scholarly journals An African Swine Fever Virus ERV1-ALRHomologue, 9GL, Affects Virion Maturation and Viral Growth in Macrophages and Viral Virulence in Swine

2000 ◽  
Vol 74 (3) ◽  
pp. 1275-1285 ◽  
Author(s):  
T. Lewis ◽  
L. Zsak ◽  
T. G. Burrage ◽  
Z. Lu ◽  
G. F. Kutish ◽  
...  
Keyword(s):  
Time Course ◽  
Gene Deletion ◽  
African Swine Fever Virus ◽  
Lethal Dose ◽  
Protein A ◽  
African Swine Fever ◽  
Fever Virus ◽  
Growth Defect ◽  
Synthesis Inhibitor ◽  
Viral Virulence

ABSTRACT The African swine fever virus (ASFV) genome contains a gene,9GL, with similarity to yeast ERV1 andALR genes. ERV1 has been shown to function in oxidative phosphorylation and in cell growth, while ALR has hepatotrophic activity. 9GL encodes a protein of 119 amino acids and was highly conserved at both nucleotide and amino acid levels among all ASFV field isolates examined. Monospecific rabbit polyclonal antibody produced to a glutathione S-transferase–9GL fusion protein specifically immunoprecipitated a 14-kDa protein from macrophage cell cultures infected with the ASFV isolate Malawi Lil-20/1 (MAL). Time course analysis and viral DNA synthesis inhibitor experiments indicated that p14 was a late viral protein. A9GL gene deletion mutant of MAL (Δ9GL), exhibited a growth defect in macrophages of approximately 2 log10 units and had a small-plaque phenotype compared to either a revertant (9GL-R) or the parental virus. 9GL affected normal virion maturation; virions containing acentric nucleoid structures comprised 90 to 99% of all virions observed in Δ9GL-infected macrophages. The Δ9GL virus was markedly attenuated in swine. In contrast to 9GL-R infection, where mortality was 100%, all Δ9GL-infected animals survived infection. With the exception of a transient fever response in some animals, Δ9GL-infected animals remained clinically normal and exhibited significant 100- to 10,000-fold reductions in viremia titers. All pigs previously infected with Δ9GL survived infection when subsequently challenged with a lethal dose of virulent parental MAL. Thus, ASFV9GL gene deletion mutants may prove useful as live-attenuated ASF vaccines.

scholarly journals African Swine Fever Virus Multigene Family 360 Genes Affect Virus Replication and Generalization of Infection in Ornithodoros porcinus Ticks

2004 ◽  
Vol 78 (5) ◽  
pp. 2445-2453 ◽  
Author(s):  
T. G. Burrage ◽  
Z. Lu ◽  
J. G. Neilan ◽  
D. L. Rock ◽  
L. Zsak
Keyword(s):  
Host Range ◽  
Virus Replication ◽  
Multigene Family ◽  
Deletion Mutant ◽  
Gene Deletion ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Reproductive Organs ◽  
Fever Virus ◽  
Growth Defect

ABSTRACT Recently, we reported that African swine fever virus (ASFV) multigene family (MGF) 360 and 530 genes are significant swine macrophage host range determinants that function by promoting infected-cell survival. To examine the function of these genes in ASFV's arthropod host, Ornithodoros porcinus porcinus, an MGF360/530 gene deletion mutant (Pr4Δ35) was constructed from an ASFV isolate of tick origin, Pr4. Pr4Δ35 exhibited a significant growth defect in ticks. The deletion of six MGF360 and two MGF530 genes from Pr4 markedly reduced viral replication in infected ticks 100- to 1,000-fold. To define the minimal set of MGF360/530 genes required for tick host range, additional gene deletion mutants lacking individual or multiple MGF genes were constructed. The deletion mutant Pr4Δ3-C2, which lacked three MGF360 genes (3HL, 3Il, and 3LL), exhibited reduced viral growth in ticks. Pr4Δ3-C2 virus titers in ticks were significantly reduced 100- to 1,000-fold compared to control values at various times postinfection. In contrast to the parental virus, with which high levels of virus replication were observed in the tissues of infected adults, Pr4Δ3-C2 replication was not detected in the midgut, hemolymph, salivary gland, coxal gland, or reproductive organs at 15 weeks postinfection. These data indicate that ASFV MGF360 genes are significant tick host range determinants and that they are required for efficient virus replication and generalization of infection. The impaired virus replication of Pr4Δ3-C2 in the tick midgut likely accounts for the absence of the generalized infection that is necessary for the natural transmission of virus from ticks to pigs.

