Coast-to-Coast Spread of SARS-CoV-2 during the Early Epidemic in the United States.

The novel coronavirus SARS-CoV-2 was first detected in the Pacific Northwest region of the United States in January 2020, with subsequent COVID-19 outbreaks detected in all 50 states by early March. To uncover the sources of SARS-CoV-2 introductions and patterns of spread within the United States, we sequenced nine viral genomes from early reported COVID-19 patients in Connecticut. Our phylogenetic analysis places the majority of these genomes with viruses sequenced from Washington state. By coupling our genomic data with domestic and international travel patterns, we show that early SARS-CoV-2 transmission in Connecticut was likely driven by domestic introductions. Moreover, the risk of domestic importation to Connecticut exceeded that of international importation by mid-March regardless of our estimated effects of federal travel restrictions. This study provides evidence of widespread sustained transmission of SARS-CoV-2 within the United States and highlights the critical need for local surveillance.

Resurgence of Ebola virus in 2021 in Guinea suggests a new paradigm for outbreaks.

Seven years after the declaration of the first epidemic of Ebola virus disease in Guinea, the country faced a new outbreak-between 14 February and 19 June 2021-near the epicentre of the previous epidemic1,2. Here we use next-generation sequencing to generate complete or near-complete genomes of Zaire ebolavirus from samples obtained from 12 different patients. These genomes form a well-supported phylogenetic cluster with genomes from the previous outbreak, which indicates that the new outbreak was not the result of a new spillover event from an animal reservoir. The 2021 lineage shows considerably lower divergence than would be expected during sustained human-to-human transmission, which suggests a persistent infection with reduced replication or a period of latency. The resurgence of Zaire ebolavirus from humans five years after the end of the previous outbreak of Ebola virus disease reinforces the need for long-term medical and social care for patients who survive the disease, to reduce the risk of re-emergence and to prevent further stigmatization.

Author Correction: Nanopore native RNA sequencing of a human poly(A) transcriptome.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Virus genomes reveal factors that spread and sustained the Ebola epidemic.

The 2013-2016 West African epidemic caused by the Ebola virus was of unprecedented magnitude, duration and impact. Here we reconstruct the dispersal, proliferation and decline of Ebola virus throughout the region by analysing 1,610 Ebola virus genomes, which represent over 5% of the known cases. We test the association of geography, climate and demography with viral movement among administrative regions, inferring a classic 'gravity' model, with intense dispersal between larger and closer populations. Despite attenuation of international dispersal after border closures, cross-border transmission had already sown the seeds for an international epidemic, rendering these measures ineffective at curbing the epidemic. We address why the epidemic did not spread into neighbouring countries, showing that these countries were susceptible to substantial outbreaks but at lower risk of introductions. Finally, we reveal that this large epidemic was a heterogeneous and spatially dissociated collection of transmission clusters of varying size, duration and connectivity. These insights will help to inform interventions in future epidemics.

Genomic epidemiology reveals multiple introductions of Zika virus into the United States.

Zika virus (ZIKV) is causing an unprecedented epidemic linked to severe congenital abnormalities. In July 2016, mosquito-borne ZIKV transmission was reported in the continental United States; since then, hundreds of locally acquired infections have been reported in Florida. To gain insights into the timing, source, and likely route(s) of ZIKV introduction, we tracked the virus from its first detection in Florida by sequencing ZIKV genomes from infected patients and Aedes aegypti mosquitoes. We show that at least 4 introductions, but potentially as many as 40, contributed to the outbreak in Florida and that local transmission is likely to have started in the spring of 2016-several months before its initial detection. By analysing surveillance and genetic data, we show that ZIKV moved among transmission zones in Miami. Our analyses show that most introductions were linked to the Caribbean, a finding corroborated by the high incidence rates and traffic volumes from the region into the Miami area. Our study provides an understanding of how ZIKV initiates transmission in new regions.

