RASCL: Rapid Assessment Of SARS-CoV-2 Clades Through Molecular Sequence Analysis (preprint)

An important component of efforts to manage the ongoing COVID19 pandemic is the Rapid Assessment of how natural selection contributes to the emergence and proliferation of potentially dangerous SARS-CoV-2 lineages and CLades (RASCL). The RASCL pipeline enables continuous comparative phylogenetics-based selection analyses of rapidly growing clade-focused genome surveillance datasets, such as those produced following the initial detection of potentially dangerous variants. From such datasets RASCL automatically generates down-sampled codon alignments of individual genes/ORFs containing contextualizing background reference sequences, analyzes these with a battery of selection tests, and outputs results as both machine readable JSON files, and interactive notebook-based visualizations.

Conserved recombination patterns across coronavirus subgenera (preprint)

Recombination contributes to the genetic diversity found in coronaviruses and is known to be a prominent mechanism whereby they evolve. It is apparent, both from controlled experiments and in genome sequences sampled from nature, that patterns of recombination in coronaviruses are non-random and that this is likely attributable to a combination of sequence features that favour the occurrence of recombination breakpoints at specific genomic sites, and selection disfavouring the survival of recombinants within which favourable intra-genome interactions have been disrupted. Here we leverage available whole-genome sequence data for six coronavirus subgenera to identify specific patterns of recombination that are conserved between multiple subgenera and then identify the likely factors that underlie these conserved patterns. Specifically, we confirm the non-randomness of recombination breakpoints across all six tested coronavirus subgenera, locate conserved recombination hot- and cold-spots, and determine that the locations of transcriptional regulatory sequences are likely major determinants of conserved recombination breakpoint hot-spot locations. We find that while the locations of recombination breakpoints are not uniformly associated with degrees of nucleotide sequence conservation, they display significant tendencies in multiple coronavirus subgenera to occur in low guanine-cytosine content genome regions, in non-coding regions, at the edges of genes, and at sites within the Spike gene that are predicted to be minimally disruptive of Spike protein folding. While it is apparent that sequence features such as transcriptional regulatory sequences are likely major determinants of where the template-switching events that yield recombination breakpoints most commonly occur, it is evident that selection against misfolded recombinant proteins also strongly impacts observable recombination breakpoint distributions in coronavirus genomes sampled from nature.

Recent zoonotic spillover and tropism shift of a Canine Coronavirus is associated with relaxed selection and putative loss of function in NTD subdomain of spike protein. (preprint)

A recent study reported the occurrence of Canine Coronavirus (CCoV) in nasopharyngeal swabs from a small number of patients hospitalized with pneumonia during a 2017-18 period in Sarawak, Malaysia. Because the genome sequence for one of these isolates is available, we conducted comparative evolutionary analyses of the spike gene of this strain (CCoV-HuPn-2018), with other available Alphacoronavirus 1 spike sequences. The most N-terminus subdomain (0-domain) of the CCoV-HuPn-2018 spike protein has sequence similarity to Transmissible Gastroenteritis Virus (TGEV) and CCoV2b strains, but not to other members of the type II Alphacoronaviruses (i.e., CCoV2a and Feline CoV2-FCoV2). This 0-domain in CCoV-HuPn-2018 has evidence for relaxed selection pressure, an increased rate of molecular evolution, and a number of unique amino acid substitutions relative to CCoV2b and TGEV sequences. A region of the 0-domain determined to be key to sialic acid binding and pathogenesis in TGEV had clear differences in amino acid sequences in CCoV-HuPn-2018 relative to both CCoV2b (enteric) and TGEV (enteric and respiratory). The 0-domain of CCoV-HuPn-2018 also had several sites inferred to be under positive diversifying selection, including sites within the signal peptide. Downstream of the 0-domain, FCoV2 shared sequence similarity to the CCoV2b and TGEV sequences, with analyses of this larger alignment identifying positively selected sites in the putative Receptor Binding Domain (RBD) and Connector Domain (CD). Recombination analyses strongly implicated a particular FCoV2 strain in the recombinant history of CCoV-HuPn-2018 with molecular divergence times estimated at around 60 years ago. We hypothesize that CCoV-HuPn-2018 had an enteric origin, but that it has lost that particular tropism, because of mutations in the sialic acid binding region of the spike 0-domain. As selection pressure on this region was reduced, the virus evolved a respiratory tropism, analogous to other Alphacoronavirus 1, such as Porcine Respiratory Coronavirus (PRCV), that have lost this region entirely. We also suggest that signals of positive selection in the signal peptide as well as other changes in the 0-domain of CCoV-HuPn-2018 could represent an adaptive role in this new host and that this could be in part due to the different spatial distribution of the N-linked glycan repertoire for this strain.