scholarly journals Combined Short and Long-Read Sequencing Reveals a Complex Transcriptomic Architecture of African Swine Fever Virus

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 579
Author(s):  
Gábor Torma ◽  
Dóra Tombácz ◽  
Zsolt Csabai ◽  
Norbert Moldován ◽  
István Mészáros ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Current View ◽  
Preparation Methods ◽  
Oxford Nanopore ◽  
Long Read ◽  
Nested Genes ◽  
Low Coverage

African swine fever virus (ASFV) is a large DNA virus belonging to the Asfarviridae family. Despite its agricultural importance, little is known about the fundamental molecular mechanisms of this pathogen. Short-read sequencing (SRS) can produce a huge amount of high-precision sequencing reads for transcriptomic profiling, but it is inefficient for comprehensively annotating transcriptomes. Long-read sequencing (LRS) can overcome some of SRS’s limitations, but it also has drawbacks, such as low-coverage and high error rate. The limitations of the two approaches can be surmounted by the combined use of these techniques. In this study, we used Illumina SRS and Oxford Nanopore Technologies LRS platforms with multiple library preparation methods (amplified and direct cDNA sequencings and native RNA sequencing) for constructing the ASFV transcriptomic atlas. This work identified many novel transcripts and transcript isoforms and annotated the precise termini of previously described RNAs. This study identified a novel species of ASFV transcripts, the replication origin-associated RNAs. Additionally, we discovered several nested genes embedded into larger canonical genes. In contrast to the current view that the ASFV transcripts are monocistronic, we detected a significant extent of polycistronism. A multifaceted meshwork of transcriptional overlaps was also discovered.

scholarly journals Combined short and long-read sequencing reveals a complex transcriptomic architecture of African swine fever virus

2020 ◽  
Author(s):  
Gábor Torma ◽  
Dóra Tombácz ◽  
Zsolt Csabai ◽  
Norbert Moldován ◽  
István Mészáros ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
African Swine Fever Virus ◽  
Genetic Regulation ◽  
African Swine Fever ◽  
Fever Virus ◽  
Current View ◽  
Preparation Methods ◽  
Rna Molecules ◽  
Oxford Nanopore ◽  
Long Read

ABSTRACTAfrican swine fever virus (ASFV) is a large DNA virus belonging to the Asfarviridae family. Despite its agricultural importance, little is known about the fundamental molecular mechanisms of this pathogen. Understanding of genetic regulation provides new insights into the virus pathogenicity, which can help prevent epidemics. Short-read sequencing (SRS) is able to produce a huge amount of high-precision sequencing reads for transcriptomic profiling, but it is inefficient for the comprehensive annotation of transcriptomes. Long-read sequencing (LRS) is able to overcome some of the limitations of SRS, but they also have drawbacks, such as low-coverage and high error rate. The limitations of the two approaches can be surmounted by the combined use of these techniques. In this study, we used Illumina SRS and Oxford Nanopore Technologies LRS platforms with multiple library preparation methods (amplified and direct cDNA sequencings and native RNA sequencing) for constructing the transcriptomic atlas of ASFV. This work identified a large number of novel genes, transcripts and RNA isoforms, and annotated the precise termini of previously described RNA molecules. In contrast to the current view that the ASFV transcripts are monocistronic, we detected a significant extent of polycistronism. A multifaceted meshwork of transcriptional overlaps is also discovered.

scholarly journals Short and Long-read Sequencing Survey of the Dynamic Transcriptomes of African Swine Fever Virus and its Host

2020 ◽  
Author(s):  
Ferenc Olasz ◽  
Dóra Tombácz ◽  
Gábor Torma ◽  
Zsolt Csabai ◽  
Norbert Moldován ◽  
...  
Keyword(s):  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Economic Losses ◽  
Swine Industry ◽  
Rna Molecules ◽  
Oxford Nanopore ◽  
Combined Use ◽  
Long Read ◽  
Low Coverage

AbstractAfrican swine fever virus (ASFV) is an important animal pathogen causing substantial economic losses in the swine industry globally. At present, little is known about the molecular biology of ASFV, including its transcriptome organization. In this study, we applied cutting-edge sequencing approaches, namely the Illumina short-read sequencing (SRS) and the Oxford Nanopore Technologies long-read sequencing (LRS) techniques, together with several library preparation chemistries to analyze the ASFV dynamic transcriptome. SRS can generate a large amount of high-precision sequencing reads, but it is inefficient for identifying long RNA molecules, transcript isoforms and overlapping transcripts. LRS can overcome these limitations, but this approach also has shortcomings, such as its high error rate and the low coverage. Amplification-based LRS techniques produce relatively high read counts but also high levels of spurious transcripts, whereas the non-amplified cDNA and direct RNA sequencing techniques are more precise but achieve lower throughput. The drawbacks of the various technologies can be circumvented by the combined use of these approaches.

