Understanding the origin of COVID-19 requires to change the paradigm on zoonotic emergence from the spillover to the circulation model.

While the COVID-19 pandemic continues to spread with currently more than 117 million cumulated cases and 2.6 million deaths worldwide as per March 2021, its origin is still debated. Although several hypotheses have been proposed, there is still no clear explanation about how its causative agent, SARS-CoV-2, emerged in human populations. Today, scientifically-valid facts that deserve to be debated still coexist with unverified statements blurring thus the knowledge on the origin of COVID-19. Our retrospective analysis of scientific data supports the hypothesis that SARS-CoV-2 is indeed a naturally occurring virus. However, the spillover model considered today as the main explanation to zoonotic emergence does not match the virus dynamics and somehow misguided the way researches were conducted. We conclude this review by proposing a change of paradigm and model and introduce the circulation model for explaining the various aspects of the dynamic of SARS-CoV-2 emergence in humans.

Pre-existing T cell-mediated cross-reactivity to SARS-CoV-2 cannot solely be explained by prior exposure to endemic human coronaviruses.

T-cell-mediated immunity to SARS-CoV-2-derived peptides in individuals unexposed to SARS-CoV-2 has been previously reported. This pre-existing immunity was suggested to largely derive from prior exposure to 'common cold' endemic human coronaviruses (HCoVs). To test this, we characterised the sequence homology of SARS-CoV-2-derived T-cell epitopes reported in the literature across the full proteome of the Coronaviridae family. 54.8% of these epitopes had no homology to any of the HCoVs. Further, the proportion of SARS-CoV-2-derived epitopes with any level of sequence homology to the proteins encoded by any of the coronaviruses tested is well-predicted by their alignment-free phylogenetic distance to SARS-CoV-2 (Pearson's r = -0.958). No coronavirus in our dataset showed a significant excess of T-cell epitope homology relative to the proportion of expected random matches, given their genetic similarity to SARS-CoV-2. Our findings suggest that prior exposure to human or animal-associated coronaviruses cannot completely explain the T-cell repertoire in unexposed individuals that recognise SARS-CoV-2 cross-reactive epitopes.

Evolutionary insights into the furin cleavage sites of SARS-CoV-2 variants from humans and animals.

The SARS-CoV-2 spike protein Q677P/H mutation and furin cleavage site (FCS) have been shown to affect cell tropism and virus transmissibility. Here, we analyzed the frequency of Q677P/H and FCS point mutations in 1,144,793 human and 1042 animal spike protein sequences and from those of the emergent variants B.1.1.7, B.1.351, P.1, B.1.429 + B.1.427, and B.1.525, which were deposited in the database of the GISAID Initiative. Different genetic polymorphisms, particularly P681H and A688V, were detected in the FCS, mainly in human isolates, and otherwise, only pangolin and bat sequences had these mutations. Multiple FCS amino acid deletions such as Δ680SPRRA684 and Δ685RSVA688 were only detected in eight and four human isolates, respectively. Surprisingly, deletion of the entire FCS motif as Δ680SPRRARSVA688 and Δ680SPRRARSVAS689 was detected only in three human isolates. On the other hand, analysis of FCS from emergent variants showed no deletions in the FCS except for spike P681del, which was detected in seven B.1.1.7 isolates from the USA. Spike Q677P was detected only once in variant, B.1.1.7, whereas Q677H was detected in all variants, i.e., B.1.1.7 (n = 1938), B.1.351 (n = 28), P.1 (n = 9), B.1.429 + B.1.427 (n = 132), and B.1.525 (n = 1584). Structural modeling predicted that mutations or deletions at or near the FCS significantly alter the cleavage loop structure and would presumably affect furin binding. Taken together, our results show that Q677H and FCS point mutations are prevalent and may have various biological effects on the circulating variants. Therefore, we recommend urgent monitoring and surveillance of the investigated mutations, as well as laboratory assessment of their pathogenicity and transmissibility.

