The Department of Defense recently began an effort to improve and standardize virus challenge materials and efficacy determination strategies for testing therapeutics and vaccines. This includes stabilization of virus genome sequences in cDNA form where appropriate, use of human-derived virus isolates, and noninvasive strategies for determination of challenge virus replication. Eventually, it is desired that these approaches will satisfy the FDA \"Animal Rule\" for licensure, which substitutes animal efficacy data when human data are unlikely to be available. To this end, we created and examined the virulence phenotype of cDNA clones of prototypic human infection-derived strains of the alphaviruses, Venezuelan (VEEV INH9813), eastern (EEEV V105) and western (WEEV Fleming) equine encephalitis viruses, and created fluorescent and luminescent reporter expression vectors for evaluation of replication characteristics in vitro and in vivo. Sequences of minimally passaged isolates of each virus were used to synthesize full-length cDNA clones along with a T7 transcription promoter-based bacterial propagation vector. Viruses generated from the cDNA clones were compared with other \"wild type\" strains derived from cDNA clones and GenBank sequences to identify and eliminate putative tissue culture artifacts accumulated in the cell passaged biological stocks. This was followed by examination of aerosol and subcutaneous infection and disease in mouse models. A mutation that increased heparan sulfate binding was identified in the VEEV INH9813 biological isolate sequence and eliminated from the cDNA clone. Viruses derived from the new human isolate cDNA clones showed similar mouse virulence to existing clone-derived viruses after aerosol or subcutaneous inoculation.

Nitric oxide (NO) is a reactive nitrogen species (RNS) that plays a vital role in regulating inflammatory processes. Under abnormal conditions, excessive NO levels can promote the oxidation of cellular components, which may cause or exacerbate diseases such as hypertension, cardiovascular dysfunction, and inflammatory bowel disease (IBD). Previous studies have shown that reducing NO levels in the lumen can attenuate the clinical symptoms of IBD. Thus, we aimed to identify bacteria that can reduce RNS and that can be used as valuable probiotics. In this study, we isolated bacteria resistant to nitrite stress from human feces and used 16S and whole-genome sequencing to identify them as Lactiplantibacillus plantarum LP7 (LP7). The ability to survive at high nitrite levels and to decrease them was greater in the LP7 strain than in the reference strain L. plantarum ATCC14917 (ATCC14917). To characterize the LP7 genome in more detail, we performed a comparative genome analysis. However, the unique genes that directly confer the ability to withstand nitrite stress were not present in the LP7 genome. Furthermore, we performed transcriptomic analysis of LP7 and ATCC14917 cells treated with nitrite. We found that the expression levels of genes involved in the cell division process were induced in LP7, which showed a more regular rod-shape than ATCC14917. This could explain why LP7 can survive better than ATCC14917 under nitrite stress. Based on its ability to survive better in nitrite stress and decrease nitrite concentration, we suggest that LP7 could be a valuable probiotic strain.

Cutibacterium modestum is a new species coined in 2020 as the fifth species of genus Cutibacterium, which includes Cutibacterium acnes. The species is predicted as a minor but common member of skin microbiome and includes a group tentatively named as \"Propionibacterium humerusii\". The description of the species has been provided only with a single strain. To establish the characteristics of C. modestum and search for possible disease-related subtypes, we investigated the biochemical characteristics of eight live strains and performed in silico comparison of nine genomes. The common features, which included the morphology of Gram-stain positive short rods, the negativity of phenylalanine arylamidase, and several unique MALDI-TOF MS spectral peaks, were considered useful in laboratory identification. Pairwise comparisons of the genomes by in silico DNA-DNA hybridization showed similarity values of 98.1% or larger, which were far higher than the subspecies cutoff of 79-80%. The 16S rRNA gene sequences of thirteen isolates and genomes were identical. Their recA gene sequences were identical except for two strains, HM-510 (HL037PA2) and Marseille-P5998, which showed unique one-nucleotide polymorphisms. The biochemical features using API kits were slightly different among the isolates but far closer than those of the nearest other species, C. acnes and Cutibacterium namnetense. Spectra of MALDI-TOF mass spectrometry showed slight differences in the presence of m/z 10,512 (10 kD chaperonin GroS) and three other peaks, further clustering the eight isolates into three subtypes. These results indicated that these isolates did not separate to form subspecies-level clusters, but subtyping is possible by using recA gene sequences or MALDI-TOF mass spectrometry spectra. Moreover, this work has confirmed that a group \"P. humerusii\" is included in C. modestum.

