Engineering topics

Ramirez-Medina, Elizabeth

Publications and source records attributed to Ramirez-Medina, Elizabeth.

At least 19 records

DOE OSTI2025

Evaluation of Cross-Protection of African Swine Fever Vaccine ASFV-G-ΔI177L Between ASFV Biotypes

Background/Objectives: Vaccine development for the prevention of ASF has been very challenging due to the extensive genetic and largely unknown antigenic diversity. Inactivated vaccines, using different inactivation methods and a variety of adjuvants, have been consistently inefficacious. Historically, animals recovering from an infection with an attenuated virus became protected from the development of a clinical disease caused by an antigenically related strain. Therefore, immunization of susceptible animals with attenuathe ted virus strains has become a common method of vaccination with the first two commercially available vaccines based on recombinant live-attenuated viruses (LAVs). An important limitation is that the efficacy of the LAV is restricted to those strains that are antigenically related and, in most cases, only provide protection against homologous strains. Due to the unknown antigenic heterogeneity among all ASFV field isolates, the development of broad-spectrum vaccines is a challenge. Besides the anecdotal data, there is not a large amount of information describing patterns of cross-protection between different ASFV strains. Methods: We evaluated the cross-protection induced by the ASFV live-attenuated vaccine ASFV-G-ΔI177L against different biotypes of ASFV and compared their genomic sequences to determine potential genetic mutations that could cause the lack of cross-protection. Results: Results presented here demonstrate different patterns of protection when ASFV-G-ΔI177L vaccinated pigs were challenged with six different ASFV field isolates belonging to different biotypes. Conclusions: The presence of cross-protection cannot be predicted solely by the classical methodology for genotyping-based B646L ORF only. Biotyping, considering the entire virus proteome, appears to be a more promising prediction tool, although additional gathering of experimental data will be necessary to fully validate it; until then, the presence of cross-protection needs to be confirmed in efficacy trials challenging vaccinated animals.

Immunology↗

DOE OSTI2025

Assessment of the Reversion to Virulence and Protective Efficacy in Pigs Receiving the Live Attenuated Classical Swine Fever Recombinant Vaccine Candidate FlagT4G

Background/Objectives: Control of classical swine fever virus (CSFV) in endemic countries relies on vaccination using live attenuated vaccines (LAVs). Most of these LAVs do not allow for the differentiation of vaccinated animals from infected animals (DIVA) based on their serological response. FlagT4G vaccine is a novel candidate that confers robust protective immunity early after vaccination and shows DIVA capabilities. Methods: This report presents the characterization of FlagT4G virus in terms of the stability of its genomic and attenuated phenotypes assessed by a reversion to virulence protocol, as well as its protective efficacy by determining the minimal protective dose. Results: Results presented here demonstrate that after five consecutive passages in groups of 5-week-old susceptible domestic pigs, FlagT4G virus remains genetically stable, and its attenuated phenotype remains unaltered. In terms of efficacy, FlagT4G virus induced solid protection against the intranasal challenge with 105 tissue culture infectious dose (TCID50) of virulent field isolate Brescia virus, even with a vaccine dose as low as 102 TCID50. Conclusions: Results presented here indicate that the FlagT4G vaccine may be a useful tool for CSFV control.

Immunology↗

DOE OSTI2025

Deletion of the African Swine Fever Virus Gen I196L in the Georgia2010 Isolate Genome Does Not Affect Virus Replication or Virulence in Domestic Pigs

African swine fever (ASF) is a lethal disease of domestic pigs that is currently challenging swine production in large areas of Eurasia and the Caribbean. The causative agent, ASF virus (ASFV), is a large, double-stranded, and structurally complex virus. The ASFV genome encodes for more than 160 proteins; however, the functions of most of them are still in the process of being characterized. Recently, ASFV gene I196L has been reported as being critically involved in disease production in domestic pigs. We report here that a recombinant virus derived from the Georgia 2010 isolate (ASFV-G) lacking the I196L gene, ASFV-G-∆I196L, had the same ability to replicate in primary cultures of swine macrophage and, when experimentally inoculated in pigs, produced a fatal form of the disease similar to that caused by the parental virulent ASFV-G. Therefore, deletion of the I196L gene does not significantly affect virus replication and virulence in domestic pigs of the ASFV Georgia 2010 isolate.

