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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 55 records · Page 3
All sourcesNASA NTRSDOE OSTI
NASA NTRS2005

Analysis of Pdeudomonas aeruginosa Growth and Virulence in Modelled Microgravity

Stress, radiation and microgravity cause astronauts to experience secondary immunosuppression. Spaceflight conditions enhance bacterial growth and alter antimicrobial susceptibility. Clinostats are used to model microgravity effects at lxg. In controls rotated on the vertical axis, the g-vector acts on cells as in static cultures. Salmonella enterica serovar Typhimurium virulence genes are up-regulated in modelled microgravity (MMG); a MMG regulon has been postulated. We hypothesize that the virulence of P. aeruginosa (PA) may be affected similarly by microgravity, which could be observed in MMG. This study focused on regulation of the ETA protein by PA during growth in MMG. PA103 was grown in an ETA production medium at 37 C. One series of media was inoculated with frozen cultures and grown using horizontal (MMG) or static incubation. Another series inoculated with refrigerated cultures included vertical rotating controls. Analyses included optical density (OD), agar plate counts (PC) on R2A, ETA ELISA, and protein expression by 2-D gel analyses. Growth and ETA results differed depending on inoculum, with minor effects of MMG. Proteomic analysis of 2-D gels indicate differences in protein expression with MMG. Growth and ETA results show that consistent methodology is critical when studying environmental effects. This study provides information on the relationships between environmental changes and virulence regulation, especially for flight experiments, when ground experiments are used to predict potential spaceflight effects.

Guadarrama, Seratna↗
NASA NTRS2007

Effect Of Spaceflight On Microbial Gene Expression And Virulence: Preliminary Results From Microbe Payload Flown On-Board STS-115

Human presence in space, whether permanent or temporary, is accompanied by the presence of microbes. However, the extent of microbial changes in response to spaceflight conditions and the corresponding changes to infectious disease risk is unclear. Previous studies have indicated that spaceflight weakens the immune system in humans and animals. In addition, preflight and in-flight monitoring of the International Space Station (ISS) and other spacecraft indicates the presence of opportunistic pathogens and the potential of obligate pathogens. Altered antibiotic resistance of microbes in flight has also been shown. As astronauts and cosmonauts live for longer periods in a closed environment, especially one using recycled water and air, there is an increased risk to crewmembers of infectious disease events occurring in-flight. Therefore, understanding how the space environment affects microorganisms and their disease potential is critically important for spaceflight missions and requires further study. The goal of this flight experiment, operationally called MICROBE, is to utilize three model microbial pathogens, Salmonella typhimurium, Pseudomonas aeruginosa, and Candida albicans to examine the global effects of spaceflight on microbial gene expression and virulence attributes. Specifically, the aims are (1) to perform microarray-mediated gene expression profiling of S. typhimurium, P. aeruginosa, and C. albicans, in response to spaceflight in comparison to ground controls and (2) to determine the effect of spaceflight on the virulence potential of these microorganisms immediately following their return from spaceflight using murine models. The model microorganisms were selected as they have been isolated from preflight or in-flight monitoring, represent different degrees of pathogenic behavior, are well characterized, and have sequenced genomes with available microarrays. In particular, extensive studies of S. typhimurium by the Principal Investigator, Dr. Nickerson, using ground-based analog systems demonstrate important changes in the genotypic, phenotypic, and virulence characteristics of this pathogen resulting from exposure to a flight-like environment (i.e. modeled microgravity).

Wilson, J. W.↗
NASA NTRS2016

Spaceflight and Simulated Microgravity Increases Virulence of the Known Bacterial Pathogen S. Marcescens

