46 results on '"Netherton CL"'
Search Results
2. Six adenoviral vectored African swine fever virus genes protect against fatal disease caused by genotype I challenge.
- Author
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Portugal R, Goldswain H, Moore R, Tully M, Harris K, Corla A, Flannery J, Dixon LK, and Netherton CL
- Subjects
- Animals, Swine, Antibodies, Viral blood, Antibodies, Viral immunology, Vaccines, Subunit immunology, Vaccines, Subunit genetics, Antigens, Viral immunology, Antigens, Viral genetics, African Swine Fever Virus genetics, African Swine Fever Virus immunology, African Swine Fever prevention & control, African Swine Fever virology, African Swine Fever immunology, Viral Vaccines immunology, Viral Vaccines genetics, Viral Vaccines administration & dosage, Genetic Vectors genetics, Genotype, Adenoviridae genetics, Adenoviridae immunology
- Abstract
African swine fever virus causes a lethal hemorrhagic disease in domestic swine and wild boar for which currently licensed commercial vaccines are only available in Vietnam. Development of subunit vaccines is complicated by the lack of information on protective antigens as well as suitable delivery systems. Our previous work showed that a pool of eight African swine fever virus genes vectored using an adenovirus prime and modified vaccinia virus boost could prevent fatal disease after challenge with a virulent genotype I isolate of the virus. Here, we identify antigens within this pool of eight that are essential for the observed protection and demonstrate that adenovirus-prime followed by adenovirus-boost can also induce protective immune responses against genotype I African swine fever virus. Immunization with a pool of adenoviruses expressing individual African swine fever virus genes partially tailored to genotype II virus did not protect against challenge with genotype II Georgia 2007/1 strain, suggesting that different antigens may be required to induce cross-protection for genetically distinct viruses., Importance: African swine fever virus causes a lethal hemorrhagic disease in domestic pigs and has killed millions of animals across Europe and Asia since 2007. Development of safe and effective subunit vaccines against African swine fever has been problematic due to the complexity of the virus and a poor understanding of protective immunity. In a previous study, we demonstrated that a complex combination of eight different virus genes delivered using two different viral vector vaccine platforms protected domestic pigs from fatal disease. In this study, we show that three of the eight genes are required for protection and that one viral vector is sufficient, significantly reducing the complexity of the vaccine. Unfortunately, this combination did not protect against the current outbreak strain of African swine fever virus, suggesting that more work to identify immunogenic and protective viral proteins is required to develop a truly effective African swine fever vaccine., Competing Interests: C.L.N. and L.D.K. are named inventors on the patent WO/2021/019232 "African swine fever vaccine". The other authors declare no conflict of interest. The funders had no role in the design of the study, collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
- Published
- 2024
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3. Investigation of activation-induced markers (AIM) in porcine T cells by flow cytometry.
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Moorton M, Tng PYL, Inoue R, Netherton CL, Gerner W, and Schmidt S
- Abstract
Activation-induced markers (AIMs) are frequently analyzed to identify re-activated human memory T cells. However, in pigs the analysis of AIMs is still not very common. Based on available antibodies, we designed a multi-color flow cytometry panel comprising pig-specific or cross-reactive antibodies against CD25, CD69, CD40L (CD154), and ICOS (CD278) combined with lineage/surface markers against CD3, CD4, and CD8α. In addition, we included an antibody against tumor necrosis factor alpha (TNF-α), to study the correlation of AIM expression with the production of this abundant T cell cytokine. The panel was tested on peripheral blood mononuclear cells (PBMCs) stimulated with phorbol 12-myristate 13-acetate (PMA)/ionomycin, Staphylococcus enterotoxin B (SEB) or PBMCs from African swine fever virus (ASFV) convalescent pigs, restimulated with homologous virus. PMA/ionomycin resulted in a massive increase of CD25/CD69 co-expressing T cells of which only a subset produced TNF-α, whereas CD40L expression was largely associated with TNF-α production. SEB stimulation triggered substantially less AIM expression than PMA/ionomycin but also here CD25/CD69 expressing T cells were identified which did not produce TNF-α. In addition, CD40L-single positive and CD25
+ CD69+ CD40L+ TNF-α- T cells were identified. In ASFV restimulated T cells TNF-α production was associated with a substantial proportion of AIM expressing T cells but also here ASFV-reactive CD25+ CD69+ TNF-α- T cells were identified. Within CD8α+ CD4 T cells, several CD25/CD40L/CD69/ICOS defined phenotypes expanded significantly after ASFV restimulation. Hence, the combination of AIMs tested will allow the identification of primed T cells beyond the commonly used cytokine panels, improving capabilities to identify the full breadth of antigen-specific T cells in pigs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Moorton, Tng, Inoue, Netherton, Gerner and Schmidt.)- Published
- 2024
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4. African swine fever virus NAM P1/95 is a mixture of genotype I and genotype VIII viruses.
- Author
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Goatley LC, Freimanis GL, Tennakoon C, Bastos A, Heath L, and Netherton CL
- Abstract
African swine fever virus causes a lethal hemorrhagic disease of domestic pigs. The NAM P1/1995 isolate was originally described as B646L genotype XVIII; however, full genome sequencing revealed that this assignment was incorrect., Competing Interests: The authors declare no conflict of interest.
- Published
- 2024
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5. The transcriptomic insight into the differential susceptibility of African Swine Fever in inbred pigs.
- Author
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Banabazi MH, Freimanis G, Goatley LC, Netherton CL, and de Koning DJ
- Subjects
- Swine, Animals, Transcriptome, Gene Expression Profiling, Immunization, African Swine Fever, African Swine Fever Virus genetics
- Abstract
African swine fever (ASF) is a global threat to animal health and food security. ASF is typically controlled by strict biosecurity, rapid diagnosis, and culling of affected herds. Much progress has been made in developing modified live virus vaccines against ASF. There is host variation in response to ASF infection in the field and under controlled conditions. To better understand the dynamics underlying this host differential morbidity, whole transcriptome profiling was carried out in twelve immunized and five sham immunized pigs. Seventeen MHC homozygous inbred Large white Babraham pigs were sampled at three time points before and after the challenge. The changes in the transcriptome profiles of infected animals were surveyed over time. In addition, the immunization effect on the host response was studied as well among the contrasts of all protection subgroups. The results showed two promising candidate genes to distinguish between recovered and non-recovered pigs after infection with a virulent African swine fever virus (ASFV) pre-infection: HTRA3 and GFPT2 (padj < 0.05). Variant calling on the transcriptome assemblies showed a two-base pair insertion into the ACOX3 gene closely located to HTRA3 that may regulate its expression as a putative genomic variant for ASF. Several significant DGEs, enriched gene ontology (GO) terms, and KEGG pathways at 1 day and 7 days post-infection, compared to the pre-infection, indicate a significant inflammation response immediately after ASF infection. The presence of the virus was confirmed by the mapping of RNA-Seq reads on two whole viral genome sequences. This was concordant with a higher virus load in the non-recovered animals 7 days post-infection. There was no transcriptome signature on the immunization at pre-infection and 1 day post-infection. More samples and data from additional clinical trials may support these findings., (© 2024. The Author(s).)
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- 2024
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6. 2023 International African Swine Fever Workshop: Critical Issues That Need to Be Addressed for ASF Control.
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Wang L, Ganges L, Dixon LK, Bu Z, Zhao D, Truong QL, Richt JA, Jin M, Netherton CL, Benarafa C, Summerfield A, Weng C, Peng G, Reis AL, Han J, Penrith ML, Mo Y, Su Z, Vu Hoang D, Pogranichniy RM, Balaban-Oglan DA, Li Y, Wang K, Cai X, and Shi J
- Subjects
- Swine, Animals, Humans, Asia, China epidemiology, Africa epidemiology, Sus scrofa, Disease Outbreaks veterinary, African Swine Fever prevention & control, African Swine Fever epidemiology, African Swine Fever Virus
- Abstract
The 2023 International African Swine Fever Workshop (IASFW) took place in Beijing, China, on 18-20 September 2023. It was jointly organized by the U.S.-China Center for Animal Health (USCCAH) at Kansas State University (KSU) and the Chinese Veterinary Drug Association (CVDA) and sponsored by the United States Department of Agriculture Foreign Agricultural Service (USDA-FAS), Harbin Veterinary Research Institute, and Zoetis Inc. The objective of this workshop was to provide a platform for ASF researchers around the world to unite and share their knowledge and expertise on ASF control and prevention. A total of 24 outstanding ASF research scientists and experts from 10 countries attended this meeting. The workshop included presentations on current ASF research, opportunities for scientific collaboration, and discussions of lessons and experiences learned from China/Asia, Africa, and Europe. This article summarizes the meeting highlights and presents some critical issues that need to be addressed for ASF control and prevention in the future.
- Published
- 2023
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7. Capsid-Specific Antibody Responses of Domestic Pigs Immunized with Low-Virulent African Swine Fever Virus.
