Wide Host Range Research Articles

P-24. Toward the clinical development of ATI-1701, a genetically defined live attenuated tularemia vaccine

Abstract Background Tularemia is a life-threatening disease caused by the bacterium Francisella tularensis. While rare, the disease is notable for its wide host range, low infectious dose, high case fatality rate, and having been previously weaponized. Type A (F. t. tularensis) strains are found predominantly in North America and are generally more virulent than Type B (F. t. holarctica) strains. Both a killed whole cell (KWC) and a live attenuated (LA) vaccine were initially developed and underwent clinical testing in the pre-antibiotic era. While both types of vaccines generated strong antibody responses, later human challenge studies demonstrated that the KWC vaccine failed to protect against aerosol exposure. The LA strain designated LVS, which provided better protection against aerosol challenge but less robust protection against type A strains, was used until recently to immunize laboratory workers. Obstacles for approval of LVS include concerns regarding its undefined attenuation and potential for reversion to virulence. No acellular vaccines have proven to be as effective as LA vaccines. Methods ATI-1701 is a next-generation LA vaccine with a targeted, well-characterized mechanism of attenuation. ATI-1701 carries a clpB gene deletion, which is thought to inhibit its ability to escape from phagosomes, thereby limiting replication in host macrophages. Results Mice immunized ID with 10 CFU of ATI-1701 were fully protected from lethal challenge with the SCHU S4 strain. Rats vaccinated with one dose of ATI-1701 and challenged via aerosol exposure to 5,600 LD50 of virulent SCHU S4 survived infection up to one year post-challenge, whereas all unvaccinated rats died. Regardless of immunizing dose (103-109 CFU), spleens from vaccinated rats contained 103-105 CFU of ATI-1701 14 days after vaccination, and bacteria were cleared from the spleen prior to day 42. Immunization of cynomolgus macaques with a single dose of 105 CFU ATI-1701 protected 80-100% of animals challenged with up to 157,000 LD50 of SCHU S4 via aerosol exposure up to three months after vaccination. One year after vaccination approximately 30% of vaccinated macaques survived challenge whereas control animals died. Conclusion ATI-1701 is a promising tularemia vaccine candidate and is being positioned for a Phase 1 clinical trial. Disclosures All Authors: No reported disclosures

First Report of Neoscytalidium dimidiatum Causing Fruit Rot on Fig in China.

Fig (Ficus carica L.) holds economic significance in Atushi, Xinjiang, but as fig cultivation expands, disease prevalence has risen. In July 2024, approximately 22% of harvested fig (cv. Xinjiang Zaohuang) from 20 commercial orchards (covering 40 hectares) in Atushi (39°39'37.65" N, 76°14'3.62" E) showed varying degrees of fruit rot symptoms. The initial symptoms were characterized by the appearance of small, brown lesions on the fruit surface. These lesions rapidly progressed into water-soaked spots, which expanded quickly. As the disease advanced, the affected areas became covered with dense, white, fluffy mycelia, accompanied by prominent black sporulation. In later stages, the infected tissues softened further, ultimately resulting in the complete decay of the fruit. Twenty diseased fig were collected from the sampling site. Tissue samples (5×5×5 mm) were cut at the diseased-healthy junction, surface-sterilized in 0.5% NaClO for 1 minute, rinsed twice in sterile distilled water, air-dried, and transferred aseptically onto potato dextrose agar (PDA), and incubated at 25°C for 5 days with a 12-hour photoperiod. Fifteen isolates were obtained from the infected tissues, with two representative isolates (WH 12 and WH 23) selected for further study due to morphological similarity. The fungal colonies initially appeared as white mycelium, later turning olive green to grayish-black. Colony growth was rapid (32 mm/day). Arthrospores were colorless to light brown, short columnar, aseptate, with a truncated base, 0 to 1 septate, averaging 11.9±2.3×3.6±0.8 μm (n = 50), and sometimes formed arthric chains. Chlamydospores were dark brown, round or oval, 0 to 1 septate, averaging 7.26±1.7×5.05±1.0 μm (n = 50). Genomic DNA was extracted from the two isolates. The internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF1-α), and beta-tubulin (TUB2) genes were amplified using primers ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone & Kohn. 1999), and BT2a/BT2b (Glass & Donaldson. 1995), respectively, and sequences were deposited in GenBank (ITS: PQ555020, PQ555021; TUB2: PQ557519, PQ557521; TEF1-α: PQ557520, PQ557522). BLAST analysis revealed 99-100% similarity to Neoscytalidium dimidiatum Arp2-D (ITS: MK813852; TUB2: MK816354; TEF1-α: MK816355). Phylogenetic analysis using IQ-Tree and MrBayes3.2.7 based on concatenated ITS-TEF1-TUB sequences showed WH 12 and WH 23 clustering with N. dimidiatum Arp2-D (99% bootstrap). Morphological and molecular data identified the isolates as N. dimidiatum (Penz.) Crous & Slippers (Crous et al. 2006). Pathogenicity tests were conducted on 20 healthy fig (cv. Xinjiang Zaohuang) by inoculating each fruit with 10 µl of a WH 12 conidial suspension (1 × 10⁶ conidia/ml) using sterile needles. The Control were treated with 10 µl of sterile distilled water. All fruits were placed in sterile plastic containers and incubated at 25 ± 1°C, 90% relative humidity, and a 12-hour light cycle. This experiment was performed twice. On the 1st day post-inoculation, brown lesions began to develop on the fruit. By the 4th day post-inoculation, the entire fruit was completely decayed and covered with white mycelia and black spores, while the control fruit showed no symptoms. The fungus was successfully reisolated from the inoculated fruits and identified as N. dimidiatum following the methods described above, fulfilling Koch's postulates. N. dimidiatum has been reported to have a wide range of hosts in China, such as Jacaranda mimosifolia, Hylocereus megalanthus, Hylocereus undatus, and Styphnolobium japonicum (Li et al. 2024; Zeng et al. 2024; Lan et al. 2022; Luo et al. 2024). To our knowledge, this study is the first report of N. dimidiatum as the causal agent of fruit rot in fig in China. Our findings have expanded the host range of N. dimidiatum in China and provide a theoretical basis for the diagnosis and treatment of the disease.

