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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Deletion of the A137R Gene from the Pandemic Strain of African Swine Fever Virus Attenuates the Strain and Offers Protection against the Virulent Pandemic Virus

African swine fever virus (ASFV) is causing a devastating pandemic in domestic and wild swine within an extended geographical area from Central Europe to East Asia, resulting in economic losses for the regional swine industry. There are no commercial vaccines; therefore, disease control relies on identification and culling of infected animals. We report here that the deletion of the ASFV gene A137R from the highly virulent ASFV-Georgia2010 (ASFV-G) isolate induces a significant attenuation of virus virulence in swine. A recombinant virus lacking the A137R gene, ASFV-G-DA137R, was developed to assess the role of this gene in ASFV virulence in domestic swine. Animals inoculated intramuscularly with 102 50% hemadsorption doses (HAD50) of ASFV-G-DA137R remained clinically healthy during the 28-day observational period. All animals inoculated with ASFV-G-DA137R had medium to high viremia titers and developed a strong virus-specific antibody response. Importantly, all ASFV-G-DA137Rinoculated animals were protected when challenged with the virulent parental strain ASFV-G. No evidence of replication of challenge virus was observed in the ASFV-GDA137R-inoculated animals. Therefore, ASFV-G-DA137R is a novel potential live attenuated vaccine candidate and one of the few experimental vaccine strains reported to induce protection against the highly virulent ASFV Georgia virus that is the cause of the current Eurasian pandemic.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Single-Dose Immunogenic DNA Vaccines Coding for Live-Attenuated Alpha- and Flaviviruses

Single-dose, immunogenic DNA (iDNA) vaccines coding for whole live-attenuated viruses are reviewed. This platform, sometimes called immunization DNA, has been used for vaccine development for flavi- and alphaviruses. An iDNA vaccine uses plasmid DNA to launch live-attenuated virus vaccines in vitro or in vivo. When iDNA is injected into mammalian cells in vitro or in vivo, the RNA genome of an attenuated virus is transcribed, which starts replication of a defined, live-attenuated vaccine virus in cell culture or the cells of a vaccine recipient. In the latter case, an immune response to the live virus vaccine is elicited, which protects against the pathogenic virus. Unlike other nucleic acid vaccines, such as mRNA and standard DNA vaccines, iDNA vaccines elicit protection with a single dose, thus providing major improvement to epidemic preparedness. Still, iDNA vaccines retain the advantages of other nucleic acid vaccines. In summary, the iDNA platform combines the advantages of reverse genetics and DNA immunization with the high immunogenicity of live-attenuated vaccines, resulting in enhanced safety and immunogenicity. This vaccine platform has expanded the field of genetic DNA and RNA vaccines with a novel type of immunogenic DNA vaccines that encode entire live-attenuated viruses.

60 APPLIED LIFE SCIENCES↗
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↗
NASA NTRS2012

Paramyxovirus Infection Mimics In Vivo Cellular Dynamics in Three-Demensional Human Bronchio-Epithelial Tissue-Like Assemblies