SARS-CoV-2 Testing in the Community: Testing Positive Samples with the TaqMan SARS-CoV-2 Mutation Panel To Find Variants in Real Time.

Genome sequencing is a powerful tool for identifying SARS-CoV-2 variant lineages; however, there can be limitations due to sequence dropout when used to identify specific key mutations. Recently, ThermoFisher Scientific has developed genotyping assays to help bridge the gap between testing capacity and sequencing capability to generate real-time genotyping results based on specific variants. Over a 6-week period during the months of April and May 2021, we set out to assess the ThermoFisher TaqMan mutation panel genotyping assay, initially for three mutations of concern and then for an additional two mutations of concern, against SARS-CoV-2-positive clinical samples and the corresponding COVID-19 Genomics UK Consortium (COG-UK) sequencing data. We demonstrate that genotyping is a powerful in-depth technique for identifying specific mutations, is an excellent complement to genome sequencing, and has real clinical health value potential, allowing laboratories to report and take action on variants of concern much more quickly.

Nanopore native RNA sequencing of a human poly(A) transcriptome.

High-throughput complementary DNA sequencing technologies have advanced our understanding of transcriptome complexity and regulation. However, these methods lose information contained in biological RNA because the copied reads are often short and modifications are not retained. We address these limitations using a native poly(A) RNA sequencing strategy developed by Oxford Nanopore Technologies. Our study generated 9.9 million aligned sequence reads for the human cell line GM12878, using thirty MinION flow cells at six institutions. These native RNA reads had a median length of 771 bases, and a maximum aligned length of over 21,000 bases. Mitochondrial poly(A) reads provided an internal measure of read-length quality. We combined these long nanopore reads with higher accuracy short-reads and annotated GM12878 promoter regions to identify 33,984 plausible RNA isoforms. We describe strategies for assessing 3' poly(A) tail length, base modifications and transcript haplotypes.

Genomic Surveillance of Yellow Fever Virus Epizootic in São Paulo, Brazil, 2016 - 2018.

São Paulo, a densely inhabited state in southeast Brazil that contains the fourth most populated city in the world, recently experienced its largest yellow fever virus (YFV) outbreak in decades. YFV does not normally circulate extensively in São Paulo, so most people were unvaccinated when the outbreak began. Surveillance in non-human primates (NHPs) is important for determining the magnitude and geographic extent of an epizootic, thereby helping to evaluate the risk of YFV spillover to humans. Data from infected NHPs can give more accurate insights into YFV spread than when using data from human cases alone. To contextualise human cases, identify epizootic foci and uncover the rate and direction of YFV spread in São Paulo, we generated and analysed virus genomic data and epizootic case data from NHPs in São Paulo. We report the occurrence of three spatiotemporally distinct phases of the outbreak in São Paulo prior to February 2018. We generated 51 new virus genomes from YFV positive cases identified in 23 different municipalities in São Paulo, mostly sampled from NHPs between October 2016 and January 2018. Although we observe substantial heterogeneity in lineage dispersal velocities between phylogenetic branches, continuous phylogeographic analyses of generated YFV genomes suggest that YFV lineages spread in São Paulo at a mean rate of approximately 1km per day during all phases of the outbreak. Viral lineages from the first epizootic phase in northern São Paulo subsequently dispersed towards the south of the state to cause the second and third epizootic phases there. This alters our understanding of how YFV was introduced into the densely populated south of São Paulo state. Our results shed light on the sylvatic transmission of YFV in highly fragmented forested regions in São Paulo state and highlight the importance of continued surveillance of zoonotic pathogens in sentinel species.

Real-time, portable genome sequencing for Ebola surveillance.

