Sequence analyses of RNA virus genomes remain challenging owing to the exceptional genetic plasticity of these viruses. Because of high mutation and recombination rates, genome replication by viral RNA-dependent RNA polymerases leads to populations of closely related viruses, so-called "quasispecies." Standard (short-read) sequencing technologies are ill-suited to reconstruct large numbers of full-length haplotypes of (1) RNA virus genomes and (2) subgenome-length (sg) RNAs composed of noncontiguous genome regions. Here, we used a full-length, direct RNA sequencing (DRS) approach based on nanopores to characterize viral RNAs produced in cells infected with a human coronavirus. By using DRS, we were able to map the longest (∼26-kb) contiguous read to the viral reference genome. By combining Illumina and Oxford Nanopore sequencing, we reconstructed a highly accurate consensus sequence of the human coronavirus (HCoV)-229E genome (27.3 kb). Furthermore, by using long reads that did not require an assembly step, we were able to identify, in infected cells, diverse and novel HCoV-229E sg RNAs that remain to be characterized. Also, the DRS approach, which circumvents reverse transcription and amplification of RNA, allowed us to detect methylation sites in viral RNAs. Our work paves the way for haplotype-based analyses of viral quasispecies by showing the feasibility of intra-sample haplotype separation. Even though several technical challenges remain to be addressed to exploit the potential of the nanopore technology fully, our work illustrates that DRS may significantly advance genomic studies of complex virus populations, including predictions on long-range interactions in individual full-length viral RNA haplotypes.
Use of PET/MR in children has not previously been reported, to the best of our knowledge. Children with systemic malignancies may benefit from the reduced radiation exposure offered by PET/MR. We report our initial experience with PET/MR hybrid imaging and our current established sequence protocol after 21 PET/MR studies in 15 children with multifocal malignant diseases. The effective dose of a PET/MR scan was only about 20% that of the equivalent PET/CT examination. Simultaneous acquisition of PET and MR data combines the advantages of the two previously separate modalities. Furthermore, the technique also enables whole-body diffusion-weighted imaging (DWI) and statements to be made about the biological cellularity and nuclear/cytoplasmic ratio of tumours. Combined PET/MR saves time and resources. One disadvantage of PET/MR is that in order to have an effect, a significantly longer examination time is needed than with PET/CT. In our initial experience, PET/MR has turned out to be an unexpectedly stable and reliable hybrid imaging modality, which generates a complementary diagnostic study of great additional value.
Sequence analyses of RNA virus genomes remain challenging due to the exceptional genetic plasticity of these viruses. Because of high mutation and recombination rates, genome replication by viral RNA-dependent RNA polymerases leads to populations of closely related viruses, so-called 'quasispecies'. Standard (short-read) sequencing technologies are ill-suited to reconstruct large numbers of full-length haplotypes of (i) RNA virus genomes and (ii) subgenome-length (sg) RNAs comprised of noncontiguous genome regions. Here, we used a full-length, direct RNA sequencing (DRS) approach based on nanopores to characterize viral RNAs produced in cells infected with a human coronavirus. Using DRS, we were able to map the longest (∼26 kb) contiguous read to the viral reference genome. By combining Illumina and nanopore sequencing, we reconstructed a highly accurate consensus sequence of the human coronavirus (HCoV) 229E genome (27.3 kb). Furthermore, using long reads that did not require an assembly step, we were able to identify, in infected cells, diverse and novel HCoV-229E sg RNAs that remain to be characterized. Also, the DRS approach, which circumvents reverse transcription and amplification of RNA, allowed us to detect methylation sites in viral RNAs. Our work paves the way for haplotype-based analyses of viral quasispecies by demonstrating the feasibility of intra-sample haplotype separation. Even though several technical challenges remain to be addressed to exploit the potential of the nanopore technology fully, our work illustrates that direct RNA sequencing may significantly advance genomic studies of complex virus populations, including predictions on long-range interactions in individual full-length viral RNA haplotypes.
Metagenomic sequencing is promising for clinical applications to study microbial composition concerning disease or patient outcomes. Alterations of the vaginal microbiome are associated with adverse pregnancy outcomes, like preterm premature rupture of membranes and preterm birth. Methodologically these samples often have to deal with low relative amounts of prokaryotic DNA and high amounts of host DNA (> 90%), decreasing the overall microbial resolution. Nanopore's adaptive sampling method offers selective DNA depletion or target enrichment to directly reject or accept DNA molecules during sequencing without specialized sample preparation. Here, we demonstrate how selective ‘human host depletion’ resulted in a 1.70 fold (± 0.27 fold) increase in total sequencing depth, providing higher taxonomic profiling sensitivity. At the same time, the microbial composition remains consistent with the control experiments. The complete removal of all human host sequences is not yet possible and should be considered as an ethical approval statement might still be necessary. Adaptive sampling increased microbial sequencing yield in all 15 sequenced clinical routine vaginal samples, making it a valuable tool for clinical surveillance and medical-based research, which can be used in addition to other host depletion methods before sequencing.
