Isolation of SARS-CoV-2-related coronavirus from Malayan pangolins

Original Article: Xiao K. et al., Isolation of SARS-CoV-2-related coronavirus from Malayan pangolins, Nature.

Author of summary: Valeria Montis; Reviewer: Giulia Poggi

Original Article Published on May 7th, 2020

Independent studies suggest that  the virus responsible of the current CoViD-19 pandemic originated from a genetic reshuffling of pangolin and bat CoV-like viruses. While bats may be the reservoir host for various Coronaviruses, this study further indicates that  pangolins are likely to have acted as an intermediate host of SARS-CoV-2. These animals, illegally smuggled from and to various Countries, could represent a potential future threat to public health if the wildlife trade is not efficiently controlled.

Step 1: Identification and characterisation of Pangolin-CoV

Via RT-PCR with primers for a conservative region of β-CoVs, a SARS-related Coronavirus (SARSr-CoV) was detected in lung tissue of 17 out of 25 Malayan pangolins (Manis javanica).

The PCR-positive Malayan pangolins showed clinical signs and histological changes typical of a respiratory disease, and 14 of 17 infected pangolins died. A subsequent ELISA test clearly showed that some of the pangolins were naturally infected with a SARS-CoV-2-like virus.

Viral particles isolated from the lung of three dead Malayan pangolins showed typical Coronavirus morphology. The partial protein spike (S) and RNA-dependent RNA polymerase (RdRp) genes, amplified via RT-PCR, showed respectively ~84.5% and 92.2% nucleotide sequence identity with the corresponding gene sequences in SARS-CoV-2.

NGS technology provided the complete genome sequence (29,825 bp). The nearly full similarity between S gene sequences alignments in PCR-positive samples indicated the presence of only one type of Coronavirus in the batch of study samples, designated as Pangolin-CoV.

Step 2. Evolutionary relationship with other known CoVs

Comparative genetic analysis showed that Pangolin-CoV is similar to SARS-CoV-2 and highly similar to Bat-CoV RaTG13, except for the S gene (Fig. 1).

Closer studies of the S gene sequences on the SARSr-CoVs analyzed revealed that the initial part of the Pangolin-CoV S sequence is more similar to Bat-CoVs S gene (specifically, Bat SARSr-CoV ZXC21 and Bat SARSr-CoV ZC45), whereas the receptor binding domain (RBD, essential for the infection of the host cell) is virtually identical to the one of SARS-CoV-2 (Figure 2A of the original paper). This suggests that recombination events occurred among these viruses.

Analysis of evolutionary relationships based on full-genome sequences showed that Pangolin-CoV clusters with SARS-CoV-2 and Bat-CoV RaTG13. Within this cluster, SARS-CoV-2 and Bat-CoV RaTG13 form a subclade  (Figure 2C of the original paper).

In contrast, phylogenetic analysis solely on the S gene showed that Pangolin-CoV and SARS-CoV-2 RBD portions grouped together. Notably, conflicts in cluster formation among phylogenetic analyses of different regions of the genome are strong indications of genetic recombination
(Figure 2B of the original paper).

Step 3. ACE2 binding simulations

The RBD of SARS-CoV efficiently bands ACE2 of humans and civets and it also appears to be capable of binding ACE2 of pangolins. In contrast, the S proteins of SARS-CoV-2 and Pangolin-CoV can potentially only recognize ACE2 of humans and pangolins.

Pangolin-CoV identified in the present study is genetically related to SARS-CoV-2. However, given the substantial differences detected when comparing the whole-genome sequences, it is unlikely that Pangolin-CoV is directly linked to the CoViD-19 outbreak. Indeed, SARS-CoV-2 shows higher sequence identity with one of the bat CoVs (i.e. Bat-CoV RaTG13) at the whole-genome level.

In line with other independent studies performed on SARS-CoV-2 (link), this study provided further evidence that pangolins are unlikely to be a zoonotic reservoir of SARS-CoV-2. Yet, given their clinical manifestations upon CoVs infection, pangolins are likely to have served as intermediate hosts of SARS-CoV-2.  It is reasonable to conclude that bats are native hosts of SARS-CoV-2, as previously suggested for SARS-CoV and MERS-CoV. Subsequently, a SARSr-CoV from bats might have received a RBD sequence from a virus related to Pangolin-CoV, making the newborn SARS-CoV-2 capable of infecting the human species. Pangolins and bats share overlapping ecological niches, which make pangolins an ideal intermediate host for some SARSr-CoVs having in bats a natural reservoir.

Therefore, more systematic and long-term monitoring of SARSr-CoVs in bats and in various intermediate host animals like pangolins, and a stronger ban of illegal wildlife trafficking and consumption of game meat are essential to protect endangered species as well as to prevent other major outbreaks caused by SARSr-CoVs.