The evolutionary journey of Omicron.

A never-ending story?

Zoonoses are human infections caused by microbes originating from the animal world. More than 60% of the 400 emerging human diseases identified since 1943 are zoonotic (The Lancet).

Many kinds of microbe cause zoonosis. Bats (order Chiroptera) occupy a privileged position among mammalian animals. Viruses stand out among microbes. I highlight the importance of the coronaviruses-bats binomial in veterinary and human medicine. It is an inseparable couple since the beginning of time. Long before animal domestication.

But we only know the tip of the iceberg. A 2017 study by PREDICT (EcoHealth Alliance) described the active search for viruses in nature. The investigators analyzed bats, rodents, monkeys, and humans in Bangladesh. The work led to the detection of more than 100 different coronaviruses, 98% of which were in bats. The authors suppose there must be more than 3,000 coronaviruses. Most of them are still undiscovered. They live among the more than 1,420 cataloged species of bats. Although only a few dominate. Bats of the genus Rhinolophus are the most frequent, but not the only ones.

After rodents, bats are the most abundant (22%) mammals. Not to be overlooked, distracted by bats, the role of rodents in this story. And if the coronaviruses-bats pairing were not enough, there are about 5,500 known species of mammals. Many may be candidates for receiving and exporting coronaviruses. To individuals of the same or different species. Including Homo sapiens.

Comparative genomics and protein structural analysis permit to do profound studies (PNAS, June 2020). Among 410 vertebrates analyzed, 252 (61.4%) of which were mammals, only these (mammals) are at high risk of receiving coronavirus, especially bats. The reason lies in the affinity of coronaviruses for a region of the cellular receptor ACE2.

Mathematical models estimate 11.5 times more coronavirus-host associations. Also, more than 30 times more potential SARS-CoV-2 recombination hosts. And more than 40 times more host species with four or more different subgenera of coronaviruses have been observed so far. We have known some of the candidates (mammalian) for years. Covid-19 pandemic now occupies thousands of web pages, scientific journals, and newspapers. But little or nothing is known about many animals (Figure 1) and their relationship to coronaviruses.

It is a matter of time and scientific and political interest in finding out. Even so, nature warning to humanity with SARS-CoV-2 arrival should serve to organize the world in the face of pandemic(s) to come.

The current coronavirus pandemic is of zoonotic origin.

SARS-CoV-2 is a zoonotic virus. Not only because of its phylogenetic origin. Also, it infects many animal species. Coronavirus pandemic species belong to the order Nidovirales, family Coronoviridae, and subfamily Coronovirinae. The last have four genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. It is well known that Alphacoronaviruses and Betacoronaviruses infect mammals, including humans, causing respiratory and intestinal infections. Likewise, Gammacoronavirus and Deltacoronavirus infect birds (and some mammals).

Coronaviruses can colonize mammals and infect them. Also, they can spread among the same or different species. And they may not affect the host (asymptomatic carriers). Or cause diseases of varying severity (diarrhea, bronchitis, peritonitis, hepatitis) — even death. The pathogenic event occurs among the animals in isolation (rare sporadic cases). But it is usually a collective problem (epizootics or epidemics in animals).

Sometimes, coronaviruses pass or jump from an animal to a human (it is the species jump). They do so from the reservoir animal (usually various bats). Or through intermediate mammals. For example, palm civets, Arabian dromedaries, pangolins, mink, wild and laboratory rodents, domestic pets, zoo felids. Sometimes the infection is round-trip: animal-human-animal or human-animal-human. The latter scenario exists in American and European minks — also, zoos animals such as tigers and lions (Asia, North America). And in 40% of white-tailed deer from four North American states.

According to the DBatVir database, coronaviruses account for 31% of bats virome. Bats occupy almost all Earth and inhabit dark and remote caves. They are more abundant in some geographic regions. Southeast and southwest China and some neighboring countries are impressive nurseries of Chiroptera. Also, of coronaviruses. In Southeast Asia, around 400,000 people are infected each year by SARS-related coronaviruses (SARSr-CoV), according to a September 2021 preprint study.

