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

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.

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

10.1101/2020.02.27.967695 ◽

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.

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Modulation of type I interferon signaling by African swine fever virus (ASFV) of different virulence L60 and NHV in macrophage host cells

Veterinary Microbiology ◽

10.1016/j.vetmic.2018.02.008 ◽

2018 ◽

Vol 216 ◽

pp. 132-141 ◽

Cited By ~ 6

Author(s):

Raquel Portugal ◽

Alexandre Leitão ◽

Carlos Martins

Keyword(s):

Type I Interferon ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus ◽

Host Cells ◽

Type I ◽

Interferon Signaling

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African swine fever virus structural protein p17 inhibits cGAS-STING signaling pathway through interacting with STING

10.1101/2021.06.02.446854 ◽

2021 ◽

Author(s):

Wanglong Zheng ◽

Nengwen Xia ◽

Jia Luo ◽

Sen Jiang ◽

Jiajia Zhang ◽

...

Keyword(s):

Signaling Pathway ◽

Transmembrane Domain ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus ◽

Host Cells ◽

Structural Protein ◽

Swine Industry ◽

Lack Of Information ◽

Sting Pathway

African swine fever (ASF) is highly contagious, causes high mortality in domestic and feral swine, and has a significant economic impact on the global swine industry due to the lack of a vaccine or an effective treatment. African swine fever virus (ASFV) encodes more than 150 polypeptides, which may have intricate and delicate interactions with the host for the benefit of the virus to evade the host’s defenses. However, currently, there is still a lack of information regarding the roles of the viral proteins in host cells. Here, our data demonstrated that the p17, encoded by D117L gene could suppress porcine cGAS-STING signaling pathway, exhibiting the inhibitions of TBK1 and IRF3 phosphorylations, downstream promoter activities, cellular mRNA transcriptions and ISG56 induction, and antiviral responses. Further, we found that p17 was located in endoplasmic reticulum (ER) and Golgi apparatus, and interacted with STING, perturbing it in the recruitment of TBK1 and IKKϵ Additionally, it appeared that the transmembrane domain (amino acids 39–59) of p17 could be required for interacting with STING and inhibiting cGAS-STING pathway. Taken together, p17 could inhibit the cGAS-STING pathway through its interaction with STING and interference with STING in the recruitment of TBK1 and IKKϵ

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Induction of Robust Immune Responses in Swine by Using a Cocktail of Adenovirus-Vectored African Swine Fever Virus Antigens

Clinical and Vaccine Immunology ◽

10.1128/cvi.00395-16 ◽

2016 ◽

Vol 23 (11) ◽

pp. 888-900 ◽

Cited By ~ 17

Author(s):

Shehnaz Lokhandwala ◽

Suryakant D. Waghela ◽

Jocelyn Bray ◽

Cameron L. Martin ◽

Neha Sangewar ◽

...

Keyword(s):

Immune Responses ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus ◽

Host Cells ◽

Hemorrhagic Disease ◽

Virulent Strains ◽

Challenge Study ◽

Cellular Immune

ABSTRACTThe African swine fever virus (ASFV) causes a fatal hemorrhagic disease in domestic swine, and at present no treatment or vaccine is available. Natural and gene-deleted, live attenuated strains protect against closely related virulent strains; however, they are yet to be deployed and evaluated in the field to rule out chronic persistence and a potential for reversion to virulence. Previous studies suggest that antibodies play a role in protection, but induction of cytotoxic T lymphocytes (CTLs) could be the key to complete protection. Hence, generation of an efficacious subunit vaccine depends on identification of CTL targets along with a suitable delivery method that will elicit effector CTLs capable of eliminating ASFV-infected host cells and confer long-term protection. To this end, we evaluated the safety and immunogenicity of an adenovirus-vectored ASFV (Ad-ASFV) multiantigen cocktail formulated in two different adjuvants and at two immunizing doses in swine. Immunization with the cocktail rapidly induced unprecedented ASFV antigen-specific antibody and cellular immune responses against all of the antigens. The robust antibody responses underwent rapid isotype switching within 1 week postpriming, steadily increased over a 2-month period, and underwent rapid recall upon boost. Importantly, the primed antibodies strongly recognized the parental ASFV (Georgia 2007/1) by indirect fluorescence antibody (IFA) assay and Western blotting. Significant antigen-specific gamma interferon-positive (IFN-γ+) responses were detected postpriming and postboosting. Furthermore, this study is the first to demonstrate induction of ASFV antigen-specific CTL responses in commercial swine using Ad-ASFV multiantigens. The relevance of the induced immune responses in regard to protection needs to be evaluated in a challenge study.

