Autochthonous Human Dirofilaria repens Infection in Austria

Purpose This report describes a rare autochthonous case of human D. repens infection in Austria. Dirofilariosis is a mosquito-borne parasitic infection that predominantly affects dogs. Human D. repens infections have primarily been reported in Mediterranean countries, but are emerging throughout Central and Northern Europe. Methods The worm was removed surgically and identified using PCR and DNA sequencing. The consensus sequences were compared against reference sequences of Dirofilaria repens from GenBank. Results The 56-year-old woman acquired the infection, which presented as a subcutaneous nodule, in Vienna, Austria. This is the second autochthonous case of human D. repens infection in Austria. Conclusion The reasons for the emergence of D. repens and other parasitic infections in Central and Northern Europe are manifold, including climate change and globalization. This case demonstrates that with the growing number of D. repens infections, health care professionals must place further emphasis on emerging infectious diseases to ensure appropriate diagnostics and treatment in the future.

Comparing species detection success between molecular markers in DNA metabarcoding of coastal macroinvertebrates

DNA metabarcoding has great potential to improve marine biomonitoring programs by providing a rapid and accurate assessment of species composition in zoobenthic communities. However, some methodological improvements are still required, especially regarding failed detections, primers efficiency and incompleteness of databases. Here we assessed the efficiency of two different marker loci (COI and 18S) and three primer pairs in marine species detection through DNA metabarcoding of the macrozoobenthic communities colonizing three types of artificial substrates (slate, PVC and granite), sampled between 3 and 15 months of deployment. To accurately compare detection success between markers, we also compared the representativeness of the detected species in public databases and revised the reliability of the taxonomic assignments. Globally, we recorded extensive complementarity in the species detected by each marker, with 69% of the species exclusively detected by either 18S or COI. Individually, each of the three primer pairs recovered, at most, 52% of all species detected on the samples, showing also different abilities to amplify specific taxonomic groups. Most of the detected species have reliable reference sequences in their respective databases (82% for COI and 72% for 18S), meaning that when a species was detected by one marker and not by the other, it was most likely due to faulty amplification, and not by lack of matching sequences in the database. Overall, results showed the impact of marker and primer applied on species detection ability and indicated that, currently, if only a single marker or primer pair is employed in marine zoobenthos metabarcoding, a fair portion of the diversity may be overlooked.

ReGSP: a visualized application for homology-based gene searching and plotting using multiple reference sequences

The massively parallel nature of next-generation sequencing technologies has contributed to the generation of massive sequence data in the last two decades. Deciphering the meaning of each generated sequence requires multiple analysis tools, at all stages of analysis, from the reads stage all the way up to the whole-genome level. Homology-based approaches based on related reference sequences are usually the preferred option for gene and transcript prediction in newly sequenced genomes, resulting in the popularity of a variety of BLAST and BLAST-based tools. For organelle genomes, a single-reference–based gene finding tool that uses grouping parameters for BLAST results has been implemented in the Genome Search Plotter (GSP). However, this tool does not accept multiple and user-customized reference sequences required for a broad homology search. Here, we present multiple Reference–based Gene Search and Plot (ReGSP), a simple and convenient web tool that accepts multiple reference sequences for homology-based gene search. The tool incorporates cPlot, a novel dot plot tool, for illustrating nucleotide sequence similarity between the query and the reference sequences. ReGSP has an easy-to-use web interface and is freely accessible at https://ds.mju.ac.kr/regsp.

