The new coronavirus SARS-CoV-2 caused the most destructive pandemic in nearly a century, COVID-19, and one of the unresolved scientific problems is the natural source of this virus. Recently, from Wuhan Institute of Virology, Chinese Academy of Sciences, Chinese Academy of Sciences University researchers submitted a paper on the preprint website BioRxiv, entitled “Identification of a novel lineage bat SARS-related coronaviruses that use bat ACE2 receptor”. They reported the new SARS-CoV-2 related virus lineages identified in bats found in Tongguan Town, Mojiang County, Yunnan Province. These viruses were sampled in 2015. The location and the research team previously reported were from the Chinese chrysanthemum bat, and The RaTG13 line with 96.2% genomic identity of SARS-CoV-2 is in the same location.

The corresponding authors of this paper are Shi Zhengli, Director of the Research Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Deputy Director of Wuhan National Biosafety Laboratory (Level 4), and Wuhan Virus, Chinese Academy of Sciences Zhou Peng, head of the Bat Virus Infection and Immunity Group.

The research and development also pointed out that according to current research findings, none of the bat SARSr-CoV-2 or new lineage viruses that can be isolated at present cannot effectively use human ACE2. . Therefore, if there is no adaptation, the risk of spillover to humans is still relatively small. In contrast, from the perspective of the use of recipients, pangolin coronavirus has a higher risk of spillage.

They also further emphasized that although it was previously speculated that the leak of RaTG13 in the laboratory may have caused SARS-CoV-2, the existing experimental evidence does not support it. This speculation.

Shi Zhengli and others emphasized in this study that the SARS-like coronaviruses currently found in bats may be just the tip of the iceberg. In the future, more systematic and vertical monitoring should be carried out to prevent the virus from causing major spills again.

A new pedigree different from SARSr-CoV-1 and SARSr-CoV-2

wrote in the paper that in May 2015, the research team conducted bat sampling in Mojiang County. The bats were released after anal swab sampling, and the samples were stored at -80°C until use.

When tracking the origin of SARS-CoV-2 in bats, the research team previously discovered RaTG13, which has a 96.2% genome homology with SARS-CoV-2, which is The closest genome to date.

When they further investigated the bat samples collected at the same location in 2015, they found that the sequences of 8 samples were similar to SARS-CoV-2 The sequence homology is 93.5%. 7 copies are from Rhinolophus stheno, and the other is from Rhinolophus affinis.

therefore , They performed next-generation sequencing (NGS) to further analyze these coronaviruses. All 8 samples obtained the whole genome sequence. The 8 SARSr-CoV genomes are almost identical, and the sequence identity between each other is more than 99.7%. The team performed further analysis with one of the virus strains named RaTG15 as a representative.

In the seven conserved replicase domains used for the classification of coronaviruses The homology of RaTG15 with SARS-CoV-2 and SARS-CoV-1 is 95.3% and 92.5%, respectively, indicating that it belongs to the SARSr-CoV species in the Sarbecovirus subgenus of the Betacoronavirus genus of the Coronavirus family.

In addition, RaTG15 is close to the SARS-CoV-2 gene in open reading frame 1b (ORF1b). In the complete ORF1b region, RaTG15 is similar to SARSr-CoV-2 from wild bats from China and Southeast Asia The nucleotide sequence homology is 84.6%-89.0%, and the amino acid sequence homology is 95.6%-97.3%, including bat coronavirus RaTG13 and RmYN02 from Yunnan, Rc-o319 from Japan, RshSTT182 from Cambodia, Thailand’s RacCS203, and two pangolin coronavirus strains.

The study found that, in contrast, in addition to the above ORF1b, RaTG15 and SARSr-CoV-2 and SARSr- CoV-1 is significantly different in most genomes. ORF1a, M and NThe gene homology of S, ORF3, 6 and 7a/7b is less than 80%, and the homology of S, ORF3, 6 and 7a/7b is less than 70%.

According to the study, in general, the whole genome of RaTG15 has 74.4% homology with SARS-CoV-1 sequence and homology with SARS-CoV-2 sequence It was 77.6%. It is worth noting that the sequence identity of RaTG15 and SARS-CoV-1 on S, E, M, N and ORF6 proteins is higher than that between RaTG15 and SARS-CoV-2.

The research team believes that this new virus lineage including RaTG15 may be the result of the recombination of different SARSr-CoVs.

The phylogenetic analysis results also support this point. The research team pointed out that SARSr-CoVs were previously mainly composed of two sub-species, namely SARSr-CoV-1 and SARSr-CoV-2. The latter includes pangolins and SARS-CoV-2 in different species of chrysanthemum bats recently reported in many parts of Asia. In the full-length gene tree and S gene tree, RaTG15 and its related viruses are far away from the two existing sub-lineages mentioned above, thus forming a new lineage. Without further adaptation The zoonotic potential is limited

The researchers further tested the S protein sequence of RaTG15 and compared it with other SARSr-CoV-2.

They found that the receptor binding domain (RBD) of the S protein of RaTG15 is highly different from other sarbecoviruses, and its amino acid homology with SARS-CoV-2 is 72.6 %, the amino acid homology with related bat and pangolin coronavirus is 68.6%-73.3%.