scholarly journals Complete Genome Sequence of African Swine Fever Virus Isolated from a Domestic Pig in Timor-Leste, 2019

2021 ◽  
Vol 10 (26) ◽  
Author(s):  
Patrick Mileto ◽  
Felisiano da Conceição ◽  
Vittoria Stevens ◽  
David Cummins ◽  
Andrea Certoma ◽  
...  
Keyword(s):  
Genome Sequence ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Domestic Pig ◽  
Oxford Nanopore ◽  
Timor Leste ◽  
Long Read

Here, we report the complete genome sequence of the African swine fever virus (ASFV) isolate ASFV/Timor-Leste/2019/1, isolated from a domestic pig during the first outbreak of ASF in Timor-Leste in 2019. Using target enrichment short-read Illumina data combined with long-read Oxford Nanopore data, we assembled a full-length genome sequence of 192,237 bp.

scholarly journals Short and Long-Read Sequencing Survey of the Dynamic Transcriptomes of African Swine Fever Virus and the Host Cells

2020 ◽  
Vol 11 ◽  
Author(s):  
Ferenc Olasz ◽  
Dóra Tombácz ◽  
Gábor Torma ◽  
Zsolt Csabai ◽  
Norbert Moldován ◽  
...  
Keyword(s):  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Host Cells ◽  
Long Read

scholarly journals Rapid Sequence-Based Characterization of African Swine Fever Virus by Use of the Oxford Nanopore MinION Sequence Sensing Device and a Companion Analysis Software Tool

2019 ◽  
Vol 58 (1) ◽  
Author(s):  
Vivian K. O’Donnell ◽  
Frederic R. Grau ◽  
Gregory A. Mayr ◽  
Tracy L. Sturgill Samayoa ◽  
Kimberly A. Dodd ◽  
...  
Keyword(s):  
Real Time ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Full Genome Sequence ◽  
Effective Vaccine ◽  
Fast Analysis ◽  
Rapid Acquisition ◽  
Oxford Nanopore

ABSTRACT African swine fever virus (ASFV) is the causative agent of a severe and highly contagious viral disease of pigs that poses serious economic consequences to the swine industry due to the high mortality rate and impact on international trade. There is no effective vaccine to control African swine fever (ASF), and therefore, efficient disease control is dependent on early detection and diagnosis of ASFV. The large size of the ASFV genome (∼180 kb) has historically hindered efforts to rapidly obtain a full-genome sequence. Rapid acquisition of data is critical for characterization of the isolate and to support epidemiological efforts. Here, we investigated the capacity of the Oxford Nanopore MinION sequence sensing device to act as a rapid sequencing tool. When coupled with our novel companion software script, the African swine fever fast analysis sequencing tool (ASF-FAST), the analysis of output data was performed in real time. Complete ASFV genome sequences were generated from cell culture isolates and blood samples obtained from experimentally infected pigs. Removal of the host-methylated DNA from the extracted nucleic acid facilitated rapid ASFV sequence identification, with reads specific to ASFV detected within 6 min after the initiation of sequencing. Regardless of the starting material, sufficient sequence was available for complete genome resolution (up to 100%) within 10 min. Overall, this paper highlights the use of Nanopore sequencing technology in combination with the ASF-FAST software for the purpose of rapid and real-time resolution of the full ASFV genome from a diagnostic sample.

scholarly journals A Proteomic Atlas of the African Swine Fever Virus Particle

2018 ◽  
Vol 92 (23) ◽  
Author(s):  
Alí Alejo ◽  
Tania Matamoros ◽  
Milagros Guerra ◽  
Germán Andrés
Keyword(s):  
Virus Particle ◽  
Immunoelectron Microscopy ◽  
Protein Modification ◽  
Molecular Mechanisms ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Sub Saharan Africa ◽  
Future Research ◽  
Host Proteins

