Next-Generation Sequencing for Characterizing Respiratory Tract Virome and Improving Detection of Viral Pathogens in Children With Pneumonia.

BACKGROUND: Pneumonia is typically caused by a variety of pathogenic microorganisms. Traditional research often focuses on the infection of a few microorganisms, whereas metagenomic studies focus on the impact of the bacteriome and mycobiome on respiratory diseases. Reports on the virome characteristics of pediatric pneumonia remain relatively scarce. METHODS: We employed de novo assembly and combined homology- and feature-based methods to characterize the respiratory virome in whole-genome DNA sequencing samples from oropharynx (OP) swabs, nasopharynx (NP) swabs, and bronchoalveolar lavage fluids (BALF) of children with pneumonia. RESULTS: Significant differences were observed in the alpha and beta diversity indexes, as well as in the composition of the oropharyngeal virome, between pneumonia cases and controls. We identified 1137 viral operational taxonomic units (vOTUs) with significant differences, indicating a preference of pneumonia-reduced vOTUs for infecting Prevotella, Neisseria, and Veillonella, whereas pneumonia-enriched vOTUs included polyomavirus, human adenovirus, and phages targeting Staphylococcus, Streptococcus, Granulicatella, and Actinomyces. Comparative analysis revealed higher relative abundances and prevalence rates of pneumonia-enriched OP vOTUs in NP and BALF samples compared to pneumonia-reduced vOTUs. Additionally, virome analysis identified six pediatric patients with severe human adenovirus or polyomavirus infections, five of whom might have been undetected by targeted polymerase chain reaction (PCR)-based testing. CONCLUSIONS: This study offers insights into pediatric pneumonia respiratory viromes, highlighting frequent transmission of potentially pathogenic viruses and demonstrating virome analysis as a valuable adjunct for pathogen detection.

Bacillus halotolerans attenuates inflammation induced by enterotoxigenic Escherichia coli infection in vivo and in vitro based on its metabolite soyasaponin I regulating the p105-Tpl2-ERK pathway.

Soyasaponins, recognized for their anti-inflammatory and antioxidant effects, have not yet been fully explored for their role in combating enterotoxigenic Escherichia coli (ETEC) infections. Recent findings identified them in small-molecule metabolites of Bacillus, suggesting their broader biological relevance. This research screened 88 strains of B. halotolerans, identifying the strain BH M20221856 as significantly inhibitory against ETEC growth in vitro. It also reduced cellular damage and inflammatory response in IPEC-J2 cells. The antimicrobial activity of BH M20221856 was attributed to its small-molecule metabolites rather than secretory proteins. A total of 69 small molecules were identified from the metabolites of BH M20221856 using liquid chromatography mass spectrometry/mass spectrometry (LC-MS/MS). Among these, soyasaponin I (SoSa I) represented the largest multiple change in the enrichment analysis of differential metabolites and exhibited potent anti-ETEC effects in vivo. It significantly reduced the bacterial load of E. coli in mouse intestines, decreased serum endotoxin, D-lactic acid, and oxidative stress levels and alleviated intestinal pathological damage and inflammation. SoSa I enhanced immune regulation by mediating the p105-Tpl2-ERK signaling pathway. Further evaluations using transepithelial electrical resistance (TEER) and cell permeability assays showed that SoSa I alleviated ETEC-induced damage to epithelial barrier function. These results suggest that BH M20221856 and SoSa I may serve as preventative biologics against ETEC infections, providing new insights for developing strategies to prevent and control this disease.

A multi-kingdom collection of 33,804 reference genomes for the human vaginal microbiome.

The human vagina harbours diverse microorganisms-bacteria, viruses and fungi-with profound implications for women's health. Genome-level analysis of the vaginal microbiome across multiple kingdoms remains limited. Here we utilize metagenomic sequencing data and fungal cultivation to establish the Vaginal Microbial Genome Collection (VMGC), comprising 33,804 microbial genomes spanning 786 prokaryotic species, 11 fungal species and 4,263 viral operational taxonomic units. Notably, over 25% of prokaryotic species and 85% of viral operational taxonomic units remain uncultured. This collection significantly enriches genomic diversity, especially for prevalent vaginal pathogens such as BVAB1 (an uncultured bacterial vaginosis-associated bacterium) and Amygdalobacter spp. (BVAB2 and related species). Leveraging VMGC, we characterize functional traits of prokaryotes, notably Saccharofermentanales (an underexplored yet prevalent order), along with prokaryotic and eukaryotic viruses, offering insights into their niche adaptation and potential roles in the vagina. VMGC serves as a valuable resource for studying vaginal microbiota and its impact on vaginal health.

A naturally isolated symbiotic bacterium suppresses flavivirus transmission by Aedes mosquitoes.

