On the Origin Debate and Plausible Future Endeavours of COVID-19 (Commentary)

There has been an ongoing debate on the evolutionary origin of SARS-CoV-2 – whether naturally evolved or engineered in a virology laboratory. After prolonged analysis, it was evident that the RBD_Spike sequence, favorable for host receptor binding in SARS-CoV-2 is different from those in earlier variants of the coronavirus lineage. Six critical amino acid positions have been identified in the RBD_Spike of SARS-CoV-2 that effectively (i.e., physically) bind to the human host receptors (hACE-2: human Angiotensin Converting Enzyme 2) out of which five were mutated with respect to SARS-CoV (Y442 → L455, L472 → F486, N479 → Q493, D480 → S494, T487 → N501). The proponents of the natural evolution theory argue on a not fully optimal (or, non-ideal) binding of the viral Spike to the host receptor is a signature of natural evolution (Andersen et al., 2020) – which has later been somewhat rationalized by implementation of the concept of complementarity in probing the RBD_Spike-ACE-2 binding across coronavirus variants (Basu et al., 2021). Proponents of the natural evolution theory also argue for the fact that a deadlier variant from one of the other reverse genetic systems of beta-coronavirus (Dudas et al., 2018) could have been used instead, if one were to engineer the virus in a laboratory to cause ‘maximum harm’. For the progenitor of this deadly virus, the bats serve as the reservoir host. To that end, the RaTG13 virus isolated from Rhinolophus affinis bat shows 96% similarities with SARS-CoV-2 (Zhou et al., 2020). Although its RBD_Spike diverges from the RBD_Spike of SARS-CoV-2 resulting in a loose binding to hACE-2 (Wan et al., 2020). It has been recorded that the Malayan Pangolins (Monis javanica) imported into Guangdong Provinces harbors coronavirus analogous to SARS-CoV-2 (to which it shares the highest similarity) but not as much as to that of the RaTG13 virus. The polybasic cleavage site(s) and predicted O-linked glycans procured across different coronavirus variants have been characterized to undergo a series of mutations: non-synonymous substitutions, insertions and deletions at their S1/S2 junctions – which also emphasizes the natural evolution theory of SARS-CoV-2 wherein the mutations occur during the human passage of the virus.

Another viewpoint on how the coronavirus has emerged is said to be grasped by connecting the events at the places of seafood market and the virology lab. People suspect that the virus may be originated from a virus that intruded into an intermediate host and finally transmitted to human. The Wuhan seafood market is the epicenter of the virus where wild animals like bamboo rats, hedge wags, and hog badgers are sold as food items. The wild lives are raised on farms in an internally congested area where it’s easy for animals to grow viruses and spread from pillar to post among each other. The cross-species barrier is feeble in viruses which makes them enable to undergo horizontal gene transfers and genome fusions oftento mutate fast. Furthermore, the ss-RNA genome uses the virus-coded RNA-dependent RNA polymerase (Gong, 2021) as a machinary to replicate which, unlike DNA polymerases, lacks the proof-reading activity and therefore facilitates the mutational influx.

Some wildlife that is pliable to coronavirus infections is devoured in China either for food or used in traditional medicine. The very first cases reported in China infected by SARS-CoV-2 were either the vendors who worked at the wet market, the shopkeepers, or the customers. China’s south-westernmost province Yunnan is the habitat of many coronavirus-carrying bats that resides in an ancient cave (Karunarathna et al.). It is the dwelling place of the coronaviruses which closely resembles the pathogen SARS-CoV, the virus that caused the epidemic in 2003. The cave is very much congested and within it resides a large population of bats. The environment provides ample space for the viruses to blend and supply the niche for effective coronavirus hybridization. One of such hybrid viral offsprings was naturally selected to have suddenly intruded on us, the homo sapiens. Moreover, some wildlife farms are situated nearby that deliver animals to Wuhan which probably has made the virus move from a bat cave into the market transmitted by an intermediate host.