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Tng PYL, Al-Adwani L, Pauletto E, Hui JYK, and Netherton CL
- Abstract
African swine fever (ASF) is a lethal disease in pigs that has grave socio-economic implications worldwide. For the development of vaccines against the African swine fever virus (ASFV), immunogenic antigens that generate protective immune responses need to be identified. There are over 150 viral proteins-many of which are uncharacterized-and humoral immunity to ASFV has not been closely examined. To profile antigen-specific antibody responses, we developed luciferase-linked antibody capture assays (LACAs) for a panel of ASFV capsid proteins and screened sera from inbred and outbred animals that were previously immunized with low-virulent ASFV before challenge with virulent ASFV. Antibodies to B646L/p72, D117L/p17, M1249L, and E120R/p14.5 were detected in this study; however, we were unable to detect B438L-specific antibodies. Anti-B646L/p72 and B602L antibodies were associated with recovery from disease after challenges with genotype I OUR T88/1 but not genotype II Georgia 2007/1. Antibody responses against M1249L and E120R/p14.5 were observed in animals with reduced clinical signs and viremia. Here, we present LACAs as a tool for the targeted profiling of antigen-specific antibody responses to inform vaccine development.
- Published
- 2023
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8. The Effect of Temperature on the Stability of African Swine Fever Virus BA71V Isolate in Environmental Water Samples.
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Loundras EA, Netherton CL, Flannery J, Bowes MJ, Dixon L, and Batten C
- Abstract
African swine fever virus (ASFV) is known to be very stable and can remain infectious over long periods of time especially at low temperatures and within different matrices, particularly those containing animal-derived organic material. However, there are some gaps in our knowledge pertaining to the survivability and infectivity of ASFV in groundwater. This study aims to determine the stability and infectivity of the cell culture-adapted ASFV strain BA71V by plaque assay after incubation of the virus within river water samples at three different environmentally relevant temperatures (4 °C, 15 °C, and 21 °C) over the course of 42 days. The results from this study indicate that ASFV can remain stable and infectious when maintained at 4 °C in river water for more than 42 days, but as incubation temperatures are increased, the stability is reduced, and the virus is no longer able to form plaques after 28 days and 14 days, respectively, when stored at 15 °C and 21 °C. Characterizing the survivability of ASFV in groundwater can allow us to develop more appropriate inactivation and disinfection methods to support disease control and mitigate ASFV outbreaks.
- Published
- 2023
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9. Functional Landscape of African Swine Fever Virus-Host and Virus-Virus Protein Interactions.
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Dolata KM, Pei G, Netherton CL, and Karger A
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- Animals, Swine, Antiviral Agents, Microbial Interactions, Virus Replication, African Swine Fever Virus
- Abstract
Viral replication fully relies on the host cell machinery, and physical interactions between viral and host proteins mediate key steps of the viral life cycle. Therefore, identifying virus-host protein-protein interactions (PPIs) provides insights into the molecular mechanisms governing virus infection and is crucial for designing novel antiviral strategies. In the case of the African swine fever virus (ASFV), a large DNA virus that causes a deadly panzootic disease in pigs, the limited understanding of host and viral targets hinders the development of effective vaccines and treatments. This review summarizes the current knowledge of virus-host and virus-virus PPIs by collecting and analyzing studies of individual viral proteins. We have compiled a dataset of experimentally determined host and virus protein targets, the molecular mechanisms involved, and the biological functions of the identified virus-host and virus-virus protein interactions during infection. Ultimately, this work provides a comprehensive and systematic overview of ASFV interactome, identifies knowledge gaps, and proposes future research directions.
- Published
- 2023
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10. Complete genome analysis of African swine fever virus genotypes II, IX and XV from domestic pigs in Tanzania.
- Author
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Hakizimana JN, Yona C, Makange MR, Kasisi EA, Netherton CL, Nauwynck H, and Misinzo G
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- Swine, Animals, Sus scrofa, Tanzania epidemiology, Genotype, African Swine Fever Virus genetics, African Swine Fever
- Abstract
African swine fever (ASF) caused by ASF virus (ASFV) is an infectious transboundary animal disease notifiable to the World Organization for Animal Health causing high mortality in domestic pigs and wild boars threatening the global domestic pig industry. To date, twenty-four ASFV genotypes have been described and currently genotypes II, IX, X, XV and XVI are known to be circulating in Tanzania. Despite the endemic status of ASF in Tanzania, only one complete genome of ASFV from the country has been described. This study describes the first complete genome sequence of ASFV genotype XV. In addition, the first Tanzanian complete genome of ASFV genotype IX and three ASFV strains belonging to genotype II collected during ASF outbreaks in domestic pigs in Tanzania were determined in this study using Illumina sequencing and comparative genomics analysis. The generated ASFV complete genome sequences ranged from 171,004 to 184,521 base pairs in length with an average GC content of 38.53% and encoded 152 to 187 open reading frames. The results of this study provide insights into the genomic structure of ASFV and can be used to monitor changes within the ASFV genome and improve our understanding of ASF transmission dynamics., (© 2023. The Author(s).)
- Published
- 2023
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11. African Swine Fever Virus Host-Pathogen Interactions.
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Netherton CL, Shimmon GL, Hui JYK, Connell S, and Reis AL
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- Swine, Animals, Viral Proteins genetics, Host-Pathogen Interactions, Virus Replication, African Swine Fever Virus genetics, African Swine Fever metabolism
- Abstract
African swine fever virus is a complex double-stranded DNA virus that exhibits tropism for cells of the mononuclear phagocytic system. Virus replication is a multi-step process that involves the nucleus of the host cell as well the formation of large perinuclear sites where progeny virions are assembled prior to transport to, and budding through, the plasma membrane. Like many viruses, African swine fever virus reorganises the cellular architecture to facilitate its replication and has evolved multiple mechanisms to avoid the potential deleterious effects of host cell stress response pathways. However, how viral proteins and virus-induced structures trigger cellular stress pathways and manipulate the subsequent responses is still relatively poorly understood. African swine fever virus alters nuclear substructures, modulates autophagy, apoptosis and the endoplasmic reticulum stress response pathways. The viral genome encodes for at least 150 genes, of which approximately 70 are incorporated into the virion. Many of the non-structural genes have not been fully characterised and likely play a role in host range and modifying immune responses. As the field moves towards approaches that take a broader view of the effect of expression of individual African swine fever genes, we summarise how the different steps in virus replication interact with the host cell and the current state of knowledge on how it modulates the resulting stress responses., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)
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- 2023
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12. Cellular and Humoral Immune Responses after Immunisation with Low Virulent African Swine Fever Virus in the Large White Inbred Babraham Line and Outbred Domestic Pigs.
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Goatley LC, Nash RH, Andrews C, Hargreaves Z, Tng P, Reis AL, Graham SP, and Netherton CL
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- Animals, Immunity, Humoral, Immunization, Swine, Swine, Miniature, African Swine Fever, African Swine Fever Virus, Viral Vaccines
- Abstract
African swine fever virus is currently present in all of the world's continents apart from Antarctica, and efforts to control the disease are hampered by the lack of a commercially available vaccine. The Babraham large white pig is a highly inbred line that could represent a powerful tool to improve our understanding of the protective immune responses to this complex pathogen; however, previous studies indicated differential vaccine responses after the African swine fever virus challenge of inbred minipigs with different swine leukocyte antigen haplotypes. Lymphocyte numbers and African swine fever virus-specific antibody and T-cell responses were measured in inbred and outbred animals after inoculation with a low virulent African swine fever virus isolate and subsequent challenge with a related virulent virus. Surprisingly, diminished immune responses were observed in the Babraham pigs when compared to the outbred animals, and the inbred pigs were not protected after challenge. Recovery of Babraham pigs after challenge weakly correlated with antibody responses, whereas protective responses in outbred animals more closely correlated with the T-cell response. The Babraham pig may, therefore, represent a useful model for studying the role of antibodies in protection against the African swine fever virus.
- Published
- 2022
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13. Novel method for sub-grouping of genotype II African swine fever viruses based on the intergenic region between the A179L and A137R genes.
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Tran HTT, Truong AD, Dang AK, Ly DV, Chu NT, Van Hoang T, Nguyen HT, Netherton CL, and Dang HV
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- Animals, DNA, Intergenic genetics, Genotype, Phylogeny, Sequence Analysis, DNA veterinary, Sus scrofa genetics, Swine, African Swine Fever epidemiology, African Swine Fever Virus genetics, Swine Diseases
- Abstract
Background: African swine fever (ASF) is a highly contagious and deadly viral disease affecting domestic and wild pigs of all ages. African swine fever virus (ASFV) has spread rapidly through Eastern and Southeastern Asia first appearing in Vietnam in 2019., Objectives: Molecular typing of African swine fever virus (ASFV) in Vietnam has identified two principal variants circulating based on the sequencing of the intergenic region (IRG) between the I73R and I329L genes. Identification of additional genetic markers would enable higher resolution tracing of outbreaks within the country., Methods: Sequence analysis suggested the IRG between the A179L and A137R genes may also exhibit variability, PCR primers were designed and samples from Vietnam were subject to Sanger sequencing., Results: We developed a novel method for sub-grouping of ASFV based on the IRG between the A179L and A137R genes of ASFV. Our results demonstrated that the finding of the insertion or deletion of an 11- nucleotide sequence (GATACAATTGT) between the A179L-A137R genes., Conclusions: The sub-grouping method may provide useful insights into the evolution of genotype II ASFV as well as providing evidence of a relationship between geographically separated outbreaks., (© 2021 The Authors. Veterinary Medicine and Science published by John Wiley & Sons Ltd.)
