, represent attractive candidates for vaccine adjuvants. This review outlines some of the recent advances in vaccine development related to the use of TLR agonists, summarizes the current knowledge regarding DIP immunogenicity, and discusses the potential applications of DIPs in vaccine adjuvantation.
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Affiliation(s)
- Andri Vasou
- Department of Biological Sciences, University of Cyprus, 1 University Avenue, Aglatzia, Nicosia 2109, Cyprus.
| | - Nazife Sultanoglu
- Department of Biological Sciences, University of Cyprus, 1 University Avenue, Aglatzia, Nicosia 2109, Cyprus.
| | - Stephen Goodbourn
- Institute for Infection and Immunity, St George's, University of London, London SW17 0RE, UK.
| | - Richard E Randall
- School of Biology, University of St Andrews, The North Haugh, St Andrews KY16 9ST, UK.
| | - Leondios G Kostrikis
- Department of Biological Sciences, University of Cyprus, 1 University Avenue, Aglatzia, Nicosia 2109, Cyprus.
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Chao M, Wang TC, Lin CC, Yung-Liang Wang R, Lin WB, Lee SE, Cheng YY, Yeh CT, Iang SB. Analyses of a whole-genome inter-clade recombination map of hepatitis delta virus suggest a host polymerase-driven and viral RNA structure-promoted template-switching mechanism for viral RNA recombination. Oncotarget 2017; 8:60841-60859. [PMID: 28977829 PMCID: PMC5617389 DOI: 10.18632/oncotarget.18339] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2016] [Accepted: 05/22/2017] [Indexed: 01/05/2023] Open
Abstract
The genome of hepatitis delta virus (HDV) is a 1.7-kb single-stranded circular RNA that folds into an unbranched rod-like structure and has ribozyme activity. HDV redirects host RNA polymerase(s) (RNAP) to perform viral RNA-directed RNA transcription. RNA recombination is known to contribute to the genetic heterogeneity of HDV, but its molecular mechanism is poorly understood. Here, we established a whole-genome HDV-1/HDV-4 recombination map using two cloned sequences coexisting in cultured cells. Our functional analyses of the resulting chimeric delta antigens (the only viral-encoded protein) and recombinant genomes provide insights into how recombination promotes the genotypic and phenotypic diversity of HDV. Our examination of crossover distribution and subsequent mutagenesis analyses demonstrated that ribozyme activity on HDV genome, which is required for viral replication, also contributes to the generation of an inter-clade junction. These data provide circumstantial evidence supporting our contention that HDV RNA recombination occurs via a replication-dependent mechanism. Furthermore, we identify an intrinsic asymmetric bulge on the HDV genome, which appears to promote recombination events in the vicinity. We therefore propose a mammalian RNAP-driven and viral-RNA-structure-promoted template-switching mechanism for HDV genetic recombination. The present findings improve our understanding of the capacities of the host RNAP beyond typical DNA-directed transcription.
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Affiliation(s)
- Mei Chao
- Department of Microbiology and Immunology, Chang Gung University, Guishan, Taoyang, Taiwan.,Division of Microbiology, Graduate Institute of Biomedical Sciences, Chang Gung University, Guishan, Taoyang, Taiwan.,Department of Hepato-Gastroenterology, Liver Research Center, Chang Gung Memorial Hospital, Guishan, Taoyang, Taiwan
| | - Tzu-Chi Wang
- Division of Microbiology, Graduate Institute of Biomedical Sciences, Chang Gung University, Guishan, Taoyang, Taiwan
| | - Chia-Chi Lin
- Division of Microbiology, Graduate Institute of Biomedical Sciences, Chang Gung University, Guishan, Taoyang, Taiwan
| | - Robert Yung-Liang Wang
- Division of Microbiology, Graduate Institute of Biomedical Sciences, Chang Gung University, Guishan, Taoyang, Taiwan.,Department of Biomedical Sciences, Chang Gung University, Guishan, Taoyang, Taiwan
| | - Wen-Bin Lin
- Division of Microbiology, Graduate Institute of Biomedical Sciences, Chang Gung University, Guishan, Taoyang, Taiwan
| | - Shang-En Lee
- Division of Microbiology, Graduate Institute of Biomedical Sciences, Chang Gung University, Guishan, Taoyang, Taiwan
| | - Ying-Yu Cheng
- Division of Microbiology, Graduate Institute of Biomedical Sciences, Chang Gung University, Guishan, Taoyang, Taiwan
| | - Chau-Ting Yeh
- Department of Hepato-Gastroenterology, Liver Research Center, Chang Gung Memorial Hospital, Guishan, Taoyang, Taiwan
| | - Shan-Bei Iang
- Division of Microbiology, Graduate Institute of Biomedical Sciences, Chang Gung University, Guishan, Taoyang, Taiwan
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49
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Jaworski E, Routh A. Parallel ClickSeq and Nanopore sequencing elucidates the rapid evolution of defective-interfering RNAs in Flock House virus. PLoS Pathog 2017; 13:e1006365. [PMID: 28475646 PMCID: PMC5435362 DOI: 10.1371/journal.ppat.1006365] [Citation(s) in RCA: 53] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2016] [Revised: 05/17/2017] [Accepted: 04/19/2017] [Indexed: 11/18/2022] Open
Abstract