From those winds this mud come.

The relationship between coronaviruses and humans is an old one. However, it became fashionable two years ago. On June 24, 2021, Current Biology published a paper proposing the existence of an epidemic of probable coronavirus (or one related virus) in ancient East Asia. It happened no less than 25,000 years ago (around 900 human generations). This event was not trivial because it caused the appearance of mutations. But not in the coronavirus, which must have had them due to its condition of RNA virus, but in the human being. As a result of this encounter, mutations appeared in the human genome in a short time. At that time there were no large human concentrations (ancient people were hunter-gatherers). Forty-two dominant genes of resistance encode proteins that interact with viruses. But that is another story.

Some research estimates the time of coronaviruses’ most recent common ancestor at around 10,000 years. Other authors go much further back. They go back no less than 300 million years. It was the evolutionary moment when mammals and birds diverged. There is no doubt that these viruses are veterans. They have lived through many evolutionary wars. In nature, the fact is not insignificant.

There is a very large time gap between then and the appearance of a modern non-epidemic (respiratory) coronavirus, which the official taxonomy named NL63. It occurred between 560 and 820 years ago (6th to 9th centuries). Another coronavirus (229E) may have appeared around 200 years earlier. OC43 about 120 years ago. Finally, HKU1 passed the species barrier around the beginning of the 1950s (Figure 2). Thus, progressively, coronaviruses entered the history of virologic science and clinical medicine.

Respiratory and epi/pandemic coronaviruses.

There are seven coronaviruses affecting humans to date. Four (Alphacoronaviruses NL63, 229E, and Betacoronaviruses OC43 and HKU1) cause upper respiratory infections. Clinical problems like colds, pharyngitis, and rhinitis in adults (15%) and immunocompetent children (35%). The conditions are usually mild (50–90%). But they can sometimes cause more severe cases (pneumonia) in children, the elderly, and immunocompromised patients.

There are three more aggressive coronaviruses (SARS-CoV-1, MERS, and SARS-CoV-2). They are of the Betacoronavirus genus (with four subgenera). They have caused two epidemics, SARS in 2002–2003 and MERS in 2012. And the COVID-19 pandemic that was born for science in December 2019. Betacoronavirus SARS-CoV-2 of the Sarbecovirus subgenus is the cause of COVID-19.

The dates of onset of epi/pandemics and the emergence’s dates of the many variants of concern (VOC) and interest (VOI) described so far are very relative. Or, better, they are not always accurate. MERS coronavirus, surged in September 2012 in Jeddah (Saudi Arabia), circulated in Somalia in 1983, i.e., 19 years before its discovery in 2012. SARS-CoV-2 had been around in China and other countries for months before it skyrocketed in Wuhan (China) in December 2019.

The detection of Omicron, the lineage now dominating the world epidemiological scenario, occurred in November 2021. It happened in South Africa and Botswana. But the non-detected mutant virus entered circulation in the spring-summer of 2020, despite the high genetic sequencing potential of the African country and the significant number of mutations. In other words, while reading this article, some accidental spread of coronaviruses is happening in nature, surging many mutations and possible genomic recombination. This fact occur because RNA viruses carry these genetic characteristics in the suitcases of evolution.

For increase uncertainty, thousands of coronaviruses are not even known yet by scientists. But, based on data, investigators do not doubt their existence. All coronaviruses are owners of a single RNA strand (Figure 3) of considerable size. More than 29,000 nucleotides or letters encode various amino acids/proteins. And mutate at a rate of two mutations per month on average. It is half the mutational rate of influenza viruses and four times lower than HIV. They have a very effective evolutionary survival mechanism. And dangerous for the host. I mean genetic recombination, i.e., the exchange of genes from two different strains or viral variants housed in the same host cell. Recombination is a common phenomenon among coronaviruses.

