NEW YORK – Pangolins may be an intermediary host for SARS-CoV-2, the virus causing the ongoing COVID-19 pandemic, a new sequencing analysis has found.

While bats are thought to be the likely reservoir of SARS-CoV-2, experts suspect another host mediated the transfer of the virus from bats to humans. 

An international team of researchers led by Shantou University's Yi Guan analyzed tissue samples obtained from Malayan pangolins (Manis javanica) that had been seized by anti-smuggling operations. Pangolins, which are considered critically endangered mammals, are illegally trafficked for food and for use in traditional Chinese medicine.

According to their analysis, which was published yesterday in Nature, a number of pangolins in their sample harbored coronaviruses related to SARS-CoV-2. In particular, they uncovered two sub-lineages of SARS-CoV-2-related coronaviruses, one of which had high sequence similarity with SARS-CoV-2 at the receptor-binding domain. 

"The discovery of multiple lineages of pangolin coronavirus and their similarity to SARS-CoV-2 suggests that pangolins should be considered as possible hosts in the emergence of novel coronaviruses and should be removed from wet markets to prevent zoonotic transmission," the researchers wrote in their paper.

The researchers sequenced RNA isolated from multiple frozen tissue samples from 18 Malayan pangolins collected between August 2017 and January 2018 by anti-smuggling officials from Guangxi Customs in southern China. This uncovered the presence of coronaviruses in six of the 43 samples analyzed, and the researchers were able to generate six complete or nearly complete viral genome sequences.

A phylogenetic analysis placed these pangolin coronaviruses within the same lineage as SARS-CoV-2. 

After designing qPCR primers, the researchers tested tissues from a further 12 pangolins — ones caught in smuggling operations in Guangxi between May and July of 2018 — for the coronavirus. Three lung tissue samples from three pangolins tested positive for coronavirus. 

The researchers also sequenced samples from five smuggled pangolins that were caught more recently, in March 2019 by the Guangzhou Customs Technology Center, and analyzed diseased pangolins from Guangdong, finding the presence of SARS-CoV-2-like coronaviruses in both cases.

These novel pangolin coronaviruses share between 85.5 percent and 92.4 percent sequence similarity with SARS-CoV-2. They also represent two SARS-CoV-2-related sub-lineages on the viral phylogenetic tree, one of which falls close to the SARS-CoV-2 lineage.

Members of the Sarbecovirus subgenus are known to have undergone broad recombination and, here the researchers found that the bat coronaviruses ZC45 and ZXC21 are likely recombinants that harbor sequences from multiple SARS-CoV linages and SARS-CoV-2 lineages, including from pangolins. 

SARS-CoV-2 and the Guangdong pangolin coronavirus have highly similar receptor-binding domain sequences — a 97.4 percent amino acid similarity. In particular, the two viruses share identical amino acids at the first five key RBD residues. The rest of the SARS-CoV-2 genome, though, more closely resembles that of the bat RaTG13 virus. They noted that the similarity between SARS-CoV-2 and Guangdong pangolin coronavirus RBD could potentially be due to convergent evolution rather than recombination, as either scenario fits the current data.

An analysis published earlier this month likewise noted similarities between coronaviruses found in pangolins and SARS-CoV-2, particularly at the receptor-binding domain but also noted a high overall concordance between the bat RaTG13 virus and SARS-CoV-2.

Still, these findings indicate that pangolins are possible viral hosts, leading the researchers to suggest that they should be handled with care and that their sale at live animal markets should not be allowed.

"Further surveillance on pangolins in the natural environment in China and Southeast Asia are clearly needed to understand their role in the emergence of coronaviruses and the risk of future zoonotic transmission," they added.