An Efficient Extraction Method Allowing the Genetic Evaluation of Host DNA from Samples Collected for Virus Infection Diagnosis in Viral Transport Medium.

Introduction: During the COVID-19 pandemic, an extraordinary number of nasopharyngeal secretion samples inoculated in viral transport medium (VTM) were collected and analyzed to detect SARS-CoV-2 infection. In addition to viral detection, those samples can also be a source of host genomic material, providing excellent opportunities for biobanking and research. Objective: To describe a simple, in-house-developed DNA extraction method to obtain high yield and quality genomic DNA from VTM samples for host genetic analysis and assess its relative efficiency by comparing its yield and suitability to downstream applications to two different commercial DNA extraction kits. Methods: In this study, 13 VTM samples were processed by two commercial silica-based kits and compared with an in-House-developed protocol for host DNA extraction. An additional 452 samples were processed by the in-House method. The quantity and quality of the differentially extracted DNA samples were assessed by Qubit and spectrophotometric measurements. The suitability of extracted samples for downstream applications was tested by polymerase chain reaction (PCR) amplification followed by amplicon sequencing and allelic discrimination in real-time PCR. Results: The in-House method provided greater median DNA yield (0.81 µg), being significantly different from the PureLink® method (0.14 µg, p < 0.001), but not from the QIAamp® method (0.47 µg, p = 0.980). Overall satisfactory results in DNA concentrations and purity, in addition to cost, were observed using the in-House method, whose samples were able to produce clear amplification in PCR and sequencing reads, as well as effective allelic discrimination in real-time PCR TaqMan® assay. Conclusion: The described in-House method proved to be suitable and economically viable for genomic DNA extraction from VTM samples for biobanking purposes. These results are extremely valuable for the study of the COVID-19 pandemic and other emergent infectious diseases, allowing host genetic studies to be performed in samples initially collected for diagnosis.

Downregulation of Microcephaly-Causing Genes as a Mechanism for ZIKV Teratogenesis: A Meta-analysis of RNA-Seq Studies.

Zika virus (ZIKV) is a neurotropic teratogen that causes congenital Zika syndrome (CZS), characterized by brain and eye anomalies. Impaired gene expression in neural cells after ZIKV infection has been demonstrated; however, there is a gap in the literature of studies comparing whether the differentially expressed genes in such cells are similar and how it can cause CZS. Therefore, the aim of this study was to compare the differential gene expression (DGE) after ZIKV infection in neural cells through a meta-analysis approach. Through the GEO database, studies that evaluated DGE in cells exposed to the Asian lineage of ZIKV versus cells, of the same type, not exposed were searched. From the 119 studies found, five meet our inclusion criteria. Raw data of them were retrieved, pre-processed, and evaluated. The meta-analysis was carried out by comparing seven datasets, from these five studies. We found 125 upregulated genes in neural cells, mainly interferon-stimulated genes, such as IFI6, ISG15, and OAS2, involved in the antiviral response. Furthermore, 167 downregulated, involved with cellular division. Among these downregulated genes, classic microcephaly-causing genes stood out, such as CENPJ, ASPM, CENPE, and CEP152, demonstrating a possible mechanism by which ZIKV impairs brain development and causes CZS.

Neutral and outlier single nucleotide polymorphisms disentangle the evolutionary history of a coastal Solanaceae species.

Speciation begins with the isolation of some individuals or subpopulations due to drivers promoting a diverging genetic distribution. Such isolation may occur, followed by different processes and pressures. Isolation-by-distance (IBD), isolation-by-adaptation (IBA), and isolation-by-colonization (IBC) have been recognized as the main divergence patterns. Still, it is not easy to distinguish which one is the main pattern as each one may act at different points in time or even simultaneously. Using an extensive genome coverage from a Petunia species complex with coastal and inland distribution and multiple analytical approaches on population genomics and phylogeography, we showed a complex interplay between neutral and selective forces acting on the divergence process. We found 18,887 SNPs potentially neutral and 924 potentially under selection (outlier) loci. All analyses pointed that each subspecies displays its own genetic component and evolutionary history. We suggested plausible ecological drivers for such divergence in a southernmost South Atlantic coastal plain in Brazil and Uruguay and identified a connection between adaptation and environment heterogeneity.

