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

1项目概要 2越来越多的证据表明，犬冠状病毒（CCoV）可以感染人类，并与 3儿童和成人的临床（主要是急性呼吸道）疾病。CCoV的人感染与重组 4种犬-猫-猪刺突蛋白（hCFPL-CoV）已在包括海地在内的几个国家得到确认 5（2017），马来西亚（2018），美国（2014）和泰国（2003）。观察到高序列同一性（SI，99.4%）， 6来自地理上遥远的马来西亚（CCoV-HuPn-2018）和海地（HuCCoV-Z19）的hCFPL-CoV之间 7表明它们可能能够在人与人之间传播或代表时间聚集。CCoV 8种以前未被确认为人类病原体，它们对公共卫生构成的威胁尚不清楚 9、可能被低估了虽然全基因组测序表明hCFPL-CoV是犬的， 10猫重组冠状病毒，它未能确定潜在的祖先株可能是由于缺乏 11 CCoV基因组数据（GenBank中只有17个完整的基因组序列）。为了解决这个问题，我们 12将对多达200个新的CCoV（来自Decaro博士）和额外的hCFPL进行完整的基因组测序， 13冠状病毒（如果在格雷博士正在进行的研究中发现）。 图14在CCoV-HuPn的富含SR的结构域（ΔSR-N）的N蛋白中鉴定的独特的36 nt（12-aa）缺失。 15 2018年可能与最近的CCoV-HuPn-2018的人畜共患传播和某些生物学 16种功能被病毒获得或丢失。而在N蛋白富含SR区域的这种缺失并不存在。 17先前在CCoV中发现，在严重急性呼吸道疾病中证实了类似缺失的存在。 18综合征冠状病毒（SARS-CoV）菌株在出现于人群中后的早期， 19种类似SARS冠状病毒的蝙蝠菌株。这种缺失被证明与细胞定位的改变有关， 20 N-蛋白和增加携带它们的SARS-CoV株的致病性。因为SARS冠状病毒N蛋白 21在抑制I型干扰素（IFN）产生中起重要作用，其缺失可改变先天性免疫 22份针对SARS-CoV以及包括CCoV-HuPn-2018在内的其他CoV的回复。利用反向遗传学，我们 23将评估这种突变的生物学功能。我们提出以下研究的具体目标： 24 hCFPL-CoV的基因组学、进化和人类出现机制。目标1。进行全基因组 a）确定hCFPL-CoV之间的进化关系 26及其潜在祖先菌株和B）鉴定与CCoV-HuPn-2018相关的遗传特征 27对人类宿主的感染性或致病性。目标二。为了生成并使用CCoV-HuPn-2018感染性克隆， 28研究鉴定的N-缺失（ΔSR-N）对CCoV-HuPn-2018细胞定位、复制的影响。 29动力学和宿主转录组响应。这些研究将确定与以下疾病相关的基因组特征： 30 hCFPL-CoV/CCoV对人类的感染性，并产生关于常见的 31调节CoV的人畜共患病传播的机制。

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.