Evolution of the SARS-CoV-2 mutational spectrum

SARS-CoV-2 evolves rapidly in part because of its high mutation rate. Here we examine whether this mutational process itself has changed during viral evolution. To do this, we quantify the relative rates of different types of single nucleotide mutations at four-fold degenerate sites in the viral genome across millions of human SARS-CoV-2 sequences. We find clear shifts in the relative rates of several types of mutations during SARS-CoV-2 evolution. The most striking trend is a roughly two-fold decrease in the relative rate of G[->]T mutations in Omicron versus early clades, as was recently noted by Ruis et al (2022). There is also a decrease in the relative rate of C[->]T mutations in Delta, and other subtle changes in the mutation spectrum along the phylogeny. We speculate that these changes in the mutation spectrum could arise from viral mutations that affect genome replication, packaging, and antagonization of host innate-immune factors--although environmental factors could also play a role. Interestingly, the mutation spectrum of Omicron is more similar than that of earlier SARS-CoV-2 clades to the spectrum that shaped the long-term evolution of sarbecoviruses. Overall, our work shows that the mutation process is itself a dynamic variable during SARS-CoV-2 evolution, and suggests that human SARS-CoV-2 may be trending towards a mutation spectrum more similar to that of other animal sarbecoviruses.

A pseudovirus system enables deep mutational scanning of the full SARS-CoV-2 spike

A major challenge in understanding SARS-CoV-2 evolution is interpreting the antigenic and functional effects of emerging mutations in the viral spike protein. Here we describe a new deep mutational scanning platform based on non-replicative pseudotyped lentiviruses that directly quantifies how large numbers of spike mutations impact antibody neutralization and pseudovirus infection. We demonstrate this new platform by making libraries of the Omicron BA.1 and Delta spikes. These libraries each contain ~7000 distinct amino-acid mutations in the context of up to ~135,000 unique mutation combinations. We use these libraries to map escape mutations from neutralizing antibodies targeting the receptor binding domain, N-terminal domain, and S2 subunit of spike. Overall, this work establishes a high-throughput and safe approach to measure how ~105 combinations of mutations affect antibody neutralization and spike-mediated infection. Notably, the platform described here can be extended to the entry proteins of many other viruses.

Contributions of adaptation and purifying selection to SARS-CoV-2 evolution

Continued evolution and adaptation of SARS-CoV-2 has lead to more transmissible and immune-evasive variants with profound impact on the course of the pandemic. Here I analyze the evolution of the virus over 2.5 years since its emergence and estimate rates of evolution for synonymous and non-synonymous changes separately for evolution within clades - well defined mono-phyletic groups with gradual evolution - and for the pandemic overall. The rate of synonymous mutations is found to be around 6 changes per year. Synonymous rates within variants vary little from variant to variant and are compatible with the overall rate of 7 changes per year (or 7.5 x 10-4 per year and codon). In contrast, the rate at which variants accumulate amino acid changes (non-synonymous mutation) was initially around 12-16 changes per year, but in 2021 and 2022 dropped to 6-9 changes per year. The overall rate of non-synonymous evolution, that is across variants, is estimated to be about 26 amino acid changes per year (or 2.7 x 10-3 per year and codon). This strong acceleration of the overall rate compared to within clade evolution indicates that the evolutionary process that gave rise to the different variants is qualitatively different from that in typical transmission chains and likely dominated by adaptive evolution. I further quantify the spectrum of mutations and purifying selection in different SARS-CoV-2 proteins and show that the massive global sampling of SARS-CoV-2 is sufficient to estimate site specific fitness costs across the entire genome. Many accessory proteins evolve under limited evolutionary constraint with little short term purifying selection. About half of the mutations in other proteins are strongly deleterious.