Zoonosis, the natural transmission of infections from animal to human, is a far-reaching global problem. The recent outbreaks of Zika virus and Ebola virus are examples of viral zoonosis, which occur more frequently due to globalization. In case of a virus outbreak, it is helpful to know which host organism was the original carrier of the virus. Once the reservoir or intermediate host is known, it can be isolated to prevent further spreading of the viral infection. Recent approaches aim to predict a viral host based on the viral genome, often in combination with the potential host genome and using arbitrary selected features. This methods have a clear limitation in either the amount of different hosts they can predict or the accuracy of the prediction. Here, we present a fast and accurate deep learning approach for viral host prediction, which is based on the viral genome sequence only. To assure a high prediction accuracy we developed an effective selection approach for the training data, to avoid biases due to a highly unbalanced number of known sequences per virus-host combinations. We tested our deep neural network on three different virus species (influenza A virus, rabies lyssavirus, rotavirus A) and reached for each virus species a AUC between 0.94 and 0.98, outperforming previous approaches and allowing highly accurate predictions while only using fractions of the viral genome sequences. We show that deep neural networks are suitable to predict the host of a virus, even with a limited amount of sequences and highly unbalanced available data. The deep neural networks trained for this approach build the core of the virus host predicting tool VIDHOP (VIrus Deep learning HOst Prediction). doi: bioRxiv preprint shown to perform well with character sequences 28 , such as DNA or RNA sequences, potentially allowing for a furthermore increase in the prediction quality. Author Contributions
The global spread and diversification of multidrug-resistant Gram-negative (MRGN) bacteria poses major challenges to healthcare. In particular, carbapenem-resistant Klebsiella pneumoniae strains have been frequently identified in infections and hospital-wide outbreaks. The most frequently underlying resistance gene ( bla KPC ) has been spreading over the last decade in the health care setting. bla KPC seems to have rapidly diversified and has been found in various species and on different plasmid types. To review the progress and dynamics of this diversification, all currently available KPC plasmids in the NCBI database were analysed in this work. Plasmids were grouped into 257 different representative KPC plasmids, of which 79.4% could be clearly assigned to incompatibility (Inc) group or groups. In almost half of all representative plasmids, the KPC gene is located on Tn 4401 variants, emphasizing the importance of this transposon type for the transmission of KPC genes to other plasmids. The transposons also seem to be responsible for the occurrence of altered or uncommon fused plasmid types probably due to incomplete transposition. Moreover, many KPC plasmids contain genes that encode proteins promoting recombinant processes and mutagenesis; in consequence accelerating the diversification of KPC genes and other colocalized resistance genes.
Increasing evidence implicates the decline of microglial defensive responses in the progression of Alzheimer's disease (AD). Loss of function of genetic non-modifiable AD risk factors, as the triggering receptor expressed on myeloid cells 2 (TREM2) and the apolipoprotein E (APOE), associates with microglial dysfunction characterized by reduced clustering and survival around Aß plaques. However, the contribution of modifiable AD risk factors to microglial dysfunction is not known. We show here the concomitant activation of the HIF1-mediated stress response pathway and the transcription of aerobic respiration-related genes in Aß plaque-associated microglia (AßAM). We also demonstrate that AßAM mitochondria are elongated, a cellular response found in cells that maintain aerobic respiration under low nutrient and oxygen conditions, suggesting that HIF1 activation may be hijacking microglial mitochondrial metabolism.Overactivation of HIF1 induces microglial quiescence in cellulo, characterized by lower mitochondrial respiration and reduced proliferation. In vivo, overstabilization of HIF1, either genetically (von Hippel-Lindau deficient microglia) or by exposure to systemic hypoxia (mimicking vascular contributions to AD), reduces AßAM clustering and proliferation. We also observed increased Aß neuropathology in an AD mouse model exposed to hypoxia that mimics the loss of function of genetic AD risk genes. In the AD hippocampus, the upregulation of HIF1a and HIF1 target genes correlates with the presence of "nude" plaques (i.e., with reduced microglial coverage) in a hypoxia-prone brain area and the increase of Aß plaque-associated dystrophic neurites. Thus, low oxygen levels, a modifiable AD risk factor, disrupt microglial mitochondrial metabolism and converge with genetic susceptibility to cause AD microglial dysfunction.
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