After spaceflight, the number of immune cells is reduced in humans. In other research models, including Drosophila, not only is there a reduction in the number of plasmatocytes, but expression of immune-related genes is also changed after spaceflight. These observations suggest that the immune system is compromised after exposure to microgravity. It has also been reported that there is a change in virulence of some bacterial pathogens after spaceflight. We recently observed that samples of gram-negative S. marcescens retrieved from spaceflight is more virulent than ground controls, as determined by reduced survival and increased bacterial growth in the host. We were able to repeat this finding of increased virulence after exposure to simulated microgravity using the rotating wall vessel, a ground based analog to microgravity. With the ground and spaceflight samples, we looked at involvement of the Toll and Imd pathways in the Drosophila host in fighting infection by ground and spaceflight samples. We observed that Imd-pathway mutants were more susceptible to infection by the ground bacterial samples, which aligns with the known role of this pathway in fighting infections by gram-negative bacteria. When the Imd-pathway mutants were infected with the spaceflight sample, however, they exhibited the same susceptibility as seen with the ground control bacteria. Interestingly, all mutant flies show the same susceptibility to the spaceflight bacterial sample as do wild type flies. This suggests that neither humoral immunity pathway is effectively able to counter the increased pathogenicity of the space-flown S. marcescens bacteria.

immunity↗
DOE OSTI2023

cotH Genes Are Necessary for Normal Spore Formation and Virulence in Mucor lusitanicus

Mucormycosis is an invasive fungal infection caused by certain members of the fungal order of Mucorales. The species most frequently identified as the etiological agents of mucormycosis belong to the genera Rhizopus , Lichtheimia , and Mucor . The frequency of systemic mucormycosis has been increasing, mainly because of increasing numbers of susceptible patients. Furthermore, Mucorales display intrinsic resistance to the majority of routinely used antifungal agents (e.g., echinocandins and short-tailed azoles), which limits the number of possible therapeutic options. All the above-mentioned issues urge the improvement of molecular identification methods and the discovery of new antifungal targets and strategies. Spore coat proteins (CotH) constitute a kinase family present in many pathogenic bacteria and fungi and participate in the spore formation in these organisms. Moreover, some of them can act as virulence factors being receptors of the human GRP78 protein during Rhizopus delemar-induced mucormycosis. We identified 17 cotH -like genes in the Mucor lusitanicus genome database. Successful disruption of five cotH genes in Mucor was performed using the CRISPR-Cas9 system. The CotH3 and CotH4 proteins play a role in adaptation to different temperatures as well as in developing the cell wall structure. We also show CotH4 protein is involved in spore wall formation by affecting the total chitin content and, thus, the composition of the spore wall. The role of CotH3 and CotH4 proteins in virulence was confirmed in two invertebrate models and a diabetic ketoacidosis (DKA) mouse model.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

The histone demethylase KdmB is part of a trimeric protein complex and mediates virulence and mycotoxin production in Penicillium expansum

Epigenetic modification of chromosome structure has increasingly been associated with alterations in secondary metabolism and sporulation defects in filamentous fungal pathogens. Recently, the epigenetic reader protein SntB was shown to govern virulence, spore production and mycotoxin synthesis in the fruit pathogen Penicillium expansum. Through immunoprecipitation-coupled mass spectrometry, we found that SntB is a member of a protein complex with KdmB, a histone demethylase and the essential protein RpdA, a histone deacetylase. Deletion of kdmB phenocopied some but not all characteristics of the ΔsntB mutant. KdmB deletion strains exhibited reduced lesion development on Golden Delicious apples and this was accompanied by decreased production of patulin and citrinin in host tissue. In addition, ΔkdmB mutants were sensitive to several cell wall stressors which possibly contributed to the decreased virulence observed on apples. In conclusion, slight differences in spore production and germination rates of ΔkdmB mutants in vitro did not impact overall diameter growth in culture.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Chicken Production and Human Clinical Escherichia coli Isolates Differ in Their Carriage of Antimicrobial Resistance and Virulence Factors