Evaluating angiotensin-converting enzyme 2-mediated SARS-CoV-2 entry across species.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic represents a global threat, and the interaction between the virus and angiotensin-converting enzyme 2 (ACE2), the primary entry receptor for SARS-CoV-2, is a key determinant of the range of hosts that can be infected by the virus. However, the mechanisms underpinning ACE2-mediated viral entry across species remains unclear. Using infection assay, we evaluated SARS-CoV-2 entry mediated by ACE2 of 11 different animal species. We discovered that ACE2 of Rhinolophus sinicus (Chinese rufous horseshoe bat), Felis catus (domestic cat), Canis lupus familiaris (dog), Sus scrofa (wild pig), Capra hircus (goat), and Manis javanica (Malayan pangolin) facilitated SARS-CoV-2 entry into nonsusceptible cells. Moreover, ACE2 of the pangolin also mediated SARS-CoV-2 entry, adding credence to the hypothesis that SARS-CoV-2 may have originated from pangolins. However, the ACE2 proteins of Rhinolophus ferrumequinum (greater horseshoe bat), Gallus gallus (red junglefowl), Notechis scutatus (mainland tiger snake), or Mus musculus (house mouse) did not facilitate SARS-CoV-2 entry. In addition, a natural isoform of the ACE2 protein of Macaca mulatta (rhesus monkey) with the Y217N mutation was resistant to SARS-CoV-2 infection, highlighting the possible impact of this ACE2 mutation on SARS-CoV-2 studies in rhesus monkeys. We further demonstrated that the Y217 residue of ACE2 is a critical determinant for the ability of ACE2 to mediate SARS-CoV-2 entry. Overall, these results clarify that SARS-CoV-2 can use the ACE2 receptors of multiple animal species and show that tracking the natural reservoirs and intermediate hosts of SARS-CoV-2 is complex.

Asymptomatic SARS-CoV-2 infection: is it all about being refractile to innate immune sensing of viral spare-parts?-Clues from exotic animal reservoirs.

A vast proportion of coronavirus disease 2019 (COVID-19) individuals remain asymptomatic and can shed severe acute respiratory syndrome (SARS-CoV) type 2 virus to transmit the infection, which also explains the exponential increase in the number of COVID-19 cases globally. Furthermore, the rate of recovery from clinical COVID-19 in certain pockets of the globe is surprisingly high. Based on published reports and available literature, here, we speculated a few immunovirological mechanisms as to why a vast majority of individuals remain asymptomatic similar to exotic animal (bats and pangolins) reservoirs that remain refractile to disease development despite carrying a huge load of diverse insidious viral species, and whether such evolutionary advantage would unveil therapeutic strategies against COVID-19 infection in humans. Understanding the unique mechanisms that exotic animal species employ to achieve viral control, as well as inflammatory regulation, appears to hold key clues to the development of therapeutic versatility against COVID-19.

The genetic structure of SARS-CoV-2 does not rule out a laboratory origin: SARS-COV-2 chimeric structure and furin cleavage site might be the result of genetic manipulation.

Severe acute respiratory syndrome-coronavirus (SARS-CoV)-2's origin is still controversial. Genomic analyses show SARS-CoV-2 likely to be chimeric, most of its sequence closest to bat CoV RaTG13, whereas its receptor binding domain (RBD) is almost identical to that of a pangolin CoV. Chimeric viruses can arise via natural recombination or human intervention. The furin cleavage site in the spike protein of SARS-CoV-2 confers to the virus the ability to cross species and tissue barriers, but was previously unseen in other SARS-like CoVs. Might genetic manipulations have been performed in order to evaluate pangolins as possible intermediate hosts for bat-derived CoVs that were originally unable to bind to human receptors? Both cleavage site and specific RBD could result from site-directed mutagenesis, a procedure that does not leave a trace. Considering the devastating impact of SARS-CoV-2 and importance of preventing future pandemics, researchers have a responsibility to carry out a thorough analysis of all possible SARS-CoV-2 origins.