Random genetic changes occur anywhere in the long viral genome of almost 30 kb. Most clusters are in certain parts, such as the S protein (Figure 3). But there are still dark areas of knowledge. They will most likely illuminate in the coming future. For example, the uncertain role of some specific genes remains still unknown. A paper (from Weizmann Institute of Israel) demonstrated the existence of 23 ORF (Open Reading Frames) unknown until now. As it is also unknown its function.

All coronaviruses are similar, but not the same.

If we paraphrase the donkey Benjamin, a character from the Orwellian novel Animal Farm, we can say: all coronaviruses are alike, but some viruses are more alike than others. Both human and zoonotic coronaviruses and many related coronaviruses are genetically similar. But there are notable differences between genera, species, strains, and variants. For example, there is a significant genetic difference between the native human strain (Wuhan-Hu-1) and the bat reservoir strain (Rhinolophus affinis or RaTG13) from which it originated. Nevertheless, they share 96% genomic homology. The successive variants (from alpha to Omicron) with their genomic differences also can serve as a comparative model.

The complex world of bat-coronavirus interaction is dynamic. New viruses related to SARS-CoV-2 continue appearing. For example, a paper published in Cell (August 19, 2021) reports four new coronaviruses detected in the summer of 2020 in several species of bats in China’s Guangdong province. But they also fly the neighbor skies nesting in the caves from Laos, Vietnam, and other territories.

When comparing whole genomes, one of these chiropteran species, Rhinolophus pusillus or RpYN06, has 94.5% homology with SARS-CoV-2. It means it is two points less related than R. affinis/RaTG13 or it is more genetically distant from SARS-CoV-2. But it has the highest homology so far described with SARS-CoV-2 in four specific genomic regions (ORF1ab, orf7a, ORF8, N, and ORF10). If we focus on Omicron or BA.1 (with more than 50 mutations), there are two other subvariants (BA.2 and BA.3) with different molecular characteristics whose significance is unknown.

The key to the genomic differences in animal and human viruses lies in the mutational profile of the spike. And in other genes as well.

The evolutionary determinant role of gene/protein S.

The spike glycoprotein encoded by the S gene of SARS-CoV-2 determines the tropism and susceptibility of the host to infection. Because of its antigenic character, the S protein is the main target of natural and vaccine antibodies. Besides, adaptive mutations in protein S can affect host tropism and viral transmission. The S protein (Figure 3, brick color) is composed of two subunits: S1 and S2 involved in the binding and fusion of the virus to the host cell.

This domain is lacking in other non-SARS-CoV-2 betacoronaviruses. For example, in human SARS-CoV-1, bat RaTG13 coronavirus or pangolin coronavirus. Furin proteases process it. Also, transmembrane serine proteases such as TMPRSS2 and endosomes cathepsins. Finally, S1/S2 cleavage exposes the S2′ site. It is also cleaved and releases an internal fusion peptide that mediates membrane fusion. This mechanism is present in coronaviruses of several Asian and European bat species. And it is present in unrelated viruses such as HIV, Ebola, and avian influenza A (H5 and H7) viruses.

It is a natural phenomenon that has occurred on many occasions in nature. And it conditions the evolutionary behavior of the virus at different levels:

1. Seeking accommodation among new animal species. From the reservoir bat to an animal or human or an intermediate animal to humans.

2. Improving its ability to bind to host cells and the encounter between the spike (the RBM) and the cell receptor (ACE2).

3. Increasing fusion and entry into host cells by engaging the polybasic fusion site.

4. Increasing its pathogenic capacity through increased viral replication. For example, higher viral load detected in the laboratory by RT-PCR. Or a low cycle threshold (Ct) below 30: i.e., a more significant number of infective virions causing more severe disease. And greater contagiousness.

5. Cheating the complex immune defense system by total or partial immune evasion. Or escape innate immunity mediated by interferons. And from acquired immunity mediated by neutralizing antibodies and B and T lymphocytes. This fact entails the risk of reinfection.