The role of N6-methyladenosine (m6A) mRNA modifications in herpesvirus infections.

Herpesviruses, a family of large enveloped DNA viruses, establish persistent infections in a wide range of hosts. This characteristic requires an intricate network of interactions with their hosts and host cells. In recent years, the interplay between herpesviruses and the epitranscriptome-chemical modifications in transcripts that may affect mRNA biology and fate-has emerged as a novel aspect of herpesvirus-host interactions. In particular, herpesviruses display different mechanisms to modulate and usurp the most abundant mRNA modification, N6-methyladenosine or m6A. Some herpesviruses interfere with m6A methylation of transcripts, while others enhance or take advantage of m6A methylation of viral and/or cellular transcripts. In many cases, herpesviruses appear to modulate the m6A methylation process to suppress the antiviral host response. This review highlights the strategies used by members of the different herpesvirus subfamilies to manipulate host m6A mediators and how these contribute to virus replication and the antiviral host response. Research aimed at deciphering the interaction of herpesviruses with the m6A epitranscriptome not only may lead to new avenues in the design of antiviral and immunomodulatory strategies, but also provides new insights in the regulation and the role of m6A transcript methylation in general.

Bussuquara Virus: A Neglected Orthoflavivirus with Broad Distribution Across Central and South America and the Caribbean

Bussuquara virus (BSQV) was first discovered in the Brazilian Amazon in 1956. It is an arthropod-borne virus (arbovirus) in the genus Orthoflavivirus, family Flaviviridae. Since its discovery, BSQV has been sporadically detected across the South (Brazil, Columbia, and Argentina) and Central (Panama and Mexico) America and the Caribbean (Grenada), but there is minimal BSQV surveillance due to limited public health awareness and a lack of specific or sensitive diagnostics. BSQV exposure has been reported in a wide range of host and vector species, including humans. Little information is available in the literature and herein we summarize the published historical findings on BSQV and suggest a pathway for future studies to better understand its potential emergence into human populations.

Proteomic Variation in the Oral Secretion of Spodoptera exigua and Spodoptera littoralis Larvae in Response to Different food Sources