Doubts, however, have been cast on the natural evolution theory of SARS-CoV-2 over the last couple of years. The most promising alternative hypothesis (as of now) is that of a lab escape (or, lab-leak) originating from the Wuhan Institute of Virology – a pioneer institute in virology worldwide with high-security laboratories (biosafety level 4) which has been engaged in collaborative research with the most proficient and reputed scientists internationally. A search for the intermediate host was going on as it was believed that according to the theory of zoonotic transfer, animals are used as the reservoirs of pathogenic viruses. The most victimized creatures are the bats which seemed to be rupturing the species barrier after intruding into an intermediate host and passing on the viral pathogen. The same phenomenon had been observed during the previous eruptions, 2003 (SARS-CoV) and 2007 (MERS) – wherein civets and camels played the role of the intermediate hosts respectively.

Scientists targeted the Wuhan wet market in China and conducted a survey there, mainly pointing out a small number of alive animals, suspected to serve as the intermediate host. But no trace of the pathogen was found in the animals so far. They concluded that this is a direct transmission from bat to human, but it remains only speculation without any authentication (or, documentary proof) in its support. The assumption that the virus reared through a poisonous food chain is extremely unlikely. These failures piled up indirect support in favor of the lab escape hypothesis (Balaram, 2021). Scientists from the Wuhan biosafety laboratories working on the ‘gain of functions’ mutation of SARS-CoV (2003) frequently visited the bat caves and collected samples for their ongoing experiment (Dance, 2021). An obvious argument in favor of a possible lab-leak hypothesis at the Wuhan Institute hence is that these people might have got pre-exposed to the virus during such visits and that the viral emission this way, allowed the human passage through inter-individual transmission. People, much fond of conspiracy theories hence called the virus a ‘Chinese virus’. ‘Gain of functions’ studies of virus relies on repetitive passage of the virus through altering animal cultures and the evolutionary selection pressure to eventually hit the desired target. To that end, the American scientific establishment and eminent scientists (proponents of the natural evolution theory) postulated that the virus acquired its detrimental arms through adaptation, undergoing slow mutations due to the raised selection pressure, made to work in favor of an evolved pernicious human pathogen from its bat origin. Scientists allegedly claimed that the sequence of the SARS-CoV-2 glycoprotein is the possible cause behind its high pathogenicity. The wall of resistance collapsed when a deeply scrutinized article was published in the Bulletin of Atomic scientists by Nicholas Wade (The origin of COVID: Did people or nature open Pandora’s box at Wuhan?, 2021), a long serving-man of science journalism. Examining Furin cleavage site, the legendary biochemist David Baltimore also said that it was the smoking gun containing the arginine codons (Balaram, 2021).