Dynamically evolving novel overlapping gene as a factor in the SARS-CoV-2 pandemic.

Understanding the emergence of novel viruses requires an accurate and comprehensive annotation of their genomes. Overlapping genes (OLGs) are common in viruses and have been associated with pandemics but are still widely overlooked. We identify and characterize ORF3d, a novel OLG in SARS-CoV-2 that is also present in Guangxi pangolin-CoVs but not other closely related pangolin-CoVs or bat-CoVs. We then document evidence of ORF3d translation, characterize its protein sequence, and conduct an evolutionary analysis at three levels: between taxa (21 members of Severe acute respiratory syndrome-related coronavirus), between human hosts (3978 SARS-CoV-2 consensus sequences), and within human hosts (401 deeply sequenced SARS-CoV-2 samples). ORF3d has been independently identified and shown to elicit a strong antibody response in COVID-19 patients. However, it has been misclassified as the unrelated gene ORF3b, leading to confusion. Our results liken ORF3d to other accessory genes in emerging viruses and highlight the importance of OLGs.

COVID19: an announced pandemic.

A severe upper respiratory tract syndrome caused by the new coronavirus has now spread to the entire world as a highly contagious pandemic. The large scale explosion of the disease is conventionally traced back to January of this year in the Chinese province of Hubei, the wet markets of the principal city of Wuhan being assumed to have been the specific causative locus of the sudden explosion of the infection. A number of findings that are now coming to light show that this interpretation of the origin and history of the pandemic is overly simplified. A number of variants of the coronavirus would in principle have had the ability to initiate the pandemic well before January of this year. However, even if the COVID-19 had become, so to say, ready, conditions in the local environment would have had to prevail to induce the loss of the biodiversity's "dilution effect" that kept the virus under control, favoring its spillover from its bat reservoir to the human target. In the absence of these appropriate conditions only abortive attempts to initiate the pandemic could possibly occur: a number of them did indeed occur in China, and probably elsewhere as well. These conditions were unfortunately present at the wet marked in Wuhan at the end of last year.

Molecular Basis of Pathogenesis of Coronaviruses: A Comparative Genomics Approach to Planetary Health to Prevent Zoonotic Outbreaks in the 21st Century.

In the first quarter of the 21st century, we are already facing the third emergence of a coronavirus outbreak, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) responsible for the coronavirus disease 2019 (COVID-19) pandemic. Comparative genomics can inform a deeper understanding of the pathogenesis of COVID-19. Previous strains of coronavirus, SARS-CoV, and Middle-East respiratory syndrome-coronavirus (MERS-CoV), have been known to cause acute lung injuries in humans. SARS-CoV-2 shares genetic similarity with SARS-CoV with some modification in the S protein leading to their enhanced binding affinity toward the angiotensin-converting enzyme 2 (ACE2) receptors of human lung cells. This expert review examines the features of all three coronaviruses through a conceptual lens of comparative genomics. In particular, the life cycle of SARS-CoV-2 that enables its survival within the host is highlighted. Susceptibility of humans to coronavirus outbreaks in the 21st century calls for comparisons of the transmission history, hosts, reservoirs, and fatality rates of these viruses so that evidence-based and effective planetary health interventions can be devised to prevent future zoonotic outbreaks. Comparative genomics offers new insights on putative and novel viral targets with an eye to both therapeutic innovation and prevention. We conclude the expert review by (1) articulating the lessons learned so far, whereas the research is still being actively sought after in the field, and (2) the challenges and prospects in deciphering the linkages among multiomics biological variability and COVID-19 pathogenesis.

Natural selection versus creation: a review on the origin of SARS-COV-2.