Genomic Epidemiology of SARS-CoV-2 Divulge B.1, B.1.36, and B.1.1.7 as the Most Dominant Lineages in First, Second, and Third Wave of SARS-CoV-2 Infections in Pakistan

The present study aims to investigate the genomic variability and epidemiology of SARS-CoV-2 in Pakistan along with its role in the spread and severity of infection during the three waves of COVID-19. A total of 453 genomic sequences of Pakistani SARS-CoV-2 were retrieved from GISAID and subjected to MAFFT-based alignment and QC check which resulted in removal of 53 samples. The remaining 400 samples were subjected to Pangolin-based genomic lineage identification. And to infer our SARS-CoV-2 time-scaled and divergence phylogenetic trees, 3804 selected global reference sequences plus 400 Pakistani samples were used for the Nextstrain analysis with Wuhan/Hu-1/2019, as reference genome. Finally, maximum likelihood based phylogenetic tree was built by using the Nextstrain and coverage map was created by employing Nextclade. By using the amino acid substitutions, the maximum likelihood phylogenetic trees were developed for each wave, separately. Our results reveal the circulation of 29 lineages, belonging to following seven clades G, GH, GR, GRY, L, O, and S in the three waves. From first wave, 16 genomic lineages of SARS-CoV-2 were identified with B.1(24.7%), B.1.36(18.8%), and B.1.471(18.8%) as the most prevalent lineages respectively. The second wave data showed 18 lineages, 10 of which were overlapping with the first wave suggesting that those variants could not be contained during the first wave. In this wave, a new lineage, AE.4, was reported from Pakistan for the very first time in the world. However, B.1.36 (17.8%), B.1.36.31 (11.9%), B.1.1.7 (8.5%), and B.1.1.1 (5.9%) were the major lineages in second wave. Third wave data showed the presence of nine lineages with Alpha/B.1.1.7 (72.7%), Beta/B.1.351 (12.99%), and Delta/B.1.617.2 (10.39%) as the most predominant variants. It is suggested that these VOCs should be contained at the earliest in order to prevent any devastating outbreak of SARS-CoV-2 in the country.

isomiRs-specific differential expression is the rule, not the exception: Are we missing hundreds of species in microRNA analysis?

MicroRNAs (miRNAs) are small RNA molecules that act as regulators of gene expression through targeted mRNA degradation. They are involved in many biological and pathophysiological processes and are widely studied as potential biomarkers and therapeutics agents for human diseases, including cardiovascular disorders. Recently discovered isoforms of miRNAs (isomiRs) exist in high quantities and are very diverse. Despite having few differences with their corresponding reference miRNAs, they display specific functions and expression profiles, across tissues and conditions. However, they are still overlooked and understudied, as we lack a comprehensive view on their condition-specific regulation and impact on differential expression analysis. Here, we show that isomiRs can have major effects on differential expression analysis results, as their expression is independent of their host miRNA genes or reference sequences. We present two miRNA-seq datasets from human umbilical vein endothelial cells, and assess isomiR expression in response to senescence and compartment-specificity (nuclear/cytosolic) under hypoxia. We compare three different methods for miRNA analysis, including isomiR-specific analysis, and show that ignoring isomiRs induces major biases in differential expression. Moreover, isomiR analysis permits higher resolution of complex signal dissection, such as the impact of hypoxia on compartment localization, and differential isomiR type enrichments between compartments. Finally, we show important distribution differences across conditions, independently of global miRNA expression signals. Our results raise concerns over the quasi exclusive use of miRNA reference sequences in miRNA-seq processing and experimental assays. We hope that our work will guide future isomiR expression studies, which will correct some biases introduced by golden standard analysis, improving the resolution of such assays and the biological significance of their downstream studies.

Emergence of Multiple Novel Inter-Genotype Recombinant Strains of Human Astroviruses Detected in Pediatric Patients With Acute Gastroenteritis in Thailand

Objective: Human astrovirus (HAstV) is recognized as an important cause of acute gastroenteritis in children. Recombination between different genotypes of HAstV can contribute to diversity and evolution of the virus. This study aimed to investigate the emergence of HAstV recombinant strains in pediatric patients hospitalized with acute gastroenteritis in Chiang Mai, Thailand, spanning 2011–2020.Methods: A total of 92 archival HAstV strains collected from pediatric patients with acute gastroenteritis during 2011–2020 were further characterized to identify the recombinant strains. The ORF1b and ORF2 junction region of each strain was amplified and sequenced. The obtained sequences were analyzed in comparison with the reference sequences retrieved from GenBank database. Their genotypes were assigned using MEGA X software based on the partial ORF1b (RdRp) and ORF2 (capsid) regions, and the recombination breakpoints of recombinant strains were determined by SimPlot and RDP4 analyses.Results: Five inter-genotype recombinant strains with three recombination patterns of ORF1b/ORF2 of classic HAstV, HAstV8/HAstV1, HAstV8/HAstV3, and HAstV3/HAstV2, were detected. The recombination breakpoints of all strains were located at the 3′-end region of ORF1b close to the ORF1b/ORF2 junction.Conclusion: Several novel inter-genotype recombinant strains of classic HAstV genotypes were detected in pediatric patients with acute gastroenteritis in Chiang Mai, Thailand, during the period of 10 years from 2011 to 2020.

