Study population and setting
Frozen tissue samples (lung, intestine, blood; 43 total) from 18 Malayan pangolins seized by Guangxi Customs between August 2017 and January 2018 during anti-smuggling operations in Guangxi, southern China. Additional testing was performed on archived Malayan pangolin tissue samples (intestine, lung; 19 total) from 12 animals sampled between May 2018 and July 2018 by researchers at the Guangxi Medical University and five archived pangolin tissue samples (skin swabs, scale) from anti-smuggling operations in March 2019 from the Guangzhou Customs Technology Center, Guangzhou, southern China.
Summary of Main Findings
6 of 43 tissue samples from 5 of 18 of the individual pangolins sampled between August 2017 and January 2018 from Guangxi were positive for coronavirus RNA following high-throughput metagenomic sequencing; 3 of 19 archived tissue samples from 3 of 12 pangolins from Guangxi were also positive for coronaviruses based on quantitative PCR using primers designed from assembled genomes; one archived scale sample from Guangzhou was coronavirus-positive by metagenomic sequencing. Six complete or nearly complete genomes were assembled from metagenomic reads; phylogenetic analysis places the pangolin coronavirus genomes into the same lineage of Betacoronavirus as SARS-CoV-2 and related viruses from Rhinolophus spp. bats. Including pangolin coronaviruses from a previous study by Liu et al. (https://doi.org/10.3390/v11110979) the pangolin viruses form two lineages related to SARS-CoV-2, with the genomes from pangolins in Guandong from the Liu et al. paper forming the closest cluster to SARS-CoV-2 and the bat coronavirus RaTG13; these genomes from Guandong were less closely related to SARS-CoV-2 than RaTG13 across the whole genome, but more closely related than RaTG13 within the receptor binding domain of the spike protein.
The study benefits from previously collected samples of various tissue types to investigate the distribution of coronaviruses in infected pangolins. A combination of unbiased metagenomic sequencing and newly designed qPCR primers for this lineage of betacoronaviruses provides sensitive detection of viruses in infected tissues.
The similarity between the pangolin coronaviruses and SARS-CoV-2 is still not sufficiently high to conclusively implicate pangolins as intermediate hosts of SARS-CoV-2 for transmission to humans; it cannot yet be ruled out that pangolins acquired these viruses independently from bats. Additionally, the pangolins were trafficked animals likely from Southeast Asia and were not collected in or near Wuhan where the virus outbreak was first detected.
This study provides important new information on the wild animal hosts of coronaviruses related to SARS-CoV-2, including both bats and pangolins.