- Published
- 2022
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14. Adaptive Cellular Immunity against African Swine Fever Virus Infections.
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Schäfer A, Franzoni G, Netherton CL, Hartmann L, Blome S, and Blohm U
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African swine fever virus (ASFV) remains a threat to global pig populations. Infections with ASFV lead to a hemorrhagic disease with up to 100% lethality in Eurasian domestic and wild pigs. Although myeloid cells are the main target cells for ASFV, T cell responses are impacted by the infection as well. The complex responses remain not well understood, and, consequently, there is no commercially available vaccine. Here, we review the current knowledge about the induction of antiviral T cell responses by cells of the myeloid lineage, as well as T cell responses in infected animals, recent efforts in vaccine research, and T cell epitopes present in ASFV.
- Published
- 2022
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15. Primary Macrophage Culture from Porcine Blood and Lungs.
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Goatley LC, Nash R, and Netherton CL
- Subjects
- Animals, Cells, Cultured, Lung, Macrophages, Swine, African Swine Fever, African Swine Fever Virus
- Abstract
Primary cultures represent the most reliable method to isolate and propagate field isolates of African swine fever virus (ASFV ). Within the pig ASFV predominantly targets the reticuloendothelial system for replication; therefore, primary macrophage cell cultures are commonly used to isolate, propagate, and study the virus life cycle in the laboratory. In this chapter we will describe methods for the direct isolation of pulmonary alveolar macrophages by lung lavage and the culture of monocyte-derived macrophages from pig blood. We also include a method for the positive selection of CD14+ monocytes as a source for monocyte-derived macrophages from pig blood using microbeads., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
- Published
- 2022
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16. Laboratory Diagnosis and Quantification of African Swine Fever Virus Using Real-Time Polymerase Chain Reaction.
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Netherton CL, Goatley LC, Flannery J, Ashby M, and Batten C
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- Animals, Clinical Laboratory Techniques, Real-Time Polymerase Chain Reaction methods, Sensitivity and Specificity, Swine, African Swine Fever diagnosis, African Swine Fever Virus genetics
- Abstract
Real-time polymerase chain reaction (PCR) for the detection of African swine fever virus (ASFV) is the tool of choice for the diagnostic laboratory and is a robust and easily scalable method for the researcher analyzing viral replication both in vitro and in vivo. In this chapter, we describe protocols for both quantitative real-time polymerase chain reactions (qPCR) and non-quantitative real-time polymerase chain reactions (real-time PCR) for the detection of African swine fever virus genome in a range of samples., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
- Published
- 2022
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17. Purification of African Swine Fever Virus.
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Shimmon GL, Shah PNM, Fry E, Stuart DI, Hawes P, and Netherton CL
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- Animals, Cell Line, Cells, Cultured, DNA Viruses, Swine, Viral Proteins, African Swine Fever, African Swine Fever Virus
- Abstract
African swine fever virus is a cytolytic virus that leads to the apoptosis of both cultured cells and primary macrophages. Cell culture supernatants of virus-infected cells are routinely used for virological and immunological studies, despite differences in the biological behavior between such preparations and highly purified virus. In addition, more recent data suggests that exosomes containing viral proteins may be secreted from infected cells. While African swine fever virus can be purified through a number of methods, in our hands Percoll provides the most robust method of separating virus from cellular contaminants., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
- Published
- 2022
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18. Identification and Characterization of a Novel Epitope of ASFV-Encoded dUTPase by Monoclonal Antibodies.
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Zhang S, Wang R, Zhu X, Jin J, Lu W, Zhao X, Wan B, Liao Y, Zhao Q, Netherton CL, Zhuang G, Sun A, and Zhang G
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- African Swine Fever virology, Amino Acid Sequence, Animals, Antibodies, Viral immunology, Antigens, Viral immunology, Epitope Mapping, Epitopes genetics, Recombinant Proteins, Swine, Virus Replication, African Swine Fever Virus genetics, African Swine Fever Virus immunology, Antibodies, Monoclonal immunology, Epitopes isolation & purification, Pyrophosphatases genetics
- Abstract
Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) of African swine fever virus (ASFV) is an essential enzyme required for efficient virus replication. Previous crystallography data have indicated that dUTPase (E165R) may serve as a therapeutic target for inhibiting ASFV replication; however, the specificity of the targeting site(s) in ASFV dUTPase remains unclear. In this study, 19 mouse monoclonal antibodies (mAbs) were produced, in which four mAbs showed inhibitory reactivity against E165R recombinant protein. Epitope mapping studies indicated that E165R has three major antigenic regions: 100-120 aa, 120-140 aa, and 140-165 aa. Three mAbs inhibited the dUTPase activity of E165R by binding to the highly conserved 149-RGEGRFGSTG-158 amino acid sequence. Interestingly, 8F6 mAb specifically recognized ASFV dUTPase but not Sus scrofa dUTPase, which may be due to structural differences in the amino acids of F151, R153, and F154 in the motif V region. In summary, we developed anti-E165R-specific mAbs, and identified an important antibody-binding antigenic epitope in the motif V of ASFV dUTPase. Our study provides a comprehensive analysis of mAbs that target the antigenic epitope of ASFV dUTPase, which may contribute to the development of novel antibody-based ASFV therapeutics.
- Published
- 2021
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19. Autophagy impairment by African swine fever virus.
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Shimmon GL, Hui JYK, Wileman TE, and Netherton CL
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- Animals, Autophagy, Chlorocebus aethiops, Swine, Vero Cells, Virulence, African Swine Fever virology, African Swine Fever Virus pathogenicity, Viral Proteins metabolism
- Abstract
African swine fever is a devastating disease of domestic swine and wild boar caused by a large double-stranded DNA virus that encodes for more than 150 open reading frames. There is no licensed vaccine for the disease and the most promising current candidates are modified live viruses that have been attenuated by deletion of virulence factors. Like many viruses African swine fever virus significantly alters the host cell machinery to benefit its replication and viral genes that modify host pathways represent promising targets for development of gene deleted vaccines. Autophagy is an important cellular pathway that is involved in cellular homeostasis, innate and adaptive immunity and therefore is manipulated by a number of different viruses. Autophagy is regulated by a complex protein cascade and here we show that African swine fever virus can block formation of autophagosomes, a critical functional step of the autophagy pathway through at least two different mechanisms. Interestingly this does not require the A179L gene that has been shown to interact with Beclin-1, an important autophagy regulator.
- Published
- 2021
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20. Unpicking the Secrets of African Swine Fever Viral Replication Sites.
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Aicher SM, Monaghan P, Netherton CL, and Hawes PC
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- African Swine Fever Virus ultrastructure, Animals, Cell Culture Techniques, Chlorocebus aethiops, Fluorescent Antibody Technique, Swine, Vero Cells, Virus Assembly, African Swine Fever virology, African Swine Fever Virus physiology, Virus Replication
- Abstract
African swine fever virus (ASFV) is a highly contagious pathogen which causes a lethal haemorrhagic fever in domestic pigs and wild boar. The large, double-stranded DNA virus replicates in perinuclear cytoplasmic replication sites known as viral factories. These factories are complex, multi-dimensional structures. Here we investigated the protein and membrane compartments of the factory using super-resolution and electron tomography. Click IT chemistry in combination with stimulated emission depletion (STED) microscopy revealed a reticular network of newly synthesized viral proteins, including the structural proteins p54 and p34, previously seen as a pleomorphic ribbon by confocal microscopy. Electron microscopy and tomography confirmed that this network is an accumulation of membrane assembly intermediates which take several forms. At early time points in the factory formation, these intermediates present as small, individual membrane fragments which appear to grow and link together, in a continuous progression towards new, icosahedral virions. It remains unknown how these membranes form and how they traffic to the factory during virus morphogenesis.
- Published
- 2021
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21. Identification of novel testing matrices for African swine fever surveillance.
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Flannery J, Ashby M, Moore R, Wells S, Rajko-Nenow P, Netherton CL, and Batten C
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- African Swine Fever Virus genetics, Animals, DNA, Viral genetics, Molecular Diagnostic Techniques standards, Molecular Diagnostic Techniques veterinary, Nucleic Acid Amplification Techniques standards, Nucleic Acid Amplification Techniques veterinary, Real-Time Polymerase Chain Reaction standards, Real-Time Polymerase Chain Reaction veterinary, Sensitivity and Specificity, Swine, African Swine Fever diagnosis, Population Surveillance methods
- Abstract
African swine fever (ASF) is a devastating viral disease of pigs and wild boar, and it threatens global food security. We aimed to identify suitable sample matrices for use in ASF surveillance programs. Six pigs inoculated with ASFV were sampled at postmortem. Blood, bone marrow, ear biopsies, and oral, nasal, and rectal swabs were taken from all pigs. All samples were analyzed using 3 real-time PCR (rtPCR) assays and a LAMP assay. ASFV was detected at > 10
7 genome copies/mL in blood; bone marrow was found to provide the highest viral load. Ct values provided by the rtPCR assays were correlated, and ASFV was detected in all oral, nasal, and rectal swabs and in all ear biopsy samples irrespective of the location from which they were taken. The LAMP assay had lower sensitivity, and detected ASFV in 54 of 66 positive samples, but delivered positive results within 17 min. We identified additional sample matrices that can be considered depending on the sampling situation: bone marrow had a high probability of detection, which could be useful for decomposed carcasses. However, ear biopsies provide an appropriate, high-throughput sample matrix to detect ASFV and may be useful during surveillance programs.- Published
- 2020
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22. Identification of a Functional Small Noncoding RNA of African Swine Fever Virus.