Respiratory syncytial virus and parainfluenza virus cause severe respiratory disease, especially in infants, children and the elderly. An in vitro model that accurately mimics infection of the human respiratory epithelium (HRE) would facilitate vaccine development greatly. Monolayer cultures traditionally used to study these viruses do not accurately and precisely differentiate the replication efficiencies of wild type and attenuated viruses. Therefore, we engineered novel three-dimensional (3D) tissue-like assemblies (TLAs) of human broncho-epithelial (HBE) cells to produce a more physiologically relevant in vitro model of the HRE. TLAs resemble HRE structurally and by expression of differentiated epithelial cell markers. Most significantly, wild type viruses exhibited a clear growth advantage over attenuated strains in TLAs unlike monolayer cultures. In addition, the TLAs responded to virus infection by secreting pro-inflammatory mediators similar to the respiratory epithelia of infected children. These characteristics make the TLA model a valuable platform technology to develop and evaluate live, attenuated respiratory virus vaccine candidates for human use. Respiratory virus diseases, the most frequent and least preventable of all infectious diseases, range in severity from the common cold to severe bronchiolitis and pneumonia . Two paramyxoviruses, respiratory syncytial virus (RSV) and parainfluenza virus type 3 (PIV3), are responsible for a majority of the most severe respiratory diseases of infants and young children. RSV causes 70% of all bronchiolitis cases and is a major cause of morbidity and mortality worldwide, especially in infants. PIV3 causes 10-15% of bronchiolitis and pneumonia during infancy, second only to RSV, and 40% of croup in infants To date, licensed vaccines are not available to prevent these respiratory diseases. At present, traditional monkey kidney (Vero and LLC-MK2) and human (HEp-2) tissue culture cells and small animal models (mouse, cotton rat, guinea pig, ferret, and hamster) fail to accurately imitate viral replication and human disease states (8). Lacking an authentic model has impeded the development and evaluation of live, attenuated vaccine candidates. Development of a physiologically relevant in vitro tissue culture model that reproduces characteristics of the HRE, the primary target of RSV and PIV3, would aid in predicting clinical attenuation and safety of vaccine candidates. Successful tissue engineering of a 3D human intestinal model using novel NASA technology inspired the development of a tri-culture 3D model for the HRE. Sequential layering of primary mesenchymal cells (comprised of normal human fibroblasts and endothelial cells) followed by BEAS-2B epithelial cells derived from human bronchi and tracheae were recapitulated on Cultisphere and/or cytodex3 microcarriers in cylindrical vessels that rotate horizontally creating an organized epithelial structure. Horizontal rotation randomizes the gravity vector modeling aspects of microgravity. Mesenchymal and epithelial cells grown under these conditions reproduce the structural organization, multi-cellular complexity, and differentiation state of the HRE. The opportunity to study respiratory viruses in a nasal epithelium model is invaluable because the most promising respiratory virus vaccine candidates are live attenuated viruses for intranasal administration. Here we characterize the interactions of respiratory viruses and epithelial cells grown under modeled microgravity in comparison to gravity-ladened monolayers. 3D HBE TLAs and traditional monolayers (2D) are infected at 35 C, the upper temperature of the upper HRE, to simulate in vivo infection conditions. Growth kinetics of wild type (wt) RSV and PIV3 viruses were compared in 2D and 3D cells to that of strains attenuated in humans or rhesus macaques. This novel 3D HBE model also offers an opportunity to study whether the epithelial cell function, especially in host defenses recapitulated by mimicking the structural organization of the HRE. In vivo, airway epithelial cells play a significant and dynamic role in host defense by blocking paracellular permeability and modulating airway function through cellular interactions or tight junctions. As regulators of the innate immune response, epithelial cells constitutively express cytokines, chemokines, and colony stimulating factors including RANTES, IL-8, IL-6, GM-CSF, and G-CSF for proactive host defense. In response to viral infection, epithelial cells induce potent immuno-modulatory and pro-inflammatory cytokines that recruit phagocytic and inflammatory cells to clear the virus and enhance protection. Although disease pathogenesis is classically attributed to the cytopathic effects of the pathogen, severe disease states associated with RSV and PIV3 are attributed to the inflammatory response, especially in infants. RSV is a potent inducer of cytokines and pro-inflammatory mediators in epithelial cells in vivo. A differentiated human epithelial model independent of the complete functional immune system will help elucidate the role of epithelial cells in respiratory disease. We reported here, virus and host cell interactions in 3D HBE TLAs are similar to that in vivo. Because the epithelial cell organization of the TLAs impacts not only the expression of airway epithelial characteristics, but also cellular communication, the TLAs represent a more physiologically relevant model of the HRE than BEAS-2B or other non-tumour monolayer models of respiratory disease. As a result, wild type respiratory viruses have a clear growth advantage over attenuated viruses in TLAs unlike traditional monolayers. In addition, the TLAs respond to wild type virus infection by secreting pro-inflammatory mediators characteristic of infected HRE. TLAs expressing microbial defense mechanisms provide an excellent model to study the interactions of respiratory pathogens with their host and to identify the innate immunity mediators. Therefore, 3D HBE TLAs offer advantages for the study of respiratory viruses and the development of viral vaccine candidates.