Rhizopus spp. are the most common etiological agents of mucormycosis, causing over 90% mortality in disseminated infections. The diagnosis relies on histopathology, culture, and/or polymerase chain reaction. For the first time, the glycosylation of rhizoferrin (RHF) was described in a Rhizopus microsporus clinical isolate by liquid chromatography and accurate tandem mass spectrometry. The fermentation broth lyophilizate contained 345.3 ± 13.5, 1.2 ± 0.03, and 0.03 ± 0.002 mg/g of RHF, imido-RHF, and bis-imido-RHF, respectively. Despite a considerable RHF secretion rate, we did not obtain conclusive RHF detection from a patient with disseminated mucormycosis caused by the same R. microsporus strain. We hypothesize that parallel antimycotic therapy, RHF biotransformation, and metabolism compromised the analysis. On the other hand, the full profile of posaconazole metabolites was retrieved by our in house software CycloBranch.

Elizabethkingia meningoseptica is an emerging, healthcare-associated pathogen causing a high mortality rate in immunocompromised patients. We report the draft genome sequence of E. meningoseptica Em3, isolated from sputum from a patient with multiple underlying diseases. The genome has a length of 4,037,922 bp, a GC-content 36.4%, and 3673 predicted protein-coding sequences. Average nucleotide identity analysis (>95%) assigned the bacterium to the species E. meningoseptica. Genome analysis showed presence of the curli formation and assembly operon and a gene encoding hemagglutinins, indicating ability to form biofilm. In vitro biofilm assays demonstrated that E. meningoseptica Em3 formed more biofilm than E. anophelis Ag1 and E. miricola Emi3, both lacking the curli operon. A gene encoding thiol-activated cholesterol-dependent cytolysin in E. meningoseptica Em3 (potentially involved in lysing host immune cells) was also absent in E. anophelis Ag1 and E. miricola Emi3. Strain Em3 showed α-hemolysin activity on blood agar medium, congruent with presence of hemolysin and cytolysin genes. Furthermore, presence of heme uptake and utilization genes demonstrated adaptations for bloodstream infections. Strain Em3 contained 12 genes conferring resistance to β-lactams, including β-lactamases class A, class B, and metallo-β-lactamases. Results of comparative genomic analysis here provide insights into the evolution of E. meningoseptica Em3 as a pathogen.

Enterococcus faecium, traditionally considered a harmless gut commensal, is emerging as an important nosocomial pathogen showing increasing rates of multidrug resistance. We report the draft genome sequence of E. faecium strain LMG 8148, isolated in 1968 from a human in Gothenburg, Sweden. The draft genome has a total length of 2,697,490 bp, a GC-content of 38.3 %, and 2,402 predicted protein-coding sequences. The isolation of this strain predates the emergence of E. faecium as a nosocomial pathogen. Consequently, its genome can be useful in comparative genomic studies investigating the evolution of E. faecium as a pathogen.

Acinetobacter baumannii is a pathogen that is becoming increasingly important and causes serious hospital-acquired infections. We sequenced the genome of A. baumannii NCTC 13423, a multidrug-resistant strain belonging to the international clone II group, isolated from a human infection in the United Kingdom in 2003. The 3,937,944 bp draft genome has a GC-content of 39.0 % and a total of 3672 predicted protein-coding sequences. The availability of genome sequences of multidrug-resistant A. baumannii isolates will fuel comparative genomic studies to help understand the worrying spread of multidrug resistance in this pathogen.

