Using big sequencing data to identify chronic SARS-Coronavirus-2 infections (preprint)

The evolution of SARS-Coronavirus-2 (SARS-CoV-2) has been characterized by the periodic emergence of highly divergent variants, many of which may have arisen during chronic infections of immunocompromised individuals. Here, we harness a global phylogeny of ~11.7 million SARS-CoV-2 genomes and search for clades composed of sequences with identical metadata (location, age, and sex) spanning more than 21 days. We postulate that such clades represent repeated sampling from the same chronically infected individual. A set of 271 such chronic-like clades was inferred, and displayed signatures of an elevated rate of adaptive evolution, in line with validated chronic infections. More than 70% of adaptive mutations present in currently circulating variants are found in BA.1 chronic-like clades that predate the circulating variants by months, demonstrating the predictive nature of such clades. We find that in chronic-like clades the probability of observing adaptive mutations is approximately 10-20 higher than that in global transmission chains. We next employ language models to find mutations most predictive of chronic infections and use them to infer hundreds of additional chronic-like clades in the absence of metadata and phylogenetic information. Our proposed approach presents an innovative method for mining extensive sequencing data and providing valuable insights into future evolutionary patterns.

NOVEL RT-qPCR ASSAYS ENABLE RAPID DETECTION AND DIFFERENTIATION BETWEEN SARS-COV-2 OMICRON (BA.1) AND BA.2 VARIANTS (preprint)

In this report, we describe the development and validation of novel SARS-COV-2 Omicron-specific reactions that enable the identification of Omicron (BA.1) and BA.2 variants. Mutations that are either shared by both BA.1 and BA.2, or are exclusive for BA.1 or for BA.2 were identified by bioinformatic analysis, and corresponding probe-based quantitative PCR reactions were developed to identify them. We show that multiplex combinations of these reactions provide a single-reaction identification of the sample as BA.1, BA.2, or as non-Omicron SARS-COV-2. All four reactions described herein have a sensitivity of less than ten copies per reaction, and are amendable for multiplexing. The results of this study suggest that the new assays may be useful for testing both clinical and environmental samples to differentiate between these two variants.

SPECIFIC DETECTION OF SARS-COV-2 B.1.1.529 (OMICRON) VARIANT BY FOUR RT-qPCR DIFFERENTIAL ASSAYS (preprint)

In this report, we describe four RT-qPCR assays that enable rapid identification of the newly emerging SARS-COV-2 Omicron (B.1.1.529) variant of concern. The assays target Omicron characteristic mutations in the nsp6 (Orf1a), spike and nucleocapsid genes. We demonstrate that the assays are straightforward to assemble and perform, are amendable for multiplexing, and may be used as a reliable first-line tool to identify B.1.1.529 suspected samples. Importantly, this is a preliminary development report. Further validation and optimization of the assays described herein will be published hereafter.

Emergence and widespread circulation of a recombinant SARS-CoV-2 lineage in North America (preprint)

Genetic recombination is an important driving force of coronavirus evolution. While some degree of virus recombination has been reported during the COVID-19 pandemic, previously detected recombinant lineages of SARS-CoV-2 have shown limited circulation and been observed only in restricted areas. Prompted by reports of unusual genetic similarities among several Pango lineages detected mainly in North and Central America, we present a detailed phylogenetic analysis of four SARS-CoV-2 lineages (B.1.627, B.1.628, B.1.631 and B.1.634) in order to investigate the possibility of virus recombination among them. Two of these lineages, B.1.628 and B.1.631, are split into two distinct clusters (here named major and minor). Our phylogenetic and recombination analyses of these lineages find well-supported phylogenetic differences between the Orf1ab region and the rest of the genome (S protein and remaining reading frames). The lineages also contain several deletions in the NSP6, Orf3a and S proteins that can augment reconstruction of reliable evolutionary histories. By reconciling the deletions and phylogenetic data, we conclude that the B.1.628 major cluster originated from a recombination event between a B.1.631 major virus and a lineage B.1.634 virus. This scenario inferred from genetic data is supported by the spatial and temporal distribution of the three lineages, which all co-circulated in the USA and Mexico during 2021, suggesting this region is where the recombination event took place. We therefore support the designation of the B.1.628 major cluster as recombinant lineage XB in the Pango nomenclature. The widespread circulation of lineage XB across multiple countries over a longer timespan than the previously designated recombinant XA lineage raises important questions regarding the role and potential effects of recombination on the evolution of SARS-CoV-2 during the ongoing COVID-19 pandemic.

The unique evolutionary dynamics of the SARS-CoV-2 Delta variant (preprint)

The SARS-Coronavirus-2 (SARS-CoV-2) driven pandemic was first recognized in late 2019, and the first few months of its evolution were relatively clock-like, dominated mostly by neutral substitutions. In contrast, the second year of the pandemic was punctuated by the emergence of several variants that bore evidence of dramatic evolution. Here, we compare and contrast evolutionary patterns of various variants, with a focus on the recent Delta variant. Most variants are characterized by long branches leading to their emergence, with an excess of non-synonymous substitutions occurring particularly in the Spike and Nucleocapsid proteins. In contrast, the Delta variant that is now becoming globally dominant, lacks the signature long branch, and is characterized by a step-wise evolutionary process that is ongoing. Contrary to the "star-like" topologies of other variants, we note the formation of several distinct clades within Delta that we denote as clades A-E. We find that sequences from the Delta D clade are dramatically increasing in frequency across different regions of the globe. Delta D is characterized by an excess of non-synonymous mutations, mostly occurring in ORF1a/b, some of which occurred in parallel in other notable variants. We conclude that the Delta surge these days is composed almost exclusively of Delta D, and discuss whether selection or random genetic drift has driven the emergence of Delta D.

The rise and fall of an emerging SARS-CoV-2 variant with the spike protein mutation L452R (preprint)

Emerging SARS-CoV-2 variants may threaten global vaccination efforts and awaited reduction in outbreak burden. In this study, we report a novel variant carrying the L452R mutation that emerged from a local B.1.362 lineage, B.1.362+L452R. The L452R mutation is associated with the Delta and Epsilon variants and was shown to cause increased infection and reduction in neutralization in pseudoviruses. Indeed, the B.1.362+L452R variant demonstrated a X4-fold reduction in neutralization capacity of sera from BNT162b2-vaccinated individuals compared to a wild-type strain. The variant infected 270 individuals in Israel between December 2020 and March 2021, until diminishing due to the gain in dominance of the Alpha variant in February 2021. This study demonstrates an independent, local emergence of a variant carrying a critical mutation, L452R, which may have the potential of becoming a variant of concern and emphasizes the importance of routine surveillance and detection of novel variants among efforts undertaken to prevent further disease spread.

A unique SARS-CoV-2 spike protein P681H strain detected in Israel (preprint)

Routine detection, surveillance and reporting of SARS-CoV-2 novel variants is important, as these threaten to hinder vaccination efforts. Herein we report a local novel strain that includes a non-synonymous mutation in the spike (S) protein - P681H and additional synonymous mutations. The P681H Israeli strain has not been associated with higher infection rates and was neutralized by sera from vaccinated individuals in comparable levels to the B.1.1.7 strain and a non-P681H strain from Israel.