- Author
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Dunn LEM, Ivens A, Netherton CL, Chapman DAG, and Beard PM
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- African Swine Fever metabolism, African Swine Fever virology, African Swine Fever Virus metabolism, Animals, Gene Expression Regulation, Genome Size, Lymphoid Tissue, Macrophages, MicroRNAs classification, MicroRNAs metabolism, Primary Cell Culture, RNA, Small Untranslated classification, RNA, Small Untranslated metabolism, RNA, Viral classification, RNA, Viral metabolism, Signal Transduction, Sus scrofa, Swine, Virus Replication, African Swine Fever genetics, African Swine Fever Virus genetics, Genome, Viral, Host-Pathogen Interactions genetics, MicroRNAs genetics, RNA, Small Untranslated genetics, RNA, Viral genetics
- Abstract
African swine fever virus (ASFV) causes a lethal hemorrhagic disease of domestic pigs, against which no vaccine is available. ASFV has a large, double-stranded DNA genome that encodes over 150 proteins. Replication takes place predominantly in the cytoplasm of the cell and involves complex interactions with host cellular components, including small noncoding RNAs (sncRNAs). A number of DNA viruses are known to manipulate sncRNA either by encoding their own or disrupting host sncRNA. To investigate the interplay between ASFV and sncRNAs, a study of host and viral small RNAs extracted from ASFV-infected primary porcine macrophages (PAMs) was undertaken. We discovered that ASFV infection had only a modest effect on host miRNAs, with only 6 miRNAs differentially expressed during infection. The data also revealed 3 potential novel small RNAs encoded by ASFV, ASFVsRNA1-3. Further investigation of ASFVsRNA2 detected it in lymphoid tissue from pigs with ASF. Overexpression of ASFVsRNA2 led to an up to 1-log reduction in ASFV growth, indicating that ASFV utilizes a virus-encoded small RNA to disrupt its own replication. IMPORTANCE African swine fever (ASF) poses a major threat to pig populations and food security worldwide. The disease is endemic to Africa and Eastern Europe and is rapidly emerging into Asia, where it has led to the deaths of millions of pigs in the last 12 months. The development of safe and effective vaccines to protect pigs against ASF has been hindered by lack of understanding of the complex interactions between ASFV and the host cell. We focused our work on characterizing the interactions between ASFV and sncRNAs. Although comparatively modest changes to host sncRNA abundances were observed upon ASFV infection, we discovered and characterized a novel functional ASFV-encoded sncRNA. The results from this study add important insights into ASFV host-pathogen interactions. This knowledge may be exploited to develop more effective ASFV vaccines that take advantage of the sncRNA system., (Copyright © 2020 Dunn et al.)
- Published
- 2020
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23. A Pool of Eight Virally Vectored African Swine Fever Antigens Protect Pigs Against Fatal Disease.
- Author
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Goatley LC, Reis AL, Portugal R, Goldswain H, Shimmon GL, Hargreaves Z, Ho CS, Montoya M, Sánchez-Cordón PJ, Taylor G, Dixon LK, and Netherton CL
- Abstract
Classical approaches to African swine fever virus (ASFV) vaccine development have not been successful; inactivated virus does not provide protection and use of live attenuated viruses generated by passage in tissue culture had a poor safety profile. Current African swine fever (ASF) vaccine research focuses on the development of modified live viruses by targeted gene deletion or subunit vaccines. The latter approach would be differentiation of vaccinated from infected animals (DIVA)-compliant, but information on which viral proteins to include in a subunit vaccine is lacking. Our previous work used DNA-prime/vaccinia-virus boost to screen 40 ASFV genes for immunogenicity, however this immunization regime did not protect animals after challenge. Here we describe the induction of both antigen and ASFV-specific antibody and cellular immune responses by different viral-vectored pools of antigens selected based on their immunogenicity in pigs. Immunization with one of these pools, comprising eight viral-vectored ASFV genes, protected 100% of pigs from fatal disease after challenge with a normally lethal dose of virulent ASFV. This data provide the basis for the further development of a subunit vaccine against this devastating disease.
- Published
- 2020
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24. A Deep-Sequencing Workflow for the Fast and Efficient Generation of High-Quality African Swine Fever Virus Whole-Genome Sequences.
- Author
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Forth JH, Forth LF, King J, Groza O, Hübner A, Olesen AS, Höper D, Dixon LK, Netherton CL, Rasmussen TB, Blome S, Pohlmann A, and Beer M
- Subjects
- African Swine Fever virology, Animals, DNA, Viral genetics, Databases, Nucleic Acid, Genetic Variation, Nanopore Sequencing methods, Swine virology, Workflow, African Swine Fever Virus genetics, Genome, Viral, High-Throughput Nucleotide Sequencing methods, Viral Proteins genetics, Whole Genome Sequencing methods
- Abstract
African swine fever (ASF) is a severe disease of suids caused by African swine fever virus (ASFV). Its dsDNA genome (170-194 kbp) is scattered with homopolymers and repeats as well as inverted-terminal-repeats (ITR), which hamper whole-genome sequencing. To date, only a few genome sequences have been published and only for some are data on sequence quality available enabling in-depth investigations. Especially in Europe and Asia, where ASFV has continuously spread since its introduction into Georgia in 2007, a very low genetic variability of the circulating ASFV-strains was reported. Therefore, only whole-genome sequences can serve as a basis for detailed virus comparisons. Here, we report an effective workflow, combining target enrichment, Illumina and Nanopore sequencing for ASFV whole-genome sequencing. Following this approach, we generated an improved high-quality ASFV Georgia 2007/1 whole-genome sequence leading to the correction of 71 sequencing errors and the addition of 956 and 231 bp at the respective ITRs. This genome, derived from the primary outbreak in 2007, can now serve as a reference for future whole-genome analyses of related ASFV strains and molecular approaches. Using both workflow and the reference genome, we generated the first ASFV-whole-genome sequence from Moldova, expanding the sequence knowledge from Eastern Europe., Competing Interests: The authors declare no conflict of interest.
- Published
- 2019
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25. Crystal Structure of African Swine Fever Virus A179L with the Autophagy Regulator Beclin.
- Author
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Banjara S, Shimmon GL, Dixon LK, Netherton CL, Hinds MG, and Kvansakul M
- Subjects
- African Swine Fever Virus chemistry, African Swine Fever Virus metabolism, Animals, Apoptosis Regulatory Proteins genetics, Apoptosis Regulatory Proteins metabolism, Beclin-1 chemistry, Chlorocebus aethiops, Crystallography, X-Ray, Humans, Mutation, Peptide Fragments chemistry, Peptide Fragments metabolism, Protein Binding, Proto-Oncogene Proteins chemistry, Proto-Oncogene Proteins metabolism, Proto-Oncogene Proteins c-bcl-2 chemistry, Proto-Oncogene Proteins c-bcl-2 metabolism, Structure-Activity Relationship, Vero Cells, Viral Proteins genetics, Viral Proteins metabolism, African Swine Fever Virus pathogenicity, Apoptosis Regulatory Proteins chemistry, Autophagy, Beclin-1 metabolism, Viral Proteins chemistry
- Abstract
Subversion of programmed cell death-based host defence systems is a prominent feature of infections by large DNA viruses. African swine fever virus (ASFV) is a large DNA virus and sole member of the Asfarviridae family that harbours the B-cell lymphoma 2 or Bcl-2 homolog A179L. A179L has been shown to bind to a range of cell death-inducing host proteins, including pro-apoptotic Bcl-2 proteins as well as the autophagy regulator Beclin. Here we report the crystal structure of A179L bound to the Beclin BH3 motif. A179L engages Beclin using the same canonical ligand-binding groove that is utilized to bind to pro-apoptotic Bcl-2 proteins. The mode of binding of Beclin to A179L mirrors that of Beclin binding to human Bcl-2 and Bcl-x
L as well as murine γ-herpesvirus 68. The introduction of bulky hydrophobic residues into the A179L ligand-binding groove via site-directed mutagenesis ablates binding of Beclin to A179L, leading to a loss of the ability of A179L to modulate autophagosome formation in Vero cells during starvation. Our findings provide a mechanistic understanding for the potent autophagy inhibitory activity of A179L and serve as a platform for more detailed investigations into the role of autophagy during ASFV infection.- Published
- 2019
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26. Identification and Immunogenicity of African Swine Fever Virus Antigens.
