Gut symbiont-derived sphingosine modulates vector competence in Aedes mosquitoes.

The main vectors of Zika virus (ZIKV) and dengue virus (DENV) are Aedes aegypti and Ae. albopictus, with Ae. aegypti being more competent. However, the underlying mechanisms remain unclear. Here, we find Ae. albopictus shows comparable vector competence to ZIKV/DENV with Ae. aegypti by blood-feeding after antibiotic treatment or intrathoracic injection. This suggests that midgut microbiota can influence vector competence. Enterobacter hormaechei_B17 (Eh_B17) is isolated from field-collected Ae. albopictus and conferred resistance to ZIKV/DENV infection in Ae. aegypti after gut-transplantation. Sphingosine, a metabolite secreted by Eh_B17, effectively suppresses ZIKV infection in both Ae. aegypti and cell cultures by blocking viral entry during the fusion step, with an IC50 of approximately 10 µM. A field survey reveals that Eh_B17 preferentially colonizes Ae. albopictus compared to Ae. aegypti. And field Ae. albopictus positive for Eh_B17 are more resistant to ZIKV infection. These findings underscore the potential of gut symbiotic bacteria, such as Eh_B17, to modulate the arbovirus vector competence of Aedes mosquitoes. As a natural antiviral agent, Eh_B17 holds promise as a potential candidate for blocking ZIKV/DENV transmission.

Seroprevalence of Chikungunya and Zika virus in nonhuman primates: A systematic review and meta-analysis.

Chikungunya virus (CHIKV) and Zika virus (ZIKV) are mosquito-borne viruses that have caused several outbreaks worldwide. Aedes mosquitoes transmit these viruses mainly through sylvatic and urban transmission cycles. In the sylvatic cycle, nonhuman primates (NHPs) can be infected with CHIKV and ZIKV and may play an essential role as reservoirs for virus transmission. To improve our knowledge on the role of NHPs in the sylvatic cycle, we performed a systematic review and meta-analysis study on the seroprevalence of CHIKV and ZIKV worldwide in NHPs. According to the PRISMA guidelines, 17 CHIKV and 16 ZIKV seroprevalence studies in NHPs from 3 online databases: PubMed, Embase, and Scopus were selected. Data were extracted, including location and study year, type of NHP, sample size, serological tests, and seropositivity. All included studies have high-quality scores, between 5 and 8, corresponding to the grading criteria. Seroprevalence estimation was pooled using the 'meta' package in the R statistical software. The estimated pooled seroprevalence of CHIKV and ZIKV in NHP was 17% (95%CI: 5-34, I2: 99%, p < 0.05) and 6% (95% CI: 2-12, I 2 : 92%, p < 0.05), respectively. Most of the NHPs tested were wild Old World monkeys. The subgroup was analyzed by continents; high seropositive CHIKV and ZIKV were found in African NHPs at 35% (95% CI 9-66.0, I 2  = 100) and 16% (95% CI 1-44, I 2  = 97), respectively. While NHPs in America have 7% (95% CI 0-28, I 2 = 99) and 2% (95% CI 1-3, I 2 = 54) against CHIKV and ZIKV. In Asia, 6% (95% CI: 5-34, I 2  = 96) CHIKV seroprevalence and 7% (95% CI 0-20, I 2  = 98) ZIKV seroprevalence were found in NHP. This study provides a comprehensive overview of the seroprevalence of CHIKV and ZIKV among NHPs in various regions.

Molecular Characterization of Seasonal Influenza A and B from Hospitalized Patients in Thailand in 2018-2019.

Influenza viruses continue to be a major public health threat due to the possible emergence of more virulent influenza virus strains resulting from dynamic changes in virus adaptability, consequent of functional mutations and antigenic drift in surface proteins, especially hemagglutinin (HA) and neuraminidase (NA). In this study, we describe the genetic and evolutionary characteristics of H1N1, H3N2, and influenza B strains detected in severe cases of seasonal influenza in Thailand from 2018 to 2019. We genetically characterized seven A/H1N1 isolates, seven A/H3N2 isolates, and six influenza B isolates. Five of the seven A/H1N1 viruses were found to belong to clade 6B.1 and were antigenically similar to A/Switzerland/3330/2017 (H1N1), whereas two isolates belonged to clade 6B.1A1 and clustered with A/Brisbane/02/2018 (H1N1). Interestingly, we observed additional mutations at antigenic sites (S91R, S181T, T202I) as well as a unique mutation at a receptor binding site (S200P). Three-dimensional (3D) protein structure analysis of hemagglutinin protein reveals that this unique mutation may lead to the altered binding of the HA protein to a sialic acid receptor. A/H3N2 isolates were found to belong to clade 3C.2a2 and 3C.2a1b, clustering with A/Switzerland/8060/2017 (H3N2) and A/South Australia/34/2019 (H3N2), respectively. Amino acid sequence analysis revealed 10 mutations at antigenic sites including T144A/I, T151K, Q213R, S214P, T176K, D69N, Q277R, N137K, N187K, and E78K/G. All influenza B isolates in this study belong to the Victoria lineage. Five out of six isolates belong to clade 1A3-DEL, which relate closely to B/Washington/02/2009, with one isolate lacking the three amino acid deletion on the HA segment at position K162, N163, and D164. In comparison to the B/Colorado/06/2017, which is the representative of influenza B Victoria lineage vaccine strain, these substitutions include G129D, G133R, K136E, and V180R for HA protein. Importantly, the susceptibility to oseltamivir of influenza B isolates, but not A/H1N1 and A/H3N2 isolates, were reduced as assessed by the phenotypic assay. This study demonstrates the importance of monitoring genetic variation in influenza viruses regarding how acquired mutations could be associated with an improved adaptability for efficient transmission.