The Ebola virus disease epidemic in West Africa is the largest on record, responsible for over 28,599 cases and more than 11,299 deaths. Genome sequencing in viral outbreaks is desirable to characterize the infectious agent and determine its evolutionary rate. Genome sequencing also allows the identification of signatures of host adaptation, identification and monitoring of diagnostic targets, and characterization of responses to vaccines and treatments. The Ebola virus (EBOV) genome substitution rate in the Makona strain has been estimated at between 0.87 × 10(-3) and 1.42 × 10(-3) mutations per site per year. This is equivalent to 16-27 mutations in each genome, meaning that sequences diverge rapidly enough to identify distinct sub-lineages during a prolonged epidemic. Genome sequencing provides a high-resolution view of pathogen evolution and is increasingly sought after for outbreak surveillance. Sequence data may be used to guide control measures, but only if the results are generated quickly enough to inform interventions. Genomic surveillance during the epidemic has been sporadic owing to a lack of local sequencing capacity coupled with practical difficulties transporting samples to remote sequencing facilities. To address this problem, here we devise a genomic surveillance system that utilizes a novel nanopore DNA sequencing instrument. In April 2015 this system was transported in standard airline luggage to Guinea and used for real-time genomic surveillance of the ongoing epidemic. We present sequence data and analysis of 142 EBOV samples collected during the period March to October 2015. We were able to generate results less than 24 h after receiving an Ebola-positive sample, with the sequencing process taking as little as 15-60 min. We show that real-time genomic surveillance is possible in resource-limited settings and can be established rapidly to monitor outbreaks.

A complete bacterial genome assembled de novo using only nanopore sequencing data.

We have assembled de novo the Escherichia coli K-12 MG1655 chromosome in a single 4.6-Mb contig using only nanopore data. Our method has three stages: (i) overlaps are detected between reads and then corrected by a multiple-alignment process; (ii) corrected reads are assembled using the Celera Assembler; and (iii) the assembly is polished using a probabilistic model of the signal-level data. The assembly reconstructs gene order and has 99.5% nucleotide identity.

Field investigation with real-time virus genetic characterisation support of a cluster of Ebola virus disease cases in Dubréka, Guinea, April to June 2015.

On 11 May 2015, the Dubréka prefecture, Guinea, reported nine laboratory-confirmed cases of Ebola virus disease (EVD). None could be epidemiologically linked to cases previously reported in the prefecture. We describe the epidemiological and molecular investigations of this event. We used the Dubréka EVD registers and the Ebola treatment centre's (ETC) records to characterise chains of transmission. Real-time field Ebola virus sequencing was employed to support epidemiological results. An epidemiological cluster of 32 cases was found, of which 27 were laboratory confirmed, 24 were isolated and 20 died. Real-time viral sequencing on 12 cases demonstrated SL3 lineage viruses with sequences differing by one to three nt inside a single phylogenetic cluster. For isolated cases, the average time between symptom onset and ETC referral was 2.8 days (interquartile range (IQR): 1-4). The average time between sample collection and molecular results' availability was 3 days (IQR: 2-5). In an area with scarce resources, the genetic characterisation supported the outbreak investigations in real time, linking cases where epidemiological investigation was limited and reassuring that the responsible strain was already circulating in Guinea. We recommend coupling thorough epidemiological and genomic investigations to control EVD clusters.

Binning metagenomic contigs by coverage and composition.

Shotgun sequencing enables the reconstruction of genomes from complex microbial communities, but because assembly does not reconstruct entire genomes, it is necessary to bin genome fragments. Here we present CONCOCT, a new algorithm that combines sequence composition and coverage across multiple samples, to automatically cluster contigs into genomes. We demonstrate high recall and precision on artificial as well as real human gut metagenome data sets.

Left Ventricular Assist Device Thrombosis Is Associated With an Increase in the Systolic-to-Diastolic Velocity Ratio Measured at the Inflow and Outflow Cannulae.

