Despite displaying broad tropism in vivo, human cytomegalovirus (CMV) contained in bodily fluids replicates inefficiently in most cultured cell types except fibroblasts. As propagation in fibroblasts leads to the accumulation of genomic changes, a number of strains were generated by serial passaging on endothelial cells. One of these, TB40/E, was shown to contain a mixture of genetically distinct virus variants, and to retain tropism for fibroblasts, endothelial and epithelial cells. Cloning of an endotheliotropic subpopulation produced the TB40-BAC4 variant, extensively used in CMV tropism studies. Because TB40-BAC4 represents only one of the different variants comprising TB40/E, we generated a series of epithelial-cell adapted stocks derived from a TB40/E mixed stock, rather than from TB40-BAC4. Within two passages on ARPE-19 cells, virus populations were produced with the ability to enter and initiate replication with similar efficiencies in both epithelial cells and fibroblasts. Although the ability to release progeny also increased, cell-free virus yields from ARPE-19 cells remained consistently two to three-logs lower than from fibroblasts, hinting at the existence of a post-entry and post-genome synthesis block in epithelial cells. Multinucleated syncytia also rapidly appeared exclusively in ARPE-19 cell cultures, where their numbers and dimensions increased with virus passage. Irrespective of the number of infected nuclei comprising each syncytium, however, only one cytoplasmic virion assembly compartment was consistently observed, leading us to speculate that improvements in entry efficiency associated with ARPE-19 cell adaptation lead to the development of syncytia, which may negatively affect progeny release by limiting the amount of resources available to maturing virions. Full article

Biomethanation is a promising solution to convert H₂ (produced from surplus electricity) and CO₂ to CH₄ by using hydrogenotrophic methanogens. In ex situ biomethanation with mixed cultures, homoacetogens and methanogens compete for H₂/CO₂. We enriched a hydrogenotrophic microbiota on CO₂ and H₂ as sole carbon and energy sources, respectively, to investigate these competing reactions. The microbial community structure and dynamics of bacteria and methanogenic archaea were evaluated through 16S rRNA and mcrA gene amplicon sequencing, respectively. Hydrogenotrophic methanogens and homoacetogens were enriched, as acetate was concomitantly produced alongside CH₄. By controlling the media composition, especially changing the reducing agent, the formation of acetate was lowered and grid quality CH₄ (≥97%) was obtained. Formate was identified as an intermediate that was produced and consumed during the bioprocess. Stirring intensities ≥ 1000 rpm were detrimental, probably due to shear force stress. The predominating methanogens belonged to the genera Methanobacterium and Methanoculleus. The bacterial community was dominated by Lutispora. The methanogenic community was stable, whereas the bacterial community was more dynamic. Our results suggest that hydrogenotrophic communities can be steered towards the selective production of CH₄ from H₂/CO₂ by adapting the media composition, the reducing agent and the stirring intensity. Full article

Cryptococcosis is an important opportunistic infection and a leading cause of meningitis in patients with HIV infection. The antifungal pharmacological treatment is limited to amphotericin B, fluconazole and 5- flucytosine. In addition to the limited pharmacological options, the high toxicity, increased resistance rate and difficulty of the currently available antifungal molecules to cross the blood–brain barrier hamper the treatment. Thus, the search for new alternatives for the treatment of cryptococcal meningitis is extremely necessary. In this review, we describe the therapeutic strategies currently available, discuss new molecules with antifungal potential in different phases of clinical trials and in advanced pre-clinical phase, and examine drug nanocarriers to improve delivery to the central nervous system. Full article

Acinetobacter baumannii is an opportunistic bacterial pathogen associated with hospital-acquired infections, including pneumonia, meningitis, bacteremia, urinary tract infection, and wound infections. Recognition of host cell surface carbohydrates plays a crucial role in adhesion and enables microbes to colonize different host niches. Here the potential glycosphingolipid receptors of A. baumannii were examined by binding of ³⁵S-labeled bacteria to glycosphingolipids on thin-layer chromatograms. Thereby a selective interaction with two non-acid glycosphingolipids of human and rabbit small intestine was found. The binding-active glycosphingolipids were isolated and, on the basis of mass spectrometry, identified as neolactotetraosylceramide (Galβ4GlcNAcβ3Galβ4Glcβ1Cer) and lactotetraosylceramide (Galβ3GlcNAcβ3Galβ4Glcβ1Cer). Further binding assays using reference glycosphingolipids showed that A. baumannii also bound to lactotriaosylceramide (GlcNAcβ3Galβ4Glcβ1Cer) demonstrating that GlcNAc was the basic element recognized. In addition, the bacteria occasionally bound to galactosylceramide, lactosylceramide with phytosphingosine and/or hydroxy fatty acids, isoglobotriaosylceramide, gangliotriaosylceramide, and gangliotetraosylceramide, in analogy with binding patterns that previously have been described for other bacteria classified as “lactosylceramide-binding”. Finally, by isolation and characterization of glycosphingolipids from human skin, the presence of neolactotetraosylceramide was demonstrated in this A. baumannii target tissue. Full article