Virology↗

DOE OSTI2024

Lyophilization of ASFV vaccine candidate ASFV-G-ΔI177L offers long term stability

Abstract For over a century African swine fever (ASF) has been causing outbreaks leading to devastating losses for the swine industry. The current pandemic of ASF has shown no signs of stopping and continues to spread causing outbreaks in additional countries. Currently control relies mostly on culling infected farms, and strict biosecurity procedures. Recently a vaccine, ASFV-G-ΔI177L was approved for use in Vietnam. In this study we evaluate the long-term stability of lyophilized ASFV-G-ΔI177L. Understanding the stability of different formulations of vaccines is information necessary for deployment of vaccines to ASF outbreak areas, particularly those that do not have a reliable well established cold chain to ensure conservation of vaccine quality. In this report, we determined that ASFV-G-ΔI177L, when lyophilized under specific conditions, is stable for up to one year at 4 °C, with similar vaccine titers after storage. Next-generation sequencing analysis also determined that lyophilization and long-term storage under these conditions had no effect on the genome of ASFV as the genome remained genetically identical to the original non-lyophilized form.

Science & Technology - Other Topics↗

DOE OSTI2024

Assessment of the Effect of Deleting the African Swine Fever Virus Gene R298L on Virus Replication and Virulence of the Georgia2010 Isolate

African swine fever (ASF) is a lethal disease of domestic pigs that is currently challenging swine production in large areas of Eurasia. The causative agent, ASF virus (ASFV), is a large, double-stranded and structurally complex virus. The ASFV genome encodes for more than 160 proteins; however, the functions of most of these proteins are still in the process of being characterized. The ASF gene R298L, which has previously been characterized as able to encode a functional serine protein kinase, is expressed late in the virus infection cycle and may be part of the virus particle. There is no description of the importance of the R298L gene in basic virus functions such as replication or virulence in the natural host. Based on its evolution, it is proposed that there are four different phenotypes of R298L of ASFV in nature, which may have potential implications for R298L functionality. We report here that a recombinant virus lacking the R298L gene in the Georgia 2010 isolate, ASFV-G-∆R298L, does not exhibit significant changes in its replication in primary cultures of swine macrophages. In addition, when experimentally inoculated in pigs, ASFV-G-∆R298L induced a fatal form of the disease similar to that caused by the parental virulent ASFV-G. Therefore, deletion of R298L does not significantly affect virus replication and virulence in domestic pigs of the ASFV Georgia 2010 isolate.

Virology↗

DOE OSTI2024

A Retrospective Analysis Reveals That the 2021 Outbreaks of African Swine Fever Virus in Ghana Were Caused by Two Distinct Genotypes

African swine fever virus (ASFV) is the causative agent of African swine fever (ASF), a highly infectious and lethal disease of domesticated swine. Outbreaks of ASF have been mostly restricted to the continent of Africa. The outbreaks that have occurred outside of Africa were controlled by extensive depopulation of the domesticated pig population. However, in 2007, an outbreak occurred in the country of Georgia, where ASFV infected wild pigs and quickly spread across eastern Europe. Since the reintroduction of ASF into Europe, variants of the current pandemic strain, ASFV Georgia 2007/01 (ASFV-G), which is classified as Genotype 2 based on p72 sequencing, have been reported in countries within western Europe, Asia, and the island of Hispaniola. Additionally, isolates collected in 2020 confirmed the presence of variants of ASFV-G in Nigeria. Recently, we reported similar variants of ASFV-G collected from domestic pigs suspected of dying of ASF in Ghana in 2022. Here, we retroactively report, based on full-length sequencing, that similar variants were present in Ghana in 2021. The SNP analysis revealed derivatives of ASFV with distinct genetic markers. Furthermore, we identified three full-length ASFV genomes as Genotype 1, indicating that there were two genotypes circulating in proximity during the 2021 ASF outbreaks in Ghana.