OBJECTIVE: To determine whether the ratio of peak systolic-to-nadir diastolic velocity (S/D ratio) measured using Doppler at the left ventricular assist device (LVAD) inflow and outflow cannulae is associated with pump thrombosis and to determine whether there is an absolute decrease in the diastolic cannula velocities in LVAD thrombosis. DESIGN: Retrospective chart review. SETTING: University hospital. PARTICIPANTS: Patients who underwent LVAD exchange. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Transesophageal echocardiograms were reviewed from all patients with the HeartMate II device (Thoratec Corporation, Pleasanton, CA) over a 6-year period and who underwent LVAD exchange for pump thrombosis. The following 3 time points were evaluated: (1) initial LVAD placement (prethrombosis), (2) thrombosis, and (3) exchanged LVAD placement (postthrombosis). Systolic and diastolic flow velocities were examined using pulse-wave spectral Doppler at the inflow and outflow cannulae, and the S/D ratio for each was determined. Statistical analysis was performed with SAS, version 9.4 (SAS Institute, Cary, NC), using 2-tailed tests and alpha = 0.05. Thirteen patients were included in the study. Significant differences were observed in S/D ratios among the 3 phases at both the inflow (p = 0.0234) and outflow (p = 0.0047) cannulae. Pairwise tests of the inflow cannulae showed that the mean S/D ratio at the time of thrombosis (mean ± standard deviation [SD], 4.29 ± 1.74) was significantly greater than the prethrombosis ratio (2.49 ± 0.65; p = 0.0069). Among outflow measurements, the mean S/D ratio at thrombosis (3.94 ± 1.34) was significantly higher than both the prethrombosis (2.63 ± 0.56; p = 0.0025) and postthrombosis (2.74 ± 0.83) (p = 0.0093) ratios. Decreases in diastolic velocities were not statistically significant at the inflow cannula. At the outflow cannula, there was a significant difference in diastolic velocity among the phases (p = 0.0233). Specifically, the postthrombosis diastolic measurements (41.50 ± 9.94) were significantly higher than both the prethrombosis (26.85 ± 10.13; p = 0.0140) and thrombosis (26.7 ± 15.35; p = 0.0151) values. CONCLUSIONS: An increased S/D ratio measured with Doppler at the LVAD inflow and outflow cannulas may be associated with pump thrombosis. Decreased diastolic cannula velocities were not observed in LVAD thrombosis.

Unusual Ebola Virus Chain of Transmission, Conakry, Guinea, 2014-2015.

In October 2015, a new case of Ebola virus disease in Guinea was detected. Case investigation, serology, and whole-genome sequencing indicated possible transmission of the virus from an Ebola virus disease survivor to another person and then to the case-patient reported here. This transmission chain over 11 months suggests slow Ebola virus evolution.

Extensive Modulation of the Fecal Metagenome in Children With Crohn's Disease During Exclusive Enteral Nutrition.

OBJECTIVES: Exploring associations between the gut microbiota and colonic inflammation and assessing sequential changes during exclusive enteral nutrition (EEN) may offer clues into the microbial origins of Crohn's disease (CD). METHODS: Fecal samples (n=117) were collected from 23 CD and 21 healthy children. From CD children fecal samples were collected before, during EEN, and when patients returned to their habitual diets. Microbiota composition and functional capacity were characterized using sequencing of the 16S rRNA gene and shotgun metagenomics. RESULTS: Microbial diversity was lower in CD than controls before EEN (P=0.006); differences were observed in 36 genera, 141 operational taxonomic units (OTUs), and 44 oligotypes. During EEN, the microbial diversity of CD children further decreased, and the community structure became even more dissimilar than that of controls. Every 10 days on EEN, 0.6 genus diversity equivalents were lost; 34 genera decreased and one increased during EEN. Fecal calprotectin correlated with 35 OTUs, 14 of which accounted for 78% of its variation. OTUs that correlated positively or negatively with calprotectin decreased during EEN. The microbiota of CD patients had a broader functional capacity than healthy controls, but diversity decreased with EEN. Genes involved in membrane transport, sulfur reduction, and nutrient biosynthesis differed between patients and controls. The abundance of genes involved in biotin (P=0.005) and thiamine biosynthesis decreased (P=0.017), whereas those involved in spermidine/putrescine biosynthesis (P=0.031), or the shikimate pathway (P=0.058), increased during EEN. CONCLUSIONS: Disease improvement following treatment with EEN is associated with extensive modulation of the gut microbiome.