Contamination of food animal products by Escherichia coli is a leading cause of foodborne disease outbreaks, hospitalizations, and deaths in humans. Chicken is the most consumed meat both in the United States and across the globe according to the U.S. Department of Agriculture. Although E. coli is a ubiquitous commensal bacterium of the guts of humans and animals, its ability to acquire antimicrobial resistance (AMR) genes and virulence factors (VFs) can lead to the emergence of pathogenic strains that are resistant to critically important antibiotics. Thus, it is important to identify the genetic factors that contribute to the virulence and AMR of E. coli. In this study, we performed in-depth genomic evaluation of AMR genes and VFs of E. coli genomes available through the National Antimicrobial Resistance Monitoring System GenomeTrackr database. Our objective was to determine the genetic relatedness of chicken production isolates and human clinical isolates. To achieve this aim, we first developed a massively parallel analytical pipeline (Reads2Resistome) to accurately characterize the resistome of each E. coli genome, including the AMR genes and VFs harbored. We used random forests and hierarchical clustering to show that AMR genes and VFs are sufficient to classify isolates into different pathogenic phylogroups and host origin. We found that the presence of key type III secretion system and AMR genes differentiated human clinical isolates from chicken production isolates. These results further improve our understanding of the interconnected role AMR genes and VFs play in shaping the evolution of pathogenic E. coli strains.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Sac1 links phosphoinositide turnover to cryptococcal virulence

Cryptococcus neoformans is an environmentally acquired fungal pathogen that causes over 140,000 deaths per year. Cryptococcal infection occurs when infectious particles are deposited into the lung, where they encounter host phagocytic cells. C. neoformans may be engulfed by these phagocytes, an important step of infection that leads to outcomes ranging from termination of infection to cryptococcal dissemination. To study this critical process, we screened approximately 4,700 cryptococcal gene deletion mutants for altered uptake, using primary mouse and human phagocytic cells. Among the hits of these two screens, we identified 93 mutants with perturbed uptake in both systems, as well as others with differences in uptake by only one cell type. We further screened the hits for changes in thickness of the capsule, a protective polysaccharide layer around the cell which is an important cryptococcal virulence factor. The combination of our three screens yielded 45 mutants, including one lacking the phosphatidylinositol-4-phosphate phosphatase Sac1. In this work, we implicate Sac1 in both host cell uptake and capsule production. We found that sac1 mutants exhibit lipid trafficking defects, reductions in secretory system function, and changes in capsule size and composition. Many of these changes occur specifically in tissue culture media, highlighting the role of Sac1 phosphatase activity in responding to the stress of host-like conditions. Overall, these findings show how genome-scale screening can identify cellular factors that contribute to our understanding of cryptococcal biology and demonstrate the role of Sac1 in determining fungal virulence.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Mining the Penicillium expansum Genome for Virulence Genes: A Functional-Based Approach to Discover Novel Loci Mediating Blue Mold Decay of Apple Fruit

Blue mold, a postharvest disease of pome fruits, is caused by the filamentous fungus Penicillium expansum. In addition to the economic losses caused by P. expansum, food safety can be compromised, as this pathogen is mycotoxigenic. In this study, forward and reverse genetic approaches were used to identify genes involved in blue mold infection in apple fruits. For this, we generated a random T-DNA insertional mutant library. A total of 448 transformants were generated and screened for the reduced decay phenotype on apples. Of these mutants, six (T-193, T-275, T-434, T-588, T-625, and T-711) were selected for continued studies and five unique genes were identified of interest. In addition, two deletion mutants (Δt-625 and Δt-588) and a knockdown strain (t-434 KD ) were generated for three loci. Data show that the Δt-588 mutant phenocopied the T-DNA insertion mutant and had virulence penalties during apple fruit decay. We hypothesize that this locus encodes a glyoxalase due to bioinformatic predictions, thus contributing to reduced colony diameter when grown in methylglyoxal (MG). This work presents novel members of signaling networks and additional genetic factors that regulate fungal virulence in the blue mold fungus during apple fruit decay.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Streptococcus pneumoniae HtrA is a dynamic and monomeric virulence factor capable of forming larger oligomeric complexes

Abstract High‐temperature requirement A (HtrA) proteases are a conserved family of serine proteases central to protein quality control and bacterial virulence. While Gram‐negative and human HtrAs are structurally well studied, Gram‐positive homologs remain essentially uncharacterized. Here, we present the first integrated structural and mechanistic analysis of a Gram‐positive HtrA, from Streptococcus pneumoniae , a virulence factor essential for adhesion and infection in vivo. Proteomic profiling of an htrA knockout and cleavage assays demonstrate that S. pneumoniae HtrA is required for protein quality control, with the PDZ domain mediating substrate recognition. Biochemically, S. pneumoniae HtrA exists exclusively as a monomer in solution, a striking divergence from canonical trimeric HtrAs that we show is shared with other Gram‐positive homologs. NMR analyses reveal that the monomer dynamically samples open and closed conformations, while cryo‐EM of a catalytic mutant identifies a hexamer stabilized by a unique LoopA–PDZ interaction. Together, these findings define S. pneumoniae HtrA as a dynamic monomer with interdomain coupling between its protease and PDZ domains, establishing Gram‐positive HtrAs as a mechanistically divergent subgroup within the HtrA family.