Deatly, Anne M.↗
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↗
DOE OSTI2022

Serum Neutralizing and Enhancing Effects on African Swine Fever Virus Infectivity in Adherent Pig PBMC

African swine fever virus (ASFV) causes hemorrhagic fever with mortality rates of up to 100% in domestic pigs. Currently, there are no commercial vaccines for the disease. Only some live-attenuated viruses have been able to protect pigs from ASFV infection. The immune mechanisms involved in the protection are unclear. Immune sera can neutralize ASFV but incompletely. The mechanisms involved are not fully understood. Currently, there is no standardized protocol for ASFV neutralization assays. In this study, a flow cytometry-based ASFV neutralization assay was developed and tested in pig adherent PBMC using a virulent ASFV containing a fluorescent protein gene as a substrate for neutralization. As with previous studies, the percentage of infected macrophages was approximately five time higher than that of infected monocytes, and nearly all infected cells displayed no staining with anti-CD16 antibodies. Sera from naïve pigs and pigs immunized with a live-attenuated ASFV and fully protected against parental virus were used in the assay. The sera displayed incomplete neutralization with MOI-dependent neutralizing efficacies. Extracellular, but not intracellular, virions suspended in naïve serum were more infectious than those in the culture medium, as reported for some enveloped viruses, suggesting a novel mechanism of ASFV infection in macrophages. Both the intracellular and extracellular virions could not be completely neutralized.

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 OSTI2022

The Presence of Virus Neutralizing Antibodies Is Highly Associated with Protection against Virulent Challenge in Domestic Pigs Immunized with ASFV live Attenuated Vaccine Candidates

African swine fever virus (ASFV) is currently producing a pandemic affecting a large area of Eurasia, and more recently, the Dominican Republic in the Western Hemisphere. ASFV is a large and structurally complex virus with a large dsDNA genome encoding for more than 150 genes. Live attenuated virus strains can induce protection in domestic swine against disease produced by homologous virulent parental viruses. The roles of the different immune mechanisms induced by the attenuated strains in protection still need to be understood. In particular, the role of ASFV neutralizing antibody in protection still is an important controversial issue to be elucidated. Here we present the development of a novel methodology to detect virus neutralizing antibodies based on the reduction of virus infectivity in a Vero cell adapted ASFV strain. The described method was used to assess levels of virus neutralizing antibodies in domestic swine inoculated with live attenuated ASFV. Results demonstrated a high association between the presence of virus neutralizing antibodies and protection in 84 animals immunized with the recombinant vaccine candidates ASFV-G-Δ9GL/ΔUK or ASFV-G-ΔI177L. To our knowledge, this is the first report demonstrating an association between virus neutralizing antibodies and protection against virulent challenge in such a large number of experimental individuals.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Deoptimization of FMDV P1 Region Results in Robust Serotype-Independent Viral Attenuation