SARS-CoV-2 has created a global disaster by infecting millions of people and causing thousands of deaths across hundreds of countries. Currently, the infection is in its exponential phase in several countries and there is no sign of immediate relief from this deadly virus. At the same time, some "conspiracy theories" have arisen on the origin of this virus due to the lack of a "definite origin". To understand if this controversy is also reflected in scientific publications, here, we reviewed the key articles published at initial stages of the COVID-19 pandemic (January 01, 2020 to April 30, 2020) related to the zoonotic origin of SARS-CoV-2 and the articles opposing the "conspiracy theories". We also provide an overview on the current knowledge on SARS-CoV-2 Spike as well as the Coronavirus research domain. Furthermore, a few important points related to the "conspiracy theories" such as "laboratory engineering" or "bioweapon" aspects of SARS-CoV-2 are also reviewed. In this article, we have only considered the peer-reviewed publications that are indexed in PubMed and other official publications, and we have directly quoted the authors' statements from their respective articles to avoid any controversy.

Genomic evolution of severe acute respiratory syndrome Coronavirus 2 in India and vaccine impact.

Recent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and subsequent containment procedures have impacted the world as never seen before. Therefore, there is considerable curiosity about the genome evolution related to the origin, transmission and vaccine impact of this virus. We have analysed genome sequences of SARS-CoV-2 isolated from Indian patients to gain an in-depth understanding of genomic evolution and transmission in India. Phylogenetic analysis and mutation profiling revealed major lineages being evolved by characteristic mutations. As the mutation frequency in spike protein is comparatively lesser, the candidate vaccines expected to have wide coverage worldwide including India.

SARS-CoV-2 ORF3b Is a Potent Interferon Antagonist Whose Activity Is Increased by a Naturally Occurring Elongation Variant.

One of the features distinguishing SARS-CoV-2 from its more pathogenic counterpart SARS-CoV is the presence of premature stop codons in its ORF3b gene. Here, we show that SARS-CoV-2 ORF3b is a potent interferon antagonist, suppressing the induction of type I interferon more efficiently than its SARS-CoV ortholog. Phylogenetic analyses and functional assays reveal that SARS-CoV-2-related viruses from bats and pangolins also encode truncated ORF3b gene products with strong anti-interferon activity. Furthermore, analyses of approximately 17,000 SARS-CoV-2 sequences identify a natural variant in which a longer ORF3b reading frame was reconstituted. This variant was isolated from two patients with severe disease and further increased the ability of ORF3b to suppress interferon induction. Thus, our findings not only help to explain the poor interferon response in COVID-19 patients but also describe the emergence of natural SARS-CoV-2 quasispecies with an extended ORF3b gene that may potentially affect COVID-19 pathogenesis.

The receptor binding domain of SARS-CoV-2 spike protein is the result of an ancestral recombination between the bat-CoV RaTG13 and the pangolin-CoV MP789.

OBJECTIVE: In December 2019 a novel coronavirus (SARS-CoV-2) that is causing the current COVID-19 pandemic was identified in Wuhan, China. Many questions have been raised about its origin and adaptation to humans. In the present work we performed a genetic analysis of the Spike glycoprotein (S) of SARS-CoV-2 and other related coronaviruses (CoVs) isolated from different hosts in order to trace the evolutionary history of this protein and the adaptation of SARS-CoV-2 to humans. RESULTS: Based on the sequence analysis of the S gene, we suggest that the origin of SARS-CoV-2 is the result of recombination events between bat and pangolin CoVs. The hybrid SARS-CoV-2 ancestor jumped to humans and has been maintained by natural selection. Although the S protein of RaTG13 bat CoV has a high nucleotide identity with the S protein of SARS-CoV-2, the phylogenetic tree and the haplotype network suggest a non-direct parental relationship between these CoVs. Moreover, it is likely that the basic function of the receptor-binding domain (RBD) of S protein was acquired by the SARS-CoV-2 from the MP789 pangolin CoV by recombination and it has been highly conserved.

Susceptibility to SARS, MERS, and COVID-19 from animal health perspective.