The commensal microbiota of the mosquito gut plays a complex role in determining the vector competence for arboviruses. In this study, we identified a bacterium from the gut of field Aedes albopictus mosquitoes named Rosenbergiella sp. YN46 (Rosenbergiella_YN46) that rendered mosquitoes refractory to infection with dengue and Zika viruses. Inoculation of 1.6 × 103 colony forming units (CFUs) of Rosenbergiella_YN46 into A. albopictus mosquitoes effectively prevents viral infection. Mechanistically, this bacterium secretes glucose dehydrogenase (RyGDH), which acidifies the gut lumen of fed mosquitoes, causing irreversible conformational changes in the flavivirus envelope protein that prevent viral entry into cells. In semifield conditions, Rosenbergiella_YN46 exhibits effective transstadial transmission in field mosquitoes, which blocks transmission of dengue virus by newly emerged adult mosquitoes. The prevalence of Rosenbergiella_YN46 is greater in mosquitoes from low-dengue areas (52.9 to ~91.7%) than in those from dengue-endemic regions (0 to ~6.7%). Rosenbergiella_YN46 may offer an effective and safe lead for flavivirus biocontrol.

Isolation and Genomic Characterization of Kadipiro Virus from Mosquitoes in Yunnan, China.

Background: Kadipiro virus (KDV) is a species of the new 12 segmented RNA virus grouped under the genus Seadornavirus within the Reoviridae family. It has previously been isolated or detected from mosquito, Odonata, and bat feces in Indonesia, China, and Denmark, respectively. Here, we describe the isolation and characterization of a viral strain from mosquitoes in Yunnan Province, China. Methods: Mosquitoes were collected overnight using light traps in Shizong county, on July 17, 2023. Virus was isolated from the mosquito homogenate and grown using baby hamster kidney and Aedes albopictus (C6/36) cells. Preliminary identification of the virus was performed by agarose gel electrophoresis (AGE). The full-genome sequences of the strain were determined by full-length amplification of cDNAs and sequenced using next-generation sequencing. Results: We isolated a viral strain (SZ_M48) from mosquitoes (Culex tritaeniorhynchus Giles) that caused cytopathogenic effects in C6/36 cells. AGE analysis indicated a genome consisting of 12 segments of double-stranded RNA that demonstrated a "6-5-1" pattern, similar to the migrating bands of KDV. Phylogenetic analysis based on the full-genome sequence revealed that SZ_M48 is more clustered with KDV isolates from Hubei and Shangdong in China than with Indonesian and Danish strains. The identity between SZ_M48 and SDKL1625 (Shandong, China) is slightly lower than that of QTM27331 (Hubei, China), and the identity with JKT-7075 (Indonesia) and 21164-6/M.dau/DK (Denmark) is the lowest. Conclusion: The full-genome sequence of the new KDV strain described in this study may be useful for surveillance of the evolutionary characteristics of KDVs. Moreover, these findings extend the knowledge about the genomic diversity, potential vectors, and the distribution of KDVs in China.

Isolation of Epizootic Hemorrhagic Disease Virus Serotype 10 from Culicoides tainanus and Associated Infections in Livestock in Yunnan, China.

Two strains of viruses, JC13C644 and JC13C673, were isolated from Culicoides tainanus collected in Jiangcheng County, Yunnan Province, situated along the border area shared by China, Laos, and Vietnam. JC13C644 and JC13C673 viruses can cause cytopathic effect (CPE) in mammalian cells BHK21 and Vero cells, and cause morbidity and mortality in suckling mice 48 h after intracerebral inoculation. Whole-genome sequencing was performed, yielding complete sequences for all 10 segments from Seg-1 (3942nt) to Seg-10 (810nt). Phylogenetic analysis of the sub-core-shell (T2) showed that the JC13C644 and JC13C673 viruses clustered with the Epizootic Hemorrhagic Disease Virus (EHDV) isolated from Japan and Australia, with nucleotide and amino acid homology of 93.1% to 98.3% and 99.2% to 99.6%, respectively, suggesting that they were Eastern group EHDV. The phylogenetic analysis of outer capsid protein (OC1) and outer capsid protein (OC2) showed that the JC13C644 and JC13C673 viruses were clustered with the EHDV-10 isolated from Japan in 1998, with the nucleotide homology of 98.3% and 98.5%, and the amino acid homology of 99.6% and 99.6-99.8%, respectively, indicating that they belong to the EHDV-10. Seroepidemiological survey results demonstrated that JC13C644 virus-neutralizing antibodies were present in 29.02% (177/610) of locally collected cattle serum and 11.32% (89/786) of goat serum, implying the virus's presence in Jiangcheng, Yunnan Province. This finding suggests that EHDV-10 circulates not only among blood-sucking insects in nature but also infects local domestic animals in China. Notably, this marks the first-ever isolation of the virus in China and its discovery outside of Japan since its initial isolation from Japanese cattle. In light of these results, it is evident that EHDV Serotype 10 exists beyond Japan, notably in the natural vectors of southern Eurasia, with the capacity to infect local cattle and goats. Therefore, it is imperative to intensify the surveillance of EHDV infection in domestic animals, particularly focusing on the detection and monitoring of new virus serotypes that may emerge in the region and pose risks to animal health.