The genetic material of SARS-CoV-2 is RNA which is injected into the host cells upon viral invasion (Masters, 2006). It is stereotypical for such invading viral pathogens to disrupt all the vital processes occurring in the living host to replicate. It’s potential to cause the disease is influenced by the Spike protein interacting specifically with the hACE-2 receptor of the lung cells through its receptor-binding domain (RBD_Spike). The interaction between the two molecules is compulsory (or, necessary) but not sufficient for viral entry (Millet and Whittaker, 2018). A kinetically driven ‘down-to-earth’ conformational change of RBD_Spike (transformation from ‘lying down’ to ‘standing up’ conformation) activates the receptor-binding domain, upon its proximity to hACE-2 which then rises to the occasion and binds to the host receptor. This is one of the key steps in the viral host cell entry which is intricately and temporally coupled to a priming event of (Furin-like) host protease mediated proteolytic cleavage at the S1/S2 junction setting free the already ‘up’ RBD_Spike for the host receptor binding. Furin and other host proteases cleave the arginine-rich polybasic FLCS_Spike in SARS-CoV-2 which remains a disordered loop in the unbound Spike and gets transiently ordered (exhibiting meta-stability) (Roy et al., 2022) by forming a dynamically interchangeable ionic bond network at the Spike – Furin interface that facilitates the cleavage immensely. Such polybasic motifs are largely absent in SARS-CoV making them only rarely cleaved by non-specific host proteases (Bertram et al., 2011; Yuan et al., 2017) and thus make a big difference in the transmissibility of the two events of viral invasion. The RBD_Spike has a β-barrel as its predominant secondary structure. Upon being cleaved and released free from the rest of the Spike, the RBD_Spike approaches the lung cell receptors and binds to them transiently by forming quasi-stable complexes due to poor electrostatic match at the RBD_Spike-hACE-2 interface. The interaction, however, is characterized by high mutual affinity due to optimal shape complementarity between the molecular partners (Basu et al., 2021). The transient nature of the interaction enables the RBD_Spike-hACE-2 binding to act as molecular switches for the subsequent membrane fusion and viral host cell entry. The standing up (or ‘up’) conformation of the RBD_Spike is preferable for binding with the befitting surface, docked to the solvent-exposed Spike binding site of hACE-2. Comparative cryo-EM studies have revealed that in the case of SARS-CoV (2003), the ‘up’ state is a prevailing state while in its younger analog, SARS-CoV-2, the ‘up’ state is only stimulated near hACE-2 (Cai et al., 2020) which otherwise helps the virus to escape the host immune surveillance (Walls et al., 2020). It is very much prominent that over time the younger homolog developed a piece of machinery facilitating the binding of RBD_Spike to hACE-2 in a more transient way (by modulating and lowering the electrostatic matching at their interface making the binding less stable) in SARS-CoV-2 in comparison with SARS-CoV. This phenomenon helps other adjoining cells to become more vulnerable to viral entries than in the earlier event (SARS-CoV). The other noticeable feature is that the virus secluded from the host’s body shows a unique Furin cleavage site (consisting of the ‘PRRAR’ motif) like never before. Thus, we come to a central question of whether these pivotal differences between SARS-CoV-2 and its ancestor leave footprints of slow natural evolution or an inevitable human intervention somewhere in the pipeline. One of the key differences between the bat coronavirus (Bat RmYN01, Bat RaTG13), responsible for the disease caused in the early 21st century and SARS-CoV-2, is indeed the ‘PRRAR’ motif, the polybasic arginine-rich lucrative bait found on the FLCS_Spike that attracts Furin to cleave effectively at the S1/S2 junction of the FLCS_Spike (Matyášek and Kovařík, 2020). One other key question is how the viruses become so well adapted to a hostile environment of human host cells from the very beginning! Usually, viruses take a long time to get accustomed by gaining mutations one by one until it gets stronger to manifest the full-bloomed infection, though, in the case of SARS-CoV-2, such a lag-phase seems almost redundant. The virus seems to be familiar to the host environment from day-1 and very much well balanced in that environment to spread infections and bring about the demolition of the biochemical machinery of the host cell. It looks as if it is already very familiar with the host cell environment, this point merits attention. Such familiarity probably hints toward pre-exposure of the virus to the human cellular environment and leaves much scope to the ‘lab escape hypothesis’ to discuss and explain the phenomena. As a matter of fact, Chinese virologist Dr. Zheng Lee used to grow her viruses in humanized mice that carry functioning human genes, and tissues. Consequently, an engineered hACE-2 receptor in the streamline of these humanized mice would result in the production of threatening viruses to cause infection in human cells as it got acquired with the host cell before (Fujiwara, 2018). Such engineered hACE-2 receptors work as a switch to check the infectivity of the pathogen by introducing a new cleavage site to enhance human infection. In earlier coronaviruses, HKU1, and OCH3, it was observed that they contain highly basic cleavage sites, RRKRR and RRSRG, respectively. There are many more other proteases instead of Furin to accelerate the virus host cell entry but Furin does this more competently. The other (non-specific) proteases, for example, TMPRSS-2, lysosomal cathepsins, NE, PR3, Cat-G, NSPH etc. secreted by activated neutrophils moving around the viral pathogen also intrudes into the host cells (Shang et al., 2020, 2; Mustafa et al., 2021) while the new virus (SARS-CoV-2) had already gained capabilities to avoid the host immune surveillance by its ‘down-to-earth’ conformation of the RBD_Spike. Furin attacks the polybasic arginine-rich region of the FLCS_Spike, mainly the ‘PRRAR’ segment that protrudes out as a lucrative bait to attract Furin. Extending from comparative genomic and epidemiological studies (Walls et al., 2020; Chan and Zhan, 2021), doubts have been casted on possible human intervention (rather than raw nature) to select for the ‘PRRAR’ segment, arguing that the insertion of the sequence is only to humanize the bat. Such intervention can easily be executed by alluring nucleotides at the gene level or by genetic engineering. The ‘PRRAR’ segment is encoded by a 15 nucleotide long ORF (5′-CTCGGCGGGCACGT-3′) wherein the two consecutive arginine residues have been encoded by CGG triplet codons which happen to be the least frequent codon used to code for arginine. But yet it is recruited to code for two adjacent arginine residues giving rise to ₆₈₁PRRAR₆₈₅ in the SARS-CoV-2 FLCS_Spike elevating its pathogenicity (Balaram, 2021). The arginine residues are therefore satirically referred to as Baltimore’s smoking gun. A new variant found in India, titled B.1.617 further bears a Furin cleavage site that has a single ‘critical’ mutation (P681 → R681) making the motif more basic and disordered (Mlcochova et al., 2021). A single turnover of a letter in the coding triplet is enough for adding this extra disorder in the Spike so that Furin can cleave at the S1/S2 function even more smoothly. People have argued that this perhaps adds a more authentic signature of human intervention in designing the virus (Balaram, 2021). It is however still not clear as to which of the two events is more critical to elevate the viral pathogenicity - the transient binding of RBD_Spike to hACE-2 or the Furin mediated cleavage at the FLCS_Spike.