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Netherton CL, Goatley LC, Reis AL, Portugal R, Nash RH, Morgan SB, Gault L, Nieto R, Norlin V, Gallardo C, Ho CS, Sánchez-Cordón PJ, Taylor G, and Dixon LK
- Subjects
- Adenoviridae immunology, Animals, CD8-Positive T-Lymphocytes immunology, Genetic Vectors immunology, Immunity, Cellular immunology, Immunity, Humoral immunology, Immunization methods, Swine, Vaccination methods, Viral Vaccines immunology, Viremia immunology, Virulence immunology, African Swine Fever immunology, African Swine Fever Virus immunology, Antigens, Viral immunology, Viral Proteins immunology
- Abstract
African swine fever (ASF) is a lethal haemorrhagic disease of domestic pigs for which there is no vaccine. Strains of the virus with reduced virulence can provide protection against related virulent strains of ASFV, but protection is not 100% and there are concerns about the safety profile of such viruses. However, they provide a useful tool for understanding the immune response to ASFV and previous studies using the low virulent isolate OUR T88/3 have shown that CD8+ cells are crucial for protection. In order to develop a vaccine that stimulates an effective anti-ASFV T-cell response we need to know which of the >150 viral proteins are recognized by the cellular immune response. Therefore, we used a gamma interferon ELIspot assay to screen for viral proteins recognized by lymphocytes from ASF-immune pigs using peptides corresponding to 133 proteins predicted to be encoded by OUR T88/3. Eighteen antigens that were recognized by ASFV-specific lymphocytes were then incorporated into adenovirus and MVA vectors, which were used in immunization and challenge experiments in pigs. We present a systematic characterization of the cellular immune response to this devastating disease and identify proteins capable of inducing ASFV-specific cellular and humoral immune responses in pigs. Pools of viral vectors expressing these genes did not protect animals from severe disease, but did reduce viremia in a proportion of pigs following ASFV challenge.
- Published
- 2019
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27. The Genetics of Life and Death: Virus-Host Interactions Underpinning Resistance to African Swine Fever, a Viral Hemorrhagic Disease.
- Author
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Netherton CL, Connell S, Benfield CTO, and Dixon LK
- Abstract
Pathogen transmission from wildlife hosts to genetically distinct species is a major driver of disease emergence. African swine fever virus (ASFV) persists in sub-Saharan Africa through a sylvatic cycle between warthogs and soft ticks that infest their burrows. The virus does not cause disease in these animals, however transmission of the virus to domestic pigs or wild boar causes a hemorrhagic fever that is invariably fatal. ASFV transmits readily between domestic pigs and causes economic hardship in areas where it is endemic. The virus is also a significant transboundary pathogen that has become established in Eastern Europe, and has recently appeared in China increasing the risk of an introduction of the disease to other pig producing centers. Although a DNA genome mitigates against rapid adaptation of the virus to new hosts, extended epidemics of African swine fever (ASF) can lead to the emergence of viruses with reduced virulence. Attenuation in the field leads to large deletions of genetic material encoding genes involved in modulating host immune responses. Therefore resistance to disease and tolerance of ASFV replication can be dependent on both virus and host factors. Here we describe the different virus-host interfaces and discuss progress toward understanding the genetic determinants of disease outcome after infection with ASFV.
- Published
- 2019
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28. Immunization of Pigs by DNA Prime and Recombinant Vaccinia Virus Boost To Identify and Rank African Swine Fever Virus Immunogenic and Protective Proteins.
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Jancovich JK, Chapman D, Hansen DT, Robida MD, Loskutov A, Craciunescu F, Borovkov A, Kibler K, Goatley L, King K, Netherton CL, Taylor G, Jacobs B, Sykes K, and Dixon LK
- Subjects
- African Swine Fever genetics, African Swine Fever prevention & control, African Swine Fever Virus genetics, Animals, Recombinant Proteins genetics, Recombinant Proteins immunology, Swine, Vaccines, DNA genetics, Vaccinia virus genetics, Viral Proteins genetics, African Swine Fever immunology, African Swine Fever Virus immunology, Immunization, Secondary, Vaccines, DNA immunology, Vaccinia virus immunology, Viral Proteins immunology
- Abstract
African swine fever virus (ASFV) causes an acute hemorrhagic fever in domestic pigs, with high socioeconomic impact. No vaccine is available, limiting options for control. Although live attenuated ASFV can induce up to 100% protection against lethal challenge, little is known of the antigens which induce this protective response. To identify additional ASFV immunogenic and potentially protective antigens, we cloned 47 viral genes in individual plasmids for gene vaccination and in recombinant vaccinia viruses. These antigens were selected to include proteins with different functions and timing of expression. Pools of up to 22 antigens were delivered by DNA prime and recombinant vaccinia virus boost to groups of pigs. Responses of immune lymphocytes from pigs to individual recombinant proteins and to ASFV were measured by interferon gamma enzyme-linked immunosorbent spot (ELISpot) assays to identify a subset of the antigens that consistently induced the highest responses. All 47 antigens were then delivered to pigs by DNA prime and recombinant vaccinia virus boost, and pigs were challenged with a lethal dose of ASFV isolate Georgia 2007/1. Although pigs developed clinical and pathological signs consistent with acute ASFV, viral genome levels were significantly reduced in blood and several lymph tissues in those pigs immunized with vectors expressing ASFV antigens compared with the levels in control pigs. IMPORTANCE The lack of a vaccine limits the options to control African swine fever. Advances have been made in the development of genetically modified live attenuated ASFV that can induce protection against challenge. However, there may be safety issues relating to the use of these in the field. There is little information about ASFV antigens that can induce a protective immune response against challenge. We carried out a large screen of 30% of ASFV antigens by delivering individual genes in different pools to pigs by DNA immunization prime and recombinant vaccinia virus boost. The responses in immunized pigs to these individual antigens were compared to identify the most immunogenic. Lethal challenge of pigs immunized with a pool of antigens resulted in reduced levels of virus in blood and lymph tissues compared to those in pigs immunized with control vectors. Novel immunogenic ASFV proteins have been identified for further testing as vaccine candidates., (Copyright © 2018 Jancovich et al.)
- Published
- 2018
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29. Deletion of the African Swine Fever Virus Gene DP148R Does Not Reduce Virus Replication in Culture but Reduces Virus Virulence in Pigs and Induces High Levels of Protection against Challenge.
- Author
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Reis AL, Goatley LC, Jabbar T, Sanchez-Cordon PJ, Netherton CL, Chapman DAG, and Dixon LK
- Subjects
- Administration, Intranasal, Africa epidemiology, African Swine Fever epidemiology, African Swine Fever virology, African Swine Fever Virus immunology, Animals, Antibodies, Viral blood, Europe epidemiology, Genome, Viral, Injections, Intramuscular, Interferon-gamma blood, Lymphocyte Activation, Russia epidemiology, Swine, Vaccines, Attenuated administration & dosage, Vaccines, Attenuated immunology, Viral Vaccines administration & dosage, Virulence genetics, African Swine Fever prevention & control, African Swine Fever Virus genetics, African Swine Fever Virus pathogenicity, Gene Deletion, Viral Vaccines immunology, Virus Replication genetics
- Abstract
Many of the approximately 165 proteins encoded by the African swine fever virus (ASFV) genome do not have significant similarity to known proteins and have not been studied experimentally. One such protein is DP148R. We showed that the DP148R gene is transcribed at early times postinfection. Deletion of this gene did not reduce virus replication in macrophages, showing that it is not essential for replication in these cells. However, deletion of this gene from a virulent isolate, Benin 97/1, producing the BeninΔDP148R virus, dramatically reduced the virulence of the virus in vivo All pigs infected with the BeninΔDP148R virus survived infection, showing only transient mild clinical signs soon after immunization. Following challenge with the parental virulent virus, all pigs immunized by the intramuscular route (11/11) and all except one immunized by the intranasal route (5/6) survived. Mild or no clinical signs were observed after challenge. As expected, control nonimmune pigs developed signs of acute African swine fever (ASF). The virus genome and infectious virus were observed soon after immunization, coincident with the onset of clinical signs (∼10
6 genome copies or 50% tissue culture infective doses/ml). The levels of the virus genome declined over an extended period up to 60 days postimmunization. In contrast, infectious virus was no longer detectable by days 30 to 35. Gamma interferon (IFN-γ) was detected in serum between days 4 and 7 postimmunization, and IFN-γ-producing cells were detected in all pigs analyzed following stimulation of immune lymphocytes with whole virus. ASFV-specific antibodies were first detected from day 10 postimmunization. IMPORTANCE African swine fever (ASF) is endemic in Africa, parts of the Trans Caucasus, the Russian Federation, and several European countries. The lack of a vaccine hinders control. Many of the ASF virus genes lack similarity to known genes and have not been characterized. We have shown that one of these, DP148R, is transcribed early during virus replication in cells and can be deleted from the virus genome without reducing virus replication. The virus with the gene deletion, BeninΔDP148R, caused mild clinical signs in pigs and induced high levels of protection against challenge with the parental virulent virus. Therefore, deletion of this gene can provide a target for the rational development of vaccines., (Copyright © 2017 Reis et al.)- Published
- 2017
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30. Survival of African Swine Fever Virus in Excretions from Pigs Experimentally Infected with the Georgia 2007/1 Isolate.
