Detection of Viral −RNA and +RNA Strands in Enterovirus-Infected Cells and Tissues

The current methods to study the distribution and dynamics of viral RNA molecules inside infected cells are not ideal, as electron microscopy and immunohistochemistry can only detect mature virions, and quantitative real-time PCR does not reveal localized distribution of RNAs. We demonstrated here the branched DNA in situ hybridization (bDNA ISH) technology to study both the amount and location of the emerging −RNA and +RNA during acute and persistent enterovirus infections. According to our results, the replication of the viral RNA started 2–3 h after infection and the translation shortly after at 3–4 h post-infection. The replication hotspots with newly emerging −RNA were located quite centrally in the cell, while the +RNA production and most likely virion assembly took place in the periphery of the cell. We also discovered that the pace of replication of −RNA and +RNA strands was almost identical, and −RNA was absent during antiviral treatments. ViewRNA ISH with our custom probes also showed a good signal during acute and persistent enterovirus infections in cell and mouse models. Considering these results, along with the established bDNA FISH protocol modified by us, the effects of antiviral drugs and the emergence of enterovirus RNAs in general can be studied more effectively

Effect of inactivated nature-derived microbial composition on mouse immune system

Introduction: The hygiene hypothesis suggests that decrease in early life infections due to increased societal-level hygiene standards subjects one to allergic and autoimmune diseases. In this report, we have studied the effect of sterilized forest soil and plant-based material on mouse immune system and gut microbiome. Methods: Inbred C57Bl/6 mice maintained in normal sterile environment were subjected to autoclaved forest soil-derived powder in their bedding for 1 h a day for 3 weeks. Immune response was measured by immune cell flow cytometry, serum cytokine enzyme-linked immunoassay (ELISA) and quantitative polymerase chain reaction (qPCR) analysis. Furthermore, the mouse gut microbiome was analyzed by sequencing. Results: When compared to control mice, mice treated with soil-derived powder had decreased level of pro-inflammatory cytokines namely interleukin (IL)-17F and IL-21 in the serum. Furthermore, splenocytes from mice treated with soil-derived powder expressed less IL-1b, IL-5, IL-6, IL-13, and tumor necrosis factor (TNF) upon cell activation. Gut microbiome appeared to be stabilized by the treatment. Conclusions: These results provide insights on the effect of biodiversity on murine immune system in sterile environment. Subjecting mice to soil-based plant and microbe structures appears to elicit immune response that could be beneficial, for example, in type 2 inflammation-related diseases, that is, allergic diseases.Peer reviewe

Detection of Viral −RNA and +RNA Strands in Enterovirus-Infected Cells and Tissues

The current methods to study the distribution and dynamics of viral RNA molecules inside infected cells are not ideal, as electron microscopy and immunohistochemistry can only detect mature virions, and quantitative real-time PCR does not reveal localized distribution of RNAs. We demonstrated here the branched DNA in situ hybridization (bDNA ISH) technology to study both the amount and location of the emerging −RNA and +RNA during acute and persistent enterovirus infections. According to our results, the replication of the viral RNA started 2–3 h after infection and the translation shortly after at 3–4 h post-infection. The replication hotspots with newly emerging −RNA were located quite centrally in the cell, while the +RNA production and most likely virion assembly took place in the periphery of the cell. We also discovered that the pace of replication of −RNA and +RNA strands was almost identical, and −RNA was absent during antiviral treatments. ViewRNA ISH with our custom probes also showed a good signal during acute and persistent enterovirus infections in cell and mouse models. Considering these results, along with the established bDNA FISH protocol modified by us, the effects of antiviral drugs and the emergence of enterovirus RNAs in general can be studied more effectively.Science, Faculty ofOther UBCNon UBCMicrobiology and Immunology, Department ofReviewedFacult

Development of functionalized SYBR green II related cyanine dyes for viral RNA detection

Fluorescent probes for sensing nucleic acids have found widespread use in the field of cell and molecular biology. However, probes combined with potential for post-synthetic conjugation, e.g. for intra-endosomal measurements of RNA, are unavailable. Herein we developed cyanine dyes that can be conjugated to viral capsid or other targets. First, we solved the crystal structure of SYBR Green II. The structural elucidation of this commonly used RNA probe provided the basis for synthesizing similar molecules with much desired function for post-synthetic conjugation. To address this need, cyanine dyes were prepared using an alternative synthesis protocol. All studied compounds showed considerable brightness upon binding to nucleic acids. However, regardless of the common chromophore on the dyes, the observed fluorescence emission intensities varied significantly, where methyl-substituted dye 1 gave values higher than SYBR Green II, whereas compounds 2–5 containing undecyl spacers had lower values. Studying the structure-activity relationship revealed the longer alkyl chains to induce slight perturbation in dye intercalation, as well as demanding larger binding area on the nucleic acid lattice, explaining these differences. To study the potential biological use of the dyes, the RNA genome of enterovirus echovirus 1 was studied in vitro with the probes. A novel method employing the low binding space requirement of 1 was developed to determine the single-to-double-stranded RNA ratio of a sample, whereas compound 4 was covalently bound to the viral capsid and used successfully to monitor the viral RNA release from within the capsid. The presented results open new possibilities for preparation and use of SYBR Green-based nucleic acid probes to further apply these compounds for increasingly demanding targeting in biological contexts.peerReviewe

Mobile genetic elements from the maternal microbiome shape infant gut microbial assembly and metabolism

The perinatal period represents a critical window for cognitive and immune system development, promoted by maternal and infant gut microbiomes and their metabolites. Here, we tracked the co-development of microbiomes and metabolomes from late pregnancy to 1 year of age using longitudinal multi-omics data from a cohort of 70 mother-infant dyads. We discovered large-scale mother-to-infant interspecies transfer of mobile genetic elements, frequently involving genes associated with diet-related adaptations. Infant gut metabolomes were less diverse than maternal but featured hundreds of unique metabolites and microbe-metabolite associations not detected in mothers. Metabolomes and serum cytokine signatures of infants who received regular—but not extensively hydrolyzed—formula were distinct from those of exclusively breastfed infants. Taken together, our integrative analysis expands the concept of vertical transmission of the gut microbiome and provides original insights into the development of maternal and infant microbiomes and metabolomes during late pregnancy and early life.Peer reviewe

Strain-Level Analysis of Mother-to-Child Bacterial Transmission during the First Few Months of Life

Bacterial community acquisition in the infant gut impacts immune education and disease susceptibility. We compared bacterial strains across and within families in a prospective birth cohort of 44 infants and their mothers, sampled longitudinally in the first months of each child’s life. We identified mother-to-child bacterial transmission events and describe the incidence of family-specific antibiotic resistance genes. We observed two inheritance patterns across multiple species, where often the mother’s dominant strain is transmitted to the child, but occasionally her secondary strains colonize the infant gut. In families where the secondary strain of B. uniformis was inherited, a starch utilization gene cluster that was absent in the mother’s dominant strain was identified in the child, suggesting the selective advantage of a mother’s secondary strain in the infant gut. Our findings reveal mother-to-child bacterial transmission events at high resolution and give insights into early colonization of the infant gut