Role of African Swine Fever Virus Proteins EP153R and EP402R in Reducing Viral Persistence in Blood and Virulence in Pigs Infected with BeninΔDP148R.

The limited knowledge on the role of many of the approximately 170 proteins encoded by African swine fever virus restricts progress toward 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 while 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 postimmunization. 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 gene 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 that the proteins act synergistically. Importantly, the infected pigs were protected following infection with the wild-type virus that kills pigs.

A quadruple fluorescence quantitative PCR method for the identification of wild strains of african swine fever and gene-deficient strains.

BACKGROUND: Originating in Africa, African swine fever (ASF) was introduced to China in 2018. This acute and highly virulent infectious disease affects domestic pigs. The World Organization for Animal Health has listed it as a statutory reportable disease, and China has listed it as a category A infectious disease. METHODS: Primers and probes were designed for four ASFV genes (B646L, EP402R, MGF505-3R, and A137R). The primers/probes were highly conserved compared with the gene sequences of 21 ASFV strains. RESULTS: After optimization, the calibration curve showed good linearity (R2 > 0.99), the minimum concentration of positive plasmids that could be detected was 50 copies/µL, and the minimum viral load detection limit was 102 HAD50/mL. Furthermore, quadruple quantitative polymerase chain reaction (qPCR) with nucleic acids from three porcine-derived DNA viruses and cDNAs from eight RNA viruses did not show amplification curves, indicating that the method was specific. In addition, 1 × 106, 1 × 105, and 1 × 104 copies/µL of mixed plasmids were used for the quadruple qPCR; the coefficient of variation for triplicate determination between groups was < 2%, indicating the method was reproducible. CONCLUSIONS: The results obtained by testing clinical samples containing detectable EP402R, MGF505-3R, and A137R strains with different combinations of gene deletions were as expected. Therefore, the established quadruple qPCR method was validated for the molecular diagnosis of ASF using gene-deleted ASFV strains.

Deletion of the EP402R Gene from the Genome of African Swine Fever Vaccine Strain ASFV-G-∆I177L Provides the Potential Capability of Differentiating between Infected and Vaccinated Animals.

The African swine fever virus (ASFV) mutant ASFV-G-∆I177L is a safe and efficacious vaccine which induces protection against the challenge of its parental virus, the Georgia 2010 isolate. Although a genetic DIVA (differentiation between infected and vaccinated animals) assay has been developed for this vaccine, still there is not a serological DIVA test for differentiating between animals vaccinated with ASFV-G-∆I177L and those infected with wild-type viruses. In this report, we describe the development of the ASFV-G-∆I177L mutant having deleted the EP402R gene, which encodes for the viral protein responsible for mediating the hemadsorption of swine erythrocytes. The resulting virus, ASFV-G-∆I177L/∆EP402R, does not have a decreased ability to replicates in swine macrophages when compared with the parental ASFV-G-∆I177L. Domestic pigs intramuscularly (IM) inoculated with either 102 or 106 HAD50 of ASFV-G-∆I177L/∆EP402R remained clinically normal, when compared with a group of mock-vaccinated animals, indicating the absence of residual virulence. Interestingly, an infectious virus could not be detected in the blood samples of the ASFV-G-∆I177L/∆EP402R-inoculated animals in either group at any of the time points tested. Furthermore, while all of the mock-inoculated animals presented a quick and lethal clinical form of ASF after the intramuscular inoculation challenge with 102 HAD50 of highly virulent parental field isolate Georgia 2010 (ASFV-G), all of the ASFV-G-∆I177L/∆EP402R-inoculated animals were protected, remaining clinically normal until the end of the observational period. Most of the ASFV-G-∆I177L/∆EP402R-inoculated pigs developed strong virus-specific antibody responses against viral antigens, reaching maximum levels at 28 days post inoculation. Importantly, all of the sera collected at that time point in the ASFV-G-∆I177L/∆EP402R-inoculated pigs did not react in a direct ELISA coated with the recombinant EP402R protein. Conversely, the EP402R protein was readily recognized by the pool of sera from the animals immunized with recombinant live attenuated vaccine candidates ASFV-G-∆I177L, ASFV-G-∆MGF, or ASFV-G-∆9GL/∆UK. Therefore, ASFV-G-∆I177L/∆EP402R is a novel, safe and efficacious candidate with potential to be used as an antigenically DIVA vaccine.

