Role of African swine fever virus (ASFV) proteins EP153R and EP402R in reducing viral persistence and virulence from attenuated BeninΔDP148R

The limited knowledge on the role of many of the approximately 170 proteins encoded by African swine fever virus restricts progress towards vaccine development. In this study we investigated the effect of deleting combinations of different genes from a previously attenuated virus, BeninΔDP148R on: virus replication in macrophages, virus persistence and clinical signs post immunization, and induction of protection against challenge. Deletion of either EP402R or EP153R genes individually or in combination from BeninΔDP148R did not reduce virus replication in vitro. However, deletion of EP402R dramatically reduced viral persistence in vivo, whilst maintaining high levels of protection against challenge. The additional deletion of EP153R (BeninΔDP148RΔEP153RΔEP402R) further attenuated the virus and no viremia or clinical signs were observed post immunization. This was associated with decreased protection and detection of moderate levels of challenge virus in blood. Interestingly, the deletion of EP153R alone from BeninΔDP148R did not result in further virus attenuation and a slight increase in virus genome copies in blood was observed at different times post immunization when compared with BeninΔDP148R. These results show that EP402R and EP153R have a synergistic role in promoting viremia, however EP153R alone does not seem to have a major impact on virus levels in blood.

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Role of African swine fever virus (ASFV) proteins EP153R and EP402R in reducing viral persistence in blood and virulence in pigs infected with BeninΔDP148R

Journal of Virology ◽

10.1128/jvi.01340-21 ◽

2021 ◽

Author(s):

Vlad Petrovan ◽

Anusyah Rathakrishnan ◽

Muneeb Islam ◽

Lynnette C. Goatley ◽

Katy Moffat ◽

...

Keyword(s):

Vaccine Development ◽

African Swine Fever Virus ◽

Clinical Signs ◽

African Swine Fever ◽

Fever Virus ◽

Genotype I ◽

Virus Persistence ◽

Challenge Virus

The limited knowledge on the role of many of the approximately 170 proteins encoded by African swine fever virus restricts progress towards vaccine development. Previously, the DP148R gene was deleted from the genome of genotype I virulent Benin 97/1 isolate. This virus, BeninΔDP148R, induced transient moderate clinical signs after immunization and high levels of protection against challenge. However, the BeninΔDP148R virus and genome persisted in blood over a prolonged period. In the current study deletion of either EP402R or EP153R genes individually or in combination from BeninΔDP148R genome was shown not to reduce virus replication in macrophages in vitro. However, deletion of EP402R dramatically reduced the period of infectious virus persistence in blood in immunized pigs from 28 to 14 days and virus genome from 59 to 14 days, whilst maintaining high levels of protection against challenge. The additional deletion of EP153R (BeninΔDP148RΔEP153RΔEP402R) further attenuated the virus and no viremia or clinical signs were observed post-immunization. This was associated with decreased protection and detection of moderate levels of challenge virus in blood. Interestingly, the deletion of EP153R alone from BeninΔDP148R did not result in further virus attenuation and did not reduce the period of virus persistence in blood. These results show that EP402R and EP153R have a synergistic role in reducing clinical signs and levels of virus in blood. Importance: African swine fever virus (ASFV) causes a disease of domestic pigs and wild boar which results in death of almost all infected animals. The disease has a high economic impact, and no vaccine is available. We investigated the role of two ASFV proteins, called EP402R and EP153R, in determining the levels and length of time virus persists in blood from infected pigs. EP402R causes ASFV particles and infected cells to bind to red blood cells. Deletion of the EP402R gene dramatically reduced virus persistence in blood but did not reduce the level of virus. Deletion of the EP153R alone did not reduce the period or level of virus persistence in blood. However, deleting both EP153R and EP402R resulted in undetectable levels of virus in blood and no clinical signs showing the proteins act synergistically. Importantly the infected pigs were protected following infection with the wildtype virus that kills pigs.

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Identification and Isolation of Two Different Subpopulations Within African Swine Fever Virus Arm/07 Stock

Vaccines ◽

10.3390/vaccines8040625 ◽

2020 ◽

Vol 8 (4) ◽

pp. 625

Author(s):

Daniel Pérez-Núñez ◽

Eva Castillo-Rosa ◽

Gonzalo Vigara-Astillero ◽

Raquel García-Belmonte ◽

Carmina Gallardo ◽

...