6. Finally, avoidance of antibodies generated by vaccines. Until now, different mRNA platforms, modified adenoviruses or viral particles. It entails the real risk of breakthrough infections.

Omicron is an evolutionary variant of the original zoonotic SARS-CoV-2.

The above long summary on zoonoses, bats, other mammals, human and zoonotic coronaviruses, and SARS-CoV-2 and its genomic variants are pertinent in the light of two recently published papers discussed below. But, before, it is time to recall the consortium of mutations. I recommend retaining this idea of consortium as equivalent to a clustering of something. Also, remembering the different behavior of Omicron about immune evasion compared to other variants. And its evolution towards a much more contagious and less severe epidemiological profile from the clinical point of view. We can say it means to contemplate a new face of the pandemic. First, let’s look at the cited articles done by two leading research groups in this field.

About the H655Y mutation.

The first one is from Cell Host and Microbe and signed by Alba Escalera et al., from Adolfo García-Sastre’s group (Mount Sinai, New York). It focuses on the H655Y mutation. It is a substitution located at the edge of the furin fusion site (Figure 3). The mutational change circulated by New York in March 2020. It is present in the gamma (P.1) and Omicron (B.1.1.529 or BA.1) variants. The H655Y substitution allows the virus to evade the neutralizing action of antibodies. Natural antibodies and stimulated by vaccines.

The animal models (hamster and American mink) select the mutation after experimental infection with the WA1 (Washington) variant in vivo. WA1 is like the Wuhan-Hu-1 strain from a mutational point of view in human respiratory cells. It supports a potential role in replication, transmissibility, and pathogenicity.

Mice, cats, and mink also select the H655Y mutation in nature. Of added interest is that Omicron harbors the P681H mutation: Note the H in the alpha variant while P681R changes from H or histidine to R or arginine. This amino acid change is present in delta and gamma, D614G (detected in almost all variants), and N679K. The combination suggests increased transmissibility of SARS-CoV-2. Also, the change at site 681 may introduce an extra furin cleavage site.

The Mount Sinai group in New York concludes by saying that the 655Y polymorphism, present in the gamma and omicron variants, is a crucial determinant of virus infection and transmission. It enhances the spike cleavage and viral growth. Moreover, S:655Y substitution is more efficient than its S:655H ancestor in hamster and mink infection models. And can outcompete S:655H in the human airway epithelium.

According to Escalera et al., the variants of concern circulating now, with high transmissible, have evolved. They acquired mutations associated with enhanced processing of the spike protein. Also, it improved transmission and increased fusogenic potential (syncytium formation). In other words, they confer an advantage in viral contagion.

About three mutations clusters.

The second paper (preprint bioRxiv of January 18, 2022) is of Darren P. Martin et al. from Tulio de Oliveira’s group (South Africa/SA). This group detected the beta variant in late 2020 and the omicron variant in November 2021. The researchers analyze the genetic changes in Omicron in depth. Of the more than 50 mutations that define Omicron BA.1, 37 are in the S gene. So, they double or triple the number of changes present in other variants. But what is most interesting is that 13 of the changes are very rare or do not exist in the different human variants. Even in bats, the putative predecessors. The SA researchers consider three groups of mutations (Figure 4):

- Group 1 consists of four mutations in the spike tip. It appears to cooperate in binding to the ACE2 receptor and does so more than other variants.

-Group 2 also has four mutations. They take part in receptor binding. Also, they allow immune evasion by evading antibodies directed against that antigenic area.

- Group 3, it have five mutations helping fusion.

This consortium/clusters of mutations is interesting from an evolutionary point of view. But it could be harmful to the virus by showing adverse selection or no evidence of selection. Their location is another aspect of interest: Group 1 and 2 mutations act at the tip or top of the spike protein, modifying its spatial conformation. The change hinders the action of some antibodies. Group 3 is in the stem of the spike — the fact that limits fusion with the cell. Omicron alters the entry pathway into the cell and penetrates by endocytosis instead of using the ACE2 receptor. This mechanism works better in the upper respiratory tract than in the lungs. That is in line with a minor respiratory severity of Omicron (article in Spanish). As a result, there is a decrease in cases of pneumonia and an increase in upper respiratory infections.

