Natural selection differences detected in key protein domains between non-pathogenic and pathogenic feline coronavirus phenotypes.

Feline coronaviruses (FCoVs) commonly cause mild enteric infections in felines worldwide (termed feline enteric coronavirus [FECV]), with around 12 per cent developing into deadly feline infectious peritonitis (FIP; feline infectious peritonitis virus [FIPV]). Genomic differences between FECV and FIPV have been reported, yet the putative genotypic basis of the highly pathogenic phenotype remains unclear. Here, we used state-of-the-art molecular evolutionary genetic statistical techniques to identify and compare differences in natural selection pressure between FECV and FIPV sequences, as well as to identify FIPV- and FECV-specific signals of positive selection. We analyzed full-length FCoV protein coding genes thought to contain mutations associated with FIPV (Spike, ORF3abc, and ORF7ab). We identified two sites exhibiting differences in natural selection pressure between FECV and FIPV: one within the S1/S2 furin cleavage site (FCS) and the other within the fusion domain of Spike. We also found fifteen sites subject to positive selection associated with FIPV within Spike, eleven of which have not previously been suggested as possibly relevant to FIP development. These sites fall within Spike protein subdomains that participate in host cell receptor interaction, immune evasion, tropism shifts, host cellular entry, and viral escape. There were fourteen sites (twelve novel sites) within Spike under positive selection associated with the FECV phenotype, almost exclusively within the S1/S2 FCS and adjacent to C domain, along with a signal of relaxed selection in FIPV relative to FECV, suggesting that furin cleavage functionality may not be needed for FIPV. Positive selection inferred in ORF7b was associated with the FECV phenotype and included twenty-four positively selected sites, while ORF7b had signals of relaxed selection in FIPV. We found evidence of positive selection in ORF3c in FCoV-wide analyses, but no specific association with the FIPV or FECV phenotype. We hypothesize that some combination of mutations in FECV may contribute to FIP development, and that it is unlikely to be one singular 'switch' mutational event. This work expands our understanding of the complexities of FIP development and provides insights into how evolutionary forces may alter pathogenesis in coronavirus genomes.

Conserved recombination patterns across coronavirus subgenera.

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 break points 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 break points 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 break-point hotspot locations. We find that while the locations of recombination break points 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 break points most commonly occur, it is evident that selection against misfolded recombinant proteins also strongly impacts observable recombination break-point 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.

A canine coronavirus (CCoV) has now been reported from two independent human samples from Malaysia (respiratory, collected in 2017-2018; CCoV-HuPn-2018) and Haiti (urine, collected in 2017); these two viruses were nearly genetically identical. In an effort to identify any novel adaptations associated with this apparent shift in tropism we carried out detailed evolutionary analyses of the spike gene of this virus in the context of related Alphacoronavirus 1 species. The spike 0-domain retains homology to CCoV2b (enteric infections) and Transmissible Gastroenteritis Virus (TGEV; enteric and respiratory). This domain is subject to relaxed selection pressure and an increased rate of molecular evolution. It contains unique amino acid substitutions, including within a region important for sialic acid binding and pathogenesis in TGEV. Overall, the spike gene is extensively recombinant, with a feline coronavirus type II strain serving a prominent role in the recombinant history of the virus. Molecular divergence time for a segment of the gene where temporal signal could be determined, was estimated at around 60 years ago. We hypothesize that the virus had an enteric origin, but that it may be losing that particular tropism, possibly because of mutations in the sialic acid binding region of the spike 0-domain.

Furin cleavage sites in the spike proteins of bat and rodent coronaviruses: Implications for virus evolution and zoonotic transfer from rodent species.

Bats and rodents comprise two of the world's largest orders of mammals and the order Chiroptera (bats) has been implicated as a major reservoir of coronaviruses in nature and a source of zoonotic transfer to humans. However, the order Rodentia (rodents) also harbors coronaviruses, with two human coronaviruses (HCoV-OC43 and HCoV-HKU1) considered to have rodent origins. The coronavirus spike protein mediates viral entry and is a major determinant of viral tropism; importantly, the spike protein is activated by host cell proteases at two distinct sites, designated as S1/S2 and S2'. SARS-CoV-2, which is considered to be of bat origin, contains a cleavage site for the protease furin at S1/S2, absent from the rest of the currently known betacoronavirus lineage 2b coronaviruses (Sarbecoviruses). This cleavage site is thought to be critical to its replication and pathogenesis, with a notable link to virus transmission. Here, we examine the spike protein across coronaviruses identified in both bat and rodent species and address the role of furin as an activating protease. Utilizing two publicly available furin prediction algorithms (ProP and PiTou) and based on spike sequences reported in GenBank, we show that the S1/S2 furin cleavage site is typically not present in bat virus spike proteins but is common in rodent-associated sequences, and suggest this may have implications for zoonotic transfer. We provide a phylogenetic history of the Embecoviruses (betacoronavirus lineage 2a), including context for the use of furin as an activating protease for the viral spike protein. From a One Health perspective, continued rodent surveillance should be an important consideration in uncovering novel circulating coronaviruses.