Tripeptide glutathione, which plays important roles in many cellular mechanisms, is also a biotechnology-oriented molecule with applications in medicine, food and cosmetic. Here, the engineering of the yeast Yarrowia lipolytica for the production of this metabolite at high titer values from various agro-industrial by-products is reported. The constitutive overexpression of the glutathione biosynthetic genes GSH1 and GSH2 encoding respectively γ-glutamylcysteine synthetase and glutathione synthetase, together with the INU1 gene from Kluyveromyces marxianus encoding inulinase yielded a glutathione titer value and a productivity of 644 nmol/mg protein and 510 µmol/g_DCW, respectively. These values were obtained during bioreactor batch cultures in a medium exclusively comprising an extract of Jerusalem artichoke tuber, used as a source of inulin, and ammonium sulfate, used as a nitrogen source. Full article

The genus Cyclobacterium belongs to the phylum Bacteroidetes and includes eight species. Our study, based on the genomic parameters in silico DNA–DNA hybridization (GGDC), average nucleotide identity (OrthoANI), and average amino acid identity (AAI), confirmed that all current species of Cyclobacterium belong to this genus and constitute a coherent phylogenomic group, but with species forming two separate branches. In addition, the genome-based analyses revealed that Cyclobacterium xiamenense and Cyclobacterium halophilum are members of the same species. Besides, we carried out a taxonomic characterization of the new strain GBPx2^T, isolated from the halophytic plant Salicornia sp. Analysis of its 16S rRNA gene sequence showed the highest sequence similarity (97.5%) to Cyclobacterium lianum HY9^T. Percentages of GGDC and OrthoANI between strain GBPx2^T and species of the genus Cyclobacterium were lower than the threshold value for species delineation. The DNA G+C content was 43.0 mol%. The polar lipids included phosphatidylethanolamine as well as one unidentified phospholipid and four unidentified lipids, and its major cellular fatty acids were iso-C_15:0 and summed feature 3 (C_16:1ω7c and/or iso-C_15:0 2-OH). The only quinone present was menaquinone 7. Based on a combination of phenotypic, chemotaxonomic, and phylogenomic features, the GBPx2^T strain represents a novel species of the genus Cyclobacterium, for which the name Cyclobacterium plantarum sp. nov. is proposed. The type strain of Cyclobacterium plantarum is GBPx2^T (= IBRC-M 10634^T = LMG 28551^T). Full article

Incidental prophylactic surgeries are performed in certain situations. Incidental prophylactic appendectomies were common practice within opened bariatric surgeries. The gut microbiota has emerged as an important actor within the homeostasis of the host. A new hypothesis has been formulated about the appendix function in relation to gut microbiota. Our objective was to study the gut microbiota profiles of patients that had suffered from an incidental prophylactic appendectomy during their bariatric surgeries, while comparing them to patients whose appendixes had remained intact. A case-control observational prospective study of 40 patients who underwent bariatric surgery, with or without an incidental prophylactic appendectomy, during 2004–2008 with an evaluation of their gut microbiota populations at the end of 2016 was conducted by sequencing the 16 S rRNA gene by Next Generation Sequencing of patients’ stools and appendix tissues. Patients with their appendix removed showed lower levels of richness and diversity of their gut microbiota populations. Odoribacter, Bilophila, Butyricimonas, and Faecalibacterium levels were increased in the Intact group, while Lachnobacterium suffered an expansion in the group without the appendix. Moreover, a linear regression model introduced the concept that Butyricimonas and Odoribacter may be implicated in insulin regulation. Thus, gut microbiota should be considered in the decisions of practical surgery, regarding the appendix as a mediator of homeostasis in the host. Butyricimonas and Odoribacter require further investigation as key bacteria implicated in insulin regulation. Full article

