scholarly journals Cholesterol Flux Is Required for Endosomal Progression of African Swine Fever Virions during the Initial Establishment of Infection

2015 ◽  
Vol 90 (3) ◽  
pp. 1534-1543 ◽  
Author(s):  
Miguel Ángel Cuesta-Geijo ◽  
Michele Chiappi ◽  
Inmaculada Galindo ◽  
Lucía Barrado-Gil ◽  
Raquel Muñoz-Moreno ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Cholesterol Efflux ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Endocytic Pathway ◽  
Productive Infection ◽  
Effective Vaccine ◽  
The Caucasus ◽  
Late Endosomes ◽  
Adenovirus 5

ABSTRACTAfrican swine fever virus (ASFV) is a major threat for porcine production that has been slowly spreading in Eastern Europe since its first appearance in the Caucasus in 2007. ASFV enters the cell by endocytosis and gains access to the cytosol to start replication from late endosomes and multivesicular bodies. Cholesterol associated with low-density lipoproteins entering the cell by endocytosis also follows a trafficking pathway similar to that of ASFV. Here we show that cholesterol plays an essential role in the establishment of infection as the virus traffics through the endocytic pathway. In contrast to the case for other DNA viruses, such as vaccinia virus or adenovirus 5, cholesterol efflux from endosomes is required for ASFV release/entry to the cytosol. Accumulation of cholesterol in endosomes impairs fusion, resulting in retention of virions inside endosomes. ASFV also remodels intracellular cholesterol by increasing its cellular uptake and redistributes free cholesterol to viral replication sites. Our analysis reveals that ASFV manipulates cholesterol dynamics to ensure an appropriate lipid flux to establish productive infection.IMPORTANCESince its appearance in the Caucasus in 2007, African swine fever (ASF) has been spreading westwards to neighboring European countries, threatening porcine production. Due to the lack of an effective vaccine, ASF control relies on early diagnosis and widespread culling of infected animals. We investigated early stages of ASFV infection to identify potential cellular targets for therapeutic intervention against ASF. The virus enters the cell by endocytosis, and soon thereafter, viral decapsidation occurs in the acid pH of late endosomes. We found that ASFV infection requires and reorganizes the cellular lipid cholesterol. ASFV requires cholesterol to exit the endosome to gain access to the cytoplasm to establish productive replication. Our results indicate that there is a differential requirement for cholesterol efflux for vaccinia virus or adenovirus 5 compared to ASFV.

scholarly journals Dynamin- and Clathrin-Dependent Endocytosis in African Swine Fever Virus Entry

2009 ◽  
Vol 84 (4) ◽  
pp. 2100-2109 ◽  
Author(s):  
Bruno Hernaez ◽  
Covadonga Alonso
Keyword(s):  
Actin Polymerization ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Cell Types ◽  
Fever Virus ◽  
Dominant Negative ◽  
Endocytic Pathway ◽  
Host Cells ◽  
Productive Infection ◽  
Cellular Compartments

ABSTRACT African swine fever virus (ASFV) is a large DNA virus that enters host cells after receptor-mediated endocytosis and depends on acidic cellular compartments for productive infection. The exact cellular mechanism, however, is largely unknown. In order to dissect ASFV entry, we have analyzed the major endocytic routes using specific inhibitors and dominant negative mutants and analyzed the consequences for ASFV entry into host cells. Our results indicate that ASFV entry into host cells takes place by clathrin-mediated endocytosis which requires dynamin GTPase activity. Also, the clathrin-coated pit component Eps15 was identified as a relevant cellular factor during infection. The presence of cholesterol in cellular membranes, but not lipid rafts or caveolae, was found to be essential for a productive ASFV infection. In contrast, inhibitors of the Na+/H+ ion channels and actin polymerization inhibition did not significantly modify ASFV infection, suggesting that macropinocytosis does not represent the main entry route for ASFV. These results suggest a dynamin-dependent and clathrin-mediated endocytic pathway of ASFV entry for the cell types and viral strains analyzed.

scholarly journals Development and clinical application of a novel CRISPR-Cas12a based assay for the detection of African swine fever virus

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Xiaoying Wang ◽  
Sheng He ◽  
Na Zhao ◽  
Xiaohong Liu ◽  
Yongchang Cao ◽  
...  
Keyword(s):  
African Swine Fever Virus ◽  
Diagnostic Technique ◽  
African Swine Fever ◽  
Cross Reactivity ◽  
Fever Virus ◽  
Effective Vaccine ◽  
Highly Sensitive ◽  
Resource Poor ◽  
Good Consistency ◽  
Virus Genotypes