ABSTRACTAfrican swine fever virus (ASFV) is a large and complex DNA virus that causes a highly lethal swine disease for which there is no vaccine available. The ASFV particle, with an icosahedral multilayered structure, contains multiple polypeptides whose identity is largely unknown. Here, we analyzed by mass spectroscopy the protein composition of highly purified extracellular ASFV particles and performed immunoelectron microscopy to localize several of the detected proteins. The proteomic analysis identified 68 viral proteins, which account for 39% of the genome coding capacity. The ASFV proteome includes essentially all the previously described virion proteins and, interestingly, 44 newly identified virus-packaged polypeptides, half of which have an unknown function. A great proportion of the virion proteins are committed to the virus architecture, including two newly identified structural proteins, p5 and p8, which are derived from the core polyproteins pp220 and pp62, respectively. In addition, the virion contains a full complement of enzymes and factors involved in viral transcription, various enzymes implicated in DNA repair and protein modification, and some proteins concerned with virus entry and host defense evasion. Finally, 21 host proteins, many of them localized at the cell surface and related to the cortical actin cytoskeleton, were reproducibly detected in the ASFV particle. Immunoelectron microscopy strongly supports the suggestion that these host membrane-associated proteins are recruited during virus budding at actin-dependent membrane protrusions. Altogether, the results of this study provide a comprehensive model of the ASFV architecture that integrates both compositional and structural information.IMPORTANCEAfrican swine fever virus causes a highly contagious and lethal disease of swine that currently affects many countries of sub-Saharan Africa, the Caucasus, the Russian Federation, and Eastern Europe and has very recently spread to China. Despite extensive research, effective vaccines or antiviral strategies are still lacking, and many basic questions on the molecular mechanisms underlying the infective cycle remain. One such gap regards the composition and structure of the infectious virus particle. In the study described in this report, we identified the set of viral and host proteins that compose the virion and determined or inferred the localization of many of them. This information significantly increases our understanding of the biological and structural features of an infectious African swine fever virus particle and will help direct future research efforts.

scholarly journals Structure of African Swine Fever Virus and Associated Molecular Mechanisms Underlying Infection and Immunosuppression: A Review

2021 ◽  
Vol 12 ◽  
Author(s):  
Yue Wang ◽  
Weifang Kang ◽  
Wenping Yang ◽  
Jing Zhang ◽  
Dan Li ◽  
...  
Keyword(s):  
Immune Response ◽  
Molecular Mechanism ◽  
Molecular Mechanisms ◽  
African Swine Fever Virus ◽  
Animal Health ◽  
African Swine Fever ◽  
Fever Virus ◽  
Economic Losses ◽  
Cell Immune Response ◽  
Effective Vaccine

African swine fever (ASF) is an acute, highly contagious, and deadly infectious disease. The mortality rate of the most acute and acute ASF infection is almost 100%. The World Organization for Animal Health [Office International des épizooties (OIE)] lists it as a legally reported animal disease and China lists it as class I animal epidemic. Since the first diagnosed ASF case in China on August 3, 2018, it has caused huge economic losses to animal husbandry. ASF is caused by the African swine fever virus (ASFV), which is the only member of Asfarviridae family. ASFV is and the only insect-borne DNA virus belonging to the Nucleocytoplasmic Large DNA Viruses (NCLDV) family with an icosahedral structure and an envelope. Till date, there are still no effective vaccines or antiviral drugs for the prevention or treatment of ASF. The complex viral genome and its sophisticated ability to regulate the host immune response may be the reason for the difficulty in developing an effective vaccine. This review summarizes the recent findings on ASFV structure, the molecular mechanism of ASFV infection and immunosuppression, and ASFV-encoded proteins to provide comprehensive proteomic information for basic research on ASFV. In addition, it also analyzes the results of previous studies and speculations on the molecular mechanism of ASFV infection, which aids the study of the mechanism of clinical pathological phenomena, and provides a possible direction for an intensive study of ASFV infection mechanism. By summarizing the findings on molecular mechanism of ASFV- regulated host cell immune response, this review provides orientations and ideas for fundamental research on ASFV and provides a theoretical basis for the development of protective vaccines against ASFV.

scholarly journals The African Swine Fever Virus with MGF360 and MGF505 Deleted Reduces the Apoptosis of Porcine Alveolar Macrophages by Inhibiting the NF-κB Signaling Pathway and Interleukin-1β

Vaccines ◽  
2021 ◽  
Vol 9 (11) ◽  
pp. 1371
Author(s):  
Qi Gao ◽  
Yunlong Yang ◽  
Weipeng Quan ◽  
Jiachen Zheng ◽  
Yizhuo Luo ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Alveolar Macrophages ◽  
Molecular Mechanisms ◽  
Vaccine Development ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Effective Vaccine ◽  
Wild Type ◽  
Mrna Transcription

African swine fever virus (ASFV) poses serious threats to the swine industry. The mortality rate of African swine fever (ASF) is 100%, and there is no effective vaccine currently available. Complex immune escape strategies of ASFV are crucial factors affecting immune prevention and vaccine development. CD2v and MGF360-505R genes have been implicated in the modulation of the immune response. The molecular mechanisms contributing to innate immunity are poorly understood. In this study, we discover the cytopathic effect and apoptosis of ΔCD2v/ΔMGF360-505R-ASFV after infection in porcine alveolar macrophages (PAMs) was significantly less than wild-type ASFV. We demonstrated that CD2v- and MGF360-505R-deficient ASFV decrease the level of apoptosis by inhibiting the NF-κB signaling pathway and IL-1β mRNA transcription. Compared with wild-type ASFV infection, the levels of phospho-NF-κB p65 and p-IκB protein decreased in CD2v- and MGF360-505R-deficient ASFV. Moreover, CD2v- and MGF360-505R-deficient ASFV induced less IL-1β production than wild-type ASFV and was attenuated in replication compared with wild-type ASFV. We further found that MGF360-12L, MGF360-13L, and MGF-505-2R suppress the promoter activity of NF-κB by reporter assays, and CD2v activates the NF-κB signaling pathway. These findings suggested that CD2v- and MGF360-505R-deficient ASFV could reduce the level of ASFV p30 and the apoptosis of PAMs by inhibiting the NF-κB signaling pathway and IL-1β mRNA transcription, which might reveal a novel strategy for ASFV to maintain the replication of the virus in the host.