A flagellate, rod–shaped bacterium designated strain M15^T was isolated from rice roots. Phylogenetic analysis based on the sequences of the 16S rRNA, housekeeping genes and genomes showed that the isolate belonged to the genus Rhizobium, with the highest 16S rRNA similarity to Rhizobium radiobacter LMG140^T (99.64%) and Rhizobium pusense NRCPB10^T (99.36%), respectively. The complete genome of the strain M15^T has a 59.28% G+C content, and the highest average nucleotide identity (ANI) and DNA-DNA relatedness (DDH) values were obtained with R. radiobacter LMG140^T (88.11%, 54.80%), R. pusense NRCPB10^T (86.00%, 53.00%) and R. nepotum 39/7^T (88.80%, 49.80%), respectively. Plant growth-promoting characteristics tests showed that the strain M15^T produced siderophore, 1–aminocyclopropane–1–carboxylate (ACC) deaminase and indole-3-acetic acid (IAA) and also produced some secondary metabolites according to the analysis of the comparative genomes. Based on the data mentioned above, we proposed that the strain M15^T represented a novel species of the genus Rhizobium, named Rhizobium oryzihabitans sp. nov. The type strain is M15^T (=JCM 32903^T  = ACCC 60121^T), and the strain M15^T can be a novel biofertilizer Rhizobium to reduce the use of synthetic fertilizers for plant growth promotion. Full article

Bacillus cereus produces many factors linked to pathogenesis and is recognized for causing gastrointestinal toxemia and infections. B. cereus also causes a fulminant and often blinding intraocular infection called endophthalmitis. We reported that the PlcR/PapR system regulates intraocular virulence, but the specific factors that contribute to B. cereus virulence in the eye remain elusive. Here, we compared gene expression in ex vivo vitreous humor with expression in Luria Bertani (LB) and Brain Heart Infusion (BHI) broth by RNA-Seq. The expression of several cytolytic toxins in vitreous was less than or similar to levels observed in BHI or LB. Regulators of virulence genes, including PlcR/PapR, were expressed in vitreous. PlcR/PapR was expressed at low levels, though we reported that PlcR-deficient B. cereus was attenuated in the eye. Chemotaxis and motility genes were expressed at similar levels in LB and BHI, but at low to undetectable levels in vitreous, although motility is an important phenotype for B. cereus in the eye. Superoxide dismutase, a potential inhibitor of neutrophil activity in the eye during infection, was the most highly expressed gene in vitreous. Genes previously reported to be important to intraocular virulence were expressed at low levels in vitreous under these conditions, possibly because in vivo cues are required for higher level expression. Genes expressed in vitreous may contribute to the unique virulence of B. cereus endophthalmitis, and future analysis of the B. cereus virulome in the eye will identify those expressed in vivo, which could potentially be targeted to arrest virulence. Full article

The conventional perception that the zone of sulfate reduction and methanogenesis are separated in high- and low-sulfate-containing marine sediments has recently been changed by studies demonstrating their co-occurrence in sediments. The presence of methanogens was linked to the presence of substrates that are not used by sulfate reducers. In the current study, we hypothesized that both groups can co-exist, consuming common substrates (H₂ and/or acetate) in sediments. We enriched butyrate-degrading communities in sediment slurries originating from the sulfate, sulfate–methane transition, and methane zone of Aarhus Bay, Denmark. Sulfate was added at different concentrations (0, 3, 20 mM), and the slurries were incubated at 10 °C and 25 °C. During butyrate conversion, sulfate reduction and methanogenesis occurred simultaneously. The syntrophic butyrate degrader Syntrophomonas was enriched both in sulfate-amended and in sulfate-free slurries, indicating the occurrence of syntrophic conversions at both conditions. Archaeal community analysis revealed a dominance of Methanomicrobiaceae. The acetoclastic Methanosaetaceae reached high relative abundance in the absence of sulfate, while presence of acetoclastic Methanosarcinaceae was independent of the sulfate concentration, temperature, and the initial zone of the sediment. This study shows that there is no vertical separation of sulfate reducers, syntrophs, and methanogens in the sediment and that they all participate in the conversion of butyrate. Full article

