Analysis of naturally occurring deletion variants of african swine fever virus: Multigene family 110 is not essential for infectivity or virulence in pigs

ABSTRACT African swine fever virus (ASFV) multigene family 360 and 530 (MGF360/530) genes affect viral growth in macrophage cell cultures and virulence in pigs (L. Zsak, Z. Lu, T. G. Burrage, J. G. Neilan, G. F. Kutish, D. M. Moore, and D. L. Rock, J. Virol. 75:3066-3076, 2001). The mechanism by which these novel genes affect virus-host interactions is unknown. To define MGF360/530 gene function, we compared macrophage transcriptional responses following infection with parental ASFV (Pr4) and an MGF360/530 deletion mutant (Pr4Δ35). A swine cDNA microarray containing 7,712 macrophage cDNA clones was used to compare the transcriptional profiles of swine macrophages infected with Pr4 and Pr4Δ35 at 3 and 6 h postinfection (hpi). While at 3 hpi most (7,564) of the genes had similar expression levels in cells infected with either virus, 38 genes had significantly increased (>2.0-fold, P < 0.05) mRNA levels in Pr4Δ35-infected macrophages. Similar up-regulation of these genes was observed at 6 hpi. Viral infection was required for this induced transcriptional response. Most Pr4Δ35 up-regulated genes were part of a type I interferon (IFN) response or were genes that are normally induced by double-stranded RNA and/or viral infection. These included monocyte chemoattractant protein, transmembrane protein 3, tetratricopeptide repeat protein 1, a ubiquitin-like 17-kDa protein, ubiquitin-specific protease ISG43, an RNA helicase DEAD box protein, GTP-binding MX protein, the cytokine IP-10, and the PKR activator PACT. Differential expression of IFN early-response genes in Pr4Δ35 relative to Pr4 was confirmed by Northern blot analysis and real-time PCR. Analysis of IFN-α mRNA and secreted IFN-α levels at 3, 8, and 24 hpi revealed undetectable IFN-α in mock- and Pr4-infected macrophages but significant IFN-α levels at 24 hpi in Pr4Δ35-infected macrophages. The absence of IFN-α in Pr4-infected macrophages suggests that MGF360/530 genes either directly or indirectly suppress a type I IFN response. An inability to suppress host type I IFN responses may account for the growth defect of Pr4Δ35 in macrophages and its attenuation in swine.

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African Swine Fever Virus Multigene Family 360 and 530 Genes Are Novel Macrophage Host Range Determinants

Journal of Virology ◽

10.1128/jvi.75.7.3066-3076.2001 ◽

2001 ◽

Vol 75 (7) ◽

pp. 3066-3076 ◽

Cited By ~ 55

Author(s):

L. Zsak ◽

Z. Lu ◽

T. G. Burrage ◽

J. G. Neilan ◽

G. F. Kutish ◽

...

Keyword(s):

Host Range ◽

Cell Cultures ◽

Multigene Family ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus ◽

Target Cells ◽

Macrophage Cell ◽

Range Function

ABSTRACT Pathogenic African swine fever virus (ASFV) isolates primarily target cells of the mononuclear-phagocytic system in infected swine and replicate efficiently in primary macrophage cell cultures in vitro. ASFVs can, however, be adapted to grow in monkey cell lines. Characterization of two cell culture-adapted viruses, MS16 and BA71V, revealed that neither virus replicated in macrophage cell cultures. Cell viability experiments and ultrastructural analysis showed that infection with these viruses resulted in early macrophage cell death, which occurred prior to viral progeny production. Genomic cosmid clones from pathogenic ASFV isolate E70 were used in marker rescue experiments to identify sequences capable of restoring MS16 and BA71V growth in macrophage cell cultures. A cosmid clone representing a 38-kbp region at the left terminus of the genome completely restored the growth of both viruses. In subsequent fine-mapping experiments, an 11-kbp subclone from this region was sufficient for complete rescue of BA71V growth. Sequence analysis indicated that both MS16 and BA71V had significant deletions in the region containing members of multigene family 360 (MGF 360) and MGF530. Deletion of this same region from highly pathogenic ASFV isolate Pr4 significantly reduced viral growth in macrophage cell cultures. These findings indicate that ASFV MGF360 and MGF530 genes perform an essential macrophage host range function(s) that involves promotion of infected-cell survival.

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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

Journal of Virology ◽

10.1128/jvi.00554-15 ◽

2015 ◽

Vol 89 (11) ◽

pp. 6048-6056 ◽

Cited By ~ 69

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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Transcriptional analysis of multigene family 110 of African swine fever virus.

Journal of Virology ◽

10.1128/jvi.66.11.6655-6667.1992 ◽

1992 ◽

Vol 66 (11) ◽

pp. 6655-6667 ◽

Cited By ~ 40

Author(s):

F Almazán ◽

J M Rodríguez ◽

G Andrés ◽

R Pérez ◽

E Viñuela ◽

...