- Author
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Davies K, Goatley LC, Guinat C, Netherton CL, Gubbins S, Dixon LK, and Reis AL
- Subjects
- Animals, DNA, Viral analysis, Half-Life, Saliva chemistry, Swine, Temperature, Urine chemistry, African Swine Fever Virus isolation & purification, Feces virology
- Abstract
African swine fever virus (ASFV) causes a lethal haemorrhagic disease of swine which can be transmitted through direct contact with infected animals and their excretions or indirect contact with contaminated fomites. The shedding of ASFV by infected pigs and the stability of ASFV in the environment will determine the extent of environmental contamination. The recent outbreaks of ASF in Europe make it essential to develop disease transmission models in order to design effective control strategies to prevent further spread of ASF. In this study, we assessed the shedding and stability of ASFV in faeces, urine and oral fluid from pigs infected with the Georgia 2007/1 ASFV isolate. The half-life of infectious ASFV in faeces was found to range from 0.65 days when stored at 4°C to 0.29 days when stored at 37°C, while in urine it was found to range from 2.19 days (4°C) to 0.41 days (37°C). Based on these half-lives and the estimated dose required for infection, faeces and urine would be estimated to remain infectious for 8.48 and 15.33 days at 4°C and 3.71 and 2.88 days at 37°C, respectively. The half-life of ASFV DNA was 8 to 9 days in faeces and 2 to 3 days in oral fluid at all temperatures. In urine, the half-life of ASFV DNA was found to be 32.54 days at 4°C decreasing to 19.48 days at 37°C. These results indicate that ASFV in excretions may be an important route of ASFV transmission., (© 2015 The Authors. Transboundary and Emerging Diseases Published by Blackwell Verlag GmbH.)
- Published
- 2017
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31. Different routes and doses influence protection in pigs immunised with the naturally attenuated African swine fever virus isolate OURT88/3.
- Author
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Sánchez-Cordón PJ, Chapman D, Jabbar T, Reis AL, Goatley L, Netherton CL, Taylor G, Montoya M, and Dixon L
- Subjects
- Administration, Intranasal, African Swine Fever virology, African Swine Fever Virus genetics, African Swine Fever Virus isolation & purification, African Swine Fever Virus pathogenicity, Animals, Antibodies, Viral blood, DNA, Viral blood, Genome, Viral, Injections, Intramuscular, Interferon-gamma genetics, Interleukin-10 genetics, Swine, Vaccination methods, Vaccines, Attenuated administration & dosage, Viral Load, African Swine Fever prevention & control, African Swine Fever Virus immunology, Vaccination veterinary, Viral Vaccines administration & dosage
- Abstract
This study compares different combinations of doses and routes of immunisation of pigs with low virulent African swine fever virus (ASFV) genotype I isolate OURT88/3, including the intramuscular and intranasal route, the latter not previously tested. Intranasal immunisations with low and moderate doses (10
3 and 104 TCID50 ) of OURT88/3 provided complete protection (100%) against challenge with virulent genotype I OURT88/1 isolate. Only mild and transient clinical reactions were observed in protected pigs. Transient moderate virus genome levels were detected in blood samples after challenge that decreased, but persisted until the end of the experiment in some animals. In contrast, pigs immunised intramuscularly with low and moderate doses (103 and 104 TCID50 ) displayed lower percentages of protection (50-66%), and low or undetectable levels of virus genome were detected in blood samples throughout the study. In addition, clinical courses observed in protected pigs were asymptomatic. In pigs that were not protected and developed acute ASF, an exacerbated increase of IL-10 sometimes accompanied by an increase of IFNγ was observed before euthanasia. These results showed that factors including delivery route and dose determine the outcome of immunisation with the naturally attenuated isolate OURT88/3., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)- Published
- 2017
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32. Sensitivity of African swine fever virus to type I interferon is linked to genes within multigene families 360 and 505.
- Author
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Golding JP, Goatley L, Goodbourn S, Dixon LK, Taylor G, and Netherton CL
- Subjects
- African Swine Fever virology, African Swine Fever Virus immunology, African Swine Fever Virus pathogenicity, Animals, Cell Line, Chlorocebus aethiops, Disease Resistance genetics, Disease Resistance immunology, Genes, Viral, Multigene Family, Swine, Vero Cells, Virulence, Virus Replication, African Swine Fever immunology, African Swine Fever Virus genetics, Interferon Type I immunology
- Abstract
African swine fever virus (ASFV) causes a lethal haemorrhagic disease of pigs. There are conflicting reports on the role of interferon in ASFV infection. We therefore analysed the interaction of ASFV with porcine interferon, in vivo and in vitro. Virulent ASFV induced biologically active IFN in the circulation of pigs from day 3-post infection, whereas low virulent OUR T88/3, which lacks genes from multigene family (MGF) 360 and MGF505, did not. Infection of porcine leucocytes enriched for dendritic cells, with ASFV, in vitro, induced high levels of interferon, suggesting a potential source of interferon in animals undergoing acute ASF. Replication of OUR T88/3, but not virulent viruses, was reduced in interferon pretreated macrophages and a recombinant virus lacking similar genes to those absent in OUR T88/3 was also inhibited. These findings suggest that as well as inhibiting the induction of interferon, MGF360 and MGF505 genes also enable ASFV to overcome the antiviral state., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2016
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33. Dynamics of African swine fever virus shedding and excretion in domestic pigs infected by intramuscular inoculation and contact transmission.
- Author
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Guinat C, Reis AL, Netherton CL, Goatley L, Pfeiffer DU, and Dixon L
- Subjects
- African Swine Fever diagnosis, African Swine Fever virology, Animals, Feces virology, Georgia (Republic), Injections, Intramuscular veterinary, Swine, United Kingdom, Urine virology, Viremia diagnosis, Viremia transmission, Viremia virology, African Swine Fever transmission, African Swine Fever Virus physiology, Viremia veterinary, Virus Shedding
- Abstract
African swine fever virus (ASFV) is a highly virulent swine pathogen that has spread across Eastern Europe since 2007 and for which there is no effective vaccine or treatment available. The dynamics of shedding and excretion is not well known for this currently circulating ASFV strain. Therefore, susceptible pigs were exposed to pigs intramuscularly infected with the Georgia 2007/1 ASFV strain to measure those dynamics through within- and between-pen transmission scenarios. Blood, oral, nasal and rectal fluid samples were tested for the presence of ASFV by virus titration (VT) and quantitative real-time polymerase chain reaction (qPCR). Serum was tested for the presence of ASFV-specific antibodies. Both intramuscular inoculation and contact transmission resulted in development of acute disease in all pigs although the experiments indicated that the pathogenesis of the disease might be different, depending on the route of infection. Infectious ASFV was first isolated in blood among the inoculated pigs by day 3, and then chronologically among the direct and indirect contact pigs, by day 10 and 13, respectively. Close to the onset of clinical signs, higher ASFV titres were found in blood compared with nasal and rectal fluid samples among all pigs. No infectious ASFV was isolated in oral fluid samples although ASFV genome copies were detected. Only one animal developed antibodies starting after 12 days post-inoculation. The results provide quantitative data on shedding and excretion of the Georgia 2007/1 ASFV strain among domestic pigs and suggest a limited potential of this isolate to cause persistent infection.
- Published
- 2014
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34. Deletion of virulence associated genes from attenuated African swine fever virus isolate OUR T88/3 decreases its ability to protect against challenge with virulent virus.
- Author
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Abrams CC, Goatley L, Fishbourne E, Chapman D, Cooke L, Oura CA, Netherton CL, Takamatsu HH, and Dixon LK
- Subjects
- African Swine Fever immunology, African Swine Fever pathology, African Swine Fever virology, Animals, Severity of Illness Index, Spleen virology, Swine, Vaccines, Attenuated administration & dosage, Vaccines, Attenuated genetics, Vaccines, Attenuated immunology, Viral Vaccines administration & dosage, Viral Vaccines genetics, Viremia immunology, Viremia prevention & control, Virulence, African Swine Fever prevention & control, African Swine Fever Virus genetics, African Swine Fever Virus immunology, Sequence Deletion, Viral Vaccines immunology, Virulence Factors genetics, Virulence Factors immunology
- Abstract
African swine fever virus (ASFV) causes an acute haemorrhagic disease of domestic pigs against which there is no effective vaccine. The attenuated ASFV strain OUR T88/3 has been shown previously to protect vaccinated pigs against challenge with some virulent strains including OUR T88/1. Two genes, DP71L and DP96R were deleted from the OUR T88/3 genome to create recombinant virus OUR T88/3ΔDP2. Deletion of these genes from virulent viruses has previously been shown to reduce ASFV virulence in domestic pigs. Groups of 6 pigs were immunised with deletion virus OUR T88/3ΔDP2 or parental virus OUR T88/3 and challenged with virulent OUR T88/1 virus. Four pigs (66%) were protected by inoculation with the deletion virus OUR T88/3ΔDP2 compared to 100% protection with the parental virus OUR T88/3. Thus the deletion of the two genes DP71L and DP96R from OUR T88/3 strain reduced its ability to protect pigs against challenge with virulent virus., (Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.)
- Published
- 2013
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35. African swine fever virus organelle rearrangements.