Tracking arboviruses, their transmission vectors and potential hosts by nanopore sequencing of mosquitoes.

The risk to human health from mosquito-borne viruses such as dengue, chikungunya and yellow fever is increasing due to increased human expansion, deforestation and climate change. To anticipate and predict the spread and transmission of mosquito-borne viruses, a better understanding of the transmission cycle in mosquito populations is needed. We present a pathogen-agnostic combined sequencing protocol for identifying vectors, viral pathogens and their hosts or reservoirs using portable Oxford Nanopore sequencing. Using mosquitoes collected in São Paulo, Brazil, we extracted RNA for virus identification and DNA for blood meal and mosquito identification. Mosquitoes and blood meals were identified by comparing cytochrome c oxidase I (COI) sequences against a curated Barcode of Life Data System (BOLD). Viruses were identified using the SMART-9N protocol, which allows amplified DNA to be prepared with native barcoding for nanopore sequencing. Kraken 2 was employed to detect viral pathogens and Minimap2 and BOLD identified the contents of the blood meal. Due to the high similarity of some species, mosquito identification was conducted using blast after generation of consensus COI sequences using RACON polishing. This protocol can simultaneously uncover viral diversity, mosquito species and mosquito feeding habits. It also has the potential to increase understanding of mosquito genetic diversity and transmission dynamics of zoonotic mosquito-borne viruses.

A culture-independent sequence-based metagenomics approach to the investigation of an outbreak of Shiga-toxigenic Escherichia coli O104:H4.

IMPORTANCE: Identification of the bacterium responsible for an outbreak can aid in disease management. However, traditional culture-based diagnosis can be difficult, particularly if no specific diagnostic test is available for an outbreak strain. OBJECTIVE: To explore the potential of metagenomics, which is the direct sequencing of DNA extracted from microbiologically complex samples, as an open-ended clinical discovery platform capable of identifying and characterizing bacterial strains from an outbreak without laboratory culture. DESIGN, SETTING, AND PATIENTS: In a retrospective investigation, 45 samples were selected from fecal specimens obtained from patients with diarrhea during the 2011 outbreak of Shiga-toxigenic Escherichia coli (STEC) O104:H4 in Germany. Samples were subjected to high-throughput sequencing (August-September 2012), followed by a 3-phase analysis (November 2012-February 2013). In phase 1, a de novo assembly approach was developed to obtain a draft genome of the outbreak strain. In phase 2, the depth of coverage of the outbreak strain genome was determined in each sample. In phase 3, sequences from each sample were compared with sequences from known bacteria to identify pathogens other than the outbreak strain. MAIN OUTCOMES AND MEASURES: The recovery of genome sequence data for the purposes of identification and characterization of the outbreak strain and other pathogens from fecal samples. RESULTS: During phase 1, a draft genome of the STEC outbreak strain was obtained. During phase 2, the outbreak strain genome was recovered from 10 samples at greater than 10-fold coverage and from 26 samples at greater than 1-fold coverage. Sequences from the Shiga-toxin genes were detected in 27 of 40 STEC-positive samples (67%). In phase 3, sequences from Clostridium difficile, Campylobacter jejuni, Campylobacter concisus, and Salmonella enterica were recovered. CONCLUSIONS AND RELEVANCE: These results suggest the potential of metagenomics as a culture-independent approach for the identification of bacterial pathogens during an outbreak of diarrheal disease. Challenges include improving diagnostic sensitivity, speeding up and simplifying workflows, and reducing costs.