scholarly journals The African Swine Fever Virus Thymidine Kinase Gene Is Required for Efficient Replication in Swine Macrophages and for Virulence in Swine

1998 ◽  
Vol 72 (12) ◽  
pp. 10310-10315 ◽  
Author(s):  
D. M. Moore ◽  
L. Zsak ◽  
J. G. Neilan ◽  
Z. Lu ◽  
D. L. Rock
Keyword(s):  
Thymidine Kinase ◽  
Gene Deletion ◽  
African Swine Fever Virus ◽  
Clinical Signs ◽  
African Swine Fever ◽  
Vero Cells ◽  
Fever Virus ◽  
Growth Defect ◽  
Kinase Gene

ABSTRACT African swine fever virus (ASFV) replicates in the cytoplasm of infected cells and contains genes encoding a number of enzymes needed for DNA synthesis, including a thymidine kinase (TK) gene. Recombinant TK gene deletion viruses were produced by using two highly pathogenic isolates of ASFV through homologous recombination with an ASFV p72 promoter–β-glucuronidase indicator cassette (p72GUS) flanked by ASFV sequences targeting the TK region. Attempts to isolate double-crossover TK gene deletion mutants on swine macrophages failed, suggesting a growth deficiency of TK− ASFV on macrophages. Two pathogenic ASFV isolates, ASFV Malawi and ASFV Haiti, partially adapted to Vero cells, were used successfully to construct TK deletion viruses on Vero cells. The selected viruses grew well on Vero cells, but both mutants exhibited a growth defect on swine macrophages at low multiplicities of infection (MOI), yielding 0.1 to 1.0% of wild-type levels. At high MOI, the macrophage growth defect was not apparent. The Malawi TK deletion mutant showed reduced virulence for swine, producing transient fevers, lower viremia titers, and reduced mortality. In contrast, 100% mortality was observed for swine inoculated with the TK+ revertant virus. Swine surviving TK− ASFV infection remained free of clinical signs of African swine fever following subsequent challenge with the parental pathogenic ASFV. The data indicate that the TK gene of ASFV is important for growth in swine macrophages in vitro and is a virus virulence factor in swine.

scholarly journals Differential Effect of the Deletion of African Swine Fever Virus Virulence-Associated Genes in the Induction of Attenuation of the Highly Virulent Georgia Strain

Viruses ◽  
2019 ◽  
Vol 11 (7) ◽  
pp. 599 ◽  
Author(s):  
Elizabeth Ramirez-Medina ◽  
Elizabeth Vuono ◽  
Vivian O’Donnell ◽  
Lauren G. Holinka ◽  
Ediane Silva ◽  
...  
Keyword(s):  
Protective Immunity ◽  
Gene Deletion ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Early Death ◽  
Fever Virus ◽  
Differential Behavior ◽  
Viral Genes ◽  
The Uk ◽  
Highly Virulent

African swine fever virus (ASFV) is the etiological agent of an often lethal disease of domestic pigs, African swine fever (ASF). The ASFV Georgia 2007 isolate (ASFV-G) is responsible for the current epidemic situation in Europe and Asia. Genetically modified ASFVs containing deletions of virulence-associated genes have produced attenuated phenotypes and induced protective immunity in swine. Here we describe the differential behavior of two viral genes, NL (DP71L) and UK (DP96R), both originally described as being involved in virus virulence. Deletion of either of these genes efficiently attenuated ASFV strain E70. We demonstrated that deletion of the UK gene from the ASFV-G genome did not decrease virulence when compared to the parental virus. Conversely, deletion of the NL gene produced a heterogeneous response, with early death in one of the animals and transient fever in the other animals. With this knowledge, we attempted to increase the safety profile of the previously reported experimental vaccine ASFV-GΔ9GL/ΔUK by deleting the NL gene. A triple gene-deletion virus was produced, ASFV-GΔ9GL/ΔNL/ΔUK. Although ASFV-GΔ9GL/ΔNL/ΔUK replicated in primary cell cultures of swine macrophages, it demonstrated a severe replication deficiency in pigs, failing to induce protection against challenge with parental ASFV-G.

scholarly journals Novel Swine Virulence Determinant in the Left Variable Region of the African Swine Fever Virus Genome

2002 ◽  
Vol 76 (7) ◽  
pp. 3095-3104 ◽  
Author(s):  
J. G. Neilan ◽  
L. Zsak ◽  
Z. Lu ◽  
G. F. Kutish ◽  
C. L. Afonso ◽  
...  
Keyword(s):  
Deletion Mutant ◽  
Gene Deletion ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Variable Region ◽  
Fever Virus ◽  
Marker Rescue ◽  
Virulence Determinant ◽  
Gene Deletion Mutant