Foot-and-mouth disease (FMD), caused by the FMD virus (FMDV), is a highly contagious disease of cloven-hoofed livestock that can have severe economic impacts. Control and prevention strategies, including the development of improved vaccines, are urgently needed to effectively control FMD outbreaks in endemic settings. Previously, we employed two distinct strategies (codon pair bias deoptimization (CPD) and codon bias deoptimization (CD)) to deoptimize various regions of the FMDV serotype A subtype A12 genome, which resulted in the development of an attenuated virus in vitro and in vivo, inducing varying levels of humoral responses. In the current study, we examined the versatility of the system by using CPD applied to the P1 capsid coding region of FMDV serotype A subtype, A24, and another serotype, Asia1. Viruses carrying recoded P1 (A24-P1Deopt or Asia1-P1Deopt) exhibited different degrees of attenuation (i.e., delayed viral growth kinetics and replication) in cultured cells. Studies in vivo using a mouse model of FMD demonstrated that inoculation with the A24-P1Deopt and Asia1-P1Deopt strains elicited a strong humoral immune response capable of offering protection against challenge with homologous wildtype (WT) viruses. However, different results were obtained in pigs. While clear attenuation was detected for both the A24-P1Deopt and Asia1-P1Deopt strains, only a limited induction of adaptive immunity and protection against challenge was detected, depending on the inoculated dose and serotype deoptimized. Our work demonstrates that while CPD of the P1 coding region attenuates viral strains of multiple FMDV serotypes/subtypes, a thorough assessment of virulence and induction of adaptive immunity in the natural host is required in each case in order to finely adjust the degree of deoptimization required for attenuation without affecting the induction of protective adaptive immune responses.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

A Single Amino Acid Substitution in the Matrix Protein (M51R) of Vesicular Stomatitis New Jersey Virus Impairs Replication in Cultured Porcine Macrophages and Results in Significant Attenuation in Pigs

In this study, we explore the virulence of vesicular stomatitis New Jersey virus (VSNJV) in pigs and its potential relationship with the virus’s ability to modulate innate responses. For this purpose, we developed a mutant of the highly virulent strain NJ0612NME6, containing a single amino acid substitution in the matrix protein (M51R). The M51R mutant of NJ0612NME6 was unable to suppress the transcription of genes associated with the innate immune response both in primary fetal porcine kidney cells and porcine primary macrophage cultures. Impaired viral growth was observed only in porcine macrophage cultures, indicating that the M51 residue is required for efficient replication of VSNJV in these cells. Furthermore, when inoculated in pigs by intradermal scarification of the snout, M51R infection was characterized by decreased clinical signs including reduced fever and development of less and smaller secondary vesicular lesions. Pigs infected with M51R had decreased levels of viral shedding and absence of RNAemia compared to the parental virus. The ability of the mutant virus to infect pigs by direct contact remained intact, indicating that the M51R mutation resulted in a partially attenuated phenotype capable of causing primary lesions and transmitting to sentinel pigs. Collectively, our results show a positive correlation between the ability of VSNJV to counteract the innate immune response in swine macrophage cultures and the level of virulence in pigs, a natural host of this virus. More studies are encouraged to evaluate the interaction of VSNJV with macrophages and other components of the immune response in pigs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Lifelong cytomegalovirus and early‐LIFE irradiation synergistically potentiate age‐related defects in response to vaccination and infection

Abstract While whole‐body irradiation (WBI) can induce some hallmarks of immune aging, (re)activation of persistent microbial infection also occurs following WBI and may contribute to immune effects of WBI over the lifespan. To test this hypothesis in a model relevant to human immune aging, we examined separate and joint effects of lifelong latent murine cytomegalovirus (MCMV) and of early‐life WBI over the course of the lifespan. In late life, we then measured the response to a West Nile virus (WNV) live attenuated vaccine, and lethal WNV challenge subsequent to vaccination. We recently published that a single dose of non‐lethal WBI in youth, on its own, was not sufficient to accelerate aging of the murine immune system, despite widespread DNA damage and repopulation stress in hematopoietic cells. However, 4Gy sub‐lethal WBI caused manifest reactivation of MCMV. Following vaccination and challenge with WNV in the old age, MCMV‐infected animals experiencing 4Gy, but not lower, dose of sub‐lethal WBI in youth had reduced survival. By contrast, old irradiated mice lacking MCMV and MCMV‐infected, but not irradiated, mice were both protected to the same high level as the old non‐irradiated, uninfected controls. Analysis of the quality and quantity of anti‐WNV immunity showed that higher mortality in MCMV‐positive WBI mice correlated with increased levels of MCMV‐specific immune activation during WNV challenge. Moreover, we demonstrate that infection, including that by WNV, led to MCMV reactivation. Our data suggest that MCMV reactivation may be an important determinant of increased late‐life mortality following early‐life irradiation and late‐life acute infection.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