The Spodoptera genus is defined as the pest-rich genus because it contains some of the most destructive lepidopteran crop pests, characterized by a wide host range. During feeding, the caterpillars release small amounts of oral secretion (OS) onto the wounded leaves. This secretion contains herbivore-induced molecular patterns (HAMPs) that activate the plant defense response, as well as effectors that may inhibit or diminish the plant’s anti-herbivory response. In this study, we explored the protein components of the OS of two Spodoptera species, Spodoptera exigua and Spodoptera littoralis. We identified 336 and 276 proteins, respectively, with a major role in digestion. Using a label-free quantitative proteomics approach, we investigated changes in protein abundance in the OS of both species after switching from a laboratory artificial diet to detached pepper and tomato leaves. Several proteins, such as various lipases, polycalin and a β-1,3-glucan binding protein, were more abundant in the OS of leaf-fed larvae in both species. Conversely, a tryptophan-aspartic acid (WD)-repeat containing protein significantly decreased upon feeding on plant leaves in both species. Phenotypic plasticity dependent on each Spodoptera-plant combination was observed for several peptidases, potentially related to the need to overcome the effects of proteinase inhibitors differentially produced by the two plant species, and for several REPAT proteins, possibly related to the specific modulation of each Spodoptera-plant interaction. Altogether, our results provide useful information for understanding the interaction of these two polyphagous Spodoptera species with the host plants, and help to identify evolutionary traits that may influence the outcome of herbivory in each of these two related species.

Dual EMCV-IRES-integrated dengue virus can express an exogenous gene and cellular Mdm2 integration suppresses the dengue viral replication

Flaviviruses transmit through a wide range of vertebrate and arthropod hosts, while the other genera in Flaviviridae replicate in a limited set of vertebrate hosts. Flaviviruses possess a 5′ cap in their genome RNA for translation, while the other genera utilize their internal ribosome entry site (IRES) sequences instead of a 5′ cap. In this study, the translational modification to add an IRES sequence was examined. An IRES sequence derived from encephalomyocarditis (EMCV) was inserted into dengue virus serotype 2 (DENV2); a non-structural (NS) polyprotein was translated by IRES separately from 5′ cap-induced structural polyprotein translation. It was revealed that the IRES-integrated DENV2 is prevented from replicating in C6/36 mosquito cells, suggesting that the 5′ cap is an advantageous mechanism for flavivirus translation in invertebrate species. I further created dual IRES-integrated DENV2, in which a non-viral gene can be expressed by the flanking IRESs. The insertion of eGFP fluorescently visualized the virus spread. The renilla luciferase (Rluc) integration enabled the viral replication quantification. It was also revealed that a cellular gene, Mdm2, which antagonizes tumor suppressor protein p53 (TP53), could terminate the viral replication in BHK21 cells. Thus, the modifications of the DENV genome with IRES and the subsequent foreign gene could be utilized for controlling viral replications.

Occurrence of Pestalotiopsis sp. on Ornamental Plants Camellia japonica L. in Romanian Public Gardens

Camellias are evergreen shrubs native to the eastern and southern regions of Asia. In Romania, camellias are popular ornamental plants grown in pots. Pestalotiopsis species have a worldwide distribution and are pathogenic to a wide range of hosts. They also represent an important group of endophytes capable of producing a variety of bioactive secondary metabolites. This paper reports the occurrence of grey blight and twig or branch dieback caused by Pestalotiopsis sp. on Camellia japonica L. grown in pots and used in the landscaping of a public garden located in Iasi city, Romania (GPS coordinates: 47°09′24.0″ N 27°35′15.6″ E). Pestalotiopsis species were identified based on morphological characteristics and Sanger sequencing. Genomic analysis of the fungal isolate coded P_CJ_24, obtained from living plants, was based on ribosomal internal transcribed spacer region amplification using the LSU primers. This first report of Pestalotiopsis species infection of ornamental plants in Romania suggests that new plant pathogens can be introduced in new environmental conditions through plant pods and their incidence increases with the widespread of plant material among gardens.

Genetic Diversity of Tobacco mosaic virus (TMV) isolates from tobacco growing fields of Western Anatolia, Türkiye

Tobacco mosaic virus (TMV) is an important plant virus that is common in agriculture. It is the first evidence of the existence of viruses in history. Studies on the genetic diversity of the CP gene of TMV, which plays a leading role in host interaction, are limited both in our country and around the world. Genetic diversity analyses were conducted on ten isolates of the full CP gene region of TMV obtained from the most intensive tobacco cultivation areas in our country, and compared with global isolates. TMV infection was detected in 32 out of 300 plants collected from the Aegean and Marmara regions (Çanakkale, Balıkesir, İzmir, Manisa, Uşak, Aydın and Denizli) between 2019 and 2020 using conventional molecular techniques. To genetically characterize the virus, 10 samples were selected from each region, and the complete CP gene region sequences were determined. The aligned CP gene region sequences of TMV from Türkiye and its global isolates exhibited nucleotide homology ratios ranging from 87.7% to100%, with amino acid ratios ranging from 88.7% to 100%. The Türkiye isolates displayed similarity rates of 98.5% to 100% at the nucleotide level and 98.7% and 100% at the amino acid level. In phylogenetic analysis, the 196 known isolates of TMV registered in GenBank, belong to the CP gene region, were divided into two main clades (I and II) and two subclades (Ia and Ib). Turkish isolates were clustered in the major branch with the main clade I and subclade Ia isolates. Therefore, genetic analyses were performed on the CP gene region isolates obtained from different parts of the world and a wide range of hosts, including the isolates obtained from Türkiye. The results showed high genetic stability, similar to many tobamoviruses.