Virology↗

DOE OSTI2024

Recombinant Vaccine Strain ASFV-G-Δ9GL/ΔUK Produced in the IPKM Cell Line Is Genetically Stable and Efficacious in Inducing Protection in Pigs Challenged with the Virulent African Swine Fever Virus Field Isolate Georgia 2010

We have previously reported that the recombinant African Swine Fever (ASF) vaccine candidate ASFV-G-Δ9GL/ΔUK efficiently induces protection in domestic pigs challenged with the virulent strain Georgia 2010 (ASFV-G). As reported, ASFV-G-Δ9GL/ΔUK induces protection, while intramuscularly (IM), administered at doses of 10 4 HAD 50 or higher, prevents ASF clinical disease in animals infected with the homologous ASFV g strain. Like other recombinant vaccine candidates obtained from ASFV field isolates, ASFV-G-Δ9GL/ΔUK stocks need to be produced in primary cultures of swine macrophages, which constitutes an important limitation in the production of large virus stocks at the industrial level. Here, we describe the development of ASFV-G-Δ9GL/ΔUK stocks using IPKM (Immortalized Porcine Kidney Macrophage) cells, which are derived from swine macrophages. We show that ten successive passages of ASFV-G-Δ9GL/ΔUK in IPKM cells induced small changes in the virus genome. The produced virus, ASFV-G-Δ9GL/ΔUKp10, presented a similar level of replication in swine macrophages cultures to that of the original ASFV-G-Δ9GL/ΔUK (ASFV-G-Δ9GL/ΔUKp0). The protective efficacy of ASFV-G-Δ9GL/ΔUKp10 was evaluated in pigs that were IM-inoculated with either 10 4 or 10 6 HAD 50 of ASFV-G-Δ9GL/ΔUKp10. While animals inoculated with 10 4 HAD 50 present a partial protection against the experimental infection with the virulent parental virus ASFV-G, those inoculated with 10 6 HAD 50 were completely protected. Therefore, as was just recently reported for another ASF vaccine candidate, ASFV-G-ΔI177L, IPKM cells are an effective alternative to produce stocks for vaccine strains which only grow in swine macrophages.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2024

Deletion of the EP402R Gene from the Genome of African Swine Fever Vaccine Strain ASFV-G-ΔI177L Provides the Potential Capability of Differentiating between Infected and Vaccinated Animals

The African swine fever virus (ASFV) mutant ASFV-G-ΔI177L 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-ΔI177L and those infected with wild-type viruses. In this report, we describe the development of the ASFV-G-ΔI177L 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-ΔI177L/ΔEP402R, does not have a decreased ability to replicates in swine macrophages when compared with the parental ASFV-G-ΔI177L. Domestic pigs intramuscularly (IM) inoculated with either 10 2 or 10 6 HAD 50 of ASFV-G-ΔI177L/ΔEP402R 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-ΔI177L/ΔEP402R-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 10 2 HAD 50 of highly virulent parental field isolate Georgia 2010 (ASFV-G), all of the ASFV-G-ΔI177L/ΔEP402R-inoculated animals were protected, remaining clinically normal until the end of the observational period. Most of the ASFV-G-ΔI177L/ΔEP402R-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-ΔI177L/ΔEP402R-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-ΔI177L, ASFV-G-ΔMGF, or ASFV-G-Δ9GL/ΔUK. Therefore, ASFV-G-ΔI177L/ΔEP402R is a novel, safe and efficacious candidate with potential to be used as an antigenically DIVA vaccine.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

The Interaction between the DOCK7 Protein and the E2 Protein of Classical Swine Fever Virus Is Not Involved with Viral Replication or Pathogenicity

The classical swine fever virus (CSFV) particle consists of three glycoproteins, all of which have been shown to be important proteins involved in many virus functions, including interaction with several host proteins. One of these proteins, E2, has been shown to be directly involved with adsorption to the host cell and important for virus virulence. Using the yeast two-hybrid system, we have previously shown that CSFV E2 specifically interacts with the (DOCK7) dedicator of cytokinesis, a scaffolding protein. In this report, the interaction between E2 and DOCK7 was evaluated. To confirm the yeast two-hybrid results and to determine that DOCK7 interacts in swine cells with E2, we performed co-immunoprecipitation and proximity ligation assay (PLA). After demonstrating the protein interaction in swine cells, E2 amino acid residues Y65, V283, and T149 were determined to be critical for interaction with Dock7 by using a random mutated library of E2 and a reverse yeast two-hybrid approach. That disruption of these three residues with mutations Y65F, V283D, and T149A abrogated the Dock7-E2 protein interaction. These mutations were then introduced into a recombinant CSFV, E2DOCK7v, by a reverse genomics approach using the highly virulent CSFV Brescia isolate as a backbone. E2DOCKv was shown to have similar growth kinetics in swine primary macrophages and SK6 cell cultures to the parental Brescia strain. Similarly, E2DOCK7v demonstrated a similar level of virulence to the parental Brescia when inoculated in domestic pigs. Animals intranasally inoculated with 10 5 TCID 50 developed a lethal form of clinical disease with virological and hematological kinetics changes indistinguishable from that produced by the parental strain. Therefore, interaction between CSFV E2 and host DOCK7 is not critically involved in the process of virus replication and disease production.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2023