Swiss public health measures associated with reduced SARS-CoV-2 transmission using genome data

Genome sequences from evolving infectious pathogens allow quantification of case introductions and local transmission dynamics. We sequenced 11,357 SARS-CoV-2 genomes from Switzerland in 2020 - the 6th largest effort globally. Using a representative subset of these data, we estimated viral introductions to Switzerland and their persistence over the course of 2020. We contrast these estimates with simple null models representing the absence of certain public health measures. We show that Switzerlands border closures de-coupled case introductions from incidence in neighboring countries. Under a simple model, we estimate an 86 - 98% reduction in introductions during Switzerlands strictest border closures. Furthermore, the Swiss 2020 partial lockdown roughly halved the time for sampled introductions to die out. Finally, we quantified local transmission dynamics once introductions into Switzerland occurred, using a novel phylodynamic model. We find that transmission slowed 35 - 63% upon outbreak detection in summer 2020, but not in fall. This finding may indicate successful contact tracing over summer before overburdening in fall. The study highlights the added value of genome sequencing data for understanding transmission dynamics. One Sentence SummaryPhylogenetic and phylodynamic methods quantify the drop in case introductions and local transmission with implementation of public health measures.

PCR assay to enhance global surveillance for SARS-CoV-2 variants of concern

With the emergence of SARS-CoV-2 variants that may increase transmissibility and/or cause escape from immune responses1-3, there is an urgent need for the targeted surveillance of circulating lineages. It was found that the B.1.1.7 (also 501Y.V1) variant first detected in the UK4,5 could be serendipitously detected by the ThermoFisher TaqPath COVID-19 PCR assay because a key deletion in these viruses, spike {Delta}69-70, would cause a "spike gene target failure" (SGTF) result. However, a SGTF result is not definitive for B.1.1.7, and this assay cannot detect other variants of concern that lack spike {Delta}69-70, such as B.1.351 (also 501Y.V2) detected in South Africa6 and P.1 (also 501Y.V3) recently detected in Brazil7. We identified a deletion in the ORF1a gene (ORF1a {Delta}3675-3677) in all three variants, which has not yet been widely detected in other SARS-CoV-2 lineages. Using ORF1a {Delta}3675-3677 as the primary target and spike {Delta}69-70 to differentiate, we designed and validated an open source PCR assay to detect SARS-CoV-2 variants of concern8. Our assay can be rapidly deployed in laboratories around the world to enhance surveillance for the local emergence spread of B.1.1.7, B.1.351, and P.1.

Estimation and worldwide monitoring of the effective reproductive number of SARS-CoV-2

The effective reproductive number Re is a key indicator of the growth of an epidemic. Since the start of the SARS-CoV-2 pandemic, many methods and online dashboards have sprung up to monitor this number through time. However, these methods are not always thoroughly tested, correctly placed in time, or are overly confident during high incidence periods. Here, we present a method for timely estimation of Re, applied to COVID-19 epidemic data from 170 countries. We thoroughly evaluate the method on simulated data, and present an intuitive web interface for interactive data exploration. We show that, in early 2020, in the majority of countries the estimated Re dropped below 1 only after the introduction of major non-pharmaceutical interventions. For Europe the implementation of non-pharmaceutical interventions was broadly associated with reductions in the estimated Re. Globally though, relaxing non-pharmaceutical interventions had more varied effects on subsequent Re estimates. Our framework is useful to inform governments and the general public on the status of epidemics in their country, and is used as the official source of Re estimates for SARS-CoV-2 in Switzerland. It further allows detailed comparison between countries and in relation to covariates such as implemented public health policies, mobility, behaviour, or weather data.

