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Discovery of novel bat sarbecoviruses similar to SARS-CoV-2 strains

Many sarbecoviruses are found in Rhinolophus colonies that live in caves in China, as well as in adjacent nations to the south. During a prospective research in Northern Laos, the research team discovered five sarbecoviruses for which they got full-length genomes, in addition to coronaviruses. Three of them (BANAL-52, -103, and -236) were thought to be comparable to SARS-CoV-2 because one of the S1 domains (NTD, RBD) or S2 was similar to SARS-CoV-2. 

They compared SARS-CoV-2 strains from the two lineages reported at the start of the COVID-19 outbreak to these novel bat sarbecoviruses and pangolin strains within the SARS-CoV-2 clade, because genomic areas vulnerable to recombination are expected to contribute to host-virus interactions. 

SARS-CoV-2 genome fragments was found in different areas of the genome in strains related to R. pusillus RpYN06, R. malayanus RmYN02, and Rhinolophus sp. PrC31 isolated in China in 2018-2019, as well as R. malayanus BANAL-52, R. pusillus BANAL-103, and R. marshalli BANAL-236 isolated in Laos in 2020. Pangolin coronaviruses appear to be more distantly linked than bat coronaviruses, and no closer viral genome has yet been found as a likely cause. They found plausible recombination sites, which allowed to reconstruct the evolutionary history of early SARS-CoV-2 strains by comparing homologous regions defined by recombination points.

The team discovered a breakpoint at the start of the SARS-CoV-2 RBD, which resulted in a downstream segment crucial for the virus tropism and host spectrum that included the RBD, the furin cleavage site, and the S2 N-terminal region. Phylogenetic reconstruction of this fragment revealed that Laotian R. malayanus BANAL-52, R. pusillus BANAL-103, and R. marshalli BANAL-236 coronaviruses are the closest ancestors of SARS-CoV-2 known to date, despite the absence of the furin site. This finding was publish in "Nature".

ORF8 between SARS-CoV-2 related genomes was substantially diverged. ORF8 from BANAL-52, -103, and -236, like RaTG13, was more closely related to SARS-CoV-2 than to pangolin strains. ORF8 encodes a protein that has been linked to immune evasion, and it has been deleted in several human SARS-CoV-2 strains that have surfaced since March 2020, similar to deletions found during the 2003 SARS outbreak. As a result, the existence of ORF8 is compatible with bats serving as a natural reservoir for SARS-CoV-2 early strains.

The RBD domain that mediates the interaction with hACE2 and the host range, as well as the primary amino acids involved, have been determined through structural and functional biology studies. The RBDs (BANAL-52, -103, and -236) are more closely related to SARS-CoV-2 than any other bat strain previously described, including RaTG13, the virus discovered in R. affinis from the Mojiang mineshaft in 2012, where pneumonia cases with clinical characteristics a posteriori interpreted as similar to COVID-19 were recorded. In comparison to SARS-CoV-2, these strains have one (H498Q (BANAL-103 and -52) or two (K493Q and H498Q (BANAL-236)) amino acids interacting with hACE2 replaced. The BANAL-236 / hACE2 interaction was not destabilised by these changes.

The authors explained "Our findings back with the theory that SARS-CoV-2 arose from a recombination of sequences already present in Rhinolophus bats inhabiting in South-East Asia's and South China's large limestone cave systems. R. malayanus and R. pusillus, for example, forage in the same cave locations. Furthermore, in the Indochinese sub-region, the distributions of R. marshalli, R. malayanus, and R. pusillus overlap, suggesting that they may share caves as roosting and foraging habitats. Understanding the emergence of SARS-CoV-2 does not require hypothesising recombination or natural selection for increased RBD affinity for hACE2 in an intermediate host like the pangolin prior to spillover, nor natural selection in humans following spillover, thanks to the novel viruses described here".

On sequences derived from original faecal swab samples, however, they discovered no furin cleavage site in any of these viruses, indicating that there is no possibility of counter selection of the furin site by amplification in Vero cells. Insufficient sampling in bats could explain the lack of a furin cleavage site. Based on sequence comparisons around the S1/S2 cleavage site, it has been proposed that the furin cleavage site present in SARS-CoV-2 may have arisen from recombination events between SARS-CoV-2-related coronaviruses co-circulating in bats, implying that BANAL-116, BANAL-247, bat RmYN022, and bat RacCS203 coronaviruses may have a common history. Alternatively, the furin cleavage site could have been obtained by virus passages in a different host or during an early undocumented circulation with few symptoms.

Because the S ectodomain of BANAL-236 has a high affinity for ACE2, pseudoviruses producing it were able to penetrate human cells expressing endogenous hACE2 utilizing an ACE2-dependent route. Alternative entrance points, particularly in cells that do not express ACE2, may still exist. A serum that neutralizes SARS-CoV-2 prevented entry. The RaTG13 strain, which was previously thought to be the closest to SARS-CoV-2, had never been isolated. In contrast to SARS-CoV-2, preliminary research shows that BANAL-236 reproduced in primate VeroE6 cells with a modest plaque phenotype. Further research could reveal which processes influence infectivity.

To summarize, their findings indicate the presence of novel bat sarbecoviruses with the same potential for infecting people as early SARS-CoV-2 strains. Tourists visiting caverns, as well as guano collectors and certain austere religious organizations who spend time in or around caves, are particularly vulnerable. More research is needed to see if such exposed populations have been infected with one of these viruses, whether symptomatically or not, and whether infection can protect against later SARS-CoV-2 infections. In this respect, it's worth noting that SARS-CoV-2 with the furin site removed replicates in hamsters and transgenic mice expressing hACE2, but causes less severe sickness and protects against SARS-CoV-2 reattack.

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