Viruses are having great time as they seem to have bogged humans down. Severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and novel coronavirus (COVID-19) are the three major coronaviruses of present-day global human and animal health concern. COVID-19 caused by SARS-CoV-2 is identified as the newest disease, presumably of bat origin. Different theories on the evolution of viruses are in circulation, yet there is no denying the fact that the animal source is the skeleton. The whole world is witnessing the terror of the COVID-19 pandemic that is following the same path of SARS and MERS, and seems to be more severe. In addition to humans, several species of animals are reported to have been infected with these life-threatening viruses. The possible routes of transmission and their zoonotic potentialities are the subjects of intense research. This review article aims to overview the link of all these three deadly coronaviruses among animals along with their phylogenic evolution and cross-species transmission. This is essential since animals as pets or food are said to pose some risk, and their better understanding is a must in order to prepare a possible plan for future havoc in both human and animal health. Although COVID-19 is causing a human health hazard globally, its reporting in animals are limited compared to SARS and MERS. Non-human primates and carnivores are most susceptible to SARS-coronavirus and SARS-CoV-2, respectively, whereas the dromedary camel is susceptible to MERS-coronavirus. Phylogenetically, the trio viruses are reported to have originated from bats and have special capacity to undergo mutation and genomic recombination in order to infect humans through its reservoir or replication host. However, it is difficult to analyze how the genomic pattern of coronaviruses occurs. Thus, increased possibility of new virus-variants infecting humans and animals in the upcoming days seems to be the biggest challenge for the future of the world. One health approach is portrayed as our best way ahead, and understanding the animal dimension will go a long way in formulating such preparedness plans.

Shell Disorder Analysis Suggests That Pangolins Offered a Window for a Silent Spread of an Attenuated SARS-CoV-2 Precursor among Humans.

A model to predict the relative levels of respiratory and fecal-oral transmission potentials of coronaviruses (CoVs) by measuring the percentage of protein intrinsic disorder (PID) of the M (Membrane) and N (Nucleoprotein) proteins in their outer and inner shells, respectively, was built before the MERS-CoV outbreak. With MPID = 8.6% and NPID = 50.2%, the 2003 SARS-CoV falls into group B, which consists of CoVs with intermediate levels of both fecal-oral and respiratory transmission potentials. Further validation of the model came with MERS-CoV (MPID = 9%, NPID = 44%) and SARS-CoV-2 (MPID = 5.5%, NPID = 48%) falling into the groups C and B, respectively. Group C contains CoVs with higher fecal-oral but lower respiratory transmission potentials. Unlike SARS-CoV, SARS-CoV-2 with MPID = 5.5% has one of the hardest outer shells among CoVs. Because the hard shell is able to resist the antimicrobial enzymes in body fluids, the infected person is able to shed large quantities of viral particles via saliva and mucus, which could account for the higher contagiousness of SARS-COV-2. Further searches have found that high rigidity of the outer shell is characteristic for the CoVs of burrowing animals, such as rabbits (MPID = 5.6%) and pangolins (MPID = 5-6%), which are in contact with the buried feces. A closer inspection of pangolin-CoVs from 2017 to 2019 reveals that pangolins provided a unique window of opportunity for the entry of an attenuated SARS-CoV-2 precursor into the human population in 2017 or earlier, with the subsequent slow and silent spread as a mild cold that followed by its mutations into the current more virulent form. Evidence of this lies in both the genetic proximity of the pangolin-CoVs to SARS-CoV-2 (∼90%) and differences in N disorder. A 2017 pangolin-CoV strain shows evidence of higher levels of attenuation and higher fecal-oral transmission associated with lower human infectivity via having lower NPID (44.8%). Our shell disorder model predicts this to be a SARS-CoV-2 vaccine strain, as lower inner shell disorder is associated with the lesser virulence in a variety of viruses.

COVID-19: Time to exonerate the pangolin from the transmission of SARS-CoV-2 to humans.