There also exist alternative perspectives on the origin of SARS-CoV-2 further adding to its complexity. Some extreemists even speculated that the virus might have been made to evolve to be utilized as biological weaponry. Again, some talk in favor of zoonotic spillover models (Plowright et al., 2017; Frutos et al., 2021) which is a multivariate complex interplay of ecological, epidemiological, behavioral and other possible determinants of pathogen exposure, as well as the human internal factors leading to the varying degree of susceptibility of individuals to infection. The latter of the two events must also involve overcoming a hierarchical series of barriers to cause spillover infections in humans (Plowright et al., 2017). Extending on the first of the two hypotheses, the obvious question was whether the virus was deliberately released to cause harm to mankind globally. Some also speculated if the leading corporate multinationals worldwide together wanted to depopulate the world this way simultaneously feeding the pharmaceutical industries for years and decades. Some rumors even fueled speculation that the viruses might have originated in Canada while a troop of Chinese agents smuggled the virus from Canada to China. The Wuhan level 4 biosafety laboratory, well known worldwide for conducting (‘gain of functions’) research on lethal viruses, is the center of all these charges as it is merely 32 km away from the Wuhan seafood market which has been tagged as the epicenter of the coronavirus outbreak. WHO along with the ‘disease control and prevention laboratory’ after a careful inspection reported that it’s not a biological weaponry. Notably, they paid more attention to the seafood and animal market in Wuhan rather than blaming a world-famous pioneer laboratory in virology. However, an allegation could not be undermined about the shipping of some deadly viruses from the Canadian lab to China and these are powerful bioweapons. A piece of news also popped up in the television media that the Chinese agents working in the lab were expelled and later found involved with smuggling the virus again (probably as an act of revenge). But the scientists threw out the claim as a trumped-up story.

Yet, another alternative hypothesis postulates that SARS-CoV-2 is a recombinant between Sarbecovirus from Malayan Pangolins and RaTG13 from R. affinis. Although there are similarities between their RBD_Spikes, there is a dispute regarding their habitation as R. affinis and Pangolins did not share the same habitat. Moreover, further research revealed that no traces of Sarbecoviruses was detected in Chinese Pangolins, Manis pentadactyla, although, the virus was found in the anal swab sample of the R. affinis bat. The Pangolins M. javanica illegally imported from the Indo-Malayan region harbors Sarbecovirus also ruling out for the chances of recombination occurring between the R. affinis bat and Chinese Pangolins.

