Identification and characterization of novel alphacoronaviruses in Tadarida brasiliensis (Chiroptera, Molossidae) from Argentina: insights into recombination as a mechanism favoring bat coronavirus cross-species transmission.

Bats are reservoirs of various coronaviruses that can jump between bat species or other mammalian hosts, including humans. This article explores coronavirus infection in three bat species (Tadarida brasiliensis, Eumops bonariensis, and Molossus molossus) of the family Molossidae from Argentina using whole viral metagenome analysis. Fecal samples of 47 bats from three semiurban or highly urbanized areas of the province of Santa Fe were investigated. After viral particle enrichment, total RNA was sequenced using the Illumina NextSeq 550 instrument; the reads were assembled into contigs and taxonomically and phylogenetically analyzed. Three novel complete Alphacoronavirus (AlphaCoV) genomes (Tb1-3) and two partial sequences were identified in T. brasiliensis (Tb4-5), and an additional four partial sequences were identified in M. molossus (Mm1-4). Phylogenomic analysis showed that the novel AlphaCoV clustered in two different lineages distinct from the 15 officially recognized AlphaCoV subgenera. Tb2 and Tb3 isolates appeared to be variants of the same virus, probably involved in a persistent infectious cycle within the T. brasiliensis colony. Using recombination analysis, we detected a statistically significant event in Spike gene, which was reinforced by phylogenetic tree incongruence analysis, involving novel Tb1 and AlphaCoVs identified in Eptesicus fuscus (family Vespertilionidae) from the U.S. The putative recombinant region is in the S1 subdomain of the Spike gene, encompassing the potential receptor-binding domain of AlphaCoVs. This study reports the first AlphaCoV genomes in molossids from the Americas and provides new insights into recombination as an important mode of evolution of coronaviruses involved in cross-species transmission. IMPORTANCE This study generated three novel complete AlphaCoV genomes (Tb1, Tb2, and Tb3 isolates) identified in individuals of Tadarida brasiliensis from Argentina, which showed two different evolutionary patterns and are the first to be reported in the family Molossidae in the Americas. The novel Tb1 isolate was found to be involved in a putative recombination event with alphacoronaviruses identified in bats of the genus Eptesicus from the U.S., whereas isolates Tb2 and Tb3 were found in different collection seasons and might be involved in persistent viral infections in the bat colony. These findings contribute to our knowledge of the global diversity of bat coronaviruses in poorly studied species and highlight the different evolutionary aspects of AlphaCoVs circulating in bat populations in Argentina.

The Activation of GPR27 Increases Cytosolic L-Lactate in 3T3 Embryonic Cells and Astrocytes.

G-protein-coupled receptors (GPCRs) represent a family with over 800 members in humans, and one-third of these are targets for approved drugs. A large number of GPCRs have unknown physiologic roles. Here, we investigated GPR27, an orphan GPCR belonging to the family of super conserved receptor expressed in the brain, with unknown functions. Cytosolic levels of L-lactate ([lactate]i), the end product of aerobic glycolysis, were measured with the Laconic fluorescence resonance energy transfer nanosensor. In single 3T3 wild-type (WT) embryonic cells, the application of 8535 (1 µM), a surrogate agonist known to activate GPR27, resulted in an increase in [lactate]i. Similarly, an increase was recorded in primary rat astrocytes, a type of neuroglial cell abundant in the brain, which contain glycogen and express enzymes of aerobic glycolysis. In CRISPR-Cas9 GPR27 knocked out 3T3 cells, the 8535-induced increase in [lactate]i was reduced compared with WT controls. Transfection of the GPR27-carrying plasmid into the 3T3KOGPR27 cells rescued the 8535-induced increase in [lactate]i. These results indicate that stimulation of GPR27 enhances aerobic glycolysis and L-lactate production in 3T3 cells and astrocytes. Interestingly, in the absence of GPR27 in 3T3 cells, resting [lactate]i was increased in comparison with controls, further supporting the view that GPR27 regulates L-lactate homeostasis.

Viral Metagenomic Data Analyses of Five New World Bat Species from Argentina: Identification of 35 Novel DNA Viruses.

Bats are natural reservoirs of a variety of zoonotic viruses, many of which cause severe human diseases. Characterizing viruses of bats inhabiting different geographical regions is important for understanding their viral diversity and for detecting viral spillovers between animal species. Herein, the diversity of DNA viruses of five arthropodophagous bat species from Argentina was investigated using metagenomics. Fecal samples of 29 individuals from five species (Tadarida brasiliensis, Molossus molossus, Eumops bonariensis, Eumops patagonicus, and Eptesicus diminutus) living at two different geographical locations, were investigated. Enriched viral DNA was sequenced using Illumina MiSeq, and the reads were trimmed and filtered using several bioinformatic approaches. The resulting nucleotide sequences were subjected to viral taxonomic classification. In total, 4,520,370 read pairs were sequestered by sequencing, and 21.1% of them mapped to viral taxa. Circoviridae and Genomoviridae were the most prevalent among vertebrate viral families in all bat species included in this study. Samples from the T. brasiliensis colony exhibited lower viral diversity than samples from other species of New World bats. We characterized 35 complete genome sequences of novel viruses. These findings provide new insights into the global diversity of bat viruses in poorly studied species, contributing to prevention of emerging zoonotic diseases and to conservation policies for endangered species.