ABSTRACT Previously we have shown that the African swine fever virus (ASFV) NL gene deletion mutant E70ΔNL is attenuated in pigs. Our recent observations that NL gene deletion mutants of two additional pathogenic ASFV isolates, Malawi Lil-20/1 and Pr4, remained highly virulent in swine (100% mortality) suggested that these isolates encoded an additional virulence determinant(s) that was absent from E70. To map this putative virulence determinant, in vivo marker rescue experiments were performed by inoculating swine with infection-transfection lysates containing E70 NL deletion mutant virus (E70ΔNL) and cosmid DNA clones from the Malawi NL gene deletion mutant (MalΔNL). A cosmid clone representing the left-hand 38-kb region (map units 0.05 to 0.26) of the MalΔNL genome was capable of restoring full virulence to E70ΔNL. Southern blot analysis of recovered virulent viruses confirmed that they were recombinant E70ΔNL genomes containing a 23- to 28-kb DNA fragment of the Malawi genome. These recombinants exhibited an unaltered MalΔNL disease and virulence phenotype when inoculated into swine. Additional in vivo marker rescue experiments identified a 20-kb fragment, encoding members of multigene families (MGF) 360 and 530, as being capable of fully restoring virulence to E70ΔNL. Comparative nucleotide sequence analysis of the left variable region of the E70ΔNL and Malawi Lil-20/1 genomes identified an 8-kb deletion in the E70ΔNL isolate which resulted in the deletion and/or truncation of three MGF 360 genes and four MGF 530 genes. A recombinant MalΔNL deletion mutant lacking three members of each MGF gene family was constructed and evaluated for virulence in swine. The mutant virus replicated normally in macrophage cell culture but was avirulent in swine. Together, these results indicate that a region within the left variable region of the ASFV genome containing the MGF 360 and 530 genes represents a previously unrecognized virulence determinant for domestic swine.

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.

scholarly journals Development of a Blocking Enzyme-Linked Immunosorbent Assay for Detection of Antibodies against African Swine Fever Virus

Pathogens ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 760
Author(s):  
Fangfeng Yuan ◽  
Vlad Petrovan ◽  
Luis Gabriel Gimenez-Lirola ◽  
Jeffrey J. Zimmerman ◽  
Raymond R. R. Rowland ◽  
...  
Keyword(s):  
Time Course ◽  
Serological Test ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Internal Quality ◽  
Enzyme Linked Immunosorbent Assay ◽  
Post Inoculation ◽  
Swine Industry ◽  
Control Serum

The incursion of African swine fever virus (ASFV) into Eurasia presents a threat to the world’s swine industry. Highly sensitive and specific diagnostic assays are urgently needed for rapid detection during an outbreak, post-outbreak investigation, and disease surveillance. In this study, a highly specific and repeatable blocking ELISA (bELISA) was developed using a recombinant p30 protein as the antigen combined with biotinylated mAb against p30 as the detection antibody. Initial test validation included sera from 810 uninfected animals and 106 animals experimentally inoculated with ASFV or recombinant alphavirus/adenovirus expressing p30. Receiver operating characteristic (ROC) analysis of the data calculated an optimal percentage of inhibition (PI) cutoff value of 45.92%, giving a diagnostic sensitivity of 98.11% and diagnostic specificity of 99.42%. The coefficient of variation of an internal quality control serum was 6.81% for between runs, 6.71% for within run, and 6.14% for within plate. A time course study of infected pigs showed that bELISA was able to detect seroconversion as early as 7 days post-inoculation. Taken together, these results demonstrate that bELISA can be used as an alternative serological test for detecting ASFV infection.

scholarly journals Macrophage Transcriptional Responses following In Vitro Infection with a Highly Virulent African Swine Fever Virus Isolate

2006 ◽  
Vol 80 (21) ◽  
pp. 10514-10521 ◽  
Author(s):  
Fuquan Zhang ◽  
Paul Hopwood ◽  
Charles C. Abrams ◽  
Alison Downing ◽  
Frazer Murray ◽  
...  
Keyword(s):  
Proinflammatory Cytokines ◽  
Time Course ◽  
African Swine Fever Virus ◽  
Expression Profiles ◽  
African Swine Fever ◽  
Fever Virus ◽  
Surface Proteins ◽  
Transcriptional Responses ◽  
Expression Levels ◽  
Infected Cells