Abstract Background As no treatment or effective vaccine for African swine fever virus (ASFV) is currently available, a rapid, highly sensitive diagnostic is urgently needed to curb the spread of ASFV. Results Here we designed a novel CRISPR-Cas12a based assay for ASFV detection. To detect different ASFV genotypes, 19 crRNAs were designed to target the conserved p72 gene in ASFV, and several crRNAs with high activity were identified that could be used as alternatives in the event of new ASFV variants. The results showed that the sensitivity of the CRISPR-Cas12a based assay is about ten times higher than either the commercial quantitative PCR (qPCR) kit or the OIE-recommended qPCR. CRISPR-Cas12a based assay could also detect ASFV specifically without cross-reactivity with other important viruses in pigs and various virus genotypes. We also found that longer incubation times increased the detection limits, which could be applied to improve assay outcomes in the detection of weakly positive samples and new ASFV variants. In addition, both the CRISPR-Cas12a based assay and commercial qPCR showed very good consistency. Conclusions In summary, the CRISPR-Cas12a based assay offers a feasible approach and a new diagnostic technique for the diagnosis of ASFV, particularly in resource-poor settings.

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

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

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

scholarly journals Three African swine fever virus genes encoding proteins with homology to putative helicases of vaccinia virus

1993 ◽  
Vol 74 (9) ◽  
pp. 1969-1974 ◽  
Author(s):  
S. A. Baylis ◽  
S. R. F. Twigg ◽  
S. Vydelingum ◽  
L. K. Dixon ◽  
G. L. Smith
Keyword(s):  
Vaccinia Virus ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Genes Encoding

scholarly journals A new method for sampling African swine fever virus genome and its inactivation in environmental samples

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Aleksandra Kosowska ◽  
Jose A. Barasona ◽  
Sandra Barroso-Arévalo ◽  
Belén Rivera ◽  
Lucas Domínguez ◽  
...  
Keyword(s):  
Early Detection ◽  
Environmental Samples ◽  
Sampling Method ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Virus Genome ◽  
Economic Losses ◽  
Effective Vaccine ◽  
Speed Up ◽  
Important Basis

AbstractAfrican swine fever (ASF) is currently the most dangerous disease for the global pig industry, causing huge economic losses, due to the lack of effective vaccine or treatment. Only the early detection of ASF virus (ASFV) and proper biosecurity measures are effective to reduce the viral expansion. One of the most widely recognized risks as regards the introduction ASFV into a country is infected animals and contaminated livestock vehicles. In order to improve ASF surveillance, we have assessed the capacity for the detection and inactivation of ASFV genome by using Dry-Sponges (3 M) pre-hydrated with a new surfactant liquid. We sampled different surfaces in ASFV-contaminated facilities, including animal skins, and the results were compared to those obtained using a traditional sampling method. The surfactant liquid successfully inactivated the virus, while ASFV DNA was well preserved for the detection. This is an effective method to systematically recover ASFV DNA from different surfaces and skin, which has a key applied relevance in surveillance of vehicles transporting live animals and greatly improves animal welfare. This method provides an important basis for the detection of ASFV genome that can be assessed without the biosafety requirements of a BSL-3 laboratory at least in ASF-affected countries, which may substantially speed up the early detection of the pathogen.

scholarly journals Endosomal proteins NPC1 and NPC2 at African swine fever virus entry/fusion

2021 ◽  
Author(s):  
Covadonga Alonso ◽  
Miguel Ángel Cuesta-Geijo ◽  
Jesús Urquiza ◽  
Ana Del Puerto ◽  
Isabel García-Dorival ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
African Swine Fever Virus ◽  
Direct Interaction ◽  
African Swine Fever ◽  
Vero Cells ◽  
Fever Virus ◽  
Endocytic Pathway ◽  
Viral Fusion ◽  
Transporter Protein ◽  
Niemann Pick

