X69R Is a Non-Essential Gene That, When Deleted from African Swine Fever, Does Not Affect Virulence in Swine

African swine fever virus (ASFV) is currently causing devastating outbreaks in Asia and Europe, and the ASFV strain Georgia (ASFV-G) is responsible for these outbreaks. ASFV-G is highly virulent and continues to be maintained in these outbreak areas, apparently without suffering significant genomic or phenotypic changes. When comparing the genome of ASFV-G to other isolates, a thus-far uncharacterized gene, X69R, is highly conserved and, interestingly, is similar to another ASFV uncharacterized gene, J64R. All sequenced ASFV isolates have one or both of these genes, X69R or J64R, suggesting that the presence of at least one of these genes may be necessary for ASFV replication and or virulence. The X69R gene is present in the ASFV-G genome while J64R is absent. To assess the importance of X69R in ASFV-G functionality, we developed a recombinant virus by deleting the X69R gene from the ASFV-G genome (ASFV-G-ΔX69R). ASFV-G-ΔX69R had the same replication kinetics in primary swine macrophage cultures as the parental ASFV-G, indicating that the X69R gene is not essential for ASFV-G viability or efficient replication in the main target cell during in vivo infection. In addition, swine intramuscularly inoculated with a low dose (102 HAD50) of ASFV-G-ΔX69R developed a clinical disease indistinguishable from that induced by the same dose of the virulent parental ASFV-G isolate. Viremia values of ASFV-G-ΔX69R did not significantly differ from those detected in animals infected with parental virus. Therefore, deletion of the X69R gene from ASFV-G does not affect virus replication or virulence in swine.

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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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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 ◽

10.3390/v11070599 ◽

2019 ◽

Vol 11 (7) ◽

pp. 599 ◽

Cited By ~ 14

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.

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

Journal of Virology ◽

10.1128/jvi.76.7.3095-3104.2002 ◽

2002 ◽

Vol 76 (7) ◽

pp. 3095-3104 ◽

Cited By ~ 34

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.

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BA71ΔCD2: a New Recombinant Live Attenuated African Swine Fever Virus with Cross-Protective Capabilities

Journal of Virology ◽

10.1128/jvi.01058-17 ◽

2017 ◽

Vol 91 (21) ◽

Cited By ~ 51

Author(s):

Paula L. Monteagudo ◽

Anna Lacasta ◽

Elisabeth López ◽

Laia Bosch ◽

Javier Collado ◽

...

Keyword(s):

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus ◽

Sub Saharan Africa ◽

Swine Industry ◽

Lethal Challenge ◽

Major Threat ◽

Attenuated Viruses ◽

Genotype Ii

ABSTRACT African swine fever is a highly contagious viral disease of mandatory declaration to the World Organization for Animal Health (OIE). The lack of available vaccines makes its control difficult; thus, African swine fever virus (ASFV) represents a major threat to the swine industry. Inactivated vaccines do not confer solid protection against ASFV. Conversely, live attenuated viruses (LAV), either naturally isolated or obtained by genetic manipulation, have demonstrated reliable protection against homologous ASFV strains, although little or no protection has been demonstrated against heterologous viruses. Safety concerns are a major issue for the use of ASFV attenuated vaccine candidates and have hampered their implementation in the field so far. While trying to develop safer and efficient ASFV vaccines, we found that the deletion of the viral CD2v (EP402R) gene highly attenuated the virulent BA71 strain in vivo. Inoculation of pigs with the deletion mutant virus BA71ΔCD2 conferred protection not only against lethal challenge with the parental BA71 but also against the heterologous E75 (both genotype I strains). The protection induced was dose dependent, and the cross-protection observed in vivo correlated with the ability of BA71ΔCD2 to induce specific CD8+ T cells capable of recognizing both BA71 and E75 viruses in vitro. Interestingly, 100% of the pigs immunized with BA71ΔCD2 also survived lethal challenge with Georgia 2007/1, the genotype II strain of ASFV currently circulating in continental Europe. These results open new avenues to design ASFV cross-protective vaccines, essential to fight ASFV in areas where the virus is endemic and where multiple viruses are circulating. IMPORTANCE African swine fever virus (ASFV) remains enzootic in most countries of Sub-Saharan Africa, today representing a major threat for the development of their swine industry. The uncontrolled presence of ASFV has favored its periodic exportation to other countries, the last event being in Georgia in 2007. Since then, ASFV has spread toward neighboring countries, reaching the European Union's east border in 2014. The lack of available vaccines against ASFV makes its control difficult; so far, only live attenuated viruses have demonstrated solid protection against homologous experimental challenges, but they have failed at inducing solid cross-protective immunity against heterologous viruses. Here we describe a new LAV candidate with unique cross-protective abilities: BA71ΔCD2. Inoculation of BA71ΔCD2 protected pigs not only against experimental challenge with BA71, the virulent parental strain, but also against heterologous viruses, including Georgia 2007/1, the genotype II strain of ASFV currently circulating in Eastern Europe.