scholarly journals African Swine Fever Virus Armenia/07 Virulent Strain Controls Interferon Beta Production through the cGAS-STING Pathway

2019 ◽  
Vol 93 (12) ◽  
Author(s):  
Raquel García-Belmonte ◽  
Daniel Pérez-Núñez ◽  
Marco Pittau ◽  
Juergen A. Richt ◽  
Yolanda Revilla
Keyword(s):  
Wild Boar ◽  
Immune Modulation ◽  
Molecular Mechanisms ◽  
African Swine Fever Virus ◽  
Virulent Strain ◽  
African Swine Fever ◽  
Fever Virus ◽  
Type I ◽  
Sting Pathway ◽  
First Time

ABSTRACT African swine fever virus (ASFV) is a complex, cytoplasmic double-stranded DNA (dsDNA) virus that is currently expanding throughout the world. Currently, circulating virulent genotype II Armenia/07-like viruses cause fatal disease in pigs and wild boar, whereas attenuated strains induce infections with various levels of chronic illness. Sensing cytosolic dsDNA, mainly by the key DNA sensor cyclic GMP-AMP synthase (cGAS), leads to the synthesis of type I interferon and involves signaling through STING, TBK1, and IRF3. After phosphorylation, STING translocates from the endoplasmic reticulum to the Golgi compartment and to the perinuclear region, acting as an indispensable adaptor connecting the cytosolic detection of DNA to the TBK1-IRF3 signaling pathway. We demonstrate here that attenuated NH/P68, but not virulent Armenia/07, activates the cGAS-STING-IRF3 cascade very early during infection, inducing STING phosphorylation and trafficking through a mechanism involving cGAMP. Both TBK1 and IRF3 are subsequently activated and, in response to this, a high level of beta interferon (IFN-β) was produced during NH/P68 infection; in contrast, Armenia/07 infection generated IFN-β levels below those of uninfected cells. Our results show that virulent Armenia/07 ASFV controls the cGAS-STING pathway, but these mechanisms are not at play when porcine macrophages are infected with attenuated NH/P68 ASFV. These findings show for the first time the involvement of the cGAS-STING-IRF3 route in ASFV infection, where IFN-β production or inhibition was found after infection by attenuated or virulent ASFV strains, respectively, thus reinforcing the idea that ASFV virulence versus attenuation may be a phenomenon grounded in ASFV-mediated innate immune modulation where the cGAS-STING pathway might play an important role. IMPORTANCE African swine fever, a devastating disease for domestic pigs and wild boar, is currently spreading in Europe, Russia, and China, becoming a global threat with huge economic and ecological consequences. One interesting aspect of ASFV biology is the molecular mechanism leading to high virulence of some strains compared to more attenuated strains, which produce subclinical infections. In this work, we show that the presently circulating virulent Armenia/07 virus blocks the synthesis of IFN-β, a key mediator between the innate and adaptive immune response. Armenia/07 inhibits the cGAS-STING pathway by impairing STING activation during infection. In contrast, the cGAS-STING pathway is efficiently activated during NH/P68 attenuated strain infection, leading to the production of large amounts of IFN-β. Our results show for the first time the relationship between the cGAS-STING pathway and ASFV virulence, contributing to uncover the molecular mechanisms of ASFV virulence and to the rational development of ASFV vaccines.

scholarly journals Complete Genome Sequence of a Virulent African Swine Fever Virus from a Domestic Pig in Ukraine

2019 ◽  
Vol 8 (42) ◽  
Author(s):  
Ganna Kovalenko ◽  
Anne-Lise Ducluzeau ◽  
Liudmyla Ishchenko ◽  
Mykola Sushko ◽  
Maryna Sapachova ◽  
...  
Keyword(s):  
Genome Sequence ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Base Pairs ◽  
Domestic Pig ◽  
Long Read ◽  
Full Length Genome

Here, we report the complete genome sequence of an African swine fever (ASF) virus (ASFV/Kyiv/2016/131) isolated from the spleen of a domestic pig in Ukraine with a lethal case of African swine fever. Using only long-read Nanopore sequences, we assembled a full-length genome of 191,911 base pairs in a single contig.

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