Very close relatives of SARS-CoV-2 identified in Laotian bats

The pandemic SARS-CoV-2 virus probably originated in bats in Southeast Asia. There are over 450 million people sharing the region with the bats that live in limestone caves in countries like Vietnam, Thailand, Myanmar and Laos. One of the predominant genera of bats in the region is the Horseshoe Bat (horseshoe bat). Bat species belonging to this genus contain an ACE2 receptor that closely resembles that of humans. ACE2 is so similar, in fact, that many coronaviruses in these bats also have the potential to infect humans.

The exact origins of the SARS-CoV-2 virus are shrouded in mystery and controversy. Recent estimates of the number of people infected with coronaviruses of fruit bat origin in Southeast Asia are between 400,000 and two million. A recent to study of the Institut Pasteur, Temmam et al., studies viruses endemic to bats in this region.

The researchers sampled 645 bats from four different Laotian sites. Bats have been classified into 46 different species. This should happen because there are over 1,400 different species of bats in the world. They extracted the coronaviruses from the blood, feces, urine and saliva of the bats.

The first finding is that up to seven different species of bats harbored the same type of coronavirus, known as sarbecovirus. It is a subgenus of beta-coronavirus, to which the SARS-CoV-2 and SARS-CoV virus species belong.

In a surprising finding, the authors report that several of the viruses isolated from bat caves in Vientiane province are very closely linked to SARS-CoV-2. In fact, they’re so closely related that the idea that SARS-CoV-2 originated from the RaTG13 coronavirus isolated from the horseshoe bat in southern China in 2013 is no longer tenable. The receptor binding domains of SARS-CoV-2 and RaTG13 differ too greatly.

The original SARS-CoV-2 Wuhan strain has 17 amino acid contact points between the virus and the ACE2 receptor.

Several of them are modified in the RaTG13 coronavirus. However, two of the viruses analyzed by Temmam et al.-BANAL-20-52 and BANAL-20-103- differ by only one of 17 amino acids. Another virus analyzed, BANAL-20-236, differs only by two amino acids. In contrast, in RaTG13 there are six amino acid modifications. We note that a change of only one or two residues in the contact points falls well within the range of the various naturally occurring variants of SARS-CoV-2, which carry up to three amino acid changes in these residues.

The authors conducted several experiments to determine whether the similarity in the receptor binding domain region of the Spike protein of BANAL-20-52, 103 and 236 resulted in a function similar to that of SARS-CoV-2. Their first question was whether the receptor binding domain of viruses is capable of infecting cells that carry the human ACE2 receptor. Using a pseudotypic virus on cultured cells, they found the answer to be a definite yes. They then showed that the entire BANAL-20-236 genome forms plaques when introduced into human cells. In fact, it formed more plaques than the Wuhan strain of SARS-CoV-2. These two observations demonstrate that not only does the bat virus receptor binding domain recognize human ACE2, but that the entire virus is capable of infecting humans.

Their second concern was to understand exactly how well the bat coronaviruses bind to ACE2 compared to SARS-CoV-2. Using binding modeling and structural analyzes, the researchers showed exactly how the binding domain of the bat virus receptor could interact with the ACE2 protein, offering a biochemical explanation for exactly how which the bat binds to. The similarities between SARS-CoV-2 and the two bat viruses extend genome-wide with one notable exception, Orf8. Among the SARS-CoV-2 variants, Orf8 is one of the most frequently mutated genes.

To understand the interaction of bat receptor binding domains with ACE2 and the functional dynamics of the relationship, the researchers performed a biolayer interferometry test with the different viruses to analyze the nanomolar range of binding affinity. . Simply put, they tested to see if the bat viruses bound to ACE2 with a seal similar to SARS-CoV-2, which they did.

Finally, to analyze the binding structure, they used X-ray crystallography at a resolution of 2.9 angstroms for SARS-CoV-2 and BANAL-20-52. The figure below shows the structural similarities and differences between the two complexes, especially the mutated residues in the receptor binding domain and the structural complexes in the helices. As expected, most of the interactions observed between the ACE2 receptor binding domain and SARS-CoV-2 were also present for BANAL-236.

Recombination is frequently observed among natural coronaviruses. Laotian bat viruses are no exception. The authors were able to decipher the recombination events among the large family of coronaviruses infecting bats, which probably contribute to the origin of SARS-CoV.

The image that emerges is a continual reassortment of genes leading to a mosaic of viruses. For SARS-CoV-2 and bat viruses, there appear to be two distinct origins of the outer Spike protein, one for the N-terminal domain and another for the receptor binding domain.

The only question that remains is the origin of the Furin cleavage site. A recent investigation viruses have found that there are many coronaviruses with independently derived Furin cleavage sites. Many genera of coronavirus, including alphacoronavirus, gammacoronavirus, and beta-coronavirus, contain viruses with the naturally occurring site. We note that some viruses as well as virus pairs that are otherwise identical along their entire length may differ in the presence or absence of a Furin cleavage site. For example, SARS-CoV-2 contains one, while SARS-CoV does not. In fact, the Furin cleavage site is often lost simply by growing SARS-CoV-2 in human tissue culture. It is likely that the Furin site occurs in some variants of SARS-CoV-2 and bat virus by natural recombination, as may have been the case for SARS-CoV-2.

The authors speculate that the Furin cleavage site could be acquired during co-infection with another bat or other exotic species. I would add here that it could have been acquired by co-infection with humans in the region previously co-infected with another bat virus that contains a Furin cleavage site in a separate recombination event,

What is the significance of this finding? As we have already experienced, the Covid-19 pandemic is not a one-off event. Humans are infected repeatedly and perhaps quite frequently infected with coronaviruses of bat origin and other species. One thinks that MERS-CoV, for example, was acquired from camels, which in turn were acquired from viruses prevalent in humans and bats in Africa. Many of the cold viruses that sweep our population are also of animal origin, such as OC43, which comes from domestic farm animals like cows and pigs. In the recent past, there have been reports of canine coronaviruses infecting children in Indonesia. Additionally, SARS-CoV-2 variants have shown that coronaviruses can continually adapt to their host, resulting in a more infectious and pathogenic virus.

With regard to the current pandemic, we need to be alert to the possibility that new, more transmissible and dangerous versions of COVID-19 may emerge. Several hundred million people have already been infected with SARS-CoV-2, as well as by domestic and wild animals. It is possible that new adaptations via animal co-infection may emerge, such as those found in mink. In addition, recombination events between SARS-CoV-2 and other coronaviruses that exist in the human population could give rise to new, more dangerous forms of the pandemic.