Keyword(s):

Multigene Family ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus ◽

Transcriptional Analysis

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Age-related viral load and severity of systemic pathological lesions in acute naturally occurring African swine fever virus Genotype II infections

Comparative Immunology Microbiology and Infectious Diseases ◽

10.1016/j.cimid.2021.101709 ◽

2021 ◽

pp. 101709

Author(s):

T. Oh ◽

D.T. Do ◽

D.C. Lai ◽

T.C. Nguyen ◽

H.V. Vo ◽

...

Keyword(s):

Viral Load ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus ◽

Virus Genotype ◽

Pathological Lesions ◽

Naturally Occurring ◽

Age Related ◽

Genotype Ii

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

Journal of Virology ◽

10.1128/jvi.78.5.2445-2453.2004 ◽

2004 ◽

Vol 78 (5) ◽

pp. 2445-2453 ◽

Cited By ~ 44

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.

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The non-haemadsorbing African swine fever virus isolate ASFV/NH/P68 provides a model for defining the protective anti-virus immune response

Journal of General Virology ◽

10.1099/0022-1317-82-3-513 ◽

2001 ◽

Vol 82 (3) ◽

pp. 513-523 ◽

Cited By ~ 100

Author(s):

Alexandre Leitão ◽

Clara Cartaxeiro ◽

Ricardo Coelho ◽

Benedita Cruz ◽

R. M. E. Parkhouse ◽

...

Keyword(s):

Nk Cell ◽

African Swine Fever Virus ◽

Cell Activity ◽

African Swine Fever ◽

Fever Virus ◽

Pokeweed Mitogen ◽

Infection Model ◽

Discrete Groups ◽

Activity Levels ◽

Naturally Occurring

African swine fever virus ASFV/NH/P68 is a naturally occurring, non-haemadsorbing and non-fatal isolate. Longitudinal clinical and immunological studies on 31 pigs inoculated oronasally or intramuscularly with this isolate defined two discrete groups of animals: those developing ASF chronic type lesions and those remaining asymptomatic. Animals developing lesions had viraemia and fever late after infection, NK activity levels close to that of control animals and high levels of anti-ASFV specific antibodies together with a marked hypergammaglobulinaemia involving IgG1, IgG2, IgM and IgA immunoglobulin isotypes. Pigs remaining asymptomatic after infection, on the other hand, did not have viraemia or fever after day 14 post-infection and had elevated NK cell activity, but normal plasma Ig concentrations and relatively low specific anti-virus antibody concentrations throughout the duration of the experiments. Importantly, the latter group of pigs virus were resistant to subsequent challenge with the highly virulent ASFV/L60 isolate and survived with no major changes in any of the parameters examined and referred to above. Finally, lymphoproliferative responses to the mitogens concanavalin A, phytohaemagglutinin and pokeweed mitogen were not depressed in either of the two clinically defined groups of pigs. Thus further studies with this infection model may provide new insights on mechanisms of protective immunity to ASFV.

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The Subcellular Distribution of Multigene Family 110 Proteins of African Swine Fever Virus Is Determined by Differences in C-Terminal KDEL Endoplasmic Reticulum Retention Motifs

Journal of Virology ◽

10.1128/jvi.78.7.3710-3721.2004 ◽

2004 ◽

Vol 78 (7) ◽

pp. 3710-3721 ◽

Cited By ~ 24

Author(s):

Christopher Netherton ◽

Isabelle Rouiller ◽

Thomas Wileman

Keyword(s):

Endoplasmic Reticulum ◽

Gene Product ◽

Multigene Family ◽

Subcellular Distribution ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus ◽

C Terminus ◽

Er Retention ◽

Infected Cells

ABSTRACT African swine fever virus (ASFV) is a large double-stranded DNA virus that replicates in discrete areas in the cytosol of infected cells called viral factories. Recent studies have shown that assembling virions acquire their internal envelopes through enwrapment by membranes derived from the endoplasmic reticulum (ER). However, the mechanisms that underlie the formation of viral factories and progenitor viral membranes are as yet unclear. Analysis of the published genome of the virus revealed a conserved multigene family that encodes proteins with hydrophobic signal sequences, indicating possible translocation into the ER lumen. Strikingly, two of these genes, XP124L and Y118L, encoded proteins with KDEL-like ER retention motifs. Analysis of XP124L and Y118L gene product by biochemical and immunofluorescence techniques showed that the proteins were localized to pre-Golgi compartments and that the KEDL motif at the C terminus of pXP124L was functional. XP124L expression, in the absence of other ASFV genes, had a dramatic effect on the contents of the ER that was dependent precisely on the C-terminal sequence KEDL. The normal subcellular distribution of a number of proteins resident to this important, cellular organelle was drastically altered in cells expressing wild-type XP124L gene product. PXP124L formed unusual perinuclear structures that contained resident ER proteins, as well as proteins of the ER-Golgi intermediate compartment. The data presented here hint at a role for MGF110 gene product in preparing the ER for its role in viral morphogenesis; this and other potential functions are discussed.

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