The Nucleocapsid Proteins of SARS-CoV-2 and Its Close Relative Bat Coronavirus RaTG13 Are Capable of Inhibiting PKR- and RNase L-Mediated Antiviral Pathways.

Coronaviruses (CoVs), including severe acute respiratory syndrome CoV (SARS-CoV), Middle East respiratory syndrome CoV (MERS-CoV), and SARS-CoV-2, produce double-stranded RNA (dsRNA) that activates antiviral pathways such as PKR and OAS/RNase L. To successfully replicate in hosts, viruses must evade such antiviral pathways. Currently, the mechanism of how SARS-CoV-2 antagonizes dsRNA-activated antiviral pathways is unknown. In this study, we demonstrate that the SARS-CoV-2 nucleocapsid (N) protein, the most abundant viral structural protein, is capable of binding to dsRNA and phosphorylated PKR, inhibiting both the PKR and OAS/RNase L pathways. The N protein of the bat coronavirus (bat-CoV) RaTG13, the closest relative of SARS-CoV-2, has a similar ability to inhibit the human PKR and RNase L antiviral pathways. Via mutagenic analysis, we found that the C-terminal domain (CTD) of the N protein is sufficient for binding dsRNA and inhibiting RNase L activity. Interestingly, while the CTD is also sufficient for binding phosphorylated PKR, the inhibition of PKR antiviral activity requires not only the CTD but also the central linker region (LKR). Thus, our findings demonstrate that the SARS-CoV-2 N protein is capable of antagonizing the two critical antiviral pathways activated by viral dsRNA and that its inhibition of PKR activities requires more than dsRNA binding mediated by the CTD. IMPORTANCE The high transmissibility of SARS-CoV-2 is an important viral factor defining the coronavirus disease 2019 (COVID-19) pandemic. To transmit efficiently, SARS-CoV-2 must be capable of disarming the innate immune response of its host efficiently. Here, we describe that the nucleocapsid protein of SARS-CoV-2 is capable of inhibiting two critical innate antiviral pathways, PKR and OAS/RNase L. Moreover, the counterpart of the closest animal coronavirus relative of SARS-CoV-2, bat-CoV RaTG13, can also inhibit human PKR and OAS/RNase L antiviral activities. Thus, the importance of our discovery for understanding the COVID-19 pandemic is 2-fold. First, the ability of SARS-CoV-2 N to inhibit innate antiviral activity is likely a factor contributing to the transmissibility and pathogenicity of the virus. Second, the bat relative of SARS-CoV-2 has the capacity to inhibit human innate immunity, which thus likely contributed to the establishment of infection in humans. The findings described in this study are valuable for developing novel antivirals and vaccines.

Reviewing findings on the polypeptide sequence of the SARS-CoV-2 S-protein to discuss the origins of the virus.

Several investigations suggested origins of SARS-CoV-2 from the recombination of coronaviruses of various animals, including the bat Rhinolophus affinis and the pangolin Manis javanica, despite the processes describing the adaptation from a reservoir of animals to human are still debated. In this perspective, I will remark two main inconsistencies on the origins of SARS-CoV-2: polypeptide sequence alignment of the S-proteins does not return the expected identity of the receptor-binding motif among most of pangolin-CoVs and SARS-CoV-2; accurate referencing for samplings and sequencing deposition of the ancestral bat coronavirus named RaTG13 was missing since the first reports on the SARS-CoV-2 coronavirus. This contribution aims to stimulate discussion about the origins of SARS-CoV-2 and considers other intermediate hosts as a reservoir for coronavirus.