A minimal complex of KHNYN and zinc-finger antiviral protein binds and degrades single-stranded RNA

Detecting viral infection is a key role of the innate immune system. The genomes of some RNA viruses have a high CpG dinucleotide content relative to most vertebrate cell RNAs, making CpGs a molecular marker of infection. The human zinc-finger antiviral protein (ZAP) recognizes CpG, mediates clearance of the foreign CpG-rich RNA, and causes attenuation of CpG-rich RNA viruses. While ZAP binds RNA, it lacks enzymatic activity that might be responsible for RNA degradation and thus requires interacting cofactors for its function. One of these cofactors, KHNYN, has a predicted nuclease domain. Using biochemical approaches, we found that the KHNYN NYN domain is a single-stranded RNA ribonuclease that does not have sequence specificity and digests RNA with or without CpG dinucleotides equivalently in vitro. We show that unlike most KH domains, the KHNYN KH domain does not bind RNA. Indeed, a crystal structure of the KH region revealed a double-KH domain with a negatively charged surface that accounts for the lack of RNA binding. Rather, the KHNYN C-terminal domain (CTD) interacts with the ZAP RNA-binding domain (RBD) to provide target RNA specificity. We define a minimal complex composed of the ZAP RBD and the KHNYN NYN-CTD and use a fluorescence polarization assay to propose a model for how this complex interacts with a CpG dinucleotide-containing RNA. In the context of the cell, this module would represent the minimum ZAP and KHNYN domains required for CpG-recognition and ribonuclease activity essential for attenuation of viruses with clusters of CpG dinucleotides.

Yeoh, Zoe C. (ORCID:0000000226949068)↗
DOE OSTI2021

Convergent use of phosphatidic acid for hepatitis C virus and SARS-CoV-2 replication organelle formation

Double membrane vesicles (DMVs) serve as replication organelles of plus-strand RNA viruses such as hepatitis C virus (HCV) and SARS-CoV-2. Viral DMVs are morphologically analogous to DMVs formed during autophagy, but lipids driving their biogenesis are largely unknown. Here we show that production of the lipid phosphatidic acid (PA) by acylglycerolphosphate acyltransferase (AGPAT) 1 and 2 in the ER is important for DMV biogenesis in viral replication and autophagy. Using DMVs in HCV-replicating cells as model, we found that AGPATs are recruited to and critically contribute to HCV and SARS-CoV-2 replication and proper DMV formation. An intracellular PA sensor accumulated at viral DMV formation sites, consistent with elevated levels of PA in fractions of purified DMVs analyzed by lipidomics. Apart from AGPATs, PA is generated by alternative pathways and their pharmacological inhibition also impaired HCV and SARS-CoV-2 replication as well as formation of autophagosome-like DMVs. These data identify PA as host cell lipid involved in proper replication organelle formation by HCV and SARS-CoV-2, two phylogenetically disparate viruses causing very different diseases, i.e. chronic liver disease and COVID-19, respectively. Host-targeting therapy aiming at PA synthesis pathways might be suitable to attenuate replication of these viruses.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Construction of chicken infectious anemia virus infectious clone and study on its pathogenicity