A halophilic archaeon, strain F20-122^T, was isolated from a marine saltern of Isla Bacuta (Huelva, Spain). Cells were Gram-stain-negative, aerobic, and coccoid in morphology. It grew at 25–50 °C (optimum 37 °C), pH 6.5–9.0 (optimum pH 8.0), and 10–30% (w/v) total salts (optimum 25% salts). The phylogenetic analyses based on the 16S rRNA and rpoB’ genes showed its affiliation with the genus Natronomonas and suggested its placement as a new species within this genus. The in silico DNA–DNA hybridization (DDH) and average nucleotide identity (ANI) analyses of this strain against closely related species supported its placement in a new taxon. The DNA G + C content of this isolate was 63.0 mol%. The polar lipids of strain F20-122^T were phosphatidylglycerol phosphate methyl ester (PGP-Me), phosphatidylglycerol (PG), and phosphatidylglycerol sulfate (PGS). Traces of biphosphatidylglycerol (BPG) and other minor phospholipids and unidentified glycolipids were also present. Based on the phylogenetic, genomic, phenotypic, and chemotaxonomic characterization, we propose strain F20-122^T (= CCM 8891^T = CECT 9564^T = JCM 33320^T) as the type strain of a new species within the genus Natronomonas, with the name Natronomonas salsuginis sp. nov. Rhodopsin-like sequence analysis of strain F20-122^T revealed the presence of haloarchaeal proton pumps, suggesting a light-mediated ATP synthesis for this strain and a maximum wavelength absorption in the green spectrum. Full article

Understanding the coral microbiome is critical for predicting the fidelity of coral symbiosis with growing surface seawater temperature (SST). However, how the coral microbiome will respond to increasing SST is still understudied. Here, we compared the coral microbiome assemblages among 73 samples across six typical South China Sea coral species in two thermal regimes. The results revealed that the composition of microbiome varied across both coral species and thermal regimes, except for Porites lutea. The tropical coral microbiome displayed stronger heterogeneity and had a more un-compacted ecological network than subtropical coral microbiome. The coral microbiome was more strongly determined by environmental factors than host specificity. γ- (32%) and α-proteobacteria (19%), Bacteroidetes (14%), Firmicutes (14%), Actinobacteria (6%) and Cyanobacteria (2%) dominated the coral microbiome. Additionally, bacteria inferred to play potential roles in host nutrients metabolism, several keystone bacteria detected in human and plant rhizospheric microbiome were retrieved in explored corals. This study not only disentangles how different host taxa and microbiome interact and how such an interaction is affected by thermal regimes, but also identifies previously unrecognized keystone bacteria in corals, and also infers the community structure of coral microbiome will be changed from a compacted to an un-compacted network under elevated SST. Full article

The type A trichothecene NX-3, produced by certain Fusarium graminearum strains, is similar to the mycotoxin deoxynivalenol (DON), with the exception that it lacks the carbonyl moiety at the C-8 position. NX-3 inhibits protein biosynthesis and induces cytotoxicity to a similar extent as DON, but so far, immunomodulatory effects have not been assessed. In the present study, we investigated the impact of NX-3 on the activity of the nuclear factor kappa B (NF-κB) signaling pathway in direct comparison to DON. Under pro-inflammatory conditions (IL-1β treatment), the impact on cytokine mRNA levels of NF-κB downstream genes was studied in human colon cell lines, comparing noncancer (HCEC-1CT) and cancer cells (HT-29). In addition, potential combinatory effects with the co-occurring Fusarium secondary metabolite aurofusarin (AURO), a dimeric naphthoquinone known to induce oxidative stress, were investigated. NX-3 and DON (1 μM, 20 h) significantly activated a NF-κB regulated reporter gene to a similar extent. Both trichothecenes also enhanced transcript levels of the known NF-κB-dependent pro-inflammatory cytokines IL-8, IL-6, TNF-α and IL-1β. Comparing the colon cancer HT-29 and noncancer HCEC-1CT cells, significant differences in cytokine signaling were identified. In contrast, AURO did not affect NF-κB pathway activity and respective cytokine expression levels at the tested concentration. Despite its pro-oxidant potency, the combination with AURO did not significantly affect the immunomodulatory effects of the tested trichothecenes. Taken together, the present study reveals comparable potency of DON and NX-3 with respect to immunomodulatory and pro-inflammatory potential. Consequently, not only DON but also NX-3 should be considered as factors contributing to intestinal inflammatory processes. Full article