SPIKE MUTATION T403R ALLOWS BAT CORONAVIRUS RaTG13 to USE HUMAN ACE2

Background: The bat coronavirus RaTG13 shares 96% sequence identity to SARS-CoV-2, the causative agent of the COVID-19 pandemic. However, the RaTG13 Spike (S) protein interacts only weakly with the human SCoV-2 receptor Angiotensin-converting Enzyme 2 (ACE2) and does not mediate efficient infection of human cells. Here, we examined which alterations are required to allow the RaTG13 S protein to use human ACE2 for efficient entry into human cells. Methods: Sequence alignments showed that SARS-CoV-2 almost invariantly encodes a positively charged amino acid at position 403 of its S protein, while RaTG13 has a neutral Threonine (T). REAX based computational modeling suggested that S R403 contributes to binding of human ACE2. Wild-type and T403R mutant RaTG13 S proteins were investigated for their ability to bind ACE2 and to mediate infection of pseudotyped VSV particles in human lung-and intestine-derived cell lines as well as hPSC-derived gut organoids. Replication-competent recombinant SCoV2 S R403T was produced and replication monitored. In addition, we mutated human ACE2 to map the interacting residue of S R403. Finally, sera of vaccinated individuals were analyzed for their neutralizing potential against various WT CoV and RaTG13 S as well as mutant S containing pseudoparticles. Results: Our results show that a single amino acid change of T403R allows the RaTG13 S to utilize human ACE2 for viral entry. Spike T403R enhanced infection of VSV-based RatG13 S pseudotypes in human lung and colon cells as well as gut-derived organoids. Vice versa R403T mutation reduced infectivity of SCoV2 S pseudotypes and recombinant SCoV2 replication. The enhancing effect of T403R in RaTG13 S depends on E37 in ACE2. RaTG13 T403R S-mediated infection was blocked by the fusion inhibitor EK-1 but not by the SCoV-2 antibody Casirivimab. SARS-CoV-2 and the T403R RaTG13 S were equally susceptible to neutralization by sera from individuals vaccinated against COVID-19. Conclusion: A positively charged amino acid at position 403 in the S protein of bat coronaviruses is critical for efficient utilization of human ACE2. Our results help to better assess the zoonotic potential of bat sarbecoviruses and suggest that COVID-19 vaccination will also protect against closely bat relatives of SARS-CoV-2 that may emerge in the future.

Insertion-and-Deletion Mutations between the Genomes of SARS-CoV, SARS-CoV-2, and Bat Coronavirus RaTG13.

The evolutional process of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) development remains inconclusive. This study compared the genome sequences of severe acute respiratory syndrome coronavirus (SARS-CoV), bat coronavirus RaTG13, and SARS-CoV-2. In total, the genomes of SARS-CoV-2 and RaTG13 were 77.9% and 77.7% identical to the genome of SARS-CoV, respectively. A total of 3.6% (1,068 bases) of the SARS-CoV-2 genome was derived from insertion and/or deletion (indel) mutations, and 18.6% (5,548 bases) was from point mutations from the genome of SARS-CoV. At least 35 indel sites were confirmed in the genome of SARS-CoV-2, in which 17 were with ≥10 consecutive bases long. Ten of these relatively long indels were located in the spike (S) gene, five in nonstructural protein 3 (Nsp3) gene of open reading frame (ORF) 1a, and one in ORF8 and noncoding region. Seventeen (48.6%) of the 35 indels were based on insertion-and-deletion mutations with exchanged gene sequences of 7-325 consecutive bases. Almost the complete ORF8 gene was replaced by a single 325 consecutive base-long indel. The distribution of these indels was roughly in accordance with the distribution of the rate of point mutation rate around the indels. The genome sequence of SARS-CoV-2 was 96.0% identical to that of RaTG13. There was no long insertion-and-deletion mutation between the genomes of RaTG13 and SARS-CoV-2. The findings of the uneven distribution of multiple indels and the presence of multiple long insertion-and-deletion mutations with exchanged consecutive base sequences in the viral genome may provide insights into SARS-CoV-2 development. IMPORTANCE The developmental mechanism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains inconclusive. This study compared the base sequence one-by-one between severe acute respiratory syndrome coronavirus (SARS-CoV) or bat coronavirus RaTG13 and SARS-CoV-2. The genomes of SARS-CoV-2 and RaTG13 were 77.9% and 77.7% identical to the genome of SARS-CoV, respectively. Seventeen of the 35 sites with insertion and/or deletion mutations between SARS-CoV-2 and SARS-CoV were based on insertion-and-deletion mutations with the replacement of 7-325 consecutive bases. Most of these long insertion-and-deletion sites were concentrated in the nonstructural protein 3 (Nsp3) gene of open reading frame (ORF) 1a, S1 domain of the spike protein, and ORF8 genes. Such long insertion-and-deletion mutations were not observed between the genomes of RaTG13 and SARS-CoV-2. The presence of multiple long insertion-and-deletion mutations in the genome of SARS-CoV-2 and their uneven distributions may provide further insights into the development of the virus.