Evaluation of the Deletion of the African Swine Fever Virus Gene O174L from the Genome of the Georgia Isolate

African swine fever virus (ASFV) is a structurally complex, double-stranded DNA virus, which causes African swine fever (ASF), a contagious disease affecting swine. ASF is currently affecting pork production in a large geographical region, including Eurasia and the Caribbean. ASFV has a large genome, which harbors more than 160 genes, but most of these genes’ functions have not been experimentally characterized. One of these genes is the O174L gene which has been experimentally shown to function as a small DNA polymerase. Here, we demonstrate that the deletion of the O174L gene from the genome of the virulent strain ASFV Georgia2010 (ASFV-G) does not significantly affect virus replication in vitro or in vivo. A recombinant virus, having deleted the O174L gene, ASFV-G-ΔO174L, was developed to study the effect of the O174L protein in replication in swine macrophages cultures in vitro and disease production when inoculated in pigs. The results demonstrated that ASFV-G-ΔO174L has similar replication kinetics to parental ASFV-G in swine macrophage cultures. In addition, animals intramuscularly inoculated with 10 2 HAD 50 of ASFV-G-ΔO174L presented a clinical form of the disease that is indistinguishable from that induced by the parental virulent strain ASFV-G. All animals developed a lethal disease, being euthanized around day 7 post-infection. Therefore, although O174L is a well-characterized DNA polymerase, its function is apparently not critical for the process of virus replication, both in vitro and in vivo, or for disease production in domestic pigs.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

African Swine Fever Vaccine Candidate ASFV-G-ΔI177L Produced in the Swine Macrophage-Derived Cell Line IPKM Remains Genetically Stable and Protective against Homologous Virulent Challenge

ASFV vaccine candidate ASFV-G-ΔI177L has been shown to be highly efficacious in inducing protection against challenges with the parental virus, the Georgia 2010 isolate, as well as against field strains isolated from Vietnam. ASFV-G-ΔI177L has been shown to produce protection even when used at low doses (10 2 HAD 50 ) and shows no residual virulence even when administered at high doses (10 6 HAD 50 ) or evaluated for a relatively long period of time (6 months). ASFV-G-ΔI177L stocks can only be massively produced in primary cell macrophages. Alternatively, its modified version (ASFV-G-ΔI177L/ΔLVR) grows in a swine-derived cell line (PIPEC), acquiring significant genomic modifications. We present here the development of ASFV-G-ΔI177L stocks in a swine macrophage cell line, IPKM, and its protective efficacy when evaluated in domestic pigs. Successive passing of ASFV-G-ΔI177L in IPKM cells produces minimal genomic changes. Interestingly, a stock of ASFV-G-ΔI177L obtained after 10 passages (ASFV-G-ΔI177Lp10) in IPKM cells showed very small genomic changes when compared with the original virus stock. ASFV-G-ΔI177Lp10 conserves similar growth kinetics in primary swine macrophage cultures than the original parental virus ASFV-G-ΔI177L. Pigs infected with 10 3 HAD 50 of ASFV-G-ΔI177Lp10 developed a strong virus-specific antibody response and were completely protected against the challenge with the parental virulent field isolate Georgia 2010. Therefore, IPKM cells could be an effective alternative for the production of ASFV vaccine stocks for those vaccine candidates exclusively growing in swine macrophages.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

The 2022 Outbreaks of African Swine Fever Virus Demonstrate the First Report of Genotype II in Ghana

African swine fever (ASF) is a lethal disease of domestic pigs that has been causing outbreaks for over a century in Africa ever since its first discovery in 1921. Since 1957, there have been sporadic outbreaks outside of Africa; however, no outbreak has been as devastating and as far-reaching as the current pandemic that originated from a 2007 outbreak in the Republic of Georgia. Derivatives with a high degree of similarity to the progenitor strain, ASFV-Georgia/2007, have been sequenced from various countries in Europe and Asia. However, the current strains circulating in Africa are largely unknown, and 24 different genotypes have been implicated in different outbreaks. In this study, ASF isolates were collected from samples from swine suspected of dying from ASF on farms in Ghana in early 2022. While previous studies determined that the circulating strains in Ghana were p72 Genotype I, we demonstrate here that the strains circulating in 2022 were derivatives of the p72 Genotype II pandemic strain. Therefore, this study demonstrates for the first time the emergence of Genotype II ASFV in Ghana.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2023