The emergence of COVID-19 has triggered many works aiming at identifying the animal intermediate potentially involved in the transmission of SARS-CoV-2 to humans. The presence of SARS-CoV-2-related viruses in Malayan pangolins, in silico analysis of the ACE2 receptor polymorphism and sequence similarities between the Receptor Binding Domain (RBD) of the spike proteins of pangolin and human Sarbecoviruses led to the proposal of pangolin as intermediary. However, the binding affinity of the pangolin ACE2 receptor for SARS-CoV-2 RBD was later on reported to be low. Here, we provide evidence that the pangolin is not the intermediate animal at the origin of the human pandemic. Moreover, data available do not fit with the spillover model currently proposed for zoonotic emergence which is thus unlikely to account for this outbreak. We propose a different model to explain how SARS-CoV-2 related coronaviruses could have circulated in different species, including humans, before the emergence of COVID-19.

Protein Structure and Sequence Reanalysis of 2019-nCoV Genome Refutes Snakes as Its Intermediate Host and the Unique Similarity between Its Spike Protein Insertions and HIV-1.

As the infection of 2019-nCoV coronavirus is quickly developing into a global pneumonia epidemic, the careful analysis of its transmission and cellular mechanisms is sorely needed. In this Communication, we first analyzed two recent studies that concluded that snakes are the intermediate hosts of 2019-nCoV and that the 2019-nCoV spike protein insertions share a unique similarity to HIV-1. However, the reimplementation of the analyses, built on larger scale data sets using state-of-the-art bioinformatics methods and databases, presents clear evidence that rebuts these conclusions. Next, using metagenomic samples from Manis javanica, we assembled a draft genome of the 2019-nCoV-like coronavirus, which shows 73% coverage and 91% sequence identity to the 2019-nCoV genome. In particular, the alignments of the spike surface glycoprotein receptor binding domain revealed four times more variations in the bat coronavirus RaTG13 than in the Manis coronavirus compared with 2019-nCoV, suggesting the pangolin as a missing link in the transmission of 2019-nCoV from bats to human.

Viral CpG Deficiency Provides No Evidence That Dogs Were Intermediate Hosts for SARS-CoV-2.

Due to the scope and impact of the COVID-19 pandemic there exists a strong desire to understand where the SARS-CoV-2 virus came from and how it jumped species boundaries to humans. Molecular evolutionary analyses can trace viral origins by establishing relatedness and divergence times of viruses and identifying past selective pressures. However, we must uphold rigorous standards of inference and interpretation on this topic because of the ramifications of being wrong. Here, we dispute the conclusions of Xia (2020. Extreme genomic CpG deficiency in SARS-CoV-2 and evasion of host antiviral defense. Mol Biol Evol. doi:10.1093/molbev/masa095) that dogs are a likely intermediate host of a SARS-CoV-2 ancestor. We highlight major flaws in Xia's inference process and his analysis of CpG deficiencies, and conclude that there is no direct evidence for the role of dogs as intermediate hosts. Bats and pangolins currently have the greatest support as ancestral hosts of SARS-CoV-2, with the strong caveat that sampling of wildlife species for coronaviruses has been limited.

Where Did SARS-CoV-2 Come From?

Identifying the origin of SARS-CoV-2, the etiological agent of the current COVID-19 pandemic, may help us to avoid future epidemics of coronavirus and other zoonoses. Several theories about the zoonotic origin of SARS-CoV-2 have recently been proposed. Although Betacoronavirus found in Rhinolophus bats from China have been broadly implicated, their genetic dissimilarity to SARS-CoV-2 is so high that they are highly unlikely to be its direct ancestors. Thus, an intermediary host is suspected to link bat to human coronaviruses. Based on genomic CpG dinucleotide patterns in different coronaviruses from different hosts, it was suggested that SARS-CoV-2 might have evolved in a canid gastrointestinal tract prior to transmission to humans. However, similar CpG patterns are now reported in coronaviruses from other hosts, including bats themselves and pangolins. Therefore, reduced genomic CpG alone is not a highly predictive biomarker, suggesting a need for additional biomarkers to reveal intermediate hosts or tissues. The hunt for the zoonotic origin of SARS-CoV-2 continues.