Crystal Structures of Bat and Human Coronavirus ORF8 Protein Ig-Like Domain Provide Insights Into the Diversity of Immune Responses.

ORF8 is a viral immunoglobulin-like (Ig-like) domain protein encoded by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA genome. It tends to evolve rapidly and interfere with immune responses. However, the structural characteristics of various coronavirus ORF8 proteins and their subsequent effects on biological functions remain unclear. Herein, we determined the crystal structures of SARS-CoV-2 ORF8 (S84) (one of the epidemic isoforms) and the bat coronavirus RaTG13 ORF8 variant at 1.62 Å and 1.76 Å resolution, respectively. Comparison of these ORF8 proteins demonstrates that the 62-77 residues in Ig-like domain of coronavirus ORF8 adopt different conformations. Combined with mutagenesis assays, the residue Cys20 of ORF8 is responsible for forming the covalent disulfide-linked dimer in crystal packing and in vitro biochemical conditions. Furthermore, immune cell-binding assays indicate that various ORF8 (SARS-CoV-2 ORF8 (L84), ORF8 (S84), and RaTG13 ORF8) proteins have different interaction capabilities with human CD14+ monocytes in human peripheral blood. These results provide new insights into the specific characteristics of various coronavirus ORF8 and suggest that ORF8 variants may influence disease-related immune responses.

DEVELOPING AN ORGANOID MODEL TO UNDERSTAND THE BAT GASTROINTESTINAL EPITHELIAL RESPONSE TO THE SEVER ACUTE RESPIRATORY CORONA VIRUS-2 (SARS-COV-2)

The ongoing COVID-19 pandemic is caused by the severe acute respiratory corona virus-2 (SARS-CoV-2) which as of right now has infected 10% of world’s population and has caused >1.5 million deaths worldwide. In addition to respiratory symptoms, COVID-19 causes nausea, vomiting and diarrhea in more than half of infected subjects. This indicates that SARS-CoV-2 not only infects the respiratory tract, but also the gastrointestinal. Bats are thought to be the original reservoir for SARS-CoV-2, since SARS-CoV-2 is 96% identical to the bat coronavirus RatG13, which was identified in horseshoe bats. However, coronaviruses fail to cause overt disease in the bats, whereas strong cytopathic effects were observed in human respiratory and gastrointestinal epithelial cells upon SARS-CoV-2 infection. The goal of our research is to compare the response of primary intestinal epithelial cells of bats and humans to SARS-CoV-2 infection in order to better understand the cellular mechanism that allow bats to harbor coronaviruses without developing disease symptoms. To study the SARS-Co-V-2 infection in bats, we have, for the first time, established organoids lines from the stomach, proximal and distal small intestine of three adult Jamaican Fruit Bats (Artibeus jamaicensis). Organoids were successfully generated from both fresh and frozen tissue and could be passaged at least 25 times and frozen and thawed with no apparent changes in growth and morphology. Microscopic analysis showed that bat gastric and intestinal organoids were composed of a simple columnar epithelium and secreted variable amounts of mucus. We also observed spontaneous development of gland and crypt structures, indicating appropriate differentiation (Fig. 1). When seeded on transwell inserts, both gastric and intestinal organoid cells consistently developed a transepithelial resistance, demonstrating intact barrier function. Using confocal microscopy, we showed that both gastric and intestinal organoids from bats expressed angiotensin I converting enzyme 2 (ACE2), a key receptor for SARS-CoV-2 entry. Our innovative experimental platform will enable us to study multiple aspects of coronavirus infection including viral evolution and determinants of spillover events in a relevant primary cell model system. Importantly, we will utilize the bat organoid model to identify nonpathogenic cellular pathways that enable tolerance to SARS-CoV-2 in the reservoir hosts for this virus, potentially informing novel treatment strategies in human COVID-19 patients.