Lee, Eunjeong [Department of Biochemistry and Mole↗
DOE OSTI2024

Rapid design of bacteriophage cocktails to suppress the burden and virulence of gut-resident carbapenem-resistant Klebsiella pneumoniae

Antibiotic use can lead to the expansion of multi-drug-resistant pathobionts within the gut microbiome that can cause life-threatening infections. Selective alternatives to conventional antibiotics are in dire need. Here, in this work, we describe a Klebsiella PhageBank for the tailored design of bacteriophage cocktails to treat multi-drug-resistant Klebsiella pneumoniae. Using a transposon library in carbapenem-resistant K. pneumoniae, we identify host factors required for phage infection in major Klebsiella phage families. Leveraging the diversity of the PhageBank, we formulate phage combinations that eliminate K. pneumoniae with minimal phage resistance. Optimized cocktails selectively suppress the burden of K. pneumoniae in the mouse gut and drive the loss of key virulence factors that act as phage receptors. Phage-mediated diversification of bacterial populations in the gut leads to co-evolution of phage variants with higher virulence and broader host range. Altogether, the Klebsiella PhageBank charts a roadmap for phage therapy against a critical multidrug-resistant human pathogen.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Structure of the master regulator Rns reveals an inhibitor of enterotoxigenic Escherichia coli virulence regulons

Enteric infections caused by the gram-negative bacteria enterotoxigenic Escherichia coli (ETEC), Vibrio cholerae, Shigella flexneri, and Salmonella enterica are among the most common and affect billions of people each year. These bacteria control expression of virulence factors using a network of transcriptional regulators, some of which are modulated by small molecules as has been shown for ToxT, an AraC family member from V. cholerae. In ETEC the expression of many types of adhesive pili is dependent upon the AraC family member Rns. We present here the 3 Å crystal structure of Rns and show it closely resembles ToxT. Rns crystallized as a dimer via an interface similar to that observed in other dimeric AraC’s. Furthermore, the structure of Rns revealed the presence of a ligand, decanoic acid, that inhibits its activity in a manner similar to the fatty acid mediated inhibition observed for ToxT and the S. enterica homologue HilD. Together, these results support our hypothesis that fatty acids regulate virulence controlling AraC family members in a common manner across a number of enteric pathogens. Furthermore, for the first time this work identifies a small molecule capable of inhibiting the ETEC Rns regulon, providing a basis for development of therapeutics against this deadly human pathogen.

99 GENERAL AND MISCELLANEOUS↗
DOE OSTI2025

Hybridization capture sequencing for Vibrio spp. and associated virulence factors