Deletion of the H240R Gene in African Swine Fever Virus Partially Reduces Virus Virulence in Swine

African swine fever (ASF) is a highly contagious disease that affects wild and domestic swine. Currently, the disease is present as a pandemic affecting pork production in Eurasia and the Caribbean region. The etiological agent of ASF is a large, highly complex structural virus (ASFV) harboring a double-stranded genome encoding for more than 160 proteins whose functions, in most cases, have not been experimentally characterized. We show here that deletion of the ASFV gene H240R from the genome of the highly virulent ASFV-Georgia2010 (ASFV-G) isolate partially decreases virus virulence when experimentally inoculated in domestic swine. ASFV-G-ΔH240R, a recombinant virus harboring the deletion of the H240R gene, was produced to evaluate the function of the gene in the development of disease in pigs. While all animals intramuscularly inoculated with 10 2 HAD 50 of ASFV-G developed a fatal form of the disease, forty percent of pigs receiving a similar dose of ASFV-G-ΔH240R survived the infection, remaining healthy during the 28-day observational period, and the remaining sixty percent developed a protracted but fatal form of the disease compared to that induced by ASFV-G. Additionally, all animals inoculated with ASFV-G-ΔH240R presented protracted viremias with reduced virus titers when compared with those found in animals inoculated with ASFV-G. Animals surviving infection with ASFV-G-ΔH240R developed a strong virus-specific antibody response and were protected against the challenge of the virulent parental ASFV-G.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

ASF Vaccine Candidate ASFV-G-ΔI177L Does Not Exhibit Residual Virulence in Long-Term Clinical Studies

African swine fever (ASF) is an important disease in swine currently producing a pandemic affecting pig production worldwide. Except in Vietnam, where two vaccines were recently approved for controlled use in the field, no vaccine is commercially available for disease control. Up to now, the most effective vaccines developed are based on the use of live-attenuated viruses. Most of these promising vaccine candidates were developed by deleting virus genes involved in the process of viral pathogenesis and disease production. Therefore, these vaccine candidates were developed via the genomic modification of parental virus field strains, producing recombinant viruses and reducing or eliminating their residual virulence. In this scenario, it is critical to confirm the absence of any residual virulence in the vaccine candidate. This report describes the assessment of the presence of residual virulence in the ASFV vaccine candidate ASFV-G-ΔI177L in clinical studies conducted under high virus loads and long-term observation periods. The results demonstrated that domestic pigs intramuscularly inoculated with 10 6 HAD 50 of ASFV-G-ΔI177L did not show the presence of any clinical sign associated with ASF when observed daily either 90 or 180 days after vaccination. In addition, necropsies conducted at the end of the experiment confirmed the absence of macroscopic internal lesions associated with the disease. These results corroborate the safety of using ASFV-G-ΔI177L as a vaccine candidate.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Classical Swine Fever Virus Structural Glycoprotein E2 Interacts with Host Protein ACADM during the Virus Infectious Cycle

The E2 glycoprotein is one of the four structural proteins of the classical swine fever virus (CSFV) particle. E2 has been shown to be involved in many virus functions, including adsorption to host cells, virus virulence and interaction with several host proteins. Using a yeast two-hybrid screen, we have previously shown that the CSFV E2 specifically interacts with swine host protein medium-chain-specific acyl-Coenzyme A dehydrogenase (ACADM), an enzyme that catalyzes the initial step of the mitochondrial fatty acid beta-oxidation pathway. Here, we show that interaction between ACADM and E2 also happens in swine cells infected with CSFV using two different procedures: coimmunoprecipitation and a proximity ligation assay (PLA). In addition, the amino acid residues in E2 critically mediating the interaction with ACADM, M49 and P130 were identified via a reverse yeast two-hybrid screen using an expression library composed of randomly mutated versions of E2. A recombinant CSFV, E2ΔACADMv, harboring substitutions at residues M49I and P130Q in E2, was developed via reverse genomics from the highly virulent Brescia isolate. E2ΔACADMv was shown to have the same kinetics growth in swine primary macrophages and SK6 cell cultures as the parental Brescia strain. Similarly, E2ΔACADMv demonstrated a similar level of virulence when inoculated to domestic pigs as the parental Brescia. Animals intranasally inoculated with 10 5 TCID 50 developed a lethal form of clinical disease with virological and hematological kinetics changes undistinguishable from those produced by the parental strain. Therefore, interaction between CSFV E2 and host ACADM is not critically involved in the processes of virus replication and disease production.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2023