Kyasanur Forest disease virus (KFDV) is a tick-borne Flavivirus that causes a severe illness in humans. Disease spectrum can vary from subclinical infection to fatal cases with hemorrhagic complications. The pathology of KFDV remains incompletely understood.
This study describes the histopathologic and immunohistochemical findings in experimentally infected infant CD-1 mice with an early passage human KFDV isolate.
Acute histological changes were primarily seen in the brain. The spectrum of changes included gliosis, inflammatory response, necrosis, neural loss, and syncytium formation in mid and hind brain structures. Microscopic lesions observed in the liver were mainly necrosis and vacuolation of hepatocytes and in small intestine, prominent epithelial cell necrosis. KFDV antigens could be stained by a sensitive immunohistochemical labeling in the same organs.
Findings from this study are suggestive of neuropathology as the main manifestation of an early passaged human KFDV isolate. Importantly, this suggests that KFDV may be causing primarily a neurologic disease and secondary organ damage could be because of disease pathology per se. The use of primary low passage human isolates and neuropathology profile could also be more apt in developing a challenge model for testing potential antivirals and therapeutic agents.

Runs of homozygosity (ROHs) are extended genomic regions of homozygous genotypes that record populations\' mating patterns in the past. We performed microarray genotyping on 15 individuals from a small isolated Tunisian community. We estimated the individual and population genome-wide level of homozygosity from data on ROH above 0.5 Mb in length. We found a high average number of ROH per individual (48.2). The smallest ROH category (0.5-1.49 Mb) represents 0.93% of the whole genome, while medium-size (1.5-4.99 Mb) and long-size ROH (≥5 Mb) cover 1.18% and 0.95%, respectively. We found that genealogical individual inbreeding coefficients (Fped ) based on three- to four-generation pedigrees are not reliable indicators of the current proportion of genome-wide homozygosity inferred from ROH (FROH ) either for 0.5 or 1.5 Mb ROH length thresholds, while identity-by-descent sharing is a function of shared coancestry. This study emphasizes the effect of reproductive isolation and a prolonged practice of consanguinity that limits the genetic heterogeneity. It also provides evidence of both recent and ancient parental relatedness contribution to the current level of genome-wide homozygosity in the studied population. These findings may be useful for evaluation of long-term effects of inbreeding on human health and for future applications of ROHs in identifying recessive susceptibility genes.

Anaplasma phagocytophilum, first identified as a pathogen of sheep in Europe, more recently has been recognized as an emerging tick-borne pathogen of humans in the U.S. and Europe. Transmission of A. phagocytophilum is reported to be by ticks, primarily of the genus Ixodes. While mechanical and transplacental transmission of the type genus organism, A. marginale, occur in addition to tick transmission, these modes of transmission have not been considered for A. phagocytophilum. Recently, we developed a sheep model for studying host-tick-pathogen interactions of the human NY-18 A. phagocytophilum isolate. Sheep were susceptible to infection with this human isolate and served as a source of infection for I. scapularis ticks, but they did not display clinical signs of disease, and the pathogen was not apparent in stained blood smears. In the course of these experiments, one sheep unexpectedly gave birth to a lamb 5 weeks after being experimentally infected by inoculation with the pathogen propagated in HL-60 cells. The lamb was depressed and not feeding and was subsequently euthanized 18 h after birth. Tissues were collected at necropsy for microscopic examination and PCR to confirm A. phagocytophilum infection. At necropsy, the stomach contained colostrum, the spleen was moderately enlarged and thickened with conspicuous lymphoid follicles, and mesenteric lymph nodes were mildly enlarged and contained moderate infiltrates of eosinophils and neutrophils. Blood, spleen, heart, skin and cervical and mesenteric lymph nodes tested positive for A. phagocytophilum by PCR, and sequence analysis confirmed that the lamb was infected with the NY-18 isolate. Transplacental transmission should therefore be considered as a means of A. phagocytophilum transmission and may likely contribute to the epidemiology of tick-borne fever in sheep and other mammals, including humans.