The evolutionary phenomenon is due to positive epistasis or the influence of some genes on others. Mutations would act in a coordinated manner and with no independence. Alone or individual, they are maladaptive mutations, whereas, as a whole, they are adaptive mutations. The fact was already known or suspected following a group 2 mutation (S/Q498R) with S/N501Y. The last is the star mutation present in the 501Y SARS-CoV-2 lineages, i.e., in the alpha, beta, and gamma variants. S/498R substitution alone hardly affects the affinity of the spike for the human ACE2 receptor. But the presence of S/501N increases the binding affinity to ACE2 approximately fourfold.

It is a shocking fact because S/Q498H (H instead of R, i.e., histidine instead of arginine at position 498) alone increases the affinity to ACE2. More than 20-fold in the experimental mouse model. But is incompatible with S/N501Y. The reason is that it inhibits the binding to the receptor (bioRxiv, December 25, 2021).

At first sight, the matter seems trivial. It is a simple change of a single letter. Now it is worth remembering the advice of Ramón y Cajal (Rules and advice on scientific research, chapter II, in Spanish): There is no superior and inferior, no accessory and principal things in nature. The issue is of great importance because of what it can mean. The exchange of a single amino acid makes it possible to broaden the spectrum of host animals. It helps the virus jump and expansion between species. In this case, the exchange between rodents and humans.

A message for those who applauded and justified the universal Omicron spread “because it is a simple cold and so if you get infected, you get vaccinated naturally.” These persons should know that SARS-CoV-2 is a new and unknown coronavirus. Furthermore, there is a potential for systemic damage. And the possibility of permanency (Long COVID) is still of uncertain scope because SARS-Cov-2 is a new member of the club of fatiguing viruses.

Nature does not stop or care about the ridiculous vicissitudes of humans. One should not lose sight of animal mammals, bats, mink, pangolins, zoo cats, deer, or mice. They are excellent receptors and disseminators of coronaviruses with epi/pandemic potential. Returning to Omicron, it is worth asking -like Martin and collaborators do- how and why there are so many maladaptive mutations. An intriguing fact in this surprising variant.

To explain this, we must first accept that Omicron has been accumulating mutations for months. They were undetected by those who search for viral changes in their sophisticated laboratories. But the molecular clock allowed to date when Omicron separated from other predecessors and its sub-lineages.

The molecular clock is a technique that dates the divergence of two species. Also, it deduces the past time from the number of differences between two DNA sequences. That is, it permits the definition of the common ancestor. Recall that Omicron has been mutating since the mid-2020 year. It means it emerged before the alpha, beta, gamma, and delta lineages. Finally, it appeared in November 2021. Three scenarios attempt to explain so amazing birth.

Possible hypotheses.

One can postulate three different explanations. There are data for and against each one and a combination of the three.

1. Genomic surveillance failure hypothesis. Mutational changes occurred in a geographical region with poor epidemiological surveillance. And with poor access to the health care system. But, with Denmark and the UK, South Africa leads the world in viral genome sequencing. They are leading a good way. Still, it may have happened. This hypothesis reinforces the drift of the BA.1 omicron variant and its two sublineages (BA.2 and BA.3). BA.2 sublineage has spread in Denmark and 54 countries. It is under WHO surveillance (it appears more contagious but not more severe). But it is still premature to venture its epidemiological and clinical significance without forgetting that it is Omicron. It again serves to remind us that the evolutionary journey of the original pandemic coronavirus is not yet over. And to reinforce the demand for massive sequential studies worldwide. That is translated into investment in scientific and healthcare personnel and material resources because epi/pandemic nightmares are recurrent.

