Saturable binding sites mediate the entry of African swine fever virus into VERO cells

Virology ◽  
1989 ◽  
Vol 168 (2) ◽  
pp. 393-398 ◽  
Author(s):  
Antonio Alcamí ◽  
Angel L. Carrascosa ◽  
Eladio Viñuela
Keyword(s):  
Binding Sites ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Vero Cells ◽  
Fever Virus ◽  
Saturable Binding

scholarly journals Identification of the principal serological immunodeterminants of African swine fever virus by screening a virus cDNA library with antibody

2002 ◽  
Vol 83 (6) ◽  
pp. 1331-1342 ◽  
Author(s):  
S. D. Kollnberger ◽  
B. Gutierrez-Castañeda ◽  
M. Foster-Cuevas ◽  
A. Corteyn ◽  
R. M. E. Parkhouse
Keyword(s):  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Vero Cells ◽  
Polyclonal Antiserum ◽  
Fever Virus ◽  
Open Reading Frames ◽  
Cdna Expression Library ◽  
Lambda Phage ◽  
Cellular Mechanisms ◽  
Expression Library

Protective immunity to African swine fever virus (ASFV) may involve a combination of both serological and cellular mechanisms. This work is focused on the identification of the possible relevant serological immunodeterminants of immunity. Thus, 14 serological immunodeterminants of ASFV have been characterized by exhaustive screening of a representative lambda phage cDNA expression library of the tissue culture-adapted Ba71V strain of ASFV. The library was constructed using RNA extracted from Vero cells infected for 3, 6, 9 and 12 h. A total of 150 clones was selected arbitrarily by antibody screening of the library with a polyclonal antiserum from a domestic pig surviving infection with the virulent Malta isolate of ASFV. Sequencing of these clones permitted identification of 14 independent viral proteins that stimulated an antibody response. These included six proteins encoded by previously unassigned open reading frames (ORFs) (B602L, C44L, CP312R, E184L, K145R and K205R) as well as some of the more well-studied structural (A104R, p10, p32, p54 and p73) and non-structural proteins (RNA reductase, DNA ligase and thymidine kinase). Immunogenicity of these proteins was confirmed by demonstrating the corresponding antibodies in sera from pigs infected either with the Malta isolate or with the OURT88/3–OURT88/1 isolate combination. Furthermore, the majority of these ORFs were also recognized by immune antiserum from the natural host, the bush pig, following secondary challenge with the virulent Malawi (SINT90/1) isolate of ASFV. Thus, it is possible that some of these determinants may be important in protection against virus infection.

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.

Intracellular African swine fever virus DNA remains unmethylated in infected Vero cells

Epigenomics ◽  
2018 ◽  
Vol 10 (3) ◽  
pp. 289-299 ◽  
Author(s):  
Stefanie Weber ◽  
Astghik Hakobyan ◽  
Hovakim Zakaryan ◽  
Walter Doerfler
Keyword(s):  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Vero Cells ◽  
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.

Scanning Electron Microscopy of African Swine Fever Virus in Tissue Culture

1977 ◽  
Vol 35 ◽  
pp. 392-393
Author(s):  
S. S. Breese ◽  
I. C. Pan
Keyword(s):  
Electron Microscopy ◽  
Tissue Culture ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Vero Cells ◽  
Fever Virus ◽  
Osmic Acid ◽  
Transmission Electron ◽  
Virus Emergence ◽  
Scanning Electron

Previous studies of African swine fever virus (ASFV) development and its hemadsorption property by transmission electron microscopy showed virus emergence from the cells (1) and the attachment of infected cells to red cells (2). The scanning electron microscope has been used in the present study to examine the surfaces of infected leucocytes and Vero cells. Leucocytes prepared from swine blood were grown in buffer in Leighton tubes containing glass microscope cover slips and infected with ASFV Lisbon '60 strain prepared from tissue culture. After 1 hr. incubation at 37°C, the inoculum fluid was removed and replaced with F15 balanced salt solution maintenance fluid and 10% foetal bovine serum. The Vero cell cultures were grown and infected in a similar manner. Observations were made starting 18 hrs. after infection.The cover slips were washed gently with Sorenson's buffer pH 7.3, fixed in 2% Glutaraldehyde in Sorenson's buffer and post-fixed in 2% osmic acid in Sorenson's buffer. Dehydration with graded alcohols was followed by immersion in amyl acetate.

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