Examine exhibits Sarbecoviruses circulating in wildlife exterior Asia can infect human cells

The 2019 coronavirus disease (COVID-19) pandemic caused by Severe Acute Respiratory Syndrome-Associated Coronavirus 2 (SARS-CoV-2) and previous SARS-CoV outbreaks were the result of a zoonotic sarbecovirus overflow from Animals to humans. Although most animal sarbecoviruses do not infect eukaryotic cells, it has been found that clade 4 sarbecoviruses recently found in China have a deletion similar to clade 3 sarbecoviruses and can bind to human ACE2.

Study: Sarbecovirus found in Russian bats uses human ACE2. Image source: Corona Borealis Studio / Shutterstock

About the study

One recently in bioRxiv. published preprint study* Server tested the receptor binding domain (RBD) of 2 sarbecoviruses found in Russian horseshoe bats – Khosta-1 in Rhinolophus ferrumequinum and Khosta-2 in R. hipposideros – and other clade 3 viruses for their ability to invade human cells. Phylogenetic analysis was performed on each Sarbecovirus spike sequence used in this study with a Genbank accession number. The amino acid sequences of the spike RBD were aligned using ClustaIW multiple sequence alignment.

Spike chimeric expression plasmids were generated by replacing the RBD from SAR-CoV-1 with the Khosta virus RBD and compared to chimeric RBDs from clade-3 viruses. Researchers used a single cycle pseudotype assay of Vesicular Stomatitis Virus (VSV) and SuperSignal Western Blot substrates for analysis.

results

The three main findings of the study are: 1) Khosta virus RBDs are different from those of human viruses, 2) Khosta virus RBDs facilitate entry into human cells, and 3) Khosta virus RBD infects human cells using the ACE2 receptor.

From March to October 2020, the Khosta 1 and 2 viruses discovered in bat samples near Sochi National Park were phylogenetically analyzed and found to be closely related to clade 3 Sarbecoviruses found in Bulgaria in 2008 are related. These viruses had a clear viral lineage compared to human zoonotic viruses. Notably, the Khosta 1 and 2 viruses differ from the Group 1 Sarbecoviruses and have structural components that enable them to interact with the ACE2 enzymes.

Biosafety Level-2 (BSL-2) compatible viral reporter pseudotypes with VSV were constructed using the spike chimeric plasmids. The chimeric spike with RBD from clade 3 viruses showed reduced incorporation and had no correlation with the viral entry phenotypes found in the later experiments. Research by the authors also indicated that the presence of the exogenous protease trypsin promoted entry of Khosta virus into human cells, suggesting that the Khosta virus RBDs require a human cell receptor to mediate infection.

A receptor tropism test was also performed to characterize the receptors potentially used by the Khosta virus by transfecting baby hamster kidney (BHK) cells with known human coronavirus receptors and then using the pseudotype panel were infected. The results showed that Khosta-1 did not infect RBD cells with any of the receptors, while Khosta 2 infected RBD cells with human ACE2 receptors. The cell entry potential of Khosta-2 RBD was similar to the RBD of RatG13 – a bat sarbecovirus – which is very similar to SARS-CoV-2 RBD.

Conclusions

This study showed that animal-derived Sarbecoviruses circulating outside of Asian wildlife have the ability to infect human cells with ACE2. Despite the clear compatibility of Khosta-2 RBD with human ACE2, the pseudotype entry was almost 10 times lower than that of SARS-CoV-1 and -2. Although both Khosta-1 and -2 RBDs infected human cells in the presence of the protease trypsin, Khosta-1 was significantly more potent than Khosta-2. In the receptor-specific assay, however, only Khosta-2 infected human cells with ACE2 in the absence of trypsin.

The majority of the animal Sarbecoviruses identified so far are only distantly related to human pathogens and pose no risk to humans. The Russian bat Khosta 1 and 2 viruses used in this study are from a particular line of SARS-CoV-1 and 2 virus and were found in a wider geographic area, demonstrating that Sarbecovirus host receptor compatibility is not limited to specific geographic locations. There is variation in host-receptor compatibility in closely related Sarbecoviruses and this suggests an evolutionary mechanism for virus persistence.

The present study identifies a new group of Sarbecoviruses with zoonotic potential such as the Sarbecovirus clade 3. Unfortunately, Sarbecovirus vaccines currently in the pipeline only cover Class 1 and Class 2 viruses and do not include Class 3 viruses. The results show that the Sarbecovirus poses a greater geographic threat than previously identified and underscores the need for advanced Sarbecovirus vaccine measures.

*Important NOTE

bioRxiv publishes preliminary scientific reports that have not been peer reviewed and therefore should not be considered conclusive, that guide clinical practice / health-related behavior or should be treated as established information.