2. Chronic infection in immunosuppressed patients. This idea explains that SARS-CoV-2 infection would have occurred in an immunosuppressed patient. Without immunological opposition, a poor or null immune response would allow the coronavirus to accumulate mutational changes. The most prominent model of intra-host evolution is that of an HIV-infected person. The high prevalence of HIV infection in South Africa, with poor access to antiretroviral treatment and pandemic coronavirus coinfection, plus other factors, support this idea. This hypothesis is quite powerful. It could convince the scientific community, but it is not proven. Furthermore, it will not be easy to prove. Of course, immunosuppression should not be limited only to HIV-infected patients. It would encompass other immunodeficiency scenarios: transplants, neoplasms, and immunosuppressive treatments. From a practical point of view, the best way to avoid the risk is to protect vulnerable patients. For example, by implementing several preventive measures. Like strict non-pharmacological protection at epidemic times and scenarios. Preferential and periodic or variant-adapted complete vaccination. To monitor the immunological vaccine response (neutralizing antibodies at valid levels). And rapid detection (screening) at the slightest contact.

3. Reverse zoonosis. It means that human infection by coronavirus passes to some mammalian animals — a journey from human to animal. The latter spreads it among its conspecifics and other species, including humans (Figure 1). The spectacular case of European minks is the best proof. Also being studied, when, how, and where free-ranging and captive white-tailed deer became infected.

It is not unreasonable to think that wild mink or captives who escaped from farms could spread the virus. And think in mice too. Omicron can adapt to mice and rats. But what seems undeniable is that the zoonotic phenomenon must be broader. And involve more species of animals, especially mammals. It makes it necessary to insist on active surveillance. And to demand political and economic support for science.

Some authors question these hypotheses, while others think that a combination of them may occur. But be that as it may, if we do not invest in research, also in animal research, it may take another 25,000 years to find answers to the question Quo Vadis, Corona?

A never-ending story?

An interesting paper preprinted (The Lancet) demonstrates coronavirus transmission via imported pet hamsters. Then the spread of the virus (delta variant) among the animals in the store, the contagion from animals to humans (a sales clerk and two customers), and later spreading among humans (customers’ relatives). Summarized, the essential data are:

Epidemiology: The animals traveled from the Netherlands to Hong Kong. Two shipments with two different stopovers: Doja in Qatar and Bangkok. And two international flights of two other different airlines. The first infected person was a 23-year-old sales clerk working in a store selling pets that sell hamsters, chinchillas, guinea pigs, mice, and rabbits. There was no evidence of contact with other infected people. The sales clerk woman received two doses of Pfizer mRNA. Next, there was an infection of a mother and her daughter, both clients and later other family members. All of them received Coronavac (3) and mRNA (1). Of the animals, only the hamsters had an infection.

Virology: The variant detected was delta (AY127 lineage). It was the virus predominant in Europe in December 2021 and January 2022.

Genomics: There were mutations in the spike (L18F, H49Y, D427G). And one substitution (T38I) in ORF8. So that indicates the possibility of enhanced binding to the ACE2 receptor and antibody evasion.

Clinical: Infected humans presented with upper respiratory symptoms and cough without evident severity.

Conclusion.

Like other viruses with a pandemic vocation, coronaviruses have swarmed in nature since the most remote times. They survive antiviral avatars thanks to their innate capacity to change, replace, lose, or incorporate genes and recombine between different species, strains, or lineages. As a result, coronaviruses have occupied larger ecological spaces among the many mammalian hosts and other vertebrate animals throughout human history.

The evolutionary journey is not over yet and never will end. James Bloom, from Seattle, says it best and clear in Nature: “The virus is still expanding in evolutionary space.” BA.1 omicron variant is one of the stars of the pandemic by SARS-CoV-2, human and animal. And it is an epidemiological phenomenon unheard of in the history of medicine. It is the penultimate evolutionary traveler.

Note: A Spanish version of this article was published in Biotech Magazine & News.