Deletion of the CD2 Gene in the Virulent ASFV Congo Strain Affects Viremia in Domestic Swine, but Not the Virulence.

African swine fever (ASF) is an infectious disease that causes the most significant losses to the pig industry. One of the effective methods for combating this disease could be the development of vaccines. To date, experimental vaccines based on the use of live attenuated strains of the ASF virus (ASFV) obtained by the deletion of viral genes responsible for virulence are the most effective. Deletion of the EP402R gene encoding a CD2-like protein led to the attenuation of various strains of the ASFV, although the degree of attenuation varies among different isolates. Here we have shown that the deletion of the EP402R gene from the genome of a high-virulent Congo isolate did not change either the virulence of the virus or its ability to replicate in the swine macrophage cell cultures in vitro. However, in vivo, animals infected with ΔCongo-v_CD2v had a delay in the onset of the disease and viremia compared to animals infected with the parental strain. Thus, deletion of the CD2 gene in different isolates of the ASFV has a different effect on the virulence of the virus, depending on its genetic background.

Molecular Characterization of African Swine Fever Virus From 2019-2020 Outbreaks in Guangxi Province, Southern China.

African swine fever virus (ASFV) causes contagious hemorrhagic disease of pigs with high morbidity and mortality. To identify the molecular characteristics of ASFV strains circulating in Guangxi province, southern China, a total of 336 tissue samples collected from 336 domestic pigs that died as a result of severe hemorrhagic disease during 2019-2020 were tested for ASFV. Furthermore, 66 ASFV strains were genetically characterized by sequence analysis of the C-terminal region of B646L (p72) gene, the complete E183L (p54) gene, the variable region of EP402R (CD2v) gene, the central variable region (CVR) of B602L gene, the full MGF505-2R gene, and the tandem repeat sequence (TRS) within intergenic region (IGR) between the I73R and I329L (I73R/I329L) genes. Phylogenetic analysis revealed that the ASFV strains from Guangxi province belonged to genotypes I and II based on the B646L (p72) and E183L (p54) genes, and there were eight different tetrameric TRS variants based on the CVR of B602L gene. Phylogenetic analysis of the EP402R (CD2v) gene revealed that these ASFV strains belonged to serogroups 4 and 8. Eight of the 66 strains belonged to genotype I and serogroup 4, and showed deletion of whole MGF505-2R gene. The sequence analysis of the IGR between the I73R/I329L genes showed that IGR II and III variants were co-circulating in Guangxi province. The results indicated that ASFV strains circulating in Guangxi province during 2019-2020 outbreaks showed high genetic diversity, of which genotypes I and II, as well as serogroups 4 and 8, were simultaneously circulating in Guangxi province, and there existed wild-type and naturally gene-deleted strains in the field. This is the first detailed report on the molecular characterization of the ASFV strains circulating in southern China, and serogroup 4 in China.

Growth Kinetics and Protective Efficacy of Attenuated ASFV Strain Congo with Deletion of the EP402 Gene.

African swine fever (ASF) is an emerging disease threat to the swine industry worldwide. There is no vaccine against ASF, and progress is hindered by a lack of knowledge concerning the extent of ASFV strain diversity and the viral antigens conferring type-specific protective immunity in pigs. We have previously demonstrated that homologous ASFV serotype-specific proteins CD2v (EP402R) and/or C-type lectin are required for protection against challenge with the virulent ASFV strain Congo (Genotype I, Serogroup 2), and we have identified T-cell epitopes on CD2v which may be associated with serotype-specific protection. Here, using a cell-culture adapted derivative of the ASFV strain Congo (Congo-a) with specific deletion of the EP402R gene (ΔCongoCD2v) in swine vaccination/challenge experiments, we demonstrated that deletion of the EP402R gene results in the failure of ΔCongoCD2v to induce protection against challenge with the virulent strain Congo (Congo-v). While ΔCongoCD2v growth kinetics in COS-1 cells and primary swine macrophage culture were almost identical to parental Congo-a, replication of ΔCongoCD2v in vivo was significantly reduced compared with parental Congo-a. Our data support the idea that the CD2v protein is important for the ability of homologous live-attenuated vaccines to induce protective immunity against the ASFV strain Congo challenge in vivo.