Coding-Complete Genome Sequences of Copenhagen and Copenhagen-Derived vP811 Strains of Vaccinia Virus Isolated from Cell Culture.

The coding-complete genomes of laboratory vaccinia virus strain Copenhagen and the Copenhagen-derived deletion strain, vP811, were determined by short-read sequencing. Relative to the NCBI reference genome M35027, seven common coding differences were revealed, including an intact copy of the vaccinia virus immunomodulator A46R in both Cop and vP811.

Multiplex MinION sequencing suggests enteric adenovirus F41 genetic diversity comparable to pre-COVID-19 era.

Human adenovirus F41 causes acute gastroenteritis in children, and has recently been associated with an apparent increase in paediatric hepatitis of unknown aetiology in the UK, with further cases reported in multiple countries. Relatively little is known about the genetic diversity of adenovirus F41 in UK children; and it is unclear what, if any, impact the COVID-19 pandemic has had on viral diversity in the UK. Methods that allow F41 to be sequenced from clinical samples without the need for viral culture are required to provide the genomic data to address these questions. Therefore, we evaluated an overlapping-amplicon method of sequencing adenovirus genomes from clinical samples using Oxford Nanopore technology. We applied this method to a small sample of adenovirus-species-F-positive extracts collected as part of standard care in the East of England region in January-May 2022. This method produced genomes with >75 % coverage in 13/22 samples and >50 % coverage in 19/22 samples. We identified two F41 lineages present in paediatric patients in the East of England in 2022. Where F41 genomes from paediatric hepatitis cases were available (n=2), these genomes fell within the diversity of F41 from the UK and continental Europe sequenced before and after the 2020-2021 phase of the COVID-19 pandemic. Our analyses suggest that overlapping amplicon sequencing is an appropriate method for generating F41 genomic data from high-virus-load clinical samples, and currently circulating F41 viral lineages were present in the UK and Europe before the COVID-19 pandemic.

Genomic epidemiology of human adenovirus F40 and F41 in coastal Kenya: A retrospective hospital-based surveillance study (2013-2022).

Human enteric adenovirus species F (HAdV-F) is a leading cause of childhood diarrhoeal deaths. The genomic analysis would be key to understanding transmission dynamics, potential drivers of disease severity, and vaccine development. However, currently, there are limited HAdV-F genomic data globally. Here, we sequenced and analysed HAdV-F from stool samples collected in coastal Kenya between 2013 and 2022. The samples were collected at Kilifi County Hospital in coastal Kenya from children <13 years of age who reported a history of three or more loose stools in the previous 24 hours. The genomes were analysed together with the data from the rest of the world by phylogenetic analysis and mutational profiling. Types and lineages were assigned based on phylogenetic clustering consistent with the previously described criteria and nomenclature. Participant clinical and demographic data were linked to genotypic data. Of ninety-one cases identified using real-time Polymerase Chain Reaction, eighty-eight near-complete genomes were assembled, and these were classified into HAdV-F40 (n = 41) and HAdV-F41 (n = 47). These types co-circulated throughout the study period. Three and four distinct lineages were observed for HAdV-F40 (Lineages 1-3) and HAdV-F41 (Lineages 1, 2A, 3A, 3C, and 3D). Types F40 and F41 coinfections were observed in five samples and F41 and B7 in one sample. Two children with F40 and 41 coinfections were also infected with rotavirus and had moderate and severe diseases as defined using the Vesikari Scoring System, respectively. Intratypic recombination was found in four HAdV-F40 sequences occurring between Lineages 1 and 3. None of the HAdV-F41 cases had jaundice. This study provides evidence of extensive genetic diversity, coinfections, and recombination within HAdV-F40 in a rural coastal Kenya that will inform public health policy, vaccine development that includes the locally circulating lineages, and molecular diagnostic assay development. We recommend future comprehensive studies elucidating on HAdV-F genetic diversity and immunity for rational vaccine development.