Feline Coronaviruses (FCoVs) commonly cause mild enteric infections in felines worldwide (termed Feline Enteric Coronavirus [FECV]), with around 12% 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, and the other within the fusion domain of Spike. We also found 15 sites subject to positive selection associated with FIPV within Spike, 11 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 14 sites (12 novel) within Spike under positive selection associated with the FECV phenotype, almost exclusively within the S1/S2 furin cleavage site and adjacent 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 24 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 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.

The Coronaviridae is a highly diverse virus family, with reservoir hosts in a variety of wildlife species that encompass bats, birds and small mammals, including rodents. Within the taxonomic group alphacoronavirus, certain sub-genera (including the luchacoviruses) have phylogenetically distinct spike proteins, which remain essentially uncharacterized. Using in vitro and computational techniques, we analyzed the spike protein of the rodent coronavirus AcCoV-JC34 from the sub-genus luchacovirus, previously identified in Apodemus chevrieri (Chevriers field mouse). We show that AcCoV-JC34, unlike the other luchacoviruses, has a putative furin cleavage site (FCS) within its spike S1 domain, close to the S1/S2 interface. The pattern of basic amino acids within the AcCoV-JC34 FCS (-RR-R-) is identical to that found in pre-variant SARS-CoV-2, which is in itself atypical for an FCS, and suboptimal for furin cleavage. Our analysis shows that, while containing an -RR-R- motif, the AcCoVJC34 spike FCS is not cleaved by furin (unlike for SARS-CoV-2), suggesting the possible presence of a progenitor sequence for viral emergence from a distinct wildlife host.

Feline Coronaviruses (FCoVs) commonly cause mild enteric infections in felines worldwide (termed Feline Enteric Coronavirus [FECV]), with around 12% 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, and the other within the fusion domain of Spike. We also found 15 sites subject to positive selection associated with FIPV within Spike, 11 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 14 sites (12 novel) within Spike under positive selection associated with the FECV phenotype, almost exclusively within the S1/S2 furin cleavage site and adjacent 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 24 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 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.

No abstract

No abstract

Proteins drive many of the biological processes in cells. To do this, they fold into complex 3D structures that are integral for their functionality. Notably, various environmental and chemical stressors can disrupt protein folding and thus disable the functions of proteins, threatening the livelihood of cells. To mitigate this stress, organisms initiate the highly‐conserved heat shock stress response. In eukaryotes, the master heat shock activator heat shock factor (HSF) is rapidly recruited to the Hsp70 heat shock protein genes and triggers the recruitment of additional co‐activator proteins that facilitate gene expression. This leads to the production of heat shock proteins that function as molecular chaperones to promote refolding of proteins, prevent aggregation and increase protein degradation pathways. Notably, activation of the heat shock response pathway, can be visualized by measuring GFP‐tagged HSF binding to the heat shock protein genes in living Drosophila salivary gland nuclei. Our lab is currently using this technique to identify novel compounds that induce the heat shock response pathway. Our pioneering experiments have shown that diamide and hydrogen peroxide, two chemicals known to cause protein misfolding and activation of the heat shock response pathway, trigger the recruitment of GFP‐HSF to the Hsp70 loci in living salivary gland cells to a similar level as HS stress. In addition, to our surprise, high levels of Dithiothreitol (DTT, 100 mM), a chemical known to cause protein misfolding and activation of the unfolded protein response pathway (UPR), results in the recruitment of GFP‐HSF to the Hsp70 gene loci. Here we describe experiments to further explore a possible dose dependent activation of HSF by DTT. Support or Funding Information Student Faculty Research Grant for Slippery Rock University

The mammalian order Eulipotyphla, including hedgehogs and shrews, represent a poorly understood reservoir of coronaviruses with zoonotic potential. Here, we carried out a bioinformatic analyses of these viruses based on the viral spike protein - to illustrate the complexity of coronavirus evolutionary history and the diversity of viruses from these host species, with a focus on the presence of possible furin cleavage sites within the spike protein. We found no evidence for cleavage by furin itself; however, certain strains of Wencheng Sm Shrew coronavirus were shown to have a predicted cleavage site for other member of the proprotein convertases, which are furin family members - suggesting their spillover potential. As the expanding urbanization and the trade of small mammals in the wet markets enhance the wildlife-human interactions, this may increase pathogen spillover risks. Therefore, we should implement broad wild animal surveillance and be vigilant of contact with these small wild mammals in light of one-health perspectives

Proteins drive many of the biological processes in cells. To do this, they fold into complex 3D structures that are integral for their functionality. Notably, various environmental and chemical stressors can disrupt protein folding and thus disable the functions of proteins, threatening the livelihood of cells. To mitigate this stress, organisms initiate the highly‐conserved heat shock stress response. In eukaryotes, the master heat shock activator heat shock factor (HSF) is rapidly recruited to the Hsp70 heat shock protein genes and triggers the recruitment of additional co‐activator proteins that facilitate gene expression. This leads to the production of heat shock proteins that function as molecular chaperones to promote refolding of proteins, prevent aggregation and increase protein degradation pathways. Notably, activation of the heat shock response pathway, can be visualized by measuring GFP‐tagged HSF binding to the heat shock protein genes in living Drosophila salivary gland nuclei. Our lab is currently using this technique to identify novel compounds that induce the heat shock response pathway. Our pioneering experiments have shown that diamide and hydrogen peroxide, two chemicals known to cause protein misfolding and activation of the heat shock response pathway, trigger the recruitment of GFP‐HSF to the Hsp70 loci in living salivary gland cells to a similar level as HS stress. In addition, to our surprise, high levels of Dithiothreitol (DTT, 100 mM), a chemical known to cause protein misfolding and activation of the unfolded protein response pathway (UPR), results in the recruitment of GFP‐HSF to the Hsp70 gene loci. Here we describe experiments to further explore a possible dose dependent activation of HSF by DTT.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.