Arbuscular mycorrhizal fungi (AMF) have been shown to reduce plant stress and improve their health and growth, making them important components of the plant-root associated microbiome, especially in stressful conditions such as petroleum hydrocarbons (PHs) contaminated environments. Purposely manipulating the root-associated AMF assemblages in order to improve plant health and modulate their interaction with the rhizosphere microbes could lead to increased agricultural crop yields and phytoremediation performance by the host plant and its root-associated microbiota. In this study, we tested whether repeated inoculations with a Proteobacteria consortium influenced plant productivity and the AMF assemblages associated with the root and rhizosphere of four plant species growing either in non-contaminated natural soil or in sediments contaminated with petroleum hydrocarbons. A mesocosm experiment was performed in a randomized complete block design in four blocks with two factors: (1) substrate contamination (contaminated or not contaminated), and (2) inoculation (or not) with a bacterial consortium composed of ten isolates of Proteobacteria. Plants were grown in a greenhouse over four months, after which the effect of treatments on plant biomass and petroleum hydrocarbon concentrations in the substrate were determined. MiSeq amplicon sequencing, targeting the 18S rRNA gene, was used to assess AMF community structures in the roots and rhizosphere of plants growing in both contaminated and non-contaminated substrates. We also investigated the contribution of plant identity and biotope (plant roots and rhizospheric soil) in shaping the associated AMF assemblages. Our results showed that while inoculation caused a significant shift in AMF communities, the substrate contamination had a much stronger influence on their structure, followed by the biotope and plant identity to a lesser extent. Moreover, inoculation significantly increased plant biomass production and was associated with a decreased petroleum hydrocarbons dissipation in the contaminated soil. The outcome of this study provides knowledge on the factors influencing the diversity and community structure of AMF associated with indigenous plants following repeated inoculation of a bacterial consortium. It highlights the dominance of soil chemical properties, such as petroleum hydrocarbon presence, over biotic factors and inputs, such as plant species and microbial inoculations, in determining the plant-associated arbuscular mycorrhizal fungi communities. Full article

Pleurotus ostreatus is a species of white-rot fungi that effectively degrades lignin. In this study, we aimed to efficiently express the lac-2 gene of Pleurotus ostreatus in the Pichia pastoris X33 yeast strain. The enzymatic properties of recombinant yeast were determined, and its ability to degrade corn stover lignin was determined. The results showed the optimum pH values of recombinant laccase for 2,2’-Azinobis-3-ethylbenzothiazoline-6-sulfonic acid, 2,6-dimethoxyphenol, and 2-methoxyphenol were 3.0, 3.0, and 3.5, respectively. The optimum reaction temperature was 50 °C, and it had good thermal stability and acid and alkali resistance. The degradation rate of lignin in corn stover by recombinant laccase was 18.36%, and the native Pleurotus ostreatus degradation rate was 14.05%, the difference between them is significant (p < 0.05). This experiment lays a foundation for the study of the degradation mechanism of lignin by laccase. Full article

During the winemaking process, alcoholic fermentation is carried out by a consortium of yeasts in which interactions occurs. The consequences of these interactions on the wine matrix have been widely described for several years with the aim of controlling the winemaking process as well as possible. In this review, we highlight the wide diversity of methodologies used to study these interactions, and their underlying mechanisms and consequences on the final wine composition and characteristics. The wide variety of matrix parameters, yeast couples, and culture conditions have led to contradictions between the results of the different studies considered. More recent aspects of modifications in the composition of the matrix are addressed through different approaches that have not been synthesized recently. Non-volatile and volatile metabolomics, as well as sensory analysis approaches are developed in this paper. The description of the matrix composition modification does not appear sufficient to explain interaction mechanisms, making it vital to take an integrated approach to draw definite conclusions on them. Full article

Vector-borne flaviviruses (VBFs) affect human health worldwide, but no approved drugs are available specifically to treat VBF-associated infections. Here, we performed in silico screening of a library of U.S. Food and Drug Administration-approved antiviral drugs for their interaction with Zika virus proteins. Twelve hit drugs were identified by the docking experiments and tested in cell-based antiviral assay systems. Efavirenz, tipranavir, and dasabuvir at micromolar concentrations were identified to inhibit all VBFs tested; i.e., two representatives of mosquito-borne flaviviruses (Zika and West Nile viruses) and one representative of flaviviruses transmitted by ticks (tick-borne encephalitis virus). The results warrant further research into these drugs, either individually or in combination, as possible pan-flavivirus inhibitors. Full article

