Emergence and spread of SARS-CoV-2 variants from farmed mink to humans and back during the epidemic in Denmark, June-November 2020.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) not only caused the COVID-19 pandemic but also had a major impact on farmed mink production in several European countries. In Denmark, the entire population of farmed mink (over 15 million animals) was culled in late 2020. During the period of June to November 2020, mink on 290 farms (out of about 1100 in the country) were shown to be infected with SARS-CoV-2. Genome sequencing identified changes in the virus within the mink and it is estimated that about 4000 people in Denmark became infected with these mink virus variants. However, the routes of transmission of the virus to, and from, the mink have been unclear. Phylogenetic analysis revealed the generation of multiple clusters of the virus within the mink. Detailed analysis of changes in the virus during replication in mink and, in parallel, in the human population in Denmark, during the same time period, has been performed here. The majority of cases in mink involved variants with the Y453F substitution and the H69/V70 deletion within the Spike (S) protein; these changes emerged early in the outbreak. However, further introductions of the virus, by variants lacking these changes, from the human population into mink also occurred. Based on phylogenetic analysis of viral genome data, we estimate, using a conservative approach, that about 17 separate examples of mink to human transmission occurred in Denmark but up to 59 such events (90% credible interval: (39-77)) were identified using parsimony to count cross-species jumps on transmission trees inferred using Bayesian methods. Using the latter approach, 136 jumps (90% credible interval: (117-164)) from humans to mink were found, which may underlie the farm-to-farm spread. Thus, transmission of SARS-CoV-2 from humans to mink, mink to mink, from mink to humans and between humans were all observed.

Network medicine-based epistasis detection in complex diseases: ready for quantum computing.

Most heritable diseases are polygenic. To comprehend the underlying genetic architecture, it is crucial to discover the clinically relevant epistatic interactions (EIs) between genomic single nucleotide polymorphisms (SNPs)1-3. Existing statistical computational methods for EI detection are mostly limited to pairs of SNPs due to the combinatorial explosion of higher-order EIs. With NeEDL (network-based epistasis detection via local search), we leverage network medicine to inform the selection of EIs that are an order of magnitude more statistically significant compared to existing tools and consist, on average, of five SNPs. We further show that this computationally demanding task can be substantially accelerated once quantum computing hardware becomes available. We apply NeEDL to eight different diseases and discover genes (affected by EIs of SNPs) that are partly known to affect the disease, additionally, these results are reproducible across independent cohorts. EIs for these eight diseases can be interactively explored in the Epistasis Disease Atlas (https://epistasis-disease-atlas.com). In summary, NeEDL is the first application that demonstrates the potential of seamlessly integrated quantum computing techniques to accelerate biomedical research. Our network medicine approach detects higher-order EIs with unprecedented statistical and biological evidence, yielding unique insights into polygenic diseases and providing a basis for the development of improved risk scores and combination therapies.

Discovery of drug-omics associations in type 2 diabetes with generative deep-learning models.

The application of multiple omics technologies in biomedical cohorts has the potential to reveal patient-level disease characteristics and individualized response to treatment. However, the scale and heterogeneous nature of multi-modal data makes integration and inference a non-trivial task. We developed a deep-learning-based framework, multi-omics variational autoencoders (MOVE), to integrate such data and applied it to a cohort of 789 people with newly diagnosed type 2 diabetes with deep multi-omics phenotyping from the DIRECT consortium. Using in silico perturbations, we identified drug-omics associations across the multi-modal datasets for the 20 most prevalent drugs given to people with type 2 diabetes with substantially higher sensitivity than univariate statistical tests. From these, we among others, identified novel associations between metformin and the gut microbiota as well as opposite molecular responses for the two statins, simvastatin and atorvastatin. We used the associations to quantify drug-drug similarities, assess the degree of polypharmacy and conclude that drug effects are distributed across the multi-omics modalities.

Molecular Characterization of Highly Pathogenic Avian Influenza Viruses H5N6 Detected in Denmark in 2018-2019.

Beginning in late 2017, highly pathogenic avian influenza (HPAI) H5N6 viruses caused outbreaks in wild birds and poultry in several European countries. H5N6 viruses were detected in 43 wild birds found dead throughout Denmark. Most of the Danish virus-positive dead birds were found in the period from February to April 2018. However, unlike the rest of Europe, sporadic HPAI H5N6-positive dead wild birds were detected in Denmark in July, August, September, and December 2018, with the last positive bird being found in January 2019. HPAI viruses were not detected in active surveillance of apparently healthy wild birds. In this study, we use full genome sequencing and phylogenetic analysis to investigate the wild bird HPAI H5N6 viruses found in Denmark. The Danish viruses were found to be closely related to those of contemporary HPAI H5N6 viruses detected in Europe. Their sequences formed two clusters indicating that at least two or more introductions of H5N6 into Denmark occurred. Notably, all viruses detected in the latter half of 2018 and in 2019 grouped into the same cluster. The H5N6 viruses appeared to have been maintained undetected in the autumn 2018.