Genotypic diversity and biological characterization of Alphabaculovirus mabrassicae isolates from a wide host range.

Baculoviruses are ideal biological insecticides, providing long-lasting pest control and environmental benefits. Alphabaculovirus mabrassicae stains, with their broad host range, have been effective in agricultural pest management. Various A. mabrassicae isolates (MbMNPV-CHb1/CTa/K1/QD, MyseMNPV-Hb, HearMNPV and MacoNPV-B) have been identified in different hosts. Identifying more effective A. mabrassicae strains with detailed genetic information is crucial for commercial use. Laboratory bioassays showed that the median lethal concentration (LC50) of MyseMNPV-Hb against Mythimna separata was significantly lower than those against Helicoverpa armigera and Spodoptera exigua, but higher than the LC50 of MbMNPV-CHb1, MbMNPV-QD and HearMNPV against H. armigera or S. exigua. Comparative genomic analysis revealed significant differences in genomic composition and single-nucleotide polymorphisms between MyseMNPV-Hb and the other isolates. A piggyBac-like element, likely to have been from Alcis repandata (Lepidoptera: Geometridae), was identified in the genomes of these isolates. Eight genes in the A. mabrassicae genomes were found to be under positive selection. Alphabaculovirus mabrassicae isolates exhibit different infectivity in various pests, indicating the need for selecting appropriate isolates specific target pests. This study elucidates the genetic factors contributing to the differential infectivity of A. mabrassicae isolates and extends knowledge on its population characteristics. © 2025 Society of Chemical Industry.

First report of Ralstonia syzygii causing bacteria wilt on eucalyptus in Brazil.

The Ralstonia solanacearum Species Complex (RSSC) is the most significant plant pathogen group with a wide host range. It is genetically related but displays distinct biological features, such as restrictive geography occurrence. The RSSC comprises three species: Ralstonia pseudosolanacearum (phylotype I and III), Ralstonia solanacearum (phylotype IIA and IIB), and Ralstonia syzygii (phylotype IV) (Fegan and Prior 2005). In Brazil, eucalyptus production has been growing as one of the leading industries to produce cellulose, energy, and other products from renewable sources, covering approximately 7.8 million hectares, representing 76% of all planted forests in 2023 (IBÁ, 2024). The eucalyptus bacterial wilt (BW), caused by Ralstonia solanacearum and Ralstonia pseudosolanacearum, members of RSSC (Lopes and Rossato 2018), is one of the most important eucalyptus diseases, causing damage to nurseries and eucalyptus plantations (Alfenas et al., 2009). We collected Eucalyptus urophylla clone plants showing typical wilt symptoms in a commercial nursery with an average size of 400 m2 harboring 300k cuttings plant in rooting, showing 5% symptom incidence in February 2024 in the Bahia State, Brazil. Affected plants showed wilting of leaves and branches, reduced growth, and death at a higher level of disease severity. The vascular tissues showed darkening and intense bacterial exudation. Next, we aimed to determine the presence of RSSC bacteria in these plants by using the isolation method. For this, the stem macerate of symptomatic plants was plated on a semi-selective SMSA medium (ELPHINSTONE et al. 1996) and incubated for 72h at 28oC. It was possible to isolate bacteria with typical morphology of RSSC in all samples. Using multiplex PCR (Fegan & Prior, 2005), 96.77% of isolates were classified as phylotype II and 2.58% as phylotype I. Notably, we identified one isolate (0,65%), BR19, which exhibited an amplification pattern characteristic of phylotype IV. This isolate demonstrated typical RSSC growth, forming a slimy colony with a red internal region on CPG medium supplemented with tetrazolium. To fulfill Koch's postulates, eight plants of Eucalyptus urophylla were inoculated with bacterial suspension (20 µL adjusted to 108 CFU/mL of saline solution), and eight plants injected with distilled water were used as controls. The bacterial inoculum or the distilled water was injected at the base of the stem, and the plants were kept in a greenhouse (28 ± 2 °C), according to the methodology described by Fonseca et al. (2016). Then, after 30 days of post-inoculation with the bacterium, all plants showed wilt symptoms, and it was re-isolated from plating stem tissue macerates on SMSA medium. The identity of the bacteria was confirmed using RSSC species phylotype multiplex PCR (Fegan & Prior, 2005). Using Long-read Nanopore technology, we were able to sequence the draft complete genome composed of a chromosome and a megaplasmid, which were deposited at GenBank with assembly accession number GCF_042494895.1. The completeness of the BR19 genome was found to be 92.1%, according to the BUSCO program. Phylogenomic analysis was performed according to Subedi et al. (2024), revealing that BR19 is genetically closer to two other isolates of Ralstonia syzygy subsp. syzygii (GenBank assemblies: ASM1591059v1 and ASM2921996v1) isolated from clove (Syzygium aromaticum) in Indonesia. It is the first report of the detection of R. syzygii subsp. syzygii phylotype IV away from Southeast Asia and the first report of R. syzygii subsp. syzygii infecting eucalyptus. This result expands our knowledge about the geographic distribution and host range of RSSC.