African swine fever virus (ASFV) infectious cycle starts with the viral adsorption and entry into the host cell. The virus is internalized via clathrin/dynamin mediated endocytosis and macropinocytosis. As several other viruses, ASF virion is then internalized and incorporated into the endocytic pathway. Endosomal maturation entails luminal acidification and the lowering of pH acting on the multi-layered virion structure dissolves the outer capsid. Upon decapsidation, the inner viral membrane is exposed to interact with the limiting membrane of the late endosome for fusion. Egress from endosome is related to cholesterol efflux, but it remains an intriguing process albeit essential for infection, specifically for the viral nucleic acid exit to the cytoplasm for replication. ASFV proteins E248R and E199L, with structural homology to the VACV proteins of the fusion complex, seem to have similar functions in ASFV. A direct interaction between these ASFV proteins with the cholesterol transporter protein NPC1 (Niemann-Pick C type 1) was observed, which was also shared by the E248R homologous protein L1R of VACV. Binding occurs between the transmembrane domain of E248R with the loop C of NPC1 at the same domain than EBOV binding site. These interactions suggest that these ASFV proteins are crucial for membrane fusion. CRISPR NPC1 KO Vero cells lacking NPC1 protein that were resistant to EBOV, reduced ASFV infection levels significantly. Reductions on ASFV infectivity and replication in NPC1 KO cells were accompanied by lesser viral factories of smaller size and lacking the typical cohesive morphology between endosomes and viral proteins.  We observed a compensatory effect in NPC1 KO cells, elevating NPC2 levels while silencing NPC2 in Vero cells with shRNA, also reduced ASFV infection. Our findings pave the way to understand the role of these proteins at the membrane viral fusion step for several viruses.

scholarly journals Subunit Vaccine Approaches for African Swine Fever Virus

Vaccines ◽  
2019 ◽  
Vol 7 (2) ◽  
pp. 56 ◽  
Author(s):  
Natasha N. Gaudreault ◽  
Juergen A. Richt
Keyword(s):  
Western Europe ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Swine Industry ◽  
Fatal Disease ◽  
The Caucasus ◽  
Caucasus Region ◽  
Highly Virulent ◽  
Genotype Ii

African swine fever virus (ASFV) is the cause of a highly fatal disease in swine, for which there is no available vaccine. The disease is highly contagious and poses a serious threat to the swine industry worldwide. Since its introduction to the Caucasus region in 2007, a highly virulent, genotype II strain of ASFV has continued to circulate and spread into Eastern Europe and Russia, and most recently into Western Europe, China, and various countries of Southeast Asia. This review summarizes various ASFV vaccine strategies that have been investigated, with focus on antigen-, DNA-, and virus vector-based vaccines. Known ASFV antigens and the determinants of protection against ASFV versus immunopathological enhancement of infection and disease are also discussed.

Vaccinia virus-mediated expression of african swine fever virus genes

Virology ◽  
1991 ◽  
Vol 181 (2) ◽  
pp. 778-782 ◽  
Author(s):  
Jef M. Hammond ◽  
Linda K. Dixon
Keyword(s):  
Vaccinia Virus ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus

scholarly journals African Swine Fever Virus dUTPase Is a Highly Specific Enzyme Required for Efficient Replication in Swine Macrophages

1999 ◽  
Vol 73 (11) ◽  
pp. 8934-8943 ◽  
Author(s):  
Mariano Oliveros ◽  
Ramón García-Escudero ◽  
Alí Alejo ◽  
Eladio Viñuela ◽  
María L. Salas ◽  
...  
Keyword(s):  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Vero Cells ◽  
Fever Virus ◽  
Parental Virus ◽  
Productive Infection ◽  
Genome Replication ◽  
Specific Enzyme ◽  
Efficient Replication ◽  
Dividing Cells

ABSTRACT The African swine fever virus (ASFV) gene E165R, which is homologous to dUTPases, has been characterized. A multiple alignment of dUTPases showed the conservation in ASFV dUTPase of the motifs that define this protein family. A biochemical analysis of the purified recombinant enzyme showed that the virus dUTPase is a trimeric, highly specific enzyme that requires a divalent cation for activity. The enzyme is most probably complexed with Mg2+, the preferred cation, and has an apparent Km for dUTP of 1 μM. Northern and Western blotting, as well as immunofluorescence analyses, indicated that the enzyme is expressed at early and late times of infection and is localized in the cytoplasm of the infected cells. On the other hand, an ASFV dUTPase-deletion mutant (vΔE165R) has been obtained. Growth kinetics showed that vΔE165R replicates as efficiently as parental virus in Vero cells but only to 10% or less of parental virus in swine macrophages. Our results suggest that the dUTPase activity is dispensable for virus replication in dividing cells but is required for productive infection in nondividing swine macrophages, the natural host cell for the virus. The viral dUTPase may play a role in lowering the dUTP concentration in natural infections to minimize misincorporation of deoxyuridine into the viral DNA and ensure the fidelity of genome replication.

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