Co-circulation of multiple influenza A reassortants in swine harboring genes from seasonal human and swine influenza viruses.

Since the influenza pandemic in 2009, there has been an increased focus on swine influenza A virus (swIAV) surveillance. This paper describes the results of the surveillance of swIAV in Danish swine from 2011 to 2018. In total, 3800 submissions were received with a steady increase in swIAV-positive submissions, reaching 56% in 2018. Full-genome sequences were obtained from 129 swIAV-positive samples. Altogether, 17 different circulating genotypes were identified including six novel reassortants harboring human seasonal IAV gene segments. The phylogenetic analysis revealed substantial genetic drift and also evidence of positive selection occurring mainly in antigenic sites of the hemagglutinin protein and confirmed the presence of a swine divergent cluster among the H1pdm09Nx (clade 1A.3.3.2) viruses. The results provide essential data for the control of swIAV in pigs and emphasize the importance of contemporary surveillance for discovering novel swIAV strains posing a potential threat to the human population.

A subset of lung cancer cases shows robust signs of homologous recombination deficiency associated genomic mutational signatures.

PARP inhibitors are approved for the treatment of solid tumor types that frequently harbor alterations in the key homologous recombination (HR) genes, BRCA1/2. Other tumor types, such as lung cancer, may also be HR deficient, but the frequency of such cases is less well characterized. Specific DNA aberration profiles (mutational signatures) are induced by homologous recombination deficiency (HRD) and their presence can be used to assess the presence or absence of HR deficiency in a given tumor biopsy even in the absence of an observed alteration of an HR gene. We derived various HRD-associated mutational signatures from whole-genome and whole-exome sequencing data in the lung adenocarcinoma and lung squamous carcinoma cases from TCGA, and in a patient of ours with stage IVA lung cancer with exceptionally good response to platinum-based therapy, and in lung cancer cell lines. We found that a subset of the investigated cases, both with and without biallelic loss of BRCA1 or BRCA2, showed robust signs of HR deficiency. The extreme platinum responder case also showed a robust HRD-associated genomic mutational profile. HRD-associated mutational signatures were also associated with PARP inhibitor sensitivity in lung cancer cell lines. Consequently, lung cancer cases with HRD, as identified by diagnostic mutational signatures, may benefit from PARP inhibitor therapy.

Strand Orientation Bias Detector to determine the probability of FFPE sequencing artifacts.

Formalin-fixed paraffin-embedded tissue, the most common tissue specimen stored in clinical practice, presents challenges in the analysis due to formalin-induced artifacts. Here, we present Strand Orientation Bias Detector (SOBDetector), a flexible computational platform compatible with all the common somatic SNV-calling pipelines, designed to assess the probability whether a given detected mutation is an artifact. The underlying predictor mechanism is based on the posterior distribution of a Bayesian logistic regression model trained on The Cancer Genome Atlas whole exomes. SOBDetector is a freely available cross-platform program, implemented in Java 1.8.

Human endogenous retroviruses form a reservoir of T cell targets in hematological cancers.

Human endogenous retroviruses (HERV) form a substantial part of the human genome, but mostly remain transcriptionally silent under strict epigenetic regulation, yet can potentially be reactivated by malignant transformation or epigenetic therapies. Here, we evaluate the potential for T cell recognition of HERV elements in myeloid malignancies by mapping transcribed HERV genes and generating a library of 1169 potential antigenic HERV-derived peptides predicted for presentation by 4 HLA class I molecules. Using DNA barcode-labeled MHC-I multimers, we find CD8+ T cell populations recognizing 29 HERV-derived peptides representing 18 different HERV loci, of which HERVH-5, HERVW-1, and HERVE-3 have more profound responses; such HERV-specific T cells are present in 17 of the 34 patients, but less frequently in healthy donors. Transcriptomic analyses reveal enhanced transcription of the HERVs in patients; meanwhile DNA-demethylating therapy causes a small and heterogeneous enhancement in HERV transcription without altering T cell recognition. Our study thus uncovers T cell recognition of HERVs in myeloid malignancies, thereby implicating HERVs as potential targets for immunotherapeutic therapies.

Substantial Antigenic Drift in the Hemagglutinin Protein of Swine Influenza A Viruses.