Landscape and climatic features drive genetic differentiation processes in a South American coastal plant.

BACKGROUND: Historical and ecological processes shape patterns of genetic diversity in plant species. Colonization to new environments and geographical landscape features determine, amongst other factors, genetic diversity within- and differentiation between-populations. We analyse the genetic diversity and population structure of Calibrachoa heterophylla to infer the influence of abiotic landscape features on the level of gene flow in this coastal species of the South Atlantic Coastal Plain. RESULTS: The C. heterophylla populations located on early-deposited coastal plain regions show higher genetic diversity than those closer to the sea. The genetic differentiation follows a pattern of isolation-by-distance. Landscape features, such as water bodies and wind corridors, and geographical distances equally explain the observed genetic differentiation, whereas the precipitation seasonality exhibits a strong signal for isolation-by-environment in marginal populations. The estimated levels of gene flow suggest that marginal populations had restricted immigration rates enhancing differentiation. CONCLUSIONS: Topographical features related to coastal plain deposition history influence population differentiation in C. heterophylla. Gene flow is mainly restricted to nearby populations and facilitated by wind fields, albeit without any apparent influence of large water bodies. Furthermore, differential rainfall regimes in marginal populations seem to promote genetic differentiation.

Secondary structure of nrDNA Internal Transcribed Spacers as a useful tool to align highly divergent species in phylogenetic studies.

Recently, it has been suggested that internal transcribed spacer (ITS) sequences are under selective constraints to preserve their secondary structure. Here, we investigate the patterns of the ITS nucleotide and secondary structure conservation across the Passiflora L. genus to evaluate the potential use of secondary structure data as a helpful tool for the alignment in taxonomically complex genera. Considering the frequent use of ITS, this study also presents a perspective on future analyses in other plant groups. The ITS1 and ITS2 sequences presented significant differences for mean values of the lowest energy state (LES) and for number of hairpins in different Passiflora subgenera. Statistical analyses for the subgenera separately support significant differences between the LES values and the total number of secondary structures for ITS. In order to evaluate whether the LES values of ITS secondary structures were related to selective constraints, we compared these results among 120 ITS sequences from Passiflora species and 120 randomly generated sequences. These analyses indicated that Passiflora ITS sequences present characteristics of a region under selective constraint to maintain the secondary structure showing to be a promising tool to improve the alignments and identify sites with non-neutral substitutions or those correlated evolutionary steps.

Secondary structure of nrDNA Internal Transcribed Spacers as a useful tool to align highly divergent species in phylogenetic studies

Abstract Recently, it has been suggested that internal transcribed spacer (ITS) sequences are under selective constraints to preserve their secondary structure. Here, we investigate the patterns of the ITS nucleotide and secondary structure conservation across the Passiflora L. genus to evaluate the potential use of secondary structure data as a helpful tool for the alignment in taxonomically complex genera. Considering the frequent use of ITS, this study also presents a perspective on future analyses in other plant groups. The ITS1 and ITS2 sequences presented significant differences for mean values of the lowest energy state (LES) and for number of hairpins in different Passiflora subgenera. Statistical analyses for the subgenera separately support significant differences between the LES values and the total number of secondary structures for ITS. In order to evaluate whether the LES values of ITS secondary structures were related to selective constraints, we compared these results among 120 ITS sequences from Passiflora species and 120 randomly generated sequences. These analyses indicated that Passiflora ITS sequences present characteristics of a region under selective constraint to maintain the secondary structure showing to be a promising tool to improve the alignments and identify sites with non-neutral substitutions or those correlated evolutionary steps.