Mitochondrial sequence diversity reveals the hybrid origin of invasive gibel carp (Carassius gibelio) populations in Hungary

Background Invasive gibel carp, Carassius gibelio (Bloch, 1782) has become well-established in the Hungarian waters and now are spreading in the European waters. On major concern now is the potential hybridization between gibel carp and the other invasive species in the Carassius auratus complex (CAC), which may further accelerate the spread of the whole invasive species complex. The identification of gibel carp and their hybrids is difficult because of its morphological similarity to the other species in CAC. Here we carry out a genomic assessment to understand the history of gibel carp invasion and its phylogenetic relationship with the other species in CAC. Three loci of the mitochondrial genome (D-loop, CoI, Cytb) were used to determine the phylogenetic origin of individuals and relarionship among six gibel carp populations and the other species in the CAC. Methodolgy A total of 132 gibel carp samples from six locations in Southern Transdanubia (Hungary) were collected after phenotypic identification to measure the genetic diversity within and among gibel carp populations of Southern Transdanubia (Hungary). The genetic background was examined by the sequences of the mitochondrial genome: D-loop, Cytochrome c oxidase I (CoI) and Cytochrome b (Cytb). Mitochondrial genetic markers are excellent tools for phylogenetic studies because they are maternally inherited. Successfully identified haplotypes were aligned and with reference sequences in nucleotide databases (i.e., NCBI-BLAST: National Centre for Biotechnology Information and BOLD: Barcode of Life Data System). The phylogenetic relationships among gibel carp populations were then analyzed together with the reference sequences to understand the relationship and the level of hybridization with the species in CAC. Results Among the 132 aligned D-loop sequences 22 haplotypes were identified. Further examination of representative individuals of the 22 haplotypes, six Cytb and four CoI sequences were detected. The largest number of haplotypes of all three loci were found in Lake Balaton, the largest shallow lake in Central Europe. Based on the NCBI-BLAST alignment of the D-loop, haplotypes of Carassius auratus auratus and Carassius a. buergeri in CAC were identified in the C. gibelio samples. Further analysis of haplotypes with the other two mitochondrial markers confirmed the occurrence of intragenus hybridization of C. gibelio in the Hungarian waters. Conclusion By using three mitochondrial markers (D-loop, Cytb, CoI), we genomically characterized a gibel carp-complex in Hungarian waters and assessed the C. gibelio phylogenetic status between them. Hybrid origin of locally invasive Carassius taxon was detected in Hungary. It points out that invasive species are not only present in Hungary but reproduce with each other in the waters, further accelerating their spread.

Rbec: a tool for analysis of amplicon sequencing data from synthetic microbial communities

Synthetic microbial communities (SynComs) constitute an emerging and powerful tool in biological, biomedical, and biotechnological research. Despite recent advances in algorithms for the analysis of culture-independent amplicon sequencing data from microbial communities, there is a lack of tools specifically designed for analyzing SynCom data, where reference sequences for each strain are available. Here we present Rbec, a tool designed for the analysis of SynCom data that accurately corrects PCR and sequencing errors in amplicon sequences and identifies intra-strain polymorphic variation. Extensive evaluation using mock bacterial and fungal communities show that our tool outperforms current methods for samples of varying complexity, diversity, and sequencing depth. Furthermore, Rbec also allows accurate detection of contaminants in SynCom experiments.