Dubosiella newyorkensis modulates immune tolerance in colitis via the L-lysine-activated AhR-IDO1-Kyn pathway.

Commensal bacteria generate immensely diverse active metabolites to maintain gut homeostasis, however their fundamental role in establishing an immunotolerogenic microenvironment in the intestinal tract remains obscure. Here, we demonstrate that an understudied murine commensal bacterium, Dubosiella newyorkensis, and its human homologue Clostridium innocuum, have a probiotic immunomodulatory effect on dextran sulfate sodium-induced colitis using conventional, antibiotic-treated and germ-free mouse models. We identify an important role for the D. newyorkensis in rebalancing Treg/Th17 responses and ameliorating mucosal barrier injury by producing short-chain fatty acids, especially propionate and L-Lysine (Lys). We further show that Lys induces the immune tolerance ability of dendritic cells (DCs) by enhancing Trp catabolism towards the kynurenine (Kyn) pathway through activation of the metabolic enzyme indoleamine-2,3-dioxygenase 1 (IDO1) in an aryl hydrocarbon receptor (AhR)-dependent manner. This study identifies a previously unrecognized metabolic communication by which Lys-producing commensal bacteria exert their immunoregulatory capacity to establish a Treg-mediated immunosuppressive microenvironment by activating AhR-IDO1-Kyn metabolic circuitry in DCs. This metabolic circuit represents a potential therapeutic target for the treatment of inflammatory bowel diseases.

Potential roles of Culicoides spp. (Culicoides imicola, Culicoides oxystoma) as biological vectors of bluetongue virus in Yuanyang of Yunnan, P. R. China.

Introduction: Culicoides plays a crucial role as an insect vector in the field of veterinary medicine. The transmission of significant viruses such as bluetongue virus (BTV) and African horse sickness virus (AHSV) by this insect poses a substantial threat, leading to the development of severe diseases in domestic animals. This study aimed to explore the Culicoides species, identify their blood-meal sources, and assess the presence of BTV and AHSV carried by Culicoides in Yuanyang County, Yunnan Province. The aim was to gain insights into the potential vectors of these two viruses and elucidate their potential roles in the transmission of pathogens. Methods: The midges were collected from cattle (Bos indicus), pig (Sus scrofa), and goat (Capra hircus) pens in Yuanyang County, Yunnan Province in June 2020. Initial identification of midges was conducted through morphological characteristics, followed by molecular identification using the cytochrome C oxidase subunit I (COI) gene. The determination of Culicoides blood-meal sources was accomplished using specific primers targeting the cytochrome b (Cyt b) gene from potential hosts. BTV and AHSV RNA were identified in Culicoides pools through the application of reverse transcriptase PCR and quantitative real-time PCR. Nucleotide homology and phylogenetic analysis were performed using MegAlign (DNAStar) and Mega 6.0 software. Results: A total of 6,300 Culicoides, consisting of C. oxystoma, C. arakawai, C. imicola, and C. innoxius, were collected from cattle, pigs, and goat pens. The engorgement rates for these species were 30.2%, 54.6%, 75%, and 66.7%, respectively. In the cattle pen, the prevailing species is C. oxystoma (100%). In the pig pen, C. arakawai dominates (70%), with C. oxystoma following at 30%. In the goat pen, C. imicola holds the majority (45.45%), trailed by C. oxystoma (25%), C. innoxius (20.45%), and C. arakawai (9.09%). These Culicoides species were identified as feeding on cattle, pigs, goats, chickens (Gallus gallus), and humans (Homo sapiens). The positivity rates for BTV were 20.00% and 11.54% in blood-fed specimens of C. imicola and C. oxystoma, respectively. Conversely, the positivity rates for BTV in non-blood-fed specimens were 0.00% and 6.67% for C. imicola and C. oxystoma, respectively. BTV was not detected in C. arakawai and C. innoxius. The specimens (YY86) from C. imicola that tested positive for BTV had the closest genetic relationship to YTS-4 isolated from Mangshi, Yunnan Province in 1996. All test results for the nucleic acid of AHSV were negative. Conclusion: The study reveals variations in the species distribution, community composition, blood sucking rate, and blood-feeding sources of Culicoides across different habitats. Notably, C. imicola and C. oxystoma emerge as potential vectors for the transmission of BTV in local animals. Accordingly, this investigation provides crucial insights that can serve as a valuable reference for the prevention and control of BTV in local animals, particularly from the perspective of vector management.