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Involvement of the Reparative DNA Polymerase Pol X of African Swine Fever Virus in the Maintenance of Viral Genome Stability In Vivo

Journal of Virology ◽

10.1128/jvi.01173-13 ◽

2013 ◽

Vol 87 (17) ◽

pp. 9780-9787 ◽

Cited By ~ 12

Author(s):

M. Redrejo-Rodriguez ◽

J. M. Rodriguez ◽

C. Suarez ◽

J. Salas ◽

M. L. Salas

Keyword(s):

Dna Polymerase ◽

Viral Genome ◽

Genome Stability ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus

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

Journal of Virology ◽

10.1128/jvi.72.4.2881-2889.1998 ◽

1998 ◽

Vol 72 (4) ◽

pp. 2881-2889 ◽

Cited By ~ 74

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.

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Clinical Course and Gross Pathological Findings in Wild Boar Infected with a Highly Virulent Strain of African Swine Fever Virus Genotype II

Pathogens ◽

10.3390/pathogens9090688 ◽

2020 ◽

Vol 9 (9) ◽

pp. 688 ◽

Cited By ~ 1

Author(s):

Antonio Rodríguez-Bertos ◽

Estefanía Cadenas-Fernández ◽

Agustín Rebollada-Merino ◽

Néstor Porras-González ◽

Francisco J. Mayoral-Alegre ◽

...

Keyword(s):

Wild Boar ◽

Clinical Course ◽

African Swine Fever Virus ◽

Virulent Strain ◽

African Swine Fever ◽

Pathological Findings ◽

Wild Boars ◽

Gross Pathology ◽

Highly Virulent ◽

Genotype Ii

African swine fever (ASF) is a notifiable disease that in recent years has spread remarkably in Europe and Asia. Eurasian wild boar (Sus scrofa) plays a key role in the maintenance and spread of the pathogen. Here we examined gross pathology of infection in wild boar with a highly virulent, hemadsorbing genotype II ASF virus (ASFV) strain. To this end, six wild boars were intramuscularly inoculated with the 10 HAD50 Arm07 ASFV strain, and 11 wild boars were allowed to come into direct contact with the inoculated animals. No animals survived the infection. Clinical course, gross pathological findings and viral genome quantification by PCR in tissues did not differ between intramuscularly inoculated or contact-infected animals. Postmortem analysis showed enlargement of liver and spleen; serosanguinous effusion in body cavities; and multiple hemorrhages in lungs, endocardium, brain, kidneys, urinary bladder, pancreas, and alimentary system. These results provide detailed insights into the gross pathology of wild boar infected with a highly virulent genotype II ASFV strain. From a didactic point of view, this detailed clinical course and macroscopic description may be essential for early postmortem detection of outbreaks in wild boar in the field and contribute to disease surveillance and prevention efforts.