Keyword(s):

Vaccine Development ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus ◽

Single Mutation ◽

High Sequence Identity ◽

Genotype I ◽

High Coverage ◽

Purification Method

No efficient vaccines exist against African swine fever virus (ASFV), which causes a serious disease in wild boars and domestic pigs that produces great industrial and ecological concerns worldwide. An extensive genetic characterization of the original ASFV stocks used to produce live attenuated vaccine (LAV) prototypes is needed for vaccine biosecurity and control. Here, we sequenced for the first time the Arm/07 stock which was obtained from an infected pig during the Armenia outbreak in 2007, using an improved viral dsDNA purification method together with high coverage analysis. There was unexpected viral heterogeneity within the stock, with two genetically distinct ASFV subpopulations. The first, represented by the Arm/07/CBM/c2 clone, displayed high sequence identity to the updated genotype II Georgia 2007/1, whereas the second (exemplified by clone Arm/07/CBM/c4) displayed a hemadsorbing phenotype and grouped within genotype I based on a central region conserved among all members of this group. Intriguingly, Arm/07/CBM/c4 contained a unique EP402R sequence, produced by a single mutation in the N-terminal region. Importantly, Arm/07/CBM/c4 showed in vitro features of attenuated strains regarding innate immune response pathway. Both Arm/07/CBM/c2 and c4 represent well-characterized viral clones, useful for different molecular and virus-host interaction studies, including virulence studies and vaccine development.

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Deletion of the African Swine Fever Virus Gene DP148R Does Not Reduce Virus Replication in Culture but Reduces Virus Virulence in Pigs and Induces High Levels of Protection against Challenge

Journal of Virology ◽

10.1128/jvi.01428-17 ◽

2017 ◽

Vol 91 (24) ◽

Cited By ~ 31

Author(s):

Ana L. Reis ◽

Lynnette C. Goatley ◽

Tamara Jabbar ◽

Pedro J. Sanchez-Cordon ◽

Christopher L. Netherton ◽

...

Keyword(s):

Virus Replication ◽

Infectious Virus ◽

African Swine Fever Virus ◽

Clinical Signs ◽

African Swine Fever ◽

Virus Genome ◽

Fever Virus ◽

Virulent Virus ◽

Ifn Γ ◽

Reduce Virus Replication

ABSTRACT Many of the approximately 165 proteins encoded by the African swine fever virus (ASFV) genome do not have significant similarity to known proteins and have not been studied experimentally. One such protein is DP148R. We showed that the DP148R gene is transcribed at early times postinfection. Deletion of this gene did not reduce virus replication in macrophages, showing that it is not essential for replication in these cells. However, deletion of this gene from a virulent isolate, Benin 97/1, producing the BeninΔDP148R virus, dramatically reduced the virulence of the virus in vivo. All pigs infected with the BeninΔDP148R virus survived infection, showing only transient mild clinical signs soon after immunization. Following challenge with the parental virulent virus, all pigs immunized by the intramuscular route (11/11) and all except one immunized by the intranasal route (5/6) survived. Mild or no clinical signs were observed after challenge. As expected, control nonimmune pigs developed signs of acute African swine fever (ASF). The virus genome and infectious virus were observed soon after immunization, coincident with the onset of clinical signs (∼106 genome copies or 50% tissue culture infective doses/ml). The levels of the virus genome declined over an extended period up to 60 days postimmunization. In contrast, infectious virus was no longer detectable by days 30 to 35. Gamma interferon (IFN-γ) was detected in serum between days 4 and 7 postimmunization, and IFN-γ-producing cells were detected in all pigs analyzed following stimulation of immune lymphocytes with whole virus. ASFV-specific antibodies were first detected from day 10 postimmunization. IMPORTANCE African swine fever (ASF) is endemic in Africa, parts of the Trans Caucasus, the Russian Federation, and several European countries. The lack of a vaccine hinders control. Many of the ASF virus genes lack similarity to known genes and have not been characterized. We have shown that one of these, DP148R, is transcribed early during virus replication in cells and can be deleted from the virus genome without reducing virus replication. The virus with the gene deletion, BeninΔDP148R, caused mild clinical signs in pigs and induced high levels of protection against challenge with the parental virulent virus. Therefore, deletion of this gene can provide a target for the rational development of vaccines.