Compelling Evidence Suggesting the Codon Usage of SARS-CoV-2 Adapts to Human After the Split From RaTG13.

SARS-CoV-2 needs to efficiently make use of the resources from hosts in order to survive and propagate. Among the multiple layers of regulatory network, mRNA translation is the rate-limiting step in gene expression. Synonymous codon usage usually conforms with tRNA concentration to allow fast decoding during translation. It is acknowledged that SARS-CoV-2 has adapted to the codon usage of human lungs so that the virus could rapidly proliferate in the lung environment. While this notion seems to nicely explain the adaptation of SARS-CoV-2 to lungs, it is unable to tell why other viruses do not have this advantage. In this study, we retrieve the GTEx RNA-seq data for 30 tissues (belonging to over 17 000 individuals). We calculate the RSCU (relative synonymous codon usage) weighted by gene expression in each human sample, and investigate the correlation of RSCU between the human tissues and SARS-CoV-2 or RaTG13 (the closest coronavirus to SARS-CoV-2). Lung has the highest correlation of RSCU to SARS-CoV-2 among all tissues, suggesting that the lung environment is generally suitable for SARS-CoV-2. Interestingly, for most tissues, SARS-CoV-2 has higher correlations with the human samples compared with the RaTG13-human correlation. This difference is most significant for lungs. In conclusion, the codon usage of SARS-CoV-2 has adapted to human lungs to allow fast decoding and translation. This adaptation probably took place after SARS-CoV-2 split from RaTG13 because RaTG13 is less perfectly correlated with human. This finding depicts the trajectory of adaptive evolution from ancestral sequence to SARS-CoV-2, and also well explains why SARS-CoV-2 rather than other viruses could perfectly adapt to human lung environment.

The genetic structure of SARS-CoV-2 is consistent with both natural or laboratory origin: Response to Tyshkovskiy and Panchin (10.1002/bies.202000325).

Tyshkovskiy and Panchin have recently published a commentary on our paper in which they outline several "points of disagreement with the Segreto/Deigin hypothesis." As our paper is titled "The genetic structure of SARS-CoV-2 does not rule out a laboratory origin," points of disagreement should provide evidence that rules out a laboratory origin. However, Tyshkovskiy and Panchin provide no such evidence and instead attempt to criticize our arguments that highlight aspects of SARS-CoV-2 that could be consistent with the lab leak hypothesis. Strikingly, Tyshkovskiy and Panchin's main point of criticism is based on a false premise that we have claimed RaTG13 to be a direct progenitor of SARS-CoV-2, and their other points of criticism are either not valid, based on flawed mathematical analysis, or are unrelated to our hypotheses. Thus, the genetic structure of SARS-CoV-2 remains consistent with both natural or laboratory origin, which means that both the zoonotic and the lab leak hypothesis need to be investigated equally thoroughly.

Binding and molecular basis of the bat coronavirus RaTG13 virus to ACE2 in humans and other species.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been spreading worldwide, causing a global pandemic. Bat-origin RaTG13 is currently the most phylogenetically related virus. Here we obtained the complex structure of the RaTG13 receptor binding domain (RBD) with human ACE2 (hACE2) and evaluated binding of RaTG13 RBD to 24 additional ACE2 orthologs. By substituting residues in the RaTG13 RBD with their counterparts in the SARS-CoV-2 RBD, we found that residue 501, the major position found in variants of concern (VOCs) 501Y.V1/V2/V3, plays a key role in determining the potential host range of RaTG13. We also found that SARS-CoV-2 could induce strong cross-reactive antibodies to RaTG13 and identified a SARS-CoV-2 monoclonal antibody (mAb), CB6, that could cross-neutralize RaTG13 pseudovirus. These results elucidate the receptor binding and host adaption mechanisms of RaTG13 and emphasize the importance of continuous surveillance of coronaviruses (CoVs) carried by animal reservoirs to prevent another spillover of CoVs.

SARS-CoV-2's claimed natural origin is undermined by issues with genome sequences of its relative strains: Coronavirus sequences RaTG13, MP789 and RmYN02 raise multiple questions to be critically addressed by the scientific community.