Solid-state nanopore analysis of human genomic DNA shows unaltered global 5-hydroxymethylcytosine content associated with early-stage breast cancer.

5-Hydroxymethylcytosine (5hmC), the first oxidized form of the well-known epigenetic modification 5-methylcytosine, is an independent regulator of gene expression and therefore a potential marker for disease. Here, we report on methods developed for a selective solid-state nanopore assay that enable direct analysis of global 5hmC content in human tissue. We first describe protocols for preparing genomic DNA derived from both healthy breast tissue and stage 1 breast tumor tissue and then use our approach to probe the net abundance of the modified base in each cohort. Then, we employ empirical data to adjust for the impact of nanopore diameter on the quantification. Correcting for variations in nanopore diameter among the devices used for analysis reveals no detectable difference in global 5hmC content between healthy and tumor tissue. These results suggest that 5hmC changes may not be associated with early-stage breast cancer and instead are a downstream consequence of the disease.

Modular affinity-labeling of the cytosine demethylation base elements in DNA.

5-methylcytosine is the most studied DNA epigenetic modification, having been linked to diverse biological processes and disease states. The elucidation of cytosine demethylation has drawn added attention the three additional intermediate modifications involved in that pathway-5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine-each of which may have distinct biological roles. Here, we extend a modular method for labeling base modifications in DNA to recognize all four bases involved in demethylation. We demonstrate both differential insertion of a single affinity tag (biotin) at the precise position of target elements and subsequent repair of the nicked phosphate backbone that remains following the procedure. The approach enables affinity isolation and downstream analyses without inducing widespread damage to the DNA.

Simple and Efficient Room-Temperature Release of Biotinylated Nucleic Acids from Streptavidin and Its Application to Selective Molecular Detection.

The biotin-streptavidin bond is the strongest noncovalent bond in nature and is thus used extensively in biotechnology applications. However, the difficulty of releasing the bond without high temperatures or corrosive solutions can be a barrier to applications involving nucleic acids and other delicate substrates. Here, room-temperature phenol is employed to release biotin-tagged DNA constructs from streptavidin rapidly and efficiently. It is demonstrated that synthetic biotinylated DNA can be recovered at yields approaching 100% from both solution-phase and bead-bound streptavidin with as little as 12% (v/v) phenol, leaving the biotin tag active and reusable after extraction. As an application of this recovery method, biotinylated DNA fragments are isolated from a mixed solution to provide selectivity for solid-state nanopore detection.

Solid-State Nanopore Analysis of Diverse DNA Base Modifications Using a Modular Enzymatic Labeling Process.

Many regulated epigenetic elements and base lesions found in genomic DNA can both directly impact gene expression and play a role in disease processes. However, due to their noncanonical nature, they are challenging to assess with conventional technologies. Here, we present a new approach for the targeted detection of diverse modified bases in DNA. We first use enzymatic components of the DNA base excision repair pathway to install an individual affinity label at each location of a selected modified base with high yield. We then probe the resulting material with a solid-state nanopore assay capable of discriminating labeled DNA from unlabeled DNA. The technique features exceptional modularity via selection of targeting enzymes, which we establish through the detection of four DNA base elements: uracil, 8-oxoguanine, T:G mismatch, and the methyladenine analog 1,N6-ethenoadenine. Our results demonstrate the potential for a quantitative nanopore assessment of a broad range of base modifications.

Sequence-Specific Recognition of MicroRNAs and Other Short Nucleic Acids with Solid-State Nanopores.

The detection and quantification of short nucleic acid sequences has many potential applications in studying biological processes, monitoring disease initiation and progression, and evaluating environmental systems, but is challenging by nature. We present here an assay based on the solid-state nanopore platform for the identification of specific sequences in solution. We demonstrate that hybridization of a target nucleic acid with a synthetic probe molecule enables discrimination between duplex and single-stranded molecules with high efficacy. Our approach requires limited preparation of samples and yields an unambiguous translocation event rate enhancement that can be used to determine the presence and abundance of a single sequence within a background of nontarget oligonucleotides.