- Author
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Netherton CL and Wileman TE
- Subjects
- Cell Membrane virology, Cell Nucleus virology, Cytoplasm virology, African Swine Fever Virus physiology, Host-Pathogen Interactions, Organelles physiology, Virus Replication
- Abstract
Like most viruses African swine fever virus (ASFV) subsumes the host cell apparatus in order to facilitate its replication. ASFV replication is a highly orchestrated process with a least four stages of transcription, immediate-early, early, intermediate and late. As the infective cycle progresses through these stages most if not all of the organelles that comprise a nucleated cell are modified, adapted or in some cases destroyed. The entry of the virus is receptor-mediated, but the precise mechanism of endocytosis is a matter of keen, current debate. Once ASFV has exited from the endosomal-lysosomal complex the virus life-cycle enters into an intimate relationship with the microtubular network. Genome replication is believed to be initiated within the nucleus and ASFV infection completely reorders the structure of this organelle. The majority of replication and assembly occurs in discrete, perinuclear regions of the cell called virus factories and finally progeny virions are transported to the plasma membrane along microtubules where they bud out or are propelled away along actin projections to infect new cells. The generation of ASFV replication sites induces profound reorganisation of the organelles that comprise the secretory pathway and may contribute to the induction of cellular stress responses that ASFV modulates. The level of organisation and complexity of virus factories are not dissimilar to those seen in cellular organelles. Like their cellular counterparts the formation of virus factories, as well as virus entry and exit, are dependent on the various components of the cytoskeleton. This review will summarise these rearrangements, the viral proteins involved and their functional consequences., (Copyright © 2013 Elsevier B.V. All rights reserved.)
- Published
- 2013
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36. Cellular immunity in ASFV responses.
- Author
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Takamatsu HH, Denyer MS, Lacasta A, Stirling CM, Argilaguet JM, Netherton CL, Oura CA, Martins C, and Rodríguez F
- Subjects
- Animals, CD4-Positive T-Lymphocytes immunology, CD8-Positive T-Lymphocytes immunology, DNA, Viral chemistry, DNA, Viral genetics, Interferon-gamma metabolism, Killer Cells, Natural immunology, Molecular Sequence Data, Sequence Analysis, DNA, Swine, T-Lymphocytes, Cytotoxic immunology, Viral Proteins genetics, Viral Proteins immunology, African Swine Fever immunology, African Swine Fever Virus immunology, Immunity, Cellular
- Abstract
African swine fever virus (ASFV) infection usually results in an acute haemorrhagic disease with a mortality rate approaching 100% in domestic pigs. However, pigs can survive infection with less-virulent isolates of ASFV and may become chronically infected. Surviving animals are resistant to challenge with homologous or, in some cases, closely related isolates of the virus indicating that pigs can develop protective immunity against ASFV. During asymptomatic, non-virulent ASFV infections natural killer cell activity increases in pigs, suggesting this cell type plays a role in ASFV immunity. Furthermore, depletion of CD8(+) lymphocytes from ASFV immune pigs demolishes protective immunity against related virulent viruses. This suggests that ASFV specific antibody alone is not sufficient for protection against ASFV infection and that there is an important role for the CD8(+) lymphocyte subset in ASFV protective immunity. These results were supported by DNA immunization studies, demonstrating a correlation between the protection afforded against lethal challenge and the detection of a large number of vaccine-induced antigen-specific CD8(+) T-cells. Peripheral blood mononuclear cells (PBMCs) from ASF immune pigs protected from clinical disease show higher proportions of ASFV specific CD4(+)CD8(high+) double positive cytotoxic T cells than PBMCs from ASF immune but clinically diseased pig. The frequency of ASFV specific IFNγ producing T cells induced by immunization correlates to the degree of protection from ASFV challenge, and this may prove to be a useful indicator of any potential cross-protection against heterologous ASFV isolates., (Copyright © 2012 Elsevier B.V. All rights reserved.)
- Published
- 2013
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37. African swine fever virus replication and genomics.
- Author
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Dixon LK, Chapman DA, Netherton CL, and Upton C
- Subjects
- DNA Replication, DNA, Circular genetics, DNA, Viral genetics, Gene Order, Genes, Viral, African Swine Fever Virus genetics, African Swine Fever Virus physiology, Genome, Viral, Virus Replication
- Abstract
African swine fever virus (ASFV) is a large icosahedral DNA virus which replicates predominantly in the cytoplasm of infected cells. The ASFV double-stranded DNA genome varies in length from about 170 to 193 kbp depending on the isolate and contains between 150 and 167 open reading frames. These are closely spaced and read from both DNA strands. The virus genome termini are covalently closed by imperfectly base-paired hairpin loops that are present in two forms that are complimentary and inverted with respect to each other. Adjacent to the termini are inverted arrays of different tandem repeats. Head to head concatemeric genome replication intermediates have been described. A similar mechanism of replication to Poxviruses has been proposed for ASFV. Virus genome transcription occurs independently of the host RNA polymerase II and virus particles contain all of the enzymes and factors required for early gene transcription. DNA replication begins in perinuclear factory areas about 6h post-infection although an earlier stage of nuclear DNA synthesis has been reported. The virus genome encodes enzymes required for transcription and replication of the virus genome and virion structural proteins. Enzymes that are involved in a base excision repair pathway may be an adaptation to enable virus replication in the oxidative environment of the macrophage cytoplasm. Other ASFV genes encode factors involved in evading host defence systems and modulating host cell function. Variation between the genomes of different ASFV isolates is most commonly due to gain or loss of members of multigene families, MGFs 100, 110, 300, 360, 505/530 and family p22. These are located within the left terminal 40kbp and right terminal 20kbp. ASFV is the only member of the Asfarviridae, which is one of the families within the nucleocytoplasmic large DNA virus superfamily., (Copyright © 2012 Elsevier B.V. All rights reserved.)
- Published
- 2013
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38. Prospects for development of African swine fever virus vaccines.
- Author
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Dixon LK, Abrams CC, Chapman DD, Goatley LC, Netherton CL, Taylor G, and Takamatsu HH
- Subjects
- Africa epidemiology, African Swine Fever epidemiology, African Swine Fever virology, African Swine Fever Virus physiology, Animals, Antibodies, Viral, Genome, Viral, Genotype, Molecular Epidemiology, Phylogeography, Research, Swine, Virus Replication, African Swine Fever prevention & control, African Swine Fever Virus immunology, Viral Vaccines immunology
- Abstract
African swine fever virus is a large DNA virus which can cause an acute haemorrhagic fever in pigs resulting in high mortality. No vaccine is available, limiting options for control. The virus encodes up to 165 genes and virus particles are multi-layered and contain more than 50 proteins. Pigs immunised with natural low virulence isolates or attenuated viruses produced by passage in tissue culture and by targeted gene deletions can be protected against challenge with virulent viruses. CD8+ cells are required for protection induced by attenuated strain OURT88/3. Passive transfer of antibodies from immune to naïve pigs can also induce protection. Knowledge of the genome sequences of attenuated and virulent strains and targeted gene deletions from virulent strains have identified a number of virus genes involved in virulence and immune evasion. This information can be used to produce rationally attenuated vaccine strains. Virus antigens that are targets for neutralising antibodies have been identified and immunisation with these recombinant proteins has been shown to induce partial protection. However knowledge of antigens which encode the dominant protective epitopes recognised by CD8+ T cells is lacking.
- Published
- 2013
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39. African swine fever virus strain Georgia 2007/1 in Ornithodoros erraticus ticks.
- Author
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Diaz AV, Netherton CL, Dixon LK, and Wilson AJ
- Subjects
- African Swine Fever Virus isolation & purification, Animals, Cells, Cultured, Disease Vectors, Likelihood Functions, Swine, Viral Load, Virus Cultivation, Virus Replication, African Swine Fever Virus physiology, Ornithodoros virology
- Published
- 2012
- Full Text
- View/download PDF
40. Virus factories, double membrane vesicles and viroplasm generated in animal cells.
- Author
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Netherton CL and Wileman T
- Subjects
- Animals, DNA Viruses genetics, Humans, RNA Viruses genetics, Viral Proteins genetics, Viral Proteins metabolism, Virus Replication, Cytoplasmic Vesicles virology, DNA Viruses physiology, Intracellular Membranes virology, RNA Viruses physiology, Virus Diseases virology
- Abstract
Many viruses reorganise cellular membrane compartments and the cytoskeleton to generate subcellular microenvironments called virus factories or 'viroplasm'. These create a platform to concentrate replicase proteins, virus genomes and host proteins required for replication and also protect against antiviral defences. There is growing interest in understanding how viruses induce such large changes in cellular organisation, and recent studies are beginning to reveal the relationship between virus factories and viroplasm and the cellular structures that house them. In this review, we discuss how three supergroups of (+)RNA viruses generate replication sites from membrane-bound organelles and highlight research on perinuclear factories induced by the nucleocytoplasmic large DNA viruses., (Copyright © 2011 Elsevier B.V. All rights reserved.)
- Published
- 2011
- Full Text
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41. Protection of European domestic pigs from virulent African isolates of African swine fever virus by experimental immunisation.