RaTG13, MP789, and RmYN02 are the strains closest to SARS-CoV-2, and their existence came to light only after the start of the pandemic. Their genomes have been used to support a natural origin of SARS-CoV-2 but after a close examination all of them exhibit several issues. We specifically address the presence in RmYN02 and closely related RacCSxxx strains of a claimed natural PAA/PVA amino acid insertion at the S1/S2 junction of their spike protein at the same position where the PRRA insertion in SARS-CoV-2 has created a polybasic furin cleavage site. We show that RmYN02/RacCSxxx instead of the claimed insertion carry a 6-nucleotide deletion in the region and that the 12-nucleotide insertion in SARS-CoV-2 remains unique among Sarbecoviruses. Also, our analysis of RaTG13 and RmYN02's metagenomic datasets found unexpected reads which could indicate possible contamination. Because of their importance to inferring SARS-CoV-2's origin, we call for a careful reevaluation of RaTG13, MP789 and RmYN02 sequencing records and assembly methods.

An update on the origin of SARS-CoV-2: Despite closest identity, bat (RaTG13) and pangolin derived coronaviruses varied in the critical binding site and O-linked glycan residues.

The initial cases of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) occurred in Wuhan, China, in December 2019 and swept the world by 23 June 2020 with 8 993 659 active cases, 469 587 deaths across 216 countries, areas or territories. This strongly implies global transmission occurred before the lockdown of China. However, the initial source's transmission routes of SARS-CoV-2 remain obscure and controversial. Research data suggest bat (RaTG13) and pangolin carried CoV were the proximal source of SARS-CoV-2. In this study, we used systematic phylogenetic analysis of Coronavirinae subfamily along with wild type human SARS-CoV, MERS-CoV, and SARS-CoV-2 strains. The key residues of the receptor-binding domain (RBD) and O-linked glycan were compared. SARS-CoV-2 strains were clustered with RaTG13 (97.41% identity), Pangolin-CoV (92.22% identity) and Bat-SL-CoV (80.36% identity), forms a new clade-2 in lineage B of beta-CoV. The alignments of RBD contact residues to ACE2 justified? Those SARS-CoV-2 strains sequences were 100% identical by each other, significantly varied in RaTG13 and pangolin-CoV. SARS-CoV-2 has a polybasic cleavage site with an inserted sequence of PRRA compared to RaTG13 and only PRR to pangolin. Only serine (Ser) in pangolin and both threonine (Thr) and serine (Ser) O-linked glycans were seen in RaTG13, suggesting that a detailed study needed in pangolin (Manis javanica) and bat (Rhinolophus affinis) related CoV.

The Rhinolophus affinis bat ACE2 and multiple animal orthologs are functional receptors for bat coronavirus RaTG13 and SARS-CoV-2.

Bat coronavirus (CoV) RaTG13 shares the highest genome sequence identity with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among all known coronaviruses, and also uses human angiotensin converting enzyme 2 (hACE2) for virus entry. Thus, SARS-CoV-2 is thought to have originated from bat. However, whether SARS-CoV-2 emerged from bats directly or through an intermediate host remains elusive. Here, we found that Rhinolophus affinis bat ACE2 (RaACE2) is an entry receptor for both SARS-CoV-2 and RaTG13, although the binding of RaACE2 to the receptor-binding domain (RBD) of SARS-CoV-2 is markedly weaker than that of hACE2. We further evaluated the receptor activities of ACE2s from additional 16 diverse animal species for RaTG13, SARS-CoV, and SARS-CoV-2 in terms of S protein binding, membrane fusion, and pseudovirus entry. We found that the RaTG13 spike (S) protein is significantly less fusogenic than SARS-CoV and SARS-CoV-2, and seven out of sixteen different ACE2s function as entry receptors for all three viruses, indicating that all three viruses might have broad host rages. Of note, RaTG13 S pseudovirions can use mouse, but not pangolin ACE2, for virus entry, whereas SARS-CoV-2 S pseudovirions can use pangolin, but not mouse, ACE2 enter cells efficiently. Mutagenesis analysis revealed that residues 484 and 498 in RaTG13 and SARS-CoV-2 S proteins play critical roles in recognition of mouse and human ACE2s. Finally, two polymorphous Rhinolophous sinicus bat ACE2s showed different susceptibilities to virus entry by RaTG13 and SARS-CoV-2 S pseudovirions, suggesting possible coevolution. Our results offer better understanding of the mechanism of coronavirus entry, host range, and virus-host coevolution.