Pseudocercospora fengxinensis, a new species causing sooty spot of kiwifruit in China.

Pseudocercospora is a large cosmopolitan genus of plant pathogenic fungi that are commonly associated with leaf and fruit spots as well as blights on a wide range of plant hosts. P. actinidiae is a member of this genus, causing sooty spot on kiwifruit worldwide. With the expansion of kiwifruit cultivation, the incidence of sooty spot has become severe in Fengxin County, Jiangxi Province, China. Based on a survey conducted in 2022 across nine kiwifruit cultivars in six local orchards, the results showed that the disease incidence was higher in 'Donghong', 'Jinyan', 'Jinkui', 'Fenghuang No. 1' and 'Jinguo'. In contrast, it was lower in 'Gold3' and 'Hongshi No. 2'. No lesions were observed on 'Kuilv' and 'Miliang No. 1'. 31 fungal strains were isolated from infected leaves using single-spore isolation. Phylogenetic analysis of combined sequence data from the internal transcribed spacer region of ribosomal DNA (ITS), partial actin (actA), translation elongation factor 1-alpha (tef1), and the second largest subunit of RNA polymerase II (rpb2) sequence data, using maximum-likelihood (ML) and Bayesian inference (BI), revealed their taxonomic placement within Pseudocercospora. Both morphological and molecular phylogenetic analysis supported Pseudocercospora fengxinensis as a new taxon within Pseudocercospora. Pathogenicity tests confirmed P. fengxinensis as the causal agent of sooty spot on kiwifruit. This study is the first report of P. fengxinensis causing sooty spot of kiwifruit worldwide, enriching our understanding of Pseudocercospora diversity in China and providing a foundation for improved disease management strategies.

Picornaviridae and Caliciviridae diversity in Madagascar fruit bats is driven by cross-continental genetic exchange.

Bats are reservoir hosts for numerous well-known zoonotic viruses, but their broader virus-hosting capacities remain understudied. Picornavirales are an order of enteric viruses known to cause disease across a wide range of mammalian hosts, including Hepatitis A in humans and foot-and-mouth disease in ungulates. Host-switching and recombination drive the diversification of Picornavirales worldwide. Divergent Caliciviridae and Picornaviridae (families within the Picornavirales ) have been described in bats across mainland Africa, but surveillance for these viruses has been rare in the Southwest Indian Ocean Islands. Bats live in close proximity to and are consumed widely as a food source by humans in Madagascar, providing opportunities for zoonotic transmission. Prior work in Madagascar has described numerous evolutionarily divergent bat viruses, some with zoonotic potential. Using metagenomic Next Generation Sequencing of urine and fecal samples obtained from three species of endemic Malagasy fruit bats ( Eidolon dupreanum , Pteropus rufus , and Rousettus madagascariensis ), we recovered 13 full-length and 37 partial-length genomic sequences within the order Picornavirales (36 Picornaviridae and 14 Caliciviridae sequences), which we identify and describe here. We find evidence that genetic exchange between mainland African bat and Madagascar bat Picornavirales likely shaped the diversification patterns of these novel sequences through recombination events between closely related Picornavirales ; thus far, high host fidelity appears to have limited these viruses from spilling over into other species.

Apple Bitter Rot: Biology, Ecology, Omics, Virulence Factors, and Management of Causal Colletotrichum Species.