- Author
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King K, Chapman D, Argilaguet JM, Fishbourne E, Hutet E, Cariolet R, Hutchings G, Oura CA, Netherton CL, Moffat K, Taylor G, Le Potier MF, Dixon LK, and Takamatsu HH
- Subjects
- African Swine Fever immunology, African Swine Fever virology, African Swine Fever Virus classification, African Swine Fever Virus isolation & purification, Animals, Antibodies, Viral blood, Benin, Immunization, Interferon-gamma biosynthesis, Portugal, Sus scrofa virology, Swine, T-Lymphocytes immunology, Uganda, African Swine Fever prevention & control, African Swine Fever Virus immunology, African Swine Fever Virus pathogenicity, Sus scrofa immunology, Viral Vaccines administration & dosage, Viral Vaccines immunology
- Abstract
African swine fever (ASF) is an acute haemorrhagic disease of domestic pigs for which there is currently no vaccine. We showed that experimental immunisation of pigs with the non-virulent OURT88/3 genotype I isolate from Portugal followed by the closely related virulent OURT88/1 genotype I isolate could confer protection against challenge with virulent isolates from Africa including the genotype I Benin 97/1 isolate and genotype X Uganda 1965 isolate. This immunisation strategy protected most pigs challenged with either Benin or Uganda from both disease and viraemia. Cross-protection was correlated with the ability of different ASFV isolates to stimulate immune lymphocytes from the OURT88/3 and OURT88/1 immunised pigs., (Copyright © 2011 Elsevier Ltd. All rights reserved.)
- Published
- 2011
- Full Text
- View/download PDF
42. Inhibition of a large double-stranded DNA virus by MxA protein.
- Author
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Netherton CL, Simpson J, Haller O, Wileman TE, Takamatsu HH, Monaghan P, and Taylor G
- Subjects
- African Swine Fever Virus genetics, African Swine Fever Virus physiology, Animals, Chlorocebus aethiops, DNA, Viral genetics, Humans, Myxovirus Resistance Proteins, Protein Biosynthesis, Swine, Transcription, Genetic, Vero Cells, Virus Assembly, African Swine Fever Virus metabolism, GTP-Binding Proteins metabolism, Virus Replication
- Abstract
Increasing evidence points to the importance of the interferon (IFN) response in determining the host range and virulence of African swine fever virus (ASFV). Infection with attenuated strains of ASFV leads to the upregulation of genes controlled by IFN pathways, including myxovirus resistance (Mx) genes that are potent effectors of the antiviral state. Mx gene products are known to inhibit the replication of many negative-sense single-stranded RNA viruses, as well as double-stranded RNA viruses, positive-sense single-stranded RNA viruses, and the reverse-transcribing DNA virus hepatitis B virus. Here, we provide data that extend the known range of viruses inhibited by Mx to include the large double-stranded DNA viruses. Stably transfected Vero cells expressing human MxA protein did not support ASFV plaque formation, and virus replication in these cells was reduced 100-fold compared with that in control cells. In contrast, ASFV replication in cells expressing MxB protein or a mutant MxA protein was similar to that in control Vero cells. There was a drastic reduction in ASFV late protein synthesis in MxA-expressing cells, correlating with the results of previous work on the effect of IFN on viral replication. Strikingly, the inhibition of ASFV replication was linked to the recruitment of MxA protein to perinuclear viral assembly sites, where the protein surrounded the virus factories. Interactions between ASFV and MxA were similar to those seen between MxA and different RNA viruses, suggesting a common inhibitory mechanism.
- Published
- 2009
- Full Text
- View/download PDF
43. The envelope of intracellular African swine fever virus is composed of a single lipid bilayer.
- Author
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Hawes PC, Netherton CL, Wileman TE, and Monaghan P
- Subjects
- Animals, Cell Membrane metabolism, Chlorocebus aethiops, Freezing, Microscopy, Electron, Microscopy, Electron, Transmission, Mitochondria metabolism, Peptides chemistry, Pressure, Vero Cells, Viral Structural Proteins, Virus Assembly, African Swine Fever Virus metabolism, Cell Membrane virology, Gene Products, env chemistry, Lipid Bilayers chemistry, Mitochondria virology
- Abstract
African swine fever virus (ASFV) is a member of a family of large nucleocytoplasmic DNA viruses that include poxviruses, iridoviruses, and phycodnaviruses. Previous ultrastructural studies of ASFV using chemical fixation and cryosectioning for electron microscopy (EM) have produced uncertainty over whether the inner viral envelope is composed of a single or double lipid bilayer. In this study we prepared ASFV-infected cells for EM using chemical fixation, cryosectioning, and high-pressure freezing. The appearance of the intracellular viral envelope was determined and compared to that of mitochondrial membranes in each sample. The best resolution of membrane structure was obtained with samples prepared by high-pressure freezing, and images suggested that the envelope of ASFV consisted of a single lipid membrane. It was less easy to interpret virus structure in chemically fixed or cryosectioned material, and in the latter case the virus envelope could be interpreted as having two membranes. Comparison of membrane widths in all three preparations indicated that the intracellular viral envelope of ASFV was not significantly different from the outer mitochondrial membrane (P < 0.05). The results support the hypothesis that the intracellular ASFV viral envelope is composed of a single lipid bilayer.
- Published
- 2008
- Full Text
- View/download PDF
44. Rapid freeze-substitution preserves membranes in high-pressure frozen tissue culture cells.
- Author
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Hawes P, Netherton CL, Mueller M, Wileman T, and Monaghan P
- Subjects
- Animals, Cells, Cultured, Chlorocebus aethiops, Cricetinae, Freezing, Hydrostatic Pressure, Immunohistochemistry, Microscopy, Immunoelectron, Organometallic Compounds, Vero Cells, Freeze Substitution methods
- Abstract
We describe a method for high-pressure freezing and rapid freeze-substitution of cells in tissue culture which provides excellent preservation of membrane detail with negligible ice segregation artefacts. Cells grown on sapphire discs were placed 'face to face' without removal of tissue culture medium and frozen without the protection of aluminium planchettes. This reduction in thermal load of the sample/holder combination resulted in freezing of cells without visible ice-crystal artefact. Freeze-substitution at -90 degrees C for 60 min in acetone containing 2% uranyl acetate, followed by warming to -50 degrees C and embedding in Lowicryl HM20 gave consistent and clear membrane detail even when imaged without section contrasting. Preliminary data indicates that the high intrinsic contrast of samples prepared in this way will be valuable for tomographic studies. Immunolabelling sensitivity of sections of samples prepared by this rapid substitution technique was poor; however, reducing the uranyl acetate concentration in the substitution medium to 0.2% resulted in improved labelling. Samples substituted in this lower concentration of uranyl acetate also gave good membrane detail when imaged after section contrasting.
- Published
- 2007
- Full Text
- View/download PDF
45. African swine fever virus causes microtubule-dependent dispersal of the trans-golgi network and slows delivery of membrane protein to the plasma membrane.
- Author
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Netherton CL, McCrossan MC, Denyer M, Ponnambalam S, Armstrong J, Takamatsu HH, and Wileman TE
- Subjects
- Animals, Chlorocebus aethiops, Microtubules physiology, Vero Cells, trans-Golgi Network ultrastructure, African Swine Fever Virus physiology, Cell Membrane metabolism, Membrane Proteins metabolism, Microtubules virology, trans-Golgi Network virology
- Abstract
Viral interference with secretory cargo is a common mechanism for pathogen immune evasion. Selective down regulation of critical immune system molecules such as major histocompatibility complex (MHC) proteins enables pathogens to mask themselves from their host. African swine fever virus (ASFV) disrupts the trans-Golgi network (TGN) by altering the localization of TGN46, an organelle marker for the distal secretory pathway. Reorganization of membrane transport components may provide a mechanism whereby ASFV can disrupt the correct secretion and/or cell surface expression of host proteins. In the study reported here, we used the tsO45 temperature-sensitive mutant of the G protein of vesicular stomatitis virus to show that ASFV significantly reduces the rate at which the protein is delivered to the plasma membrane. This is linked to a general reorganization of the secretory pathway during infection and a specific, microtubule-dependent disruption of structural components of the TGN. Golgin p230 and TGN46 are separated into distinct vesicles, whereupon TGN46 is depleted. These data suggest that disruption of the TGN by ASFV can slow membrane traffic during viral infection. This may be functionally important because infection of macrophages with virulent isolates of ASFV increased the expression of MHC class I genes, but there was no parallel increase in MHC class I molecule delivery to the plasma membrane.
- Published
- 2006
- Full Text
- View/download PDF
46. African swine fever virus inhibits induction of the stress-induced proapoptotic transcription factor CHOP/GADD153.
- Author
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Netherton CL, Parsley JC, and Wileman T
- Subjects
- Animals, Apoptosis physiology, CHO Cells, Chlorocebus aethiops, Cricetinae, Cricetulus, Immunoblotting, Microscopy, Fluorescence, Signal Transduction, Transcription Factor CHOP, Vero Cells, African Swine Fever Virus pathogenicity, CCAAT-Enhancer-Binding Proteins metabolism, Gene Expression Regulation, Transcription Factors metabolism
- Abstract
Stress signaling from mitochondria and the endoplasmic reticulum (ER) leads to the induction of the proapoptotic transcription factor CHOP/GADD153. Many viruses use the ER as a site of replication and/or envelopment, and this activity can lead to the activation of ER stress and apoptosis. African swine fever virus (ASFV) is assembled on the cytoplasmic face of the ER and ultimately enveloped by ER membrane cisternae. The virus also recruits mitochondria to sites of viral replication and induces the mitochondrial stress protein hsp60. Here we studied the effects of ASFV on the induction of CHOP/GADD153 in infected cells. Interestingly, unlike other ER-tropic viruses, ASFV did not activate CHOP and was able to inhibit the induction of CHOP/GADD153 by a number of exogenous stimuli.
- Published
- 2004
- Full Text
- View/download PDF
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