It is worth mentioning that, unlike RmYN02 and RacCS203 mentioned above, RaTG15 RBD does not have a defect, which corresponds to SARS-CoV-2 The amino acid 473-486 in the S protein is missing (deletion 2). According to previous studies, this deletion determines the use of angiotensin-converting enzyme 2 (ACE2). However, consistent with SARS-CoV-2 and RaTG13, a deletion corresponding to amino acids 444-447 (deletion 1) was found. The missing position is similar to that of RshSTT182 in Cambodia.

In addition, among the 5 amino acid residues that SARS-CoV-2 binds to the ACE2 receptor, RaTG15 shows that 4 are different (486, 493, 494 and 501). In addition, like most bat SARSr-CoVs, there is no furin restriction site at the S1-S2 junction of RaTG15.

The paper mentioned that sequence analysis showed that the RaTG15 virus may use ACE2 as an entry receptor, and the researchers also conducted further verification tests. RBD proteins from SARS-CoV-2, SARS-CoV-1, RaTG13, pangolin-CoV-GD, pangolin-CoV-GX and RaTG15 were used, as well as the outer domains of human and middle chrysanthemum bat ACE2 proteins (Figure S2A).

They found that the RaTG13 and RaTG15 RBD proteins from the middle chrysanthemum bat have weak or no binding affinity for human ACE2 (HuACE2). The RBD proteins of the two pangolins SARSr-CoVs have much higher binding affinity to HuACE2, only slightly weaker than SARS-CoV-2 RBD, but still higher than SARS-CoV-1. The binding affinity of pangolin-CoV-GX to HuACE2 is slightly weaker than that of pangolin-CoV-GD.

The researchers further verified whether the bat coronavirus RaTG13 and RaTG15 can more effectively use the chrysanthemum bat ACE2 (RaACE2) compared to the use of huACE2? The results showed that the affinity of RaTG15 RBD to RaACE2 was also significantly lower than that of SARS-CoV-2 and pangolin-CoV-GD/GX to RaACE2. As in HuACE2, the binding of RaTG13 RBD to RaACE2 is weak.

Researchers also conducted a fake virus infection test. Consistent with the protein binding analysis of RBD-ACE2, HuACE2 mediates the entry of all SARSr-CoVs except RaTG15, while ACE2 supports the entry of all SARSr-CoVs.

The study concluded that neither the SARSr-CoV-2 lineage nor the new lineage virus identified this time can effectively bind to HuACE2. The absence of RBD region seems to greatly affect the binding ability .

Researchers believe that these results indicate that if there is no further adaptation, bat-derived RaTG13, RaTG15 and other possible SARSr-CoV-2 viruses, their zoonotic The suffering potential is still limited. In contrast, from the use of cell receptors, pangolin coronavirus has a higher risk of spillage. Experimental evidence does not support laboratory leaks Leading to SARS-CoV-2

In general, the study reported a new lineage of SARSr-CoVs discovered from bats. These viruses It is closely related to SARS-CoV-2 in the RdRp region, but it is still far from any known SARSr-CoVs at the genome level.

Researchers pointed out that although several SARS-CoV-2 related coronaviruses have been detected from wild animals, none of them are related to SARS-CoV-2 at the genome level. SARS-CoV-2 is 99% genetically identical.

It should be noted that recombination events are more likely to occur between coronaviruses, which is also considered to be the potential origin of SARS-CoV-1. In previous SARS traceability studies, the SARSr-CoVs found in bats carried all SARS-CoV-1 genome fragments. Part of the genome regions of different wild animals are highly similar to the SARS-CoV-2 sequence, which also suggests that the virus may recombine during the evolution of viruses that spread across species or between species.

Researchers pointed out in the discussion section of the paper that their research results indicate that the SARSr-CoVs we now find from bats may be just the tip of the iceberg. These viruses may have undergone selection or recombination events in animal hosts, allowing the viruses to adapt to new hosts and then spread to new species until they enter human society.

They emphasized that they shouldThe identified new lineage viruses are monitored to prevent future outbreaks. Because viruses from the other two lineages of SARSr-CoV, namely SARS-CoV-1 and SARS-CoV-2, caused SARS and COVID-19, respectively.

In addition, according to the current research findings, the currently discovered bat SARSr-CoV-2 or new lineage viruses that can be isolated cannot effectively use human ACE2, so If there is no adaptation, the risk of spillover to humans is still relatively small. In contrast, viruses that can use ACE2 in the bat SARSr-CoV-1 related lineage seem to be more dangerous in terms of cross-species transmission.

The research mentioned that RaTG13 is the closest bat coronavirus to SARS-CoV-2, but it still only shows weak binding affinity to HuACE2.

They emphasized that although there were speculations that the RaTG13 leak in the laboratory may have caused SARS-CoV-2, the existing experimental evidence does not support this Speculate. In contrast, pangolin coronavirus has a strong binding ability to humans or bat ACE2, and has a high cross-species potential for humans or other species.

Researchers analyzed that under the background that SARS-CoV-2 is derived from animals, there may be a bat SARSr-CoV that uses HuACE2 more effectively than RaTG13. Or there is a pangolin coronavirus with higher genetic sequence identity.

The study finally emphasized that in the future, a more systematic and vertical sampling survey of bats, pangolins or other possible intermediate host animals is needed to prevent future SARSr-CoV related viruses. It is also possible to better understand the source of SARS-CoV-2.

Link to the paper: https://www.biorxiv.org/content/10.1101/2021.05.21.445091v1