The degree of antigenic drift in swine influenza A viruses (swIAV) has historically been regarded as minimal compared to that of human influenza A virus strains. However, as surveillance activities on swIAV have increased, more isolates have been characterized, revealing a high level of genetic and antigenic differences even within the same swIAV lineage. The objective of this study was to investigate the level of genetic drift in one enzootically infected swine herd over one year. Nasal swabs were collected monthly from sows (n = 4) and piglets (n = 40) in the farrowing unit, and from weaners (n = 20) in the nursery. Virus from 1-4 animals were sequenced per month. Analyses of the sequences revealed that the hemagglutinin (HA) gene was the main target for genetic drift with a substitution rate of 7.6 × 10-3 substitutions/site/year and evidence of positive selection. The majority of the mutations occurred in the globular head of the HA protein and in antigenic sites. The phylogenetic tree of the HA sequences displayed a pectinate typology, where only a single lineage persists and forms the ancestor for subsequent lineages. This was most likely caused by repeated selection of a single immune-escape variant, which subsequently became the founder of the next wave of infections.

Acute Influenza A virus outbreak in an enzootic infected sow herd: Impact on viral dynamics, genetic and antigenic variability and effect of maternally derived antibodies and vaccination.

Influenza A virus (IAV) is a highly contagious pathogen in pigs. Swine IAV (swIAV) infection causes respiratory disease and is thereby a challenge for animal health, animal welfare and the production economy. In Europe, the most widespread strategy for controlling swIAV is implementation of sow vaccination programs, to secure delivery of protective maternally derived antibodies (MDAs) to the newborn piglets. In this study we report a unique case, where a persistently swIAV (A/sw/Denmark/P5U4/2016(H1N1)) infected herd experienced an acute outbreak with a new swIAV subtype (A/sw/Denmark/HB4280U1/2017(H1N2)) and subsequently decided to implement a mass sow vaccination program. Clinical registrations, nasal swabs and blood samples were collected from four different batches of pigs before and after vaccination. Virus isolation, sequencing of the virus strain and hemagglutinin inhibition (HI) tests were performed on samples collected before and during the outbreak and after implementation of mass sow vaccination. After implementation of the sow mass vaccination, the time of infection was delayed and the viral load significantly decreased. An increased number of pigs, however, tested positive at two consecutive sampling times indicating prolonged shedding. In addition, a significantly smaller proportion of the 10-12 weeks old pigs were seropositive by the end of the study, indicating an impaired induction of antibodies against swIAV in the presence of MDAs. Sequencing of the herd strains revealed major differences in the hemagglutinin gene of the strain isolated before- and during the acute outbreak despite that, the two strains belonged to the same HA lineage. The HI tests confirmed a limited degree of cross-reaction between the two strains. Furthermore, the sequencing results of the hemagglutinin gene obtained before and after implementation of mass sow vaccination revealed an increased substitution rate and an increase in positively selected sites in the globular head of the hemagglutinin after vaccination.

Virus Adaptation and Selection Following Challenge of Animals Vaccinated against Classical Swine Fever Virus.

Vaccines against classical swine fever have proven very effective in protecting pigs from this deadly disease. However, little is known about how vaccination impacts the selective pressures acting on the classical swine fever virus (CSFV). Here we use high-throughput sequencing of viral genomes to investigate evolutionary changes in virus populations following the challenge of naïve and vaccinated pigs with the highly virulent CSFV strain "Koslov". The challenge inoculum contained an ensemble of closely related viral sequences, with three major haplotypes being present, termed A, B, and C. After the challenge, the viral haplotype A was preferentially located within the tonsils of naïve animals but was highly prevalent in the sera of all vaccinated animals. We find that the viral population structure in naïve pigs after infection is very similar to that in the original inoculum. In contrast, the viral population in vaccinated pigs, which only underwent transient low-level viremia, displayed several distinct changes including the emergence of 16 unique non-synonymous single nucleotide polymorphisms (SNPs) that were not detectable in the challenge inoculum. Further analysis showed a significant loss of heterogeneity and an increasing positive selection acting on the virus populations in the vaccinated pigs. We conclude that vaccination imposes a strong selective pressure on viruses that subsequently replicate within the vaccinated animal.

Author Correction: 137 ancient human genomes from across the Eurasian steppes.

with In this Article, Angela M. Taravella and Melissa A. Wilson Sayres have been added to the author list (associated with: School of Life Sciences, Center for Evolution and Medicine, The Biodesign Institute, Arizona State University, Tempe, AZ, USA). The author list and Author Information section have been corrected online.

