Recurrent SARS-CoV-2 Mutations in Immunodeficient Patients

Long-term SARS-CoV-2 infections in immunodeficient patients are an important source of variation for the virus but are understudied. Many case studies have been published which describe one or a small number of long-term infected individuals but no study has combined these sequences into a cohesive dataset. This work aims to rectify this and study the genomics of this patient group through a combination of literature searches as well as identifying new case series directly from the COG-UK dataset. The spike gene receptor binding domain (RBD) and N-terminal domains (NTD) were identified as mutation hotspots. Numerous mutations associated with variants of concern were observed to emerge recurrently. Additionally a mutation in the envelope gene, - T30I was determined to be the most recurrent frequently occurring mutation arising in persistent infections. A high proportion of recurrent mutations in immunodeficient individuals are associated with ACE2 affinity, immune escape, or viral packaging optimisation. There is an apparent selective pressure for mutations which aid intra-host transmission or persistence which are often different to mutations which aid inter-host transmission, although the fact that multiple recurrent de novo mutations are considered defining for variants of concern strongly indicates that this potential source of novel variants should not be discounted.

Context-specific emergence and growth of the SARS-CoV-2 Delta variant

The Delta variant of concern of SARS-CoV-2 has spread globally causing large outbreaks and resurgences of COVID-19 cases1-3. The emergence of Delta in the UK occurred on the background of a heterogeneous landscape of immunity and relaxation of non-pharmaceutical interventions4,5. Here we analyse 52,992 Delta genomes from England in combination with 93,649 global genomes to reconstruct the emergence of Delta, and quantify its introduction to and regional dissemination across England, in the context of changing travel and social restrictions. Through analysis of human movement, contact tracing, and virus genomic data, we find that the focus of geographic expansion of Delta shifted from India to a more global pattern in early May 2021. In England, Delta lineages were introduced >1,000 times and spread nationally as non-pharmaceutical interventions were relaxed. We find that hotel quarantine for travellers from India reduced onward transmission from importations; however the transmission chains that later dominated the Delta wave in England had been already seeded before restrictions were introduced. In England, increasing inter-regional travel drove Deltas nationwide dissemination, with some cities receiving >2,000 observable lineage introductions from other regions. Subsequently, increased levels of local population mixing, not the number of importations, was associated with faster relative growth of Delta. Among US states, we find that regions that previously experienced large waves also had faster Delta growth rates, and a model including interactions between immunity and human behaviour could accurately predict the rise of Delta there. Deltas invasion dynamics depended on fine scale spatial heterogeneity in immunity and contact patterns and our findings will inform optimal spatial interventions to reduce transmission of current and future VOCs such as Omicron.

Defining the analytical and clinical sensitivity of the ARTIC method for the detection of SARS-CoV-2

The SARS-CoV-2 ARTIC amplicon protocol is the most widely used genome sequencing method for SARS-CoV-2, accounting for over 43% of publicly-available genome sequences. The protocol utilises 98 primers to amplify [~]400bp fragments of the SARS-CoV-2 genome covering all 30,000 bases. Understanding the analytical performance metrics of this protocol will improve how the data is used and interpreted. Different concentrations of SARS-CoV-2 control material were used to establish the limit of detection (LoD) of the ARTIC protocol. Results demonstrated the LoD was a minimum of 25-50 virus particles per mL. The sensitivity of ARTIC was comparable to the published sensitivities of commercial diagnostics assays and could therefore be used to confirm diagnostic testing results. A set of over 3,600 clinical samples from three UK regions were then evaluated to compare the protocols performance to clinical diagnostic assays (Roche Lightcycler 480 II, AusDiagnostics, Roche Cobas, Hologic Panther, Corman RdRp, Roche Flow, ABI QuantStudio 5, Seegene Nimbus, Qiagen Rotorgene, Abbott M2000, Thermo TaqPath, Xpert). We developed a Python tool, RonaLDO, to perform this validation (available under the GNU GPL3 open-source licence from https://github.com/quadram-institute-bioscience/ronaldo). Positives detected by diagnostic platforms were generally supported by sequencing data; platforms that used RT-qPCR were the best predictors of whether the sample would subsequently sequence successfully. To maximise success of sample sequencing for phylogenetic analysis, samples with Ct <31 should be chosen. For diagnostic tests that do not provide a quantifiable Ct value, adding a quantification step is recommended. The ARTIC SARS-CoV-2 sequencing protocol is highly sensitive, capable of detecting SARS-CoV-2 in samples with Cts in the high 30s. However, to routinely obtain whole genome coverage, samples with Ct <31 are recommended. Comparing different virus detection methods close to their LoD was challenging and significant discordance was observed.