Deletion of CD2-Like (CD2v) and C-Type Lectin-Like (EP153R) Genes from African Swine Fever Virus Georgia-∆9GL Abrogates Its Effectiveness as an Experimental Vaccine.

African swine fever virus (ASFV) is currently the most dreaded infectious disease affecting the global swine production industry. There is no commercial vaccine available, making the culling of infected animals the current solution to control outbreaks. Effective experimental vaccines have been developed by deleting virus genes associated with virulence. Deletion of the ASFV 9GL gene (∆9GL) has resulted in the attenuation of different ASFV strains, although the degree of attenuation varies across isolates. Here, we investigated the possibility of the increased safety of the experimental vaccine strain ASFV-G-Δ9GL by deleting two additional virus genes involved in pathogenesis, CD2v, a CD2 like viral encoded gene from the EP402R open reading frame (ORF), and C-type lectin-like viral gene, encoded from the EP153R ORF. Two new recombinant viruses were developed, ASFV-G-Δ9GL/ΔCD2v and ASFV-G-Δ9GL/ΔCD2v/ΔEP153R, harboring two and three gene deletions, respectively. ASFV-G-Δ9GL/ΔCD2v/ΔEP153R, but not ASFV-G-Δ9GL/ΔCD2v, had a decreased ability to replicate in vitro in swine macrophage cultures when compared with parental ASFV-G-Δ9GL. Importantly, ASFV-G-Δ9GL/ΔCD2v and ASFV-G-Δ9GL/ΔCD2v/ΔEP153R induced almost undetectable viremia levels when inoculated into domestic pigs and failed to protect them against challenge with parental virulent ASFV-Georgia, while ASFV-G-Δ9GL offered robust protection during challenge. Therefore, the deletion of CD2-like and C-type lectin-like genes significantly decreased the protective potential of ASFV-G-Δ9GL as a vaccine candidate. This study constitutes an example of the unpredictability of genetic manipulation involving the simultaneous deletion of multiple genes from the ASFV genome.

Production of Recombinant African Swine Fever Viruses: Speeding Up the Process.

African swine fever (ASF) is a devastating disease in pigs, with no vaccines for control. The genetic manipulation of African swine fever virus (ASFV) is often tedious and time consuming. Here, we describe a method to manipulate the virus genome to produce gene deletion viruses in a much-reduced time. This method combines the conventional homologous recombination with fluorescent-activated cells sorting (FACS), to isolate and purify viruses expressing fluorescent reporter genes. With three rounds of single cell isolation via FACS and two rounds of limiting dilution, we deleted two additional genes, EP153R and EP402R, from Benin 97/1 ASFV lacking the DP148R gene. By combining different fluorescent markers, this method has the potential to greatly facilitate studies on understanding ASFV gene functions and develop candidate live-attenuated vaccines.

Attempts at the Development of a Recombinant African Swine Fever Virus Strain with Abrogated EP402R, 9GL, and A238L Gene Structure using the CRISPR/Cas9 System.

INTRODUCTION: African swine fever (ASF) is a pressing economic problem in a number of Eastern European countries. It has also depleted the Chinese sow population by 50%. Managing the disease relies on culling infected pigs or hunting wild boars as sanitary zone creation. The constraints on the development of an efficient vaccine are mainly the virus' mechanisms of host immune response evasion. The study aimed to adapt a field ASFV strain to established cell lines and to construct recombinant African swine fever virus (ASFV) strain. MATERIAL AND METHODS: The host immune response modulation genes A238L, EP402R, and 9GL were deleted using the clustered regularly interspaced short palindromic repeats/caspase 9 (CRISPR/Cas9) mutagenesis system. A representative virus isolate (Pol18/28298/Out111) from Poland was isolated in porcine primary pulmonary alveolar macrophage (PPAM) cells. Adaptation of the virus to a few established cell lines was attempted. The plasmids encoding CRISPR/Cas9 genes along with gRNA complementary to the target sequences were designed, synthesised, and transfected into ASFV-infected PPAM cells. RESULTS: The reconstituted virus showed similar kinetics of replication in comparison to the parent virus isolate. CONCLUSION: Taking into account the usefulness of the developed CRISPR/Cas9 system it has been shown that modification of the A238L, EP402R, and 9GL genes might occur with low frequency, resulting in difficulties in separation of various virus populations.