Trace elements (TEs) are vital for anaerobic digestion (AD), due to their role as cofactors in many key enzymes. The aim of this study was to evaluate the effects of specific TE deficiencies on mixed microbial communities during AD of soluble polymer-free substrates, thus focusing on AD after hydrolysis. Three mesophilic (37 °C) continuous stirred-tank biogas reactors were depleted either of Co, Ni, or a combination of Se and W, respectively, by discontinuing their supplementation. Ni and Se/W depletion led to changes in methane kinetics, linked to progressive volatile fatty acid (VFA) accumulation, eventually resulting in process failure. No significant changes occurred in the Co-depleted reactor, indicating that the amount of Co present in the substrate in absence of supplementation was sufficient to maintain process stability. Archaeal communities remained fairly stable independent of TE concentrations, while bacterial communities gradually changed with VFA accumulation in Ni- and Se-/W-depleted reactors. Despite this, the communities remained relatively similar between these two reactors, suggesting that the major shifts in composition likely occurred due to the accumulating VFAs. Overall, the results indicate that Ni and Se/W depletion primarily lead to slower metabolic activities of methanogenic archaea and their syntrophic partners, which then has a ripple effect throughout the microbial community due to a gradual accumulation of intermediate fermentation products. Full article

Objective: The aims of this study are as follows: (1) to understand the relationship between gut microbiota and the choking phenomenon in diving athletes, and (2) to regulate the gut microbiota in diving athletes by drinking yogurt containing Bifidobacterium animalis subsp. lactis BB-12 and observe changes in the choking phenomenon in diving athletes. Methods: Experiment 1: A total of 20 diving athletes were tested in low- and high-pressure situations. Gut microbiota (n = 18) composition was then determined and differences in the gut microbiota composition among diving athletes who presented choking vs. no choking were identified. Experiment 2: A total of 16 divers who presented choking were divided into a high yogurt group (n = 6) and a low yogurt group (n = 10) for 15 days. Results: (1) The content of Veillonellaceae in divers who presented choking was significantly higher when compared to divers who did not present choking (p < 0.05). Bifidobacteriaceae (r = −0.52, p < 0.05) and Lactobacillaceae (r = −0.66, p < 0.05) were negatively correlated with the choking index. (2) During experiment 2, the average daily intake of the high yogurt group was 611.78 ± 94.94 mL and the average daily intake of the low yogurt group was 338 ± 71.45 mL and the abundance of Bifidobacteriaceae was significantly higher in the high yogurt group than in the low yogurt group. After the experiment, the choking index in the high yogurt group became significantly lower than that of the low yogurt group (z = −3.26, p < 0.001). Conclusion: The intake of yogurt containing B. animalis subsp. lactis can increase the abundance of Bifidobacteriaceae in elite diving athletes and their performance under high pressure. Hence, gut microbiota may affect the choking phenomenon in elite diving athletes. Full article

The sandfly-borne Toscana phlebovirus (TOSV), a close relative of the sandfly fever Sicilian phlebovirus (SFSV), is one of the most common causes of acute meningitis or meningoencephalitis in humans in the Mediterranean Basin. However, most of human phlebovirus infections in endemic areas either are asymptomatic or cause mild influenza-like illness. To date, a vertebrate reservoir for sandfly-borne phleboviruses has not been identified. Dogs are a prime target for blood-feeding phlebotomines and are the primary reservoir of human sandfly-borne Leishmania infantum. However, there are no definitive studies to assess whether dogs play a significant role as a reservoir host for human phlebovirus survival in the environment. Here, we have evaluated the susceptibility of domestic dogs to infection by TOSV and SFSV following the direct inoculation of the infectious virus. After experimental infection, the presence of viral RNA was investigated in plasma, urine, saliva, conjunctiva, faeces, semen, and bone marrow samples from 0 to 91 days postinoculation (dpi), as well as in plasma, saliva, and tears samples at 760 dpi. None of the challenged dogs developed clinical signs of infection with either TOSV or SFSV. SFSV RNA was never detected. TOSV RNA was not in any of the specimen types, except for plasma samples that showed low viral loads, although irregularly. None of the dogs developed detectable neutralizing antibodies after a single challenge dose of either TOSV or SFSV. However, a second challenge dose of virus given 56 days later elicited neutralizing antibodies, implying that the first inoculation of virus primed the animals for an anamnestic response following the second challenge. These results demonstrated that healthy domestic dogs are not highly susceptible to infection by TOSV or SFSV and do not develop significant viremia or excrete virus following infection. Consequently, dogs are unlikely natural reservoir hosts of infection and do not appear to play a significant role in phlebovirus transmission cycles. Full article