Defective-Interfering RNAs (DI-RNAs) have long been known to play an important role in virus replication and transmission. DI-RNAs emerge during virus passaging in both cell-culture and their hosts as a result of non-homologous RNA recombination. However, the principles of DI-RNA emergence and their subsequent evolution have remained elusive. Using a combination of long- and short-read Next-Generation Sequencing, we have characterized the formation of DI-RNAs during serial passaging of Flock House virus (FHV) in cell-culture over a period of 30 days in order to elucidate the pathways and potential mechanisms of DI-RNA emergence and evolution. For short-read RNAseq, we employed 'ClickSeq' due to its ability to sensitively and confidently detect RNA recombination events with nucleotide resolution. In parallel, we used the Oxford Nanopore Technologies's (ONT) MinION to resolve full-length defective and wild-type viral genomes. Together, these accurately resolve both rare and common RNA recombination events, determine the correlation between recombination events, and quantifies the relative abundance of different DI-RNAs throughout passaging. We observe the formation of a diverse pool of defective RNAs at each stage of viral passaging. However, many of these 'intermediate' species, while present in early stages of passaging, do not accumulate. After approximately 9 days of passaging we observe the rapid accumulation of DI-RNAs with a correlated reduction in specific infectivity and with the Nanopore data find that DI-RNAs are characterized by multiple RNA recombination events. This suggests that intermediate DI-RNA species are not competitive and that multiple recombination events interact epistatically to confer 'mature' DI-RNAs with their selective advantage allowing for their rapid accumulation. Alternatively, it is possible that mature DI-RNA species are generated in a single event involving multiple RNA rearrangements. These insights have important consequences for our understanding of the mechanisms, determinants and limitations in the emergence and evolution of DI-RNAs.
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Affiliation(s)
- Elizabeth Jaworski
- Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, TX, United States of America
| | - Andrew Routh
- Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, TX, United States of America.,Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, Texas, United States of America
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50
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Piedra FA, Mei M, Avadhanula V, Mehta R, Aideyan L, Garofalo RP, Piedra PA. The interdependencies of viral load, the innate immune response, and clinical outcome in children presenting to the emergency department with respiratory syncytial virus-associated bronchiolitis. PLoS One 2017; 12:e0172953. [PMID: 28267794 PMCID: PMC5340370 DOI: 10.1371/journal.pone.0172953] [Citation(s) in RCA: 40] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2016] [Accepted: 02/13/2017] [Indexed: 11/18/2022] Open
Abstract
Respiratory syncytial virus (RSV) causes significant infant morbidity and mortality. For decades severe RSV-induced disease was thought to result from an uncontrolled host response to viral replication, but recent work suggests that a strong innate immune response early in infection is protective. To shed light on host-virus interactions and the viral determinants of disease, copy numbers of five RSV genes (NS1, NS2, N, G, F) were measured by quantitative real-time polymerase chain reaction (qPCR) in nasal wash samples from children with RSV-associated bronchiolitis. Correlations were sought with host cytokines/chemokines and biomarkers. Associations with disposition from the emergency department (hospitalized or sent home) and pulse oximetry O2 saturation levels were also sought. Additionally, RNase P copy number was measured and used to normalize nasal wash data. RSV gene copy numbers were found to significantly correlate with both cytokine/chemokine and biomarker levels; and RNase P-normalized viral gene copy numbers (NS1, NS2, N and G) were significantly higher in infants with less severe disease. Moreover, three of the normalized viral gene copy numbers (NS1, NS2, and N) correlated significantly with arterial O2 saturation levels. The data support a model where a higher viral load early in infection can promote a robust innate immune response that protects against progression into hypoxic RSV-induced lower respiratory tract illness.
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Affiliation(s)
- Felipe-Andrés Piedra
- Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, United States of America
| | - Minghua Mei
- Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, United States of America
| | - Vasanthi Avadhanula
- Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, United States of America
| | - Reena Mehta
- Allergy & Asthma Specialists, P.C., Saddle River, New Jersey, United States of America
| | - Letisha Aideyan
- Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, United States of America
| | - Roberto P. Garofalo
- Department of Pediatrics, University of Texas Medical Branch, Galveston, Texas, United States of America
| | - Pedro A. Piedra
- Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, United States of America
- Department of Pediatrics, Baylor College of Medicine, Houston, Texas, United States of America
- * E-mail:
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