There is also suspicion that SARS-CoV-2 might have originated from an unintended laboratory accident as the same thing happened in the case of SARS-CoV. But investigation says that there is no clear evidence regarding such an accident as the low mortality rate (in comparison to SARS-CoV) characteristic of asymptomatic patients could have easily buffered such an undue event (Frutos et al., 2021). Also, none of the staff members of Wuhan Institute of Virology tested positive (Fact Sheet: Activity at the Wuhan Institute of Virology). Moreover, it’s not clear whether the template for possible engineering (the RaTG13 virus) exists in reality or merely a predicted sequence, in-silico, which is unable to make a lab escape.

A covid vaccine is a vaccine that is manufactured to build up a protective shield around us and make us safe from getting affected by SARS-CoV-2. The vaccines are attributed worldwide for their role in diminishing the strength and lethality caused by COVID-19. The viral host cell entry can be prevented by blocking the RBD_Spike-hACE-2 interaction either by structurally mimicking the host-receptor (Cao et al., 2020) to entrap the viral Spike well before reaching the lung cells or by pre-occupying the receptor with the abundant use of non-reactive bio-friendly structural mimics of RBD_Spike (Basu, 2021). The RBD_Spike is very much sturdy towards acquiring mutations on its own, making it less difficult to engineer its suitable non-virulent mimics with a better electrostatic match at the RBD_Spike-hACE-2 interface, offering high-affinity binding. These mimics, being much smaller compared to the full-length Spikes carrying the viruses will hence approach faster than the native RBD_Spike towards the hACE-2 receptor, thereby, effectively inhibiting the native Spike protein binding to the host cells (Basu et al., 2021). Apart from that, David Baker and co-workers have designed S mini peptides (Cao et al., 2020) mimicking the RBD_Spike-binding ‘ACE-2 peptidase domain α1 helix’ in hACE-2 which can effectively entrap the viral Spike (via binding to RBD_Spike) much before the virus can reach out to the host (lung) cells. Also, the PRRAR motif of the FLCS_Spike uniquely present in SARS-CoV-2 and yet absent in its ancestors helps priming the virus by a host-protease mediated proteolytic cleavage that sets free the RBD_Spike – which stands out to be a major contributory factor in the enhancement of viral transmissibility in COVID-19. For these reasons, it is natural to target the RBD_Spike-hACE-2 interaction (and eventually block it) as a therapeutic measure to prevent the viral host cell entry. In parallel, virus replication and spreading are inhibited by targeting RdRP (RNA dependent RNA-polymerase) (Gong, 2021) wherein replacing one of the RNA-nucleotides with a suitable nucleotide analog that can easily fit in the same enzyme-substrate binding site, can effectively terminate the replication of the viral RNA. As alternatives to the mainstream approaches, parallel therapeutic directions have also been explored using nanobodies (Huo et al., 2020, 2), and influenza virus-like particle (VLP) vaccines (Chu et al., 2021) etc. Plenty of natural products (Antonio et al., 2020; Huang et al., 2020; Omrani et al., 2021) have also been surveyed and have shown much promise to be aided as plausible ‘herbal medicines’ for the future. Keeping in mind the multi-wave dynamics of the COVID-19 pandemic with the growing number of emerging variants, a section of the scientific community is aiming to procure apt interpretable supervised learning / artificial intelligence (AI) – based predictors which would need to be trained continually with more and more available data to predict plausible future mutations of the virus. Though, these methods are currently only in their early days of development. Another section of the community is devoting committed efforts using immuno-informatics to design and develop possible multi-epitope combination vaccines (Naz et al., 2020) which are aimed to cover a plethora of viral antigens (epitopes) and their plausible future mutations (much like the decades-long challenge in developing anti-influenza and/or anti-HIV vaccines (Enuh et al., 2018)).