Generation and transmission of inter-lineage recombinants in the SARS-CoV-2 pandemic

We present evidence for multiple independent origins of recombinant SARS-CoV-2 viruses sampled from late 2020 and early 2021 in the United Kingdom. Their genomes carry single nucleotide polymorphisms and deletions that are characteristic of the B.1.1.7 variant of concern, but lack the full complement of lineage-defining mutations. Instead, the remainder of their genomes share contiguous genetic variation with non-B.1.1.7 viruses circulating in the same geographic area at the same time as the recombinants. In four instances there was evidence for onward transmission of a recombinant-origin virus, including one transmission cluster of 45 sequenced cases over the course of two months. The inferred genomic locations of recombination breakpoints suggest that every community-transmitted recombinant virus inherited its spike region from a B.1.1.7 parental virus, consistent with a transmission advantage for B.1.1.7s set of mutations.

MAJORA: Continuous integration supporting decentralised sequencing for SARS-CoV-2 genomic surveillance

Genomic epidemiology has become an increasingly common tool for epidemic response. Recent technological advances have made it possible to sequence genomes rapidly enough to inform outbreak response, and cheaply enough to justify dense sampling of even large epidemics. With increased availability of sequencing it is possible for agile networks of sequencing facilities to collaborate on the sequencing and analysis of epidemic genomic data. In response to the ongoing SARS-CoV-2 pandemic in the United Kingdom, the COVID-19 Genomics UK (COG-UK) consortium was formed with the aim of rapidly sequencing SARS-CoV-2 genomes as part of a national-scale genomic surveillance strategy. The network consists of universities, academic institutes, regional sequencing centres and the four UK Public Health Agencies. We describe the development and deployment of Majora, an encompassing digital infrastructure to address the challenge of collecting and integrating both genomic sequencing data and sample-associated metadata produced across the COG-UK network. The system was designed and implemented pragmatically to stand up capacity rapidly in a pandemic caused by a novel virus. This approach has underpinned the success of COG-UK, which has rapidly become the leading contributor of SARS-CoV-2 genomes to international databases and has generated over 60,000 sequences to date.

Improvements to the ARTIC multiplex PCR method for SARS-CoV-2 genome sequencing using nanopore

Genome sequencing has been widely deployed to study the evolution of SARS-CoV-2 with more than 90,000 genome sequences uploaded to the GISAID database. We published a method for SARS-CoV-2 genome sequencing (https://www.protocols.io/view/ncov-2019-sequencing-protocol-bbmuik6w) online on January 22, 2020. This approach has rapidly become the most popular method for sequencing SARS-CoV-2 due to its simplicity and cost-effectiveness. Here we present improvements to the original protocol: i) an updated primer scheme with 22 additional primers to improve genome coverage, ii) a streamlined library preparation workflow which improves demultiplexing rate for up to 96 samples and reduces hands-on time by several hours and iii) cost savings which bring the reagent cost down to {pound}10 per sample making it practical for individual labs to sequence thousands of SARS-CoV-2 genomes to support national and international genomic epidemiology efforts.

Coast-to-coast spread of SARS-CoV-2 in the United States revealed by genomic epidemiology

Since its emergence and detection in Wuhan, China in late 2019, the novel coronavirus SARS-CoV-2 has spread to nearly every country around the world, resulting in hundreds of thousands of infections to date. The virus 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 U.S., 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 impacts of federal travel restrictions. This study provides evidence for widespread, sustained transmission of SARS-CoV-2 within the U.S. and highlights the critical need for local surveillance.