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The determinants of African Swine Fever Virus Virulence – the Georgia 2007/1 strain and the host macrophage response

10.1101/2021.07.26.453801 ◽

2021 ◽

Author(s):

Gwenny Cackett ◽

Raquel Portugal ◽

Dorota Matelska ◽

Linda Dixon ◽

Finn Werner

Keyword(s):

Gene Expression ◽

African Swine Fever Virus ◽

Case Fatality ◽

African Swine Fever ◽

Fever Virus ◽

Host Cells ◽

Macrophage Response ◽

Transcription Start Sites ◽

Nucleotide Resolution ◽

Cap Analysis

African swine fever virus (ASFV) has a major global economic impact. With a case fatality in domestic pigs approaching 100%, it currently presents the largest threat to animal farming. Although genomic differences between attenuated and highly virulent ASFV strains have been identified the molecular determinants for virulence at the level of gene expression have remained opaque. Here we characterise the transcriptome of ASFV genotype II Georgia 2007/1 (GRG) during infection of the physiologically relevant host cells, porcine macrophages. In this study Cap Analysis Gene Expression sequencing (CAGE-seq) was used to map the 5’ ends of mRNAs at nucleotide resolution, transcription start sites (TSSs) and the global promoter landscape of GRG at early and late times, 5 and 16 hours, post-infection. We then compared transcriptomic maps between the GRG isolate against the lab-attenuated BA71V strain. GRG-specific transcripts identified potential determinants of virulence including members of early expressed multi-gene family members (MGFs), including two we newly characterised in MGF 100 (I7L and I8L). We have importantly shown the response of the host transcriptome to infection, which highlighted a pro-inflammatory immune response with the upregulation of NF-kB activated genes, innate immunity- as well as lysosome components such as S100 proteins.

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Spatiotemporally Orchestrated Interactions between Viral and Cellular Proteins Involved in the Entry of African Swine Fever Virus

Viruses ◽

10.3390/v13122495 ◽

2021 ◽

Vol 13 (12) ◽

pp. 2495

Author(s):

Kehui Zhang ◽

Su Li ◽

Sheng Liu ◽

Shuhong Li ◽

Liang Qu ◽

...

Keyword(s):

Intracellular Transport ◽

Early Stage ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus ◽

Host Cells ◽

Hemorrhagic Disease ◽

Multilayered Structures ◽

Viral Receptor ◽

Molecular Events

African swine fever (ASF) is a highly contagious hemorrhagic disease in domestic pigs and wild boars with a mortality of up to 100%. The causative agent, African swine fever virus (ASFV), is a member of the Asfarviridae family of the nucleocytoplasmic large DNA viruses. The genome size of ASFV ranges from 170 to 194 kb, encoding more than 50 structural and 100 nonstructural proteins. ASFV virions are 260–300 nm in diameter and composed of complex multilayered structures, leading to an intricate internalization pathway to enter host cells. Currently, no commercial vaccines or antivirals are available, due to the insufficient knowledge of the viral receptor(s), the molecular events of ASFV entry into host cells, and the functions of virulence-associated genes. During the early stage of ASFV infection, the fundamental aspects of virus-host interactions, including virus internalization, intracellular transport through the endolysosomal system, and membrane fusion with endosome, are precisely regulated and orchestrated via a series of molecular events. In this review, we summarize the currently available knowledge on the pathways of ASFV entry into host cells and the functions of viral proteins involved in virus entry. Furthermore, we conclude with future perspectives and highlight areas that require further investigation. This review is expected to provide unique insights for further understanding ASFV entry and facilitate the development of vaccines and antivirals.

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

Microbiology Resource Announcements ◽

10.1128/mra.00263-21 ◽

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.

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