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Comparative efficacy of broad-spectrum antiviral agents as inhibitors of African swine fever virus replication in vitro

Antiviral Research ◽

10.1016/0166-3542(87)90003-9 ◽

1987 ◽

Vol 7 (3) ◽

pp. 151-160 ◽

Cited By ~ 18

Author(s):

Carmen Gil-Fernández ◽

Erik De Clercq

Keyword(s):

Virus Replication ◽

Broad Spectrum ◽

African Swine Fever Virus ◽

Antiviral Agents ◽

African Swine Fever ◽

Fever Virus ◽

Comparative Efficacy

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I267L Is Neither the Virulence- Nor the Replication-Related Gene of African Swine Fever Virus and Its Deletant Is an Ideal Fluorescent-Tagged Virulence Strain

Viruses ◽

10.3390/v14010053 ◽

2021 ◽

Vol 14 (1) ◽

pp. 53

Author(s):

Yanyan Zhang ◽

Junnan Ke ◽

Jingyuan Zhang ◽

Huixian Yue ◽

Teng Chen ◽

...

Keyword(s):

Fluorescent Protein ◽

African Swine Fever Virus ◽

Clinical Signs ◽

Case Fatality ◽

African Swine Fever ◽

Fever Virus ◽

Economic Losses ◽

Effective Vaccine ◽

Domestic Pigs

African swine fever virus (ASFV) is the causative agent of African swine fever (ASF) which reaches up to 100% case fatality in domestic pigs and wild boar and causes significant economic losses in the swine industry. Lack of knowledge of the function of ASFV genes is a serious impediment to the development of the safe and effective vaccine. Herein, I267L was identified as a relative conserved gene and an early expressed gene. A recombinant virus (SY18ΔI267L) with I267L gene deletion was produced by replacing I267L of the virulent ASFV SY18 with enhanced green fluorescent protein (EGFP) cassette. The replication kinetics of SY18ΔI267L is similar to that of the parental isolate in vitro. Moreover, the doses of 102.0 TCID50 (n = 5) and 105.0 TCID50 (n = 5) SY18ΔI267L caused virulent phenotype, severe clinical signs, viremia, high viral load, and mortality in domestic pigs inoculated intramuscularly as the virulent parental virus strain. Therefore, the deletion of I267L does not affect the replication or the virulence of ASFV. Utilizing the fluorescent-tagged virulence deletant can be easy to gain a visual result in related research such as the inactivation effect of some drugs, disinfectants, extracts, etc. on ASFV.

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

Journal of Virology ◽

10.1128/jvi.72.12.10310-10315.1998 ◽

1998 ◽

Vol 72 (12) ◽

pp. 10310-10315 ◽

Cited By ~ 37

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.

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Clinical Indicators of Moribundity in Swine Experimentally Inoculated with African Swine Fever Virus

Journal of the American Association for Laboratory Animal Science ◽

10.30802/aalas-jaalas-20-000032 ◽

2020 ◽

Author(s):

Benjamin J Hershey ◽

Jenna L Hagart ◽

Karyn A Havas

Keyword(s):

Vaccine Development ◽

African Swine Fever Virus ◽

Significant Risk ◽

African Swine Fever ◽

Fever Virus ◽

Complex Nature ◽

Clinical Indicators ◽

Record Data

African swine fever virus (ASFV), the causative agent of African Swine Fever (ASF), is an infectious disease of swine that is associated with high rates of morbidity and mortality in naive populations. ASFV is challenging to work with in vitro and the in vivo immune response remains an active area of study. Vaccine development, pathogenesis, and diagnostic assay development studies often require use of live swine housed in high-containment laboratories. Studies of this type are intended to obtain key data yet must minimize the pain and distress experienced by the animals. To implement humane endpoints, pigs are ideally euthanatized by barbiturate overdose prior to death from ASFV infection, as the final stages of ASF can be clinically severe. However, due to the complex nature of ASFV pathogenesis, predicting when an infected animal will become moribund and require euthanasia is difficult. The current study was intended to aid in predicting the onset of moribundity in swine. Toward this end, we performed statistical analyses of historical health record data from 103 swine experimentally infected with ASFV. Regression analysis suggested that rectal temperature has potential utility as a marker for predicting moribundity, whereas viral strain and duration of survival after inoculation were significant risk factors for death due to disease rather than euthanasia.

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DNA-Protein Vaccination Strategy Does Not Protect from Challenge with African Swine Fever Virus Armenia 2007 Strain

Vaccines ◽

10.3390/vaccines7010012 ◽

2019 ◽

Vol 7 (1) ◽

pp. 12 ◽

Cited By ~ 19

Author(s):

Sun-Young Sunwoo ◽

Daniel Pérez-Núñez ◽

Igor Morozov ◽

Elena Sánchez ◽

Natasha Gaudreault ◽

...