Emergence and spread of a SARS-CoV-2 variant through Europe in the summer of 2020

Following its emergence in late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic resulting in unprecedented efforts to reduce transmission and develop therapies and vaccines (WHO Emergency Committee, 2020; Zhu et al., 2020). Rapidly generated viral genome sequences have allowed the spread of the virus to be tracked via phylogenetic analysis (Worobey et al., 2020; Hadfield et al., 2018; Pybus et al., 2020). While the virus spread globally in early 2020 before borders closed, intercontinental travel has since been greatly reduced, allowing continent-specific variants to emerge. However, within Europe travel resumed in the summer of 2020, and the impact of this travel on the epidemic is not well understood. Here we report on a novel SARS-CoV-2 variant, 20E (EU1), that emerged in Spain in early summer, and subsequently spread to multiple locations in Europe. We find no evidence of increased transmissibility of this variant, but instead demonstrate how rising incidence in Spain, resumption of travel across Europe, and lack of effective screening and containment may explain the variants success. Despite travel restrictions and quarantine requirements, we estimate 20E (EU1) was introduced hundreds of times to countries across Europe by summertime travellers, likely undermining local efforts to keep SARS-CoV-2 cases low. Our results demonstrate how a variant can rapidly become dominant even in absence of a substantial transmission advantage in favorable epidemiological settings. Genomic surveillance is critical to understanding how travel can impact SARS-CoV-2 transmission, and thus for informing future containment strategies as travel resumes. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the first pandemic where the spread of a viral pathogen has been globally tracked in near real-time using phylogenetic analysis of viral genome sequences (Worobey et al., 2020; Hadfield et al., 2018; Pybus et al., 2020). SARS-CoV-2 genomes continue to be generated at a rate far greater than for any other pathogen and more than 500,000 full genomes are available on GISAID as of February 2020 (Shu and McCauley, 2017). In addition to tracking the viral spread, these genome sequences have been used to monitor mutations which might change the transmission, pathogenesis, or anti-genic properties of the virus. One mutation in particular, D614G in the spike protein, has received much attention. This variant (Nextstrain clade 20A) seeded large outbreaks in Europe in early 2020 and subsequently dominated the outbreaks in the Americas, thereby largely replacing previously circulating lineages. This rapid rise led to the suggestion that this variant is more transmissible, which has since been corroborated by phylogenetic (Korber et al., 2020; Volz et al., 2020) and experimental evidence (Plante et al., 2020; Yurkovetskiy et al., 2020). Following the global dissemination of SARS-CoV-2 in early 2020 (Worobey et al., 2020), intercontinental travel dropped dramatically. Within Europe, however, travel and in particular holiday travel resumed in summer (though at lower levels than in previous years) with largely uncharacterized effects on the pandemic. Here we report on a novel SARS-CoV-2 variant 20E (EU1) (S:A222V) that emerged in early summer 2020, presumably in Spain, and subsequently spread to multiple locations in Europe. Over the summer, it rose in frequency in parallel in multiple countries. As we report here, this variant, 20E (EU1), and a second variant 20A.EU2 with mutation S477N in the spike protein accounted for the majority of sequences in Europe in the autumn of 2020.

Quantifying SARS-CoV-2 spread in Switzerland based on genomic sequencing data

Pathogen genomes provide insights into their evolution and epidemic spread. We sequenced 1,439 SARS-CoV-2 genomes from Switzerland, representing 3-7% of all confirmed cases per week. Using these data, we demonstrate that no one lineage became dominant, pointing against evolution towards general lower virulence. On an epidemiological level, we report no evidence of cryptic transmission before the first confirmed case. We find many early viral introductions from Germany, France, and Italy and many recent introductions from Germany and France. Over the summer, we quantify the number of non-traceable infections stemming from introductions, quantify the effective reproductive number, and estimate the degree of undersampling. Our framework can be applied to quantify evolution and epidemiology in other locations or for other pathogens based on genomic data. One Sentence SummaryWe quantify SARS-CoV-2 spread in Switzerland based on genome sequences from our nation-wide sequencing effort.

