Origin and host adaptation of the novel canine coronavirus (CCoV-HuPn-2018) isolated from a human pneumonia patient

1 Project Summary 2 There is growing evidence that canine coronaviruses (CCoVs) can infect humans and be associated with 3 clinical (mostly acute respiratory) illness in children and adults. Human infections with CCoVs with recombinant 4 canine-feline-porcine spike proteins (hCFPL-CoVs) have been confirmed in several countries including Haiti 5 (2017), Malaysia (2018), the USA (2014) and Thailand (2003). The high sequence identity (SI, 99.4%) observed 6 between hCFPL-CoVs from geographically distant Malaysia (CCoV-HuPn-2018) and Haiti (HuCCoV-Z19) 7 suggests that they may be capable of human-to-human transmission or represents temporal clustering. CCoVs 8 have not been recognized previously as human pathogens, and the threat they pose to public health is unknown 9 and may be underappreciated. While complete genome sequencing demonstrated hCFPL-CoVs are canine- 10 feline recombinant alphacoronaviruses, it failed to identify potential ancestral strains likely due to scarcity of 11 CCoV genomic data (only 17 complete genome sequences are available in the GenBank). To address that, we 12 will conduct complete genomic sequencing of up to 200 new CCoVs (from Dr. Decaro) and additional hCFPL- 13 CoVs (if identified in Dr. Gray's ongoing study). 14 A unique 36 nt (12-aa) deletion identified in the N protein in the SR-rich domain (ΔSR-N) of CCoV-HuPn- 15 2018 may be associated with a recent zoonotic transmission of CCoV-HuPn-2018 and certain biological 16 functions acquired or lost by the virus. While such deletions in the SR-rich region of the N-protein were not 17 previously found in CCoVs, presence of a similar deletion was demonstrated in severe acute respiratory 18 syndrome coronavirus (SARS-CoV) strains early following their emergence into human population but not in 19 SARS-CoV-like bat strains. This deletion was shown to be associated with the altered cellular localization of the 20 N-protein and increased pathogenicity of the SARS-CoV strains bearing them. Because SARS-CoV N-protein 21 plays an important role in inhibition of type I interferon (IFN) production, deletions in it may alter innate immune 22 responses against SARS-CoV as well as other CoVs including CCoV-HuPn-2018. Using reverse genetics, we 23 will evaluate the biological function of this mutation. We propose the following Specific Aims to study the 24 genomics, evolution and human emergence mechanisms of hCFPL-CoVs. Aim 1. Conduct genome-wide 25 analysis of historic and current CCoV strains a) to determine the evolutionary relationship between hCFPL-CoVs 26 and their potentially ancestral strains and b) to identify genetic features associated with CCoV-HuPn-2018 27 infectivity or pathogenicity to human host. Aim 2. To generate and use CCoV-HuPn-2018 infectious clone to 28 investigate the effect of the identified N- deletion (ΔSR-N) on CCoV-HuPn-2018 cellular localization, replication 29 dynamics and the host transcriptome response. These studies will identify the genomic features associated with 30 hCFPL-CoV/CCoV infectivity to humans and generate essential fundamental knowledge regarding the common 31 mechanisms regulating zoonotic transmission of CoVs.