ABSTRACT Proliferation ofVibriospp. in aquatic ecosystems is associated with climate change and, concomitantly, increased incidence of vibriosis. They are autochthonous to aquatic environments globally, but traditional metagenomic methods for detecting and typing pathogenicVibriospp. are challenged by their presence in relatively low abundance and ability to persist in a viable but nonculturable state. In the study reported here, hybridization capture sequencing (HCS) was employed to profile low-abundanceVibriospp. in environmental samples. The HCS panel targeted a family of molecular chaperones (CPN60) specific to 69Vibriospp. and 162Vibrio-specific virulence factors. This approach was evaluated in parallel with traditional whole-community shotgun sequencing in a metagenomic analysis of water and oyster samples collected from the Chesapeake Bay. In addition,Vibrio parahaemolyticusandVibrio vulnificusstrains isolated from the samples were subjected to whole-genome sequencing to determine the genetic characteristics of pathogenicVibriospp. circulating in an aquatic environment. HCS, employed to determine the incidence and characterization of specificVibriospp., yielded significantly greater metagenomic insight, notably a variety of otherVibriospp., including detection ofVibrio cholerae,Vibrio fluvialis, andVibrio aestuarianus, in addition toVibrio parahaemolyticusandVibrio vulnificus, and also important virulence factors not detectable using traditional molecular methods. Thus, pathogenicVibriospp. in aquatic ecosystems may be far more common than currently understood. It is concluded that environmental surveillance should include HCS, a valuable tool for the detection and characterization of pathogenic agents in aquatic ecosystems, notably vibrios. IMPORTANCE The increasing prevalence of pathogenicVibriospp. in aquatic ecosystems, driven by climate change, is closely linked to a rise in cholera and vibriosis cases, emphasizing the need for improved environmental surveillance. Vibrios are naturally occurring in aquatic environments globally, but traditional metagenomic methods for detecting and typing pathogenicVibriospp. are challenged by their presence in relatively low abundance and ability to persist in a viable but nonculturable state. In the study reported here, hybridization capture sequencing was employed to profile low-abundanceVibriospp. in metagenomic samples, namely water and oysters collected from the Chesapeake Bay. This approach was evaluated in parallel with traditional whole-community shotgun sequencing and whole-genome sequencing ofVibrio parahaemolyticusandVibrio vulnificusstrains isolated from the samples. Results suggest pathogenicVibriospp. in aquatic ecosystems may be far more common than currently understood, when multiple methods are considered for environmental surveillance.

Microbiology↗
DOE OSTI2026

Transcriptomic and functional analyses uncover a conserved effector driving genotype-dependent virulence in the Sphaerulina musiva-Populus trichocarpa interaction

The introduction of invasive microbes compromises the structure, biodiversity, and function of naïve ecosystems. Sphaerulina musiva, a hemibiotrophic pathogen that causes leaf spot and stem cankers in Populus species, exemplifies an invasive fungal pathogen spread by human activities. However, the genetic mechanisms of pathogenicity and virulence are poorly understood, impeding mitigation strategies. We utilized RNA sequencing to identify fungal effectors linked to stem canker formation, informing the development of future strategies for effective disease management. Our analysis revealed 70 genes differentially expressed at 2 weeks and 110 genes at 3 weeks between inoculated trees and controls. Notably, the gene with the highest expression at 2 weeks and the second highest at 3 weeks was homologous to Extracellular protein 2 (Ecp2). Complementary genome-wide association studies linked sequence polymorphisms in this locus to phenotypic variation in disease severity. Infiltration of S. musiva Ecp2 into Populus trichocarpa leaves induced necrosis in susceptible genotypes. Gene disruption using a CRISPR-Cas9 RNP system resulted in a genotype-dependent reduction of stem canker and disease severity. Tracing the evolutionary history of this effector across the fungal kingdom, we uncovered clade-specific gene-family expansions and orthologs in new species. These findings raise questions about the function and adaptive significance of these gene families in fungal lifestyles. Our study provides the first tractable target for breeding resistant poplar genotypes, addressing the challenges of managing S. musiva and uncovering mechanisms that drive its virulence, and provides deeper insights into the evolutionary dynamics of a conserved small-secreted protein with a diversity of functions.

Sondreli, Kelsey L [Oregon State University]↗
DOE OSTI2025

Production of the light-activated elsinochrome phytotoxin in the soybean pathogen Coniothyrium glycines hints at virulence factor