Interactions between seasonal human coronaviruses and implications for the SARS-CoV-2 pandemic: A retrospective study in Stockholm, Sweden, 2009-2020

ObjectivesThe four seasonal coronaviruses 229E, NL63, OC43, and HKU1 are frequent causes of respiratory infections and show annual and seasonal variation. Increased understanding about these patterns could be informative about the epidemiology of SARS-CoV-2. MethodsResults from PCR diagnostics for the seasonal coronaviruses, and other respiratory viruses, were obtained for 55,190 clinical samples analysed at the Karolinska University Hospital, Stockholm, Sweden, between 14 September 2009 and 2 April 2020. ResultsSeasonal coronaviruses were detected in 2,130 samples (3.9%) and constituted 8.1% of all virus detections. OC43 was most commonly detected (28.4% of detections), followed by NL63 (24.0%), HKU1 (17.6%), and 229E (15.3%). The overall fraction of positive samples was similar between seasons, but at species level there were distinct biennial alternating peak seasons for the Alphacoronaviruses, 229E and NL63, and the Betacoronaviruses, OC43 and HKU1, respectively. The Betacoronaviruses peaked earlier in the winter season (Dec-Jan) than the Alphacoronaviruses (Feb-Mar). Coronaviruses were detected across all ages, but diagnostics were more frequently requested for paediatric patients than adults and the elderly. OC43 and 229E incidence was relatively constant across age strata, while that of NL63 and HKU1 decreased with age. ConclusionsBoth the Alphacoronaviruses and Betacoronaviruses showed alternating biennial winter incidence peaks, which suggests some type of immune mediated interaction. Symptomatic reinfections in adults and the elderly appear relatively common. Both findings may be of relevance for the epidemiology of SARS-CoV-2.

Cryptic transmission of SARS-CoV-2 in Washington State

Following its emergence in Wuhan, China, in late November or early December 2019, the SARS-CoV-2 virus has rapidly spread throughout the world. Genome sequencing of SARS-CoV-2 strains allows for the reconstruction of transmission history connecting these infections. Here, we analyze 346 SARS-CoV-2 genomes from samples collected between 20 February and 15 March 2020 from infected patients in Washington State, USA. We found that the large majority of SARS-CoV-2 infections sampled during this time frame appeared to have derived from a single introduction event into the state in late January or early February 2020 and subsequent local spread, indicating cryptic spread of COVID-19 before active community surveillance was implemented. We estimate a common ancestor of this outbreak clade as occurring between 18 January and 9 February 2020. From genomic data, we estimate an exponential doubling between 2.4 and 5.1 days. These results highlight the need for large-scale community surveillance for SARS-CoV-2 and the power of pathogen genomics to inform epidemiological understanding.

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.

Impact of seasonal forcing on a potential SARS-CoV-2 pandemic

A novel coronavirus (SARS-CoV-2) first detected in Wuhan, China, has spread rapidly since December 2019, causing more than 80,000 confirmed infections and 2,700 fatalities (as of Feb 27, 2020). Imported cases and transmission clusters of various sizes have been reported globally suggesting a pandemic is likely. Here, we explore how seasonal variation in transmissibility could modulate a SARS-CoV-2 pandemic. Data from routine diagnostics show a strong and consistent seasonal variation of the four endemic coronaviruses (229E, HKU1, NL63, OC43) and we parameterize our model for SARS-CoV-2 using these data. The model allows for many subpopulations of different size with variable parameters. Simulations of different scenarios show that plausible parameters result in a small peak in early 2020 in temperate regions of the Northern Hemisphere and a larger peak in winter 2020/2021. Variation in transmission and migration rates can result in substantial variation in prevalence between regions. While the uncertainty in parameters is large, the scenarios we explore show that transient reductions in the incidence rate might be due to a combination of seasonal variation and infection control efforts but do not necessarily mean the epidemic is contained. Seasonal forcing on SARS-CoV-2 should thus be taken into account in the further monitoring of the global transmission. The likely aggregated effect of seasonal variation, infection control measures, and transmission rate variation is a prolonged pandemic wave with lower prevalence at any given time, thereby providing a window of opportunity for better preparation of health care systems.