Identification of a Spike-Specific CD8+ T-Cell Epitope Following Vaccination Against the Middle East Respiratory Syndrome Coronavirus in Humans.

Licensed vaccines against the Middle East respiratory syndrome coronavirus (MERS-CoV), an emerging pathogen of concern, are lacking. The modified vaccinia virus Ankara vector-based vaccine MVA-MERS-S, expressing the MERS-CoV-spike glycoprotein (MERS-S), is one of 3 candidate vaccines in clinical development and elicits robust humoral and cellular immunity. Here, we identified for the first time a MERS-S-specific CD8+ T-cell epitope in an HLA-A*03:01/HLA-B*35:01-positive vaccinee using a screening assay, intracellular cytokine staining, and in silico epitope prediction. As evidence from MERS-CoV infection suggests a protective role of long-lasting CD8+ T-cell responses, the identification of epitopes will facilitate longitudinal analyses of vaccine-induced T-cell immunity.

Biphasic MERS-CoV Incidence in Nomadic Dromedaries with Putative Transmission to Humans, Kenya, 2022-2023.

Middle East respiratory syndrome coronavirus (MERS-CoV) is endemic in dromedaries in Africa, but camel-to-human transmission is limited. Sustained 12-month sampling of dromedaries in a Kenya abattoir hub showed biphasic MERS-CoV incidence; peak detections occurred in October 2022 and February 2023. Dromedary-exposed abattoir workers (7/48) had serologic signs of previous MERS-CoV exposure.

Structural basis of main proteases of MERS-CoV bound to antineoplastic drug carmofur.

Recurrent epidemics of coronaviruses