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.

scholarly journals Deletion of a CD2-Like Gene, 8-DR, from African Swine Fever Virus Affects Viral Infection in Domestic Swine

1998 ◽  
Vol 72 (4) ◽  
pp. 2881-2889 ◽  
Author(s):  
M. V. Borca ◽  
C. Carrillo ◽  
L. Zsak ◽  
W. W. Laegreid ◽  
G. F. Kutish ◽  
...  
Keyword(s):  
Viral Infection ◽  
Mononuclear Cells ◽  
African Swine Fever Virus ◽  
Disease Onset ◽  
African Swine Fever ◽  
Fever Virus ◽  
Parental Virus ◽  
Viral Virulence

ABSTRACT An African swine fever virus (ASFV) gene with similarity to the T-lymphocyte surface antigen CD2 has been found in the pathogenic African isolate Malawi Lil-20/1 (open reading frame [ORF] 8-DR) and a cell culture-adapted European virus, BA71V (ORF EP402R) and has been shown to be responsible for the hemadsorption phenomenon observed for ASFV-infected cells. The structural and functional similarities of the ASFV gene product to CD2, a cellular protein involved in cell-cell adhesion and T-cell-mediated immune responses, suggested a possible role for this gene in tissue tropism and/or immune evasion in the swine host. In this study, we constructed an ASFV 8-DR gene deletion mutant (Δ8-DR) and its revertant (8-DR.R) from the Malawi Lil-20/1 isolate to examine gene function in vivo. In vitro, Δ8-DR, 8-DR.R, and the parental virus exhibited indistinguishable growth characteristics on primary porcine macrophage cell cultures. In vivo,8-DR had no obvious effect on viral virulence in domestic pigs; disease onset, disease course, and mortality were similar for the mutant Δ8-DR, its revertant 8-DR.R, and the parental virus. Altered viral infection was, however, observed for pigs infected with Δ8-DR. A delay in spread to and/or replication of Δ8-DR in the draining lymph node, a delay in generalization of infection, and a 100- to 1,000-fold reduction in virus titers in lymphoid tissue and bone marrow were observed. Onset of viremia for Δ8-DR-infected animals was significantly delayed (by 2 to 5 days), and mean viremia titers were reduced approximately 10,000-fold at 5 days postinfection and 30- to 100-fold at later times; moreover, unlike in 8-DR.R-infected animals, the viremia was no longer predominantly erythrocyte associated but rather was equally distributed among erythrocyte, leukocyte, and plasma fractions. Mitogen-dependent lymphocyte proliferation of swine peripheral blood mononuclear cells in vitro was reduced by 90 to 95% following infection with 8-DR.R but remained unaltered following infection with Δ8-DR, suggesting that 8-DR has immunosuppressive activity in vitro. Together, these results suggest an immunosuppressive role for 8-DR in the swine host which facilitates early events in viral infection. This may be of most significance for ASFV infection of its highly adapted natural host, the warthog.

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.

scholarly journals African Swine Fever Virus Georgia Isolate Harboring Deletions of MGF360 and MGF505 Genes Is Attenuated in Swine and Confers Protection against Challenge with Virulent Parental Virus

2015 ◽  
Vol 89 (11) ◽  
pp. 6048-6056 ◽  
Author(s):  
Vivian O'Donnell ◽  
Lauren G. Holinka ◽  
Douglas P. Gladue ◽  
Brenton Sanford ◽  
Peter W. Krug ◽  
...  
Keyword(s):  
Multigene Family ◽  
Recombinant Virus ◽  
African Swine Fever Virus ◽  
Genetically Modified ◽  
African Swine Fever ◽  
Fever Virus ◽  
Parental Virus ◽  
First Report ◽  
Highly Virulent

ABSTRACTAfrican swine fever virus (ASFV) is the etiological agent of a contagious and often lethal disease of domestic pigs that has significant economic consequences for the swine industry. The control of African swine fever (ASF) has been hampered by the unavailability of vaccines. Experimental vaccines have been developed using genetically modified live attenuated ASFVs where viral genes involved in virus virulence were removed from the genome. Multigene family 360 (MGF360) and MGF505 represent a group of genes sharing partial sequence and structural identities that have been connected with ASFV host range specificity, blocking of the host innate response, and virus virulence. Here we report the construction of a recombinant virus (ASFV-G-ΔMGF) derived from the highly virulent ASFV Georgia 2007 isolate (ASFV-G) by specifically deleting six genes belonging to MGF360 or MGF505: MGF505-1R, MGF360-12L, MGF360-13L, MGF360-14L, MGF505-2R, and MGF505-3R. ASFV-G-ΔMGF replicates as efficiently in primary swine macrophage cell cultures as the parental virus.In vivo, ASFV-G-ΔMGF is completely attenuated in swine, since pigs inoculated intramuscularly (i.m.) with either 102or 10450% hemadsorbing doses (HAD50) remained healthy, without signs of the disease. Importantly, when these animals were subsequently exposed to highly virulent parental ASFV-G, no signs of the disease were observed, although a proportion of these animals harbored the challenge virus. This is the first report demonstrating the role of MGF genes acting as independent determinants of ASFV virulence. Additionally, ASFV-G-ΔMGF is the first experimental vaccine reported to induce protection in pigs challenged with highly virulent and epidemiologically relevant ASFV-G.IMPORTANCEThe main problem for controlling ASF is the lack of vaccines. Studies focusing on understanding ASFV virulence led to the production of genetically modified recombinant viruses that, while attenuated, are able to confer protection in pigs challenged with homologous viruses. Here we have produced an attenuated recombinant ASFV derived from highly virulent ASFV strain Georgia (ASFV-G) lacking only six of the multigene family 360 (MGF360) and MGF505 genes (ASFV-G-ΔMGF). It is demonstrated, by first time, that deleting specific MGF genes alone can completely attenuate a highly virulent field ASFV isolate. Recombinant virus ASFV-G-ΔMGF effectively confers protection in pigs against challenge with ASFV-G when delivered once via the intramuscular (i.m.) route. The protection against ASFV-G is highly effective by 28 days postvaccination. This is the first report of an experimental vaccine that induces solid protection against virulent ASFV-G.

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