Conservation analysis of SARS-CoV-2 spike suggests complicated viral adaptation history from bat to human.

BACKGROUND: The current coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome (SARS)-CoV-2, has become the most devastating public health emergency in the 21st century and one of the most influential plagues in history. Studies on the origin of SARS-CoV-2 have generally agreed that the virus probably comes from bat, closely related to a bat CoV named BCoV-RaTG13 taken from horseshoe bat (Rhinolophus affinis), with Malayan pangolin (Manis javanica) being a plausible intermediate host. However, due to the relatively low number of SARS-CoV-2-related strains available in public domain, the evolutionary history remains unclear. METHODOLOGY: Nine hundred ninety-five coronavirus sequences from NCBI Genbank and GISAID were obtained and multiple sequence alignment was carried out to categorize SARS-CoV-2 related groups. Spike sequences were analyzed using similarity analysis and conservation analyses. Mutation analysis was used to identify variations within receptor-binding domain (RBD) in spike for SARS-CoV-2-related strains. RESULTS: We identified a family of SARS-CoV-2-related strains, including the closest relatives, bat CoV RaTG13 and pangolin CoV strains. Sequence similarity analysis and conservation analysis on spike sequence identified that N-terminal domain, RBD and S2 subunit display different degrees of conservation with several coronavirus strains. Mutation analysis on contact sites in SARS-CoV-2 RBD reveals that human-susceptibility probably emerges in pangolin. CONCLUSION AND IMPLICATION: We conclude that the spike sequence of SARS-CoV-2 is the result of multiple recombination events during its transmission from bat to human, and we propose a framework of evolutionary history that resolve the relationship of BCoV-RaTG13 and pangolin coronaviruses with SARS-CoV-2. LAY SUMMARY: This study analyses whole-genome and spike sequences of coronavirus from NCBI using phylogenetic and conservation analyses to reconstruct the evolutionary history of severe acute respiratory syndrome (SARS)-CoV-2 and proposes an evolutionary history of spike in the progenitors of SARS-CoV-2 from bat to human through mammal hosts before they recombine into the current form.

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.

Lethal Pneumonia Cases in Mojiang Miners (2012) and the Mineshaft Could Provide Important Clues to the Origin of SARS-CoV-2.

With the COVID-19 pandemic reaching its worst heights, people are interested in the origin of SARS-CoV-2. This study started with two important questions: first, were there any similar atypical pneumonia outbreaks, even on a smaller level, reported between SARS in 2004 and COVID-19 in 2019/20 in China. Second, examining the beta-coronavirus most closely related to date with SARS-CoV-2 at the genome sequence level, strain RaTG13 (CoV4991), which was sampled from a horseshoe bat in Yunnan province, we asked where exactly did it come from. It was found that RaTG13/CoV4991 was collected from Tongguan mineshaft in Mojiang, Yunnan, China, in 2013. Surprisingly, the same mineshaft was also associated with a severe pneumonia-like illness in miners in 2012 killing three of the six miners. A Master's thesis (in the Chinese language) was found on the cnki.net website which described in detail the severe illness in miners. The thesis concluded that a SARS-like CoV originating from Chinese horseshoe bats (Rhinolophus) was the predicted causative agent. The cases were remotely monitored by a prominent pulmonologist in China. Retrospective analysis of the pneumonia cases shows striking similarities with COVID-19. Bilateral pneumonia, vascular complications like pulmonary thromboembolism, and secondary infections are the main similarities. The treatment regimes were similar to the current treatments for COVID-19. We propose that the Mojiang mineshaft miners' illness could provide important clues to the origin of SARS-CoV-2. These cases should be studied by various academicians, researchers, and medical professionals as many important questions are raised in this context.