Evaluation of the Function of ASFV Gene E66L in the Process of Virus Replication and Virulence in Swine

African swine fever virus (ASFV) is the etiological agent of an economically important disease of swine currently affecting large areas of Africa, Eurasia and the Caribbean. ASFV has a complex structure harboring a large dsDNA genome which encodes for more than 160 proteins. One of the proteins, E66L, has recently been involved in arresting gene transcription in the infected host cell. Here, we investigate the role of E66L in the processes of virus replication in swine macrophages and disease production in domestic swine. A recombinant ASFV was developed (ASFV-G-ΔE66L), from the virulent parental Georgia 2010 isolate (ASFV-G), harboring the deletion of the E66L gene as a tool to assess the role of the gene. ASFV-G-ΔE66L showed that the E66L gene is non-essential for ASFV replication in primary swine macrophages when compared with the parental highly virulent field isolate ASFV-G. Additionally, domestic pigs infected with ASFV-G-ΔE66L developed a clinical disease undistinguishable from that produced by ASFV-G. Therefore, E66L is not involved in virus replication or virulence in domestic pigs.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Full genome sequence for the African swine fever virus outbreak in the Dominican Republic in 1980

African swine fever is a lethal disease of domestic pigs, geographically expanding as a pandemic, that is affecting countries across Eurasia and severely damaging their swine production industry. After more than 40 years of being absent in the Western hemisphere, in 2020 ASF reappeared in the Dominican Republic and Haiti. The recent outbreak strain in the Dominican Republic has been identified as a genotype II ASFV a derivative of the ASF strain circulating in Asia and Europe. However, to date no full-length genome sequence from either the 1978–1980 Here we report the complete genome sequence of an African swine fever virus (ASFV) (DR-1980) that was previously isolated from blood collected in 1980 from the Dominican Republic at the end of the last outbreak, before culling of all swine on the island of Hispaniola and stored in the Plum Island Animal Disease Center ASFV repository. A contig representing the full-length genome (183,687 base pairs) was de novo assembled into a single contig using both Nanopore and Illumina sequences. DR-1980 was determined to belong to genotype I and, as determined by full genome comparison, a close relative to the sequenced Sardinia viruses that were causing outbreaks at this time.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2022

A Highly Effective African Swine Fever Virus Vaccine Elicits a Memory T Cell Response in Vaccinated Swine

African Swine Fever Virus (ASFV) is the causative agent of a highly contagious and lethal vector-borne disease in suids. Recently, a live attenuated virus strain, developed using the currently circulating, virulent Georgia strain (ASFV-G) with a single gene deletion (ASFV-G-ΔI177L), resulted in an effective vaccine. Nevertheless, protective immune response mechanisms induced by this candidate are poorly understood. In this study, Yorkshire crossbred swine intramuscularly vaccinated with 10 6 50% hemadsorption dose (HAD 50 ) of ASFV-G-ΔI177L or a vehicle control were challenged at 28 days post-inoculation (dpi) with 10 2 HAD 50 of ASFV-G. Analysis of purified peripheral blood mononuclear cells following inoculation and challenge revealed that CD4+, CD8+ and CD4+CD8+ central memory T cells (CD44+CD25–CD27–CD62L+CCR7+, T cm ) decreased significantly by 28 dpi in ASFV-G-ΔI177L-vaccinated swine compared to baseline and time-matched controls. Conversely, CD4+, CD8+ and CD4+CD8+ effector memory T cells (CD44+CD25–CD27–CD62–CCR7–, T em ) increased significantly among ASFV-G-ΔI177L-vaccined swine by 28 dpi compared to baseline and time-matched controls. Additionally, the percentage of natural killer (NK), CD4+ and CD4+CD8+ T em and CD8+ T cm and T em positive for IFNγ increased significantly following inoculation, surpassing that of controls by 28 dpi or earlier. These results suggest that NK and memory T cells play a role in prote

60 APPLIED LIFE SCIENCES↗