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EFFECTS OF MODERATELY VIRULENT AFRICAN SWINE FEVER VIRUS ON INTERLEUKIN-10 PRODUCTION

Veterinary Science Today ◽

10.29326/2304-196x-2019-3-30-23-28 ◽

2019 ◽

pp. 23-28 ◽

Cited By ~ 1

Author(s):

A. S. Pershin ◽

I. V. Shevchenko ◽

A. S. Igolkin ◽

Ye. V. Aronova ◽

N. N. Vlasova

Keyword(s):

Immune Response ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus ◽

Noticeable Effect ◽

Specific Antibodies ◽

Virulent Virus ◽

Virus Isolates ◽

Highly Virulent

A characteristic feature of African swine fever virus (ASFV) is the ability to escape from host immune response, affecting macrophages and replicating in them. Besides, ASFV - specific antibodies do not completely neutralize the virus. Cytokines are important factors for various viral infection pathologies. The virulence of ASFV isolates may depend on the capacity to regulate cytokine expression by macrophages. Thus, when comparing in vitro and in vivo cytokine production by macrophages, it was established that infection with low virulent virus isolates leads to an immune response with a predominance of cytokines involved in cellular immunity, such as INF-α and IL-12p40, as compared with infection with highly virulent isolates. The aim of this paper was to study the effect of African swine fever virus on the production of IL-10, a pleiotropic cytokine that inhibits synthesis of cytokines and shows a strong antiinflammatory effect. For this, 12 piglets were experimentally infected intramuscularly with a continuous cell culture-adapted ASFV isolate Vero25 at a dose of 10 HAdU per animal followed by control infection of surviving animals with the reference virus isolate Arm 07 at a dose of 1,000 HAdU per animal. Temperature measurements were taken and blood sampling to obtain serum was conducted during the experiment. IL-10 amount in blood sera was determined using Invitrogen test systems (Thermo Fisher, USA). A higher IL-10 level (15.8–173 pg/ml) was observed in blood sera of dead animals infected with a moderately virulent virus, as compared with surviving pigs (4–5 pg/ml). No correlation between the speed of appearance of specific antibodies and IL-10 serum levels has been established. No noticeable effect of the IL-10 serum level prior to infection on the survival rate of animals has been observed. Further studies are needed to establish a causal relationship, including study of the expression of various cytokines during infection with both low- and highly virulent virus isolates.

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Genetic characterisation of African swine fever virus in outbreaks in Ha Nam province, Red River Delta Region of Vietnam, and activity of antimicrobial products against virus infection in contaminated feed

Journal of Veterinary Research ◽

10.2478/jvetres-2020-0041 ◽

2020 ◽

Vol 64 (2) ◽

pp. 207-213

Author(s):

Ha Thi Thanh Tran ◽

Anh Duc Truong ◽

Duc Viet Ly ◽

Thi Hao Vu ◽

Van Tuan Hoang ◽

...

Keyword(s):

African Swine Fever Virus ◽

African Swine Fever ◽

River Delta ◽

Red River ◽

Feed Additive ◽

Porcine Alveolar Macrophage ◽

Eastern European ◽

Delta Region ◽

Red River Delta

AbstractIntroductionAfrican swine fever (ASF) was officially reported in Vietnam in February 2019 and spread across the whole country, affecting all 63 provinces and cities.Material and MethodsIn this study, ASF virus (ASFV) VN/Pig/HaNam/2019 (VN/Pig/HN/19) strain was isolated in primary porcine alveolar macrophage (PAM) cells from a sample originating from an outbreak farm in Vietnam’s Red River Delta region. The isolate was characterised using the haemadsorption (HAD) test, real-time PCR, and sequencing. The activity of antimicrobial feed products was evaluated via a contaminated ASFV feed assay.ResultsPhylogenetic analysis of the viral p72 and EP402R genes placed VN/Pig/HN/19 in genotype II and serogroup 8 and related it closely to Eastern European and Chinese strains. Infectious titres of the virus propagated in primary PAMs were 106 HAD50/ml. Our study reports the activity against ASFV VN/Pig/HN/19 strain of antimicrobial Sal CURB RM E Liquid, F2 Dry and K2 Liquid. Our feed assay findings suggest that the antimicrobial RM E Liquid has a strong effect against ASFV replication. These results suggest that among the Sal CURB products, the antimicrobial RM E Liquid may have the most potential as a mitigant feed additive for ASFV infection. Therefore, further studies on the use of antimicrobial Sal CURB RM E Liquid in vivo are required.ConclusionsOur study demonstrates the threat of ASFV and emphasises the need to control and eradicate it in Vietnam by multiple measures.

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