ABSTRACT We used a porcine microarray containing 2,880 cDNAs to investigate the response of macrophages to infection by a virulent African swine fever virus (ASFV) isolate, Malawi LIL20/1. One hundred twenty-five targets were found to be significantly altered at either or both 4 h and 16 h postinfection compared with targets after mock infection. These targets were assigned into three groups according to their temporal expression profiles. Eighty-six targets showed increased expression levels at 4 h postinfection but returned to expression levels similar to those in mock-infected cells at 16 h postinfection. These encoded several proinflammatory cytokines and chemokines, surface proteins, and proteins involved in cell signaling and trafficking pathways. Thirty-four targets showed increased expression levels at 16 h postinfection compared to levels at 4 h postinfection and in mock-infected cells. One host gene showed increased expression levels at both 4 and 16 h postinfection compared to levels in mock-infected cells. The microarray results were validated for 12 selected genes by quantitative real-time PCR. Levels of protein expression and secretion were measured for two proinflammatory cytokines, interleukin 1β and tumor necrosis factor alpha, during a time course of infection with either the virulent Malawi LIL20/1 isolate or the OUR T88/3 nonpathogenic isolate. The results revealed differences between these two ASFV isolates in the amounts of these cytokines secreted from infected cells.

scholarly journals A BIR Motif Containing Gene of African Swine Fever Virus,4CL,Is Nonessential for Growthin Vitroand Viral Virulence

Virology ◽  
1997 ◽  
Vol 230 (2) ◽  
pp. 252-264 ◽  
Author(s):  
J.G. Neilan ◽  
Z. Lu ◽  
G.F. Kutish ◽  
L. Zsak ◽  
T.G. Burrage ◽  
...  
Keyword(s):  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Viral Virulence

scholarly journals Immunization of Pigs by DNA Prime and Recombinant Vaccinia Virus Boost To Identify and Rank African Swine Fever Virus Immunogenic and Protective Proteins

2018 ◽  
Vol 92 (8) ◽  
Author(s):  
James K. Jancovich ◽  
Dave Chapman ◽  
Debra T. Hansen ◽  
Mark D. Robida ◽  
Andrey Loskutov ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
African Swine Fever Virus ◽  
Lethal Dose ◽  
African Swine Fever ◽  
Hemorrhagic Fever ◽  
Recombinant Vaccinia Virus ◽  
Fever Virus ◽  
Lethal Challenge ◽  
Recombinant Vaccinia ◽  
Safety Issues

ABSTRACTAfrican 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.IMPORTANCEThe 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.

scholarly journals A Nonessential African Swine Fever Virus Gene UK Is a Significant Virulence Determinant in Domestic Swine

1998 ◽  
Vol 72 (2) ◽  
pp. 1028-1035 ◽  
Author(s):  
L. Zsak ◽  
E. Caler ◽  
Z. Lu ◽  
G. F. Kutish ◽  
J. G. Neilan ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Cell Cultures ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Macrophage Cell ◽  
Significant Similarity ◽  
Virulence Determinant ◽  
Viral Virulence ◽  
The Right

ABSTRACT Sequence analysis of the right variable genomic region of the pathogenic African swine fever virus (ASFV) isolate E70 revealed a novel gene, UK, that is immediately upstream from the previously described ASFV virulence-associated gene NL-S(L. Zsak, Z. Lu, G. F. Kutish, J. G. Neilan, and D. L. Rock, J. Virol. 70:8865–8871, 1996). UK, transcriptionally oriented toward the right end of the genome, predicts a protein of 96 amino acids with a molecular mass of 10.7 kDa. Searches of genetic databases did not find significant similarity betweenUK and other known genes. Sequence analysis of theUK genes from several pathogenic ASFVs from Europe, the Caribbean, and Africa demonstrated that this gene was highly conserved among diverse pathogenic isolates, including those from both tick and pig sources. Polyclonal antibodies raised against the UK protein specifically precipitated a 15-kDa protein from ASFV-infected macrophage cell cultures as early as 2 h postinfection. A recombinant UK gene deletion mutant, ΔUK, and its revertant, UK-R, were constructed from the E70 isolate to study gene function. Although deletion of UK did not affect the growth characteristics of the virus in macrophage cell cultures, ΔUK exhibited reduced virulence in infected pigs. While mortality among parental E70- or UK-R-infected animals was 100%, all ΔUK-infected pigs survived infection. Fever responses were comparable in E70-, UK-R-, and ΔUK-infected groups; however, ΔUK-infected animals exhibited significant, 100- to 1,000-fold, reductions in viremia titers. These data indicate that the highly conserved UK gene of ASFV, while being nonessential for growth in macrophages in vitro, is an important viral virulence determinant for domestic pigs.

Sign in / Sign up

Export Citation Format

Share Document