137 ancient human genomes from across the Eurasian steppes.

For thousands of years the Eurasian steppes have been a centre of human migrations and cultural change. Here we sequence the genomes of 137 ancient humans (about 1× average coverage), covering a period of 4,000 years, to understand the population history of the Eurasian steppes after the Bronze Age migrations. We find that the genetics of the Scythian groups that dominated the Eurasian steppes throughout the Iron Age were highly structured, with diverse origins comprising Late Bronze Age herders, European farmers and southern Siberian hunter-gatherers. Later, Scythians admixed with the eastern steppe nomads who formed the Xiongnu confederations, and moved westward in about the second or third century BC, forming the Hun traditions in the fourth-fifth century AD, and carrying with them plague that was basal to the Justinian plague. These nomads were further admixed with East Asian groups during several short-term khanates in the Medieval period. These historical events transformed the Eurasian steppes from being inhabited by Indo-European speakers of largely West Eurasian ancestry to the mostly Turkic-speaking groups of the present day, who are primarily of East Asian ancestry.

Characterization of the enhancer and promoter landscape of inflammatory bowel disease from human colon biopsies.

Inflammatory bowel disease (IBD) is a chronic intestinal disorder, with two main types: Crohn's disease (CD) and ulcerative colitis (UC), whose molecular pathology is not well understood. The majority of IBD-associated SNPs are located in non-coding regions and are hard to characterize since regulatory regions in IBD are not known. Here we profile transcription start sites (TSSs) and enhancers in the descending colon of 94 IBD patients and controls. IBD-upregulated promoters and enhancers are highly enriched for IBD-associated SNPs and are bound by the same transcription factors. IBD-specific TSSs are associated to genes with roles in both inflammatory cascades and gut epithelia while TSSs distinguishing UC and CD are associated to gut epithelia functions. We find that as few as 35 TSSs can distinguish active CD, UC, and controls with 85% accuracy in an independent cohort. Our data constitute a foundation for understanding the molecular pathology, gene regulation, and genetics of IBD.

Bacterial whole genome-based phylogeny: construction of a new benchmarking dataset and assessment of some existing methods.

BACKGROUND: Whole genome sequencing (WGS) is increasingly used in diagnostics and surveillance of infectious diseases. A major application for WGS is to use the data for identifying outbreak clusters, and there is therefore a need for methods that can accurately and efficiently infer phylogenies from sequencing reads. In the present study we describe a new dataset that we have created for the purpose of benchmarking such WGS-based methods for epidemiological data, and also present an analysis where we use the data to compare the performance of some current methods. RESULTS: Our aim was to create a benchmark data set that mimics sequencing data of the sort that might be collected during an outbreak of an infectious disease. This was achieved by letting an E. coli hypermutator strain grow in the lab for 8 consecutive days, each day splitting the culture in two while also collecting samples for sequencing. The result is a data set consisting of 101 whole genome sequences with known phylogenetic relationship. Among the sequenced samples 51 correspond to internal nodes in the phylogeny because they are ancestral, while the remaining 50 correspond to leaves. We also used the newly created data set to compare three different online available methods that infer phylogenies from whole-genome sequencing reads: NDtree, CSI Phylogeny and REALPHY. One complication when comparing the output of these methods with the known phylogeny is that phylogenetic methods typically build trees where all observed sequences are placed as leafs, even though some of them are in fact ancestral. We therefore devised a method for post processing the inferred trees by collapsing short branches (thus relocating some leafs to internal nodes), and also present two new measures of tree similarity that takes into account the identity of both internal and leaf nodes. CONCLUSIONS: Based on this analysis we find that, among the investigated methods, CSI Phylogeny had the best performance, correctly identifying 73% of all branches in the tree and 71% of all clades. We have made all data from this experiment (raw sequencing reads, consensus whole-genome sequences, as well as descriptions of the known phylogeny in a variety of formats) publicly available, with the hope that other groups may find this data useful for benchmarking and exploring the performance of epidemiological methods. All data is freely available at: https://cge.cbs.dtu.dk/services/evolution_data.php .

Using expected sequence features to improve basecalling accuracy of amplicon pyrosequencing data.