A Preliminary Study of the Virome of the South American Free-Tailed Bats (Tadarida brasiliensis) and Identification of Two Novel Mammalian Viruses.

Bats provide important ecosystem services as pollinators, seed dispersers, and/or insect controllers, but they have also been found harboring different viruses with zoonotic potential. Virome studies in bats distributed in Asia, Africa, Europe, and North America have increased dramatically over the past decade, whereas information on viruses infecting South American species is scarce. We explored the virome of Tadarida brasiliensis, an insectivorous New World bat species inhabiting a maternity colony in Rosario (Argentina), by a metagenomic approach. The analysis of five pooled oral/anal swab samples indicated the presence of 43 different taxonomic viral families infecting a wide range of hosts. By conventional nucleic acid detection techniques and/or bioinformatics approaches, the genomes of two novel viruses were completely covered clustering into the Papillomaviridae (Tadarida brasiliensis papillomavirus type 1, TbraPV1) and Genomoviridae (Tadarida brasiliensis gemykibivirus 1, TbGkyV1) families. TbraPV1 is the first papillomavirus type identified in this host and the prototype of a novel genus. TbGkyV1 is the first genomovirus reported in New World bats and constitutes a new species within the genus Gemykibivirus. Our findings extend the knowledge about oral/anal viromes of a South American bat species and contribute to understand the evolution and genetic diversity of the novel characterized viruses.

New Insights into the Evolutionary and Genomic Landscape of Molluscum Contagiosum Virus (MCV) based on Nine MCV1 and Six MCV2 Complete Genome Sequences.

Molluscum contagiosum virus (MCV) is the sole member of the Molluscipoxvirus genus and the causative agent of molluscum contagiosum (MC), a common skin disease. Although it is an important and frequent human pathogen, its genetic landscape and evolutionary history remain largely unknown. In this study, ten novel complete MCV genome sequences of the two most common MCV genotypes were determined (five MCV1 and five MCV2 sequences) and analyzed together with all MCV complete genomes previously deposited in freely accessible sequence repositories (four MCV1 and a single MCV2). In comparison to MCV1, a higher degree of nucleotide sequence conservation was observed among MCV2 genomes. Large-scale recombination events were identified in two newly assembled MCV1 genomes and one MCV2 genome. One recombination event was located in a newly identified recombinant region of the viral genome, and all previously described recombinant regions were re-identified in at least one novel MCV genome. MCV genes comprising the identified recombinant segments have been previously associated with viral interference with host T-cell and NK-cell immune responses. In conclusion, the two most common MCV genotypes emerged along divergent evolutionary pathways from a common ancestor, and the differences in the heterogeneity of MCV1 and MCV2 populations may be attributed to the strictness of the constraints imposed by the host immune response.

Diffusion of D-glucose measured in the cytosol of a single astrocyte.

Astrocytes interact with neurons and endothelial cells and may mediate exchange of metabolites between capillaries and nerve terminals. In the present study, we investigated intracellular glucose diffusion in purified astrocytes after local glucose uptake. We used a fluorescence resonance energy transfer (FRET)-based nano sensor to monitor the time dependence of the intracellular glucose concentration at specific positions within the cell. We observed a delay in onset and kinetics in regions away from the glucose uptake compared with the region where we locally super-fused astrocytes with the D-glucose-rich solution. We propose a mathematical model of glucose diffusion in astrocytes. The analysis showed that after gradual uptake of glucose, the locally increased intracellular glucose concentration is rapidly spread throughout the cytosol with an apparent diffusion coefficient (D app) of (2.38 ± 0.41) × 10(-10) m(2) s(-1) (at 22-24 °C). Considering that the diffusion coefficient of D-glucose in water is D = 6.7 × 10(-10) m(2) s(-1) (at 24 °C), D app determined in astrocytes indicates that the cytosolic tortuosity, which hinders glucose molecules, is approximately three times higher than in aqueous solution. We conclude that the value of D app for glucose measured in purified rat astrocytes is consistent with the view that cytosolic diffusion may allow glucose and glucose metabolites to traverse from the endothelial cells at the blood-brain barrier to neurons and neighboring astrocytes.