Keyword(s):

Recombinant Proteins ◽

African Swine Fever Virus ◽

Clinical Signs ◽

African Swine Fever ◽

Vaccination Strategy ◽

Fever Virus ◽

High Morbidity ◽

Protein Vaccine ◽

Vaccine Strategy

African swine fever virus (ASFV) causes high morbidity and mortality in swine (Sus scrofa), for which there is no commercially available vaccine. Recent outbreaks of the virus in Trans-Caucasus countries, Eastern Europe, Belgium and China highlight the urgent need to develop effective vaccines against ASFV. Previously, we evaluated the immunogenicity of a vaccination strategy designed to test various combinations of ASFV antigens encoded by DNA plasmids and recombinant proteins with the aim to activate both humoral and cellular immunity. Based on our previous results, the objective of this study was to test the combined DNA-protein vaccine strategy using a cocktail of the most immunogenic antigens against virulent ASFV challenge. Pigs were vaccinated three times with a cocktail that included ASFV plasmid DNA (CD2v, p72, p32, +/−p17) and recombinant proteins (p15, p35, p54, +/−p17). Three weeks after the third immunization, all pigs were challenged with the virulent ASFV Armenia 2007 strain. The results showed that vaccinated pigs were not protected from ASFV infection or disease. Compared to the non-vaccinated controls, earlier onset of clinical signs, viremia, and death were observed for the vaccinated animals following virulent ASFV challenge. ASFV induced pathology was also enhanced in the vaccinated pigs. Furthermore, while the vaccinated pigs developed antigen-specific antibodies, immunized pig sera at the time of challenge lacked the capacity to neutralize virus, and instead was observed to enhance ASFV infection in vitro. The results of this work points to a putative immune enhancement mechanism involved in ASFV pathogenesis that warrants further investigation. This pilot study provides insight for the selection of appropriate combinations of ASFV antigens for the development of a rationally-designed, safe, and efficacious vaccine for ASF.

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Generation and Evaluation of an African Swine Fever Virus Mutant with Deletion of the CD2v and UK Genes

Vaccines ◽

10.3390/vaccines8040763 ◽

2020 ◽

Vol 8 (4) ◽

pp. 763

Author(s):

Teshale Teklue ◽

Tao Wang ◽

Yuzi Luo ◽

Rongliang Hu ◽

Yuan Sun ◽

...

Keyword(s):

African Swine Fever Virus ◽

Clinical Signs ◽

African Swine Fever ◽

Virus Genome ◽

Fever Virus ◽

Effective Vaccine ◽

Post Inoculation ◽

Virus Mutant ◽

And Control

African swine fever (ASF) is a highly contagious and often lethal disease caused by African swine fever virus (ASFV). ASF emerged in China in August 2018 and has since rapidly spread into many areas of the country. The disease has caused a significant impact on China’s pig and related industries. A safe and effective vaccine is needed to prevent and control the disease. Several gene-deleted ASFVs have been reported; however, none of them is safe enough and commercially available. In this study, we report the generation of a double gene-deleted ASFV mutant, ASFV-SY18-∆CD2v/UK, from a highly virulent field strain ASFV-SY18 isolated in China. The results showed that ASFV-SY18-∆CD2v/UK lost hemadsorption properties, and the simultaneous deletion of the two genes did not significantly affect the in vitro replication of the virus in primary porcine alveolar macrophages. Furthermore, ASFV-SY18-∆CD2v/UK was attenuated in pigs. All the ASFV-SY18-∆CD2v/UK-inoculated pigs remained healthy, and none of them developed ASF-associated clinical signs. Additionally, the ASFV-SY18-∆CD2v/UK-infected pigs developed ASFV-specific antibodies, and no virus genome was detected in blood and nasal discharges at 21 and 28 days post-inoculation. More importantly, we found that all the pigs inoculated with 104 TCID50 of ASFV-SY18-∆CD2v/UK were protected against the challenge with the parental ASFV-SY18. However, low-level ASFV DNA was detected in blood, nasal swabs, and lymphoid tissue after the challenge. The results demonstrate that ASFV-SY18-∆CD2v/UK is safe and able to elicit protective immune response in pigs and can be a potential vaccine candidate to control ASF.

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