The Dothideomycete pathogenConiothyrium glycinescauses red leaf blotch of soybean, a major disease in Africa. It is one of two fungal plant pathogens on the USDA PPQ Select Agents and Toxins list of pathogens important to the biosecurity of the United States, reflective of its potential to be highly destructive if introduced. Despite its importance, there are no published reports regarding the molecular basis of host infection. Examination of theC. glycinesgenome revealed a secondary metabolite gene cluster that is similar in gene content and organization to clusters that synthesize light-activated perylenequinone toxins, such as cercosporin. Perylenequinones are non-host specific toxins that, upon exposure to light, generate reactive oxygen species, which have near-universal toxicity to plant hosts.Coniothyrium glycinesisolates from eastern and southern Africa were cultured axenically under light and dark conditions. Light-grown cultures produced red-pink pigmentation typical of perylenequinones. Differential gene expression analysis showed that six of the eight genes in the biosynthetic gene cluster, including the polyketide synthase gene, were significantly upregulated in light. Liquid chromatography-mass spectrometry confirmed production of the perylenequinone elsinochrome A, a known virulence factor in other fungal pathogens. On leaves incubated in the dark, significantly fewer lesions formed and symptoms were delayed, compared to leaves incubated in the light. In addition, we identified orthologous gene clusters in more distantly related Dothideomycete plant pathogens where their presence was previously unknown, indicating a broader importance of these toxins to agriculture and fungal ecology. This work provides the first evidence that elsinochrome A may contribute to the virulence ofC. glycines.

Science & Technology - Other Topics↗
DOE OSTI2023

Thioredoxin VdTrx1, an unconventional secreted protein, is a virulence factor in Verticillium dahliae

Understanding how plant pathogenic fungi adapt to their hosts is of critical importance to securing optimal crop productivity. In response to pathogenic attack, plants produce reactive oxygen species (ROS) as part of a multipronged defense response. Pathogens, in turn, have evolved ROS scavenging mechanisms to undermine host defense. Thioredoxins (Trx) are highly conserved oxidoreductase enzymes with a dithiol-disulfide active site, and function as antioxidants to protect cells against free radicals, such as ROS. However, the roles of thioredoxins in Verticillium dahliae , an important vascular pathogen, are not clear. Through proteomics analyses, we identified a putative thioredoxin (VdTrx1) lacking a signal peptide. VdTrx1 was present in the exoproteome of V. dahliae cultured in the presence of host tissues, a finding that suggested that it plays a role in host-pathogen interactions. We constructed a VdTrx1 deletion mutant Δ VdTrx1 that exhibited significantly higher sensitivity to ROS stress, H 2 O 2 , and tert -butyl hydroperoxide ( t -BOOH). In vivo assays by live-cell imaging and in vitro assays by western blotting revealed that while VdTrx1 lacking the signal peptide can be localized within V. dahliae cells, VdTrx1 can also be secreted unconventionally depending on VdVps36, a member of the ESCRT-II protein complex. The Δ VdTrx1 strain was unable to scavenge host-generated extracellular ROS fully during host invasion. Deletion of VdTrx1 resulted in higher intracellular ROS levels of V. dahliae mycelium, displayed impaired conidial production, and showed significantly reduced virulence on Gossypium hirsutum , and model plants, Arabidopsis thaliana and Nicotiana benthamiana . Thus, we conclude that VdTrx1 acts as a virulence factor in V. dahliae .

Tian, Li↗
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 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 OSTI2025

Virulence and Genetic Diversity of Puccinia spp., Causal Agents of Rust on Switchgrass (Panicum virgatum L.) in the USA

Switchgrass (Panicum virgatum L.) is an important cellulosic biofuel grass native to North America. Rust, caused by Puccinia spp. is the most predominant disease of switchgrass and has the potential to impact biomass conversion. In this study, virulence patterns were determined on a set of 38 switchgrass genotypes for 14 single-spore rust isolates from 14 field samples collected in seven states. Single nucleotide polymorphism (SNP) variation was also assessed in 720 sequenced cloned amplicons representing 654 base pairs of the elongation factor 1-α gene from the field samples. Five major haplotypes were identified differing by 11 out of the 39 SNP positions identified. STRUCTURE, Principal Coordinate Analysis, and phylogenetic analyses divided the rust population into two genetic clusters. Virginia and Georgia had the highest and lowest rust genetic diversity, respectively. Only nine accessions showed a differential disease response between the 14 isolates, allowing the identification of eight races, differing by 1–3 virulence factors. Overall, the results suggested clonal reproduction of the pathogen and a North–South differentiation via local adaptation. However, similar haplotypes and races were also recovered from several states, suggesting migration events, and highlighting the need to further investigate the switchgrass rust population structure and evolution in the USA.

Bahri, Bochra A. (ORCID:0000000159055880)↗