Chicken infectious anemia virus (CIAV) can be transmitted through contaminated live poultry vaccine. However, the pathogenicity of contaminated CIAV strains is rarely reported. Previously, the chickens showed the typical symptoms of anemia after using the attenuated live fowl pox virus (FPV) vaccine. Therefore, exogenous CIAV contamination was suspected. We detected anti-CIAV antibodies in SPF chicks vaccinated with the FPV vaccine. CIAV contamination was confirmed in the FPV vaccine, and the CIAV strain was named JS2020-FPV. This study aims to rescue JS2020-FPV by reverse genetic assays and investigate its pathogenicity. Firstly, double-copies infectious clone of JS2020-FPV was constructed. For the pathogenicity study, infectious clone of JS2020-FPV was used to inoculate 1-day-old SPF chicks. The typical symptoms of anemia were observed in the JS2020-PFV group 14 days post inoculation. The hematocrit and body weight of chicks in the JS2020-PFV group were significantly lower than those in the mock group. Notably, the thymus development index and antibody levels of NDV were lower in chicks in the JS2020-PFV group than those in the mock group. Different degrees of apoptosis of MSB1 and DF-1 were observed after inoculated with the JS2020-FPV VP3 recombinant fusion protein expressed by E. coli system, indicating that VP3 induced apoptosis in the transformed cells. Overall, the pathogenicity of the CIAV detected in the contaminated vaccine was confirmed by inoculating SPF chicks with the double-copies infectious DNA clone in this study. Our findings indicate that the dangers of vaccine contamination cannot be ignored.

Wang, Jinjin↗
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 OSTI2023

A Re-Evaluation of African Swine Fever Genotypes Based on p72 Sequences Reveals the Existence of Only Six Distinct p72 Groups

The African swine fever virus (ASFV) is currently causing a world-wide pandemic of a highly lethal disease in domestic swine and wild boar. Currently, recombinant ASF live-attenuated vaccines based on a genotype II virus strain are commercially available in Vietnam. With 25 reported ASFV genotypes in the literature, it is important to understand the molecular basis and usefulness of ASFV genotyping, as well as the true significance of genotypes in the epidemiology, transmission, evolution, control, and prevention of ASFV. Historically, genotyping of ASFV was used for the epidemiological tracking of the disease and was based on the analysis of small fragments that represent less than 1% of the viral genome. The predominant method for genotyping ASFV relies on the sequencing of a fragment within the gene encoding the structural p72 protein. Genotype assignment has been accomplished through automated phylogenetic trees or by comparing the target sequence to the most closely related genotyped p72 gene. To evaluate its appropriateness for the classification of genotypes by p72, we reanalyzed all available genomic data for ASFV. We conclude that the majority of p72-based genotypes, when initially created, were neither identified under any specific methodological criteria nor correctly compared with the already existing ASFV genotypes. Based on our analysis of the p72 protein sequences, we propose that the current twenty-five genotypes, created exclusively based on the p72 sequence, should be reduced to only six genotypes. To help differentiate between the new and old genotype classification systems, we propose that Arabic numerals (1, 2, 8, 9, 15, and 23) be used instead of the previously used Roman numerals. Furthermore, we discuss the usefulness of genotyping ASFV isolates based only on the p72 gene sequence.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Discovery of Small-Molecule Inhibitors of SARS-CoV-2 Proteins Using a Computational and Experimental Pipeline

A rapid response is necessary to contain emergent biological outbreaks before they can become pandemics. The novel coronavirus (SARS-CoV-2) that causes COVID-19 was first reported in December of 2019 in Wuhan, China and reached most corners of the globe in less than two months. In just over a year since the initial infections, COVID-19 infected almost 100 million people worldwide. Although similar to SARS-CoV and MERS-CoV, SARS-CoV-2 has resisted treatments that are effective against other coronaviruses. Crystal structures of two SARS-CoV-2 proteins, spike protein and main protease, have been reported and can serve as targets for studies in neutralizing this threat. We have employed molecular docking, molecular dynamics simulations, and machine learning to identify from a library of 26 million molecules possible candidate compounds that may attenuate or neutralize the effects of this virus. The viability of selected candidate compounds against SARS-CoV-2 was determined experimentally by biolayer interferometry and FRET-based activity protein assays along with virus-based assays. In the pseudovirus assay, imatinib and lapatinib had IC 50 values below 10 μM, while candesartan cilexetil had an IC 50 value of approximately 67 µM against M pro in a FRET-based activity assay. Comparatively, candesartan cilexetil had the highest selectivity index of all compounds tested as its half-maximal cytotoxicity concentration 50 (CC 50 ) value was the only one greater than the limit of the assay (>100 μM).

59 BASIC BIOLOGICAL SCIENCES↗