BACKGROUND: Amplicon pyrosequencing targets a known genetic region and thus inherently produces reads highly anticipated to have certain features, such as conserved nucleotide sequence, and in the case of protein coding DNA, an open reading frame. Pyrosequencing errors, consisting mainly of nucleotide insertions and deletions, are on the other hand likely to disrupt open reading frames. Such an inverse relationship between errors and expectation based on prior knowledge can be used advantageously to guide the process known as basecalling, i.e. the inference of nucleotide sequence from raw sequencing data. RESULTS: The new basecalling method described here, named Multipass, implements a probabilistic framework for working with the raw flowgrams obtained by pyrosequencing. For each sequence variant Multipass calculates the likelihood and nucleotide sequence of several most likely sequences given the flowgram data. This probabilistic approach enables integration of basecalling into a larger model where other parameters can be incorporated, such as the likelihood for observing a full-length open reading frame at the targeted region. We apply the method to 454 amplicon pyrosequencing data obtained from a malaria virulence gene family, where Multipass generates 20 % more error-free sequences than current state of the art methods, and provides sequence characteristics that allow generation of a set of high confidence error-free sequences. CONCLUSIONS: This novel method can be used to increase accuracy of existing and future amplicon sequencing data, particularly where extensive prior knowledge is available about the obtained sequences, for example in analysis of the immunoglobulin VDJ region where Multipass can be combined with a model for the known recombining germline genes. Multipass is available for Roche 454 data at http://www.cbs.dtu.dk/services/MultiPass-1.0 , and the concept can potentially be implemented for other sequencing technologies as well.

Early divergent strains of Yersinia pestis in Eurasia 5,000 years ago.

The bacteria Yersinia pestis is the etiological agent of plague and has caused human pandemics with millions of deaths in historic times. How and when it originated remains contentious. Here, we report the oldest direct evidence of Yersinia pestis identified by ancient DNA in human teeth from Asia and Europe dating from 2,800 to 5,000 years ago. By sequencing the genomes, we find that these ancient plague strains are basal to all known Yersinia pestis. We find the origins of the Yersinia pestis lineage to be at least two times older than previous estimates. We also identify a temporal sequence of genetic changes that lead to increased virulence and the emergence of the bubonic plague. Our results show that plague infection was endemic in the human populations of Eurasia at least 3,000 years before any historical recordings of pandemics.

Creation of Functional Viruses from Non-Functional cDNA Clones Obtained from an RNA Virus Population by the Use of Ancestral Reconstruction.

RNA viruses have the highest known mutation rates. Consequently it is likely that a high proportion of individual RNA virus genomes, isolated from an infected host, will contain lethal mutations and be non-functional. This is problematic if the aim is to clone and investigate high-fitness, functional cDNAs and may also pose problems for sequence-based analysis of viral evolution. To address these challenges we have performed a study of the evolution of classical swine fever virus (CSFV) using deep sequencing and analysis of 84 full-length cDNA clones, each representing individual genomes from a moderately virulent isolate. In addition to here being used as a model for RNA viruses generally, CSFV has high socioeconomic importance and remains a threat to animal welfare and pig production. We find that the majority of the investigated genomes are non-functional and only 12% produced infectious RNA transcripts. Full length sequencing of cDNA clones and deep sequencing of the parental population identified substitutions important for the observed phenotypes. The investigated cDNA clones were furthermore used as the basis for inferring the sequence of functional viruses. Since each unique clone must necessarily be the descendant of a functional ancestor, we hypothesized that it should be possible to produce functional clones by reconstructing ancestral sequences. To test this we used phylogenetic methods to infer two ancestral sequences, which were then reconstructed as cDNA clones. Viruses rescued from the reconstructed cDNAs were tested in cell culture and pigs. Both reconstructed ancestral genomes proved functional, and displayed distinct phenotypes in vitro and in vivo. We suggest that reconstruction of ancestral viruses is a useful tool for experimental and computational investigations of virulence and viral evolution. Importantly, ancestral reconstruction can be done even on the basis of a set of sequences that all correspond to non-functional variants.

Rescue of the highly virulent classical swine fever virus strain "Koslov" from cloned cDNA and first insights into genome variations relevant for virulence.

Classical swine fever virus (CSFV) strain "Koslov" is highly virulent with a mortality rate of up to 100% in pigs. In this study, we modified non-functional cDNAs generated from the blood of Koslov virus infected pigs by site-directed mutagenesis, removing non-synonymous mutations step-by-step, thereby producing genomes encoding the consensus amino acid sequence. Viruses rescued from the construct corresponding to the inferred parental form were highly virulent, when tested in pigs, with infected animals displaying pronounced clinical symptoms leading to high mortality. The reconstruction therefore gave rise to a functional cDNA corresponding to the highly virulent Koslov strain of CSFV. It could be demonstrated that two single amino acid changes (S763L and P968H) in the surface structural protein E2 resulted in attenuation in the porcine infection system while another single amino acid change within the nonstructural protein NS3 (D2183G) reduced virus growth within cells in vitro.