Colletotrichum species-Domain Eukaryota, Kingdom Fungi, Phylum Ascomycota, Class Sordariomycetes, Order Glomerellales, Family Glomerellaceae, Genus Colletotrichum. Hemibiotrophic pathogen with a wide host range that establishes a biotrophic interaction where it penetrates host plants using appressoria followed by a switch to necrotrophy causing rot symptoms. Cercosporin, colletotrichins, colletotric acid, ferricrocin. The host range varies by species but largely occurs on dicotyledonous plants and is less prevalent on monocots as well as gymnosperms, ferns, mosses and animals (e.g., insects). Symptoms often manifest as flat to sunken necrotic areas on fruit. Lesions on leaves and fruit can have concentric rings with abundant pathogen sporulation. Colletotrichum spp. are primarily managed by single-site quinone outside inhibitor (Qol), methyl benzimidazole carbamate (MBC), demethylation inhibitor (DMI) fungicides, and multisite dithiocarbamate and phthalimide fungicides. Susceptibility may vary with species, strain specificity, or geographic region. Other management options include clean stock production, cultural practices, resistance breeding, and biological control through the introduction of protective or competing microorganisms.

Tn-seq profiling reveals that NodS of the beta-rhizobium Paraburkholderia phymatum is detrimental for nodulating soybean

The beta-rhizobial strain Paraburkholderia phymatum STM815T is noteworthy for its wide host range in nodulating legumes, primarily mimosoids (over 50 different species) but also some papilionoids. It cannot, however, nodulate soybean (Glycine max [L.] Merr.), one of the world’s most important crops. Here, we constructed a highly saturated genome-wide transposon library of a P. phymatum strain and employed a transposon sequencing (Tn-seq) approach to investigate the underlying genetic mechanisms of symbiotic incompatibility between P. phymatum and soybean. Soybean seedlings inoculated with the P. phymatum Tn-seq library display nodules on the roots that are mainly occupied by different mutants in a gene, nodS, coding for a methyltransferase involved in the biosynthesis of nodulation factors. The construction of a nodS deletion strain and a complemented mutant confirms that nodS is responsible for the nodulation-incompatibility of P. phymatum with soybean. Moreover, infection tests with different host plants reveal that NodS is necessary for optimal nodulation of common bean (Phaseolus vulgaris), but it is not required for nodulation of its natural host Mimosa pudica. In conclusion, our results suggest that NodS is involved in determining nodulation specificity of P. phymatum.

First report of watermelon mosaic virus infecting faba bean (Vicia faba L.) in China.

Faba bean (Vicia faba L.) is the fourth most cultivated temperate legume (Lyu et al., 2021). It is widely grown throughout the world as a nutritious legume crop. From March to April in 2023, field-grown faba bean plants with virus-like symptoms such as mosaic, discoloration, stunting, yellowing, chlorosis and leaf curling were observed at 13 different locations in Nanjing and Xinghua City in Jiangsu Province, China. A total of sixty-five leaf samples were collected from the symptomatic faba bean plants. The leaf morphology of these plants is deformed, and the growth and development are severely affected by the infected diseases. To identify the viruses that infected the faba bean plants, the samples were mixed, and the total RNA was extracted using the TRIzol reagent (Invitrogen, USA). A small RNA sequence library was constructed using the Trusses Small RNA Sample Prep Kit (Illumina, San Diego, USA) and analysed by high-throughput sequencing using an Illumina HiSeq 2500 platform (Lianchuan Biotechnology, Hangzhou, China). The obtained clean sequencing reads were mapped mainly to 11 viruses (with mapped read counts >20000). De novo assembly of the total reads was performed using ACGT101-miR and Velvet software (LCSciences, Houston, Texas, USA). The resulting assembled contigs were analysed by BLASTn and BLASTx at National Center for Biotechnology Information. Seventy-three contigs were matched to the genome sequence of watermelon mosaic virus (WMV) in the genus Potyvirus of the family Potyviridae. RT-PCR was used to detect WMV in the high-through sequencing sample using specific primers (WMV-F: 5'- TTAACCACATTGATGGTTGC-3', WMV-R: 5'- GACCCGAAATGCTAACTGTG-3') with an expected product of 924 bp. The sequencing results of PCR amplicon confirmed that the sample was infected with WMV (Fig. S1A). RT-PCR analysis of pooled samples from each location showed that samples from three locations were infected with WMV (Fig.S1B). To detect WMV in the individual sample collected from these three locations, a total of 18 samples were analysed by RT-PCR and four samples were positive for WMV (Fig.S1C). The symptoms of the four infected plants are shown in Fig.S1D. To determine the full-length sequence of the WMV isolate from faba bean, RT-PCR was performed using five overlapping primer pairs designed according to the WMV sequences obtained by high-throughput sequencing and the aligned WMV sequences. The amplified PCR products were then inserted into pMD19-T (Takara, Dalian, China) and sequenced. The full-length sequence of the faba bean WMV was assembled from the five overlapping sequences and deposited in GenBank as WMV-Fb isolate (accession number PP782053). The complete sequence of the WMV-Fb isolate has 10,044 nt and contains a 9,648 nt open reading frame encoding a polyprotein of 3,216 amino acids. It shares the highest nucleotide sequence identity (97.46%) with two WMV isolates (accession number OM948829.1 and OM948820.1) from Zucchini in Spain and had 97.40% nucleotide sequence identity with Sapindus mukorossi potyvirus strain pt164-pot-23 (accession number MN722418.1) from Sapindus mukorossi Gaertn. in Jiangsu, China at the complete genome level. To reveal the molecular evolutionary relationships between WMV-Fb and other isolates, a phylogenetic tree was constructed using the polyprotein nucleotide acid sequence of WMV-Fb and reported WMV isolates in the G1 (classical isolates), G2 and G3 (emerging isolates) groups (Bertin et al., 2020; Abdalla et al., 2021). Phylogenetic analysis showed that the WMV-Fb isolate was clustered in the G3 group (Fig.S1E). To the best of our knowledge, this is the first report of WMV infecting faba bean. WMV has a wide host range and causes severe losses in cucurbit crops, such as melon, watermelon, zucchini, and cucumber (Dong et al., 2017; Nematollahi et al., 2021). Our results provide a better understanding of the host range of WMV and suggest that WMV poses a potential threat to legume crops.

Exploring the Inhibition of Toxoplasma gondii α-Carbonic Anhydrase by Sulfonamides: Insights into Potential Drug Targeting.

Toxoplasma gondii, the causative agent of toxoplasmosis, is a protozoan parasite capable of infecting a wide range of hosts, posing significant health risks, particularly to immunocompromised individuals and congenital transmission. Current therapeutic options primarily target the active tachyzoite stage but are limited by issues such as toxicity and incomplete efficacy. As a result, there is an urgent need for alternative therapies that can selectively target parasite-specific mechanisms critical for metabolic processes and host-parasite interactions. In this context, α-carbonic anhydrase (Tg_CA), an enzyme essential for T. gondii survival has emerged as a promising drug target. Tg_CA was successfully expressed and purified to evaluate its susceptibility to sulfonamide-based inhibitors, represented by compounds 1-24 and the AAZ-HCT series. These inhibitors demonstrated a broad spectrum of activity, with KI values ranging from 17.8 to 8450 nM. Several compounds exhibited moderate to high potency against Tg_CA; however, concerns regarding selectivity arose because of the inhibition of human isoforms, particularly CA I and CA II. Thus, although some inhibitors showed strong activity against Tg_CA, optimizing selectivity remains crucial for minimizing off-target effects and improving therapeutic efficacy. Further structural modifications may enhance selectivity and advance the development of effective treatments for toxoplasmosis.

Genomic characterization of novel bat kobuviruses in Madagascar: implications for viral evolution and zoonotic risk.

Kobuviruses (family Picornaviridae , genus Kobuvirus ) are enteric viruses that infect a wide range of both human and animal hosts. Much of the evolutionary history of kobuviruses remains elusive, largely due to limited screening in wildlife. Bats have been implicated as major sources of virulent zoonoses, including coronaviruses, henipaviruses, and filoviruses, though much of the bat virome still remains uncharacterized. While most bat virus research has historically focused on immediately recognizable zoonotic clades (e.g. SARS-related coronaviruses), a handful of prior reports catalog kobuvirus infection in bats and posit the role of bats as potential progenitors of downstream kobuvirus evolution. As part of a multi-year study, we carried out metagenomic Next Generation Sequencing (mNGS) on fecal samples obtained from endemic, wild-caught Madagascar fruit bats to characterize potentially zoonotic viruses circulating within these populations. The wild bats of Madagascar represent diverse Asian and African phylogeographic histories, presenting a unique opportunity for viruses from disparate origins to mix, posing significant public health threats. Here, we report detection of kobuvirus RNA in Malagasy fruit bat ( Eidolon dupreanum ) feces and undertake phylogenetic characterization of one full genome kobuvirus sequence, which nests within the Aichivirus A clade - a kobuvirus clade known to infect a wide range of hosts including humans, rodents, canids, felids, birds, and bats. Given the propensity of kobuviruses for recombination and cross-species infection, further characterization of this clade is critical to accurate evaluation of future zoonotic threats.

Comparative Genome Analysis of Canine Frederiksenia canicola Isolates.

Background/Objectives: The One Health approach is crucial for managing and controlling the spread of antimicrobial resistance. Frederiksenia canicola is a recently identified bacterial species that seems to be a component of the oral microbiota of dogs; however, its pathogenic nature is questionable. Methods: In this study, the antibacterial susceptibility of F. canicola isolates was determined using the disk diffusion and broth microdilution methods. Genome-wide comparative analyses were performed to identify the genetic factors driving virulence and antimicrobial drug resistance (e.g., virulence factors, antimicrobial resistance genes (ARGs) and prophage-related sequences). Results: Most of the F. canicola isolates lacked virulence-associated genes. F. canicola is likely resistant to clindamycin, lincomycin and neomycin, but susceptible to penicillin, erythromycin and enrofloxacin. Antimicrobial resistance genes were not found in the F. canicola genomes, but prophage-related sequences were identified, suggesting its potential in the transfer of genes associated with drug resistance between bacteria in the oral microbiome. Conclusions: F. canicola is presumably a commensal organism with low virulence potential, as evidenced by the absence of virulence-associated genes. As F. canicola can colonize a wide range of hosts, including humans, further investigation with a greater number of isolates is needed to better understand the role of F. canicola in disease development and the spread of drug resistance.

Evolution of sympatric host-specialized lineages of the fungal plant pathogen Zymoseptoria passerinii in natural ecosystems.

The barley disease Septoria Speckled Leaf Blotch, caused by the fungus Zymoseptoria passerinii, last appeared in North America in the early 2000s. Although rare in crops, field sampling of wild grasses in the Middle East revealed the disease persistence in wild barley. Identification of Z. passerinii in various wild barley species prompted us to examine genomic signatures of host specialization and trace the emergence of the domesticated-barley-infecting lineage. Furthermore, we applied virulence assays and confocal laser microscopy to evaluate whether the disease development differs between wild and domesticated barley. Wild- and domesticated-host-infecting populations have diverged, and phylogenetic relationships support the evolution of sympatric host-specialized lineages in wild hosts. Cross-virulence assays showed that Z. passerinii from domesticated hosts infect domesticated barley and its wild ancestor, Hordeum spontaneum. However, wild isolates from Iran did not infect domesticated barley. Wild and domesticated pathosystems have similar disease timing and progression, suggesting its persistence does not depend on a shorter period of incubation. The study supports that a wide range of hosts can foster the evolution of host-specialized lineages in sympatry and provide novel insights into the evolution of understudied fungal pathogens on wild hosts.

DIAGNOSTIC INVESTIGATION OF AMERICAN ROBINS (TURDUS MIGRATORIUS) WITH FATAL ETHYLENE GLYCOL TOXICOSIS AND CONCURRENT PLASMODIUM SPP. INFECTION WITH RETROSPECTIVE REVIEW OF 44 YEARS OF DIAGNOSTIC DATA.

We describe a mortality event involving at least 44 adult American robins (Turdus migratorius) that were found dead on the morning of February 25, 2023 in a residential backyard in Natchitoches Parish, Louisiana. Five carcasses were submitted for diagnostic evaluation. All five robins were in good nutritional and feather condition with no external indicators of injury or illness. Grossly, kidneys were diffusely pale in all robins and 4/5 also had splenomegaly. Consistent histologic lesions included severe, multifocal to coalescing, renal tubular degeneration and necrosis with intraluminal, birefringent calcium oxalate crystals and calcified concretions. Toxicologic testing revealed a markedly elevated calcium concentration (10,000 parts per million) in a pooled kidney sample. Collectively, these findings support a diagnosis of ethylene glycol toxicosis. Ethylene glycol, an ingredient in antifreeze, is a hazardous toxicant in domestic and wild animals living in residential areas, although confirmed reports in songbirds are scarce. In the present case, the circumstances of ethylene glycol exposure are unknown but most likely anthropogenic in origin. Additionally, splenomegaly prompted molecular testing, which detected Plasmodium unalis in three robins and P. vaughani in one robin. Both Plasmodium spp. can infect a wide range of hosts, and although infections likely were subclinical, their detection raises awareness of their potential pathogenicity in wild birds. This mortality event underscores the need to recognize health risks posed by environmental contaminants and vector-borne pathogens to wildlife, and reinforces that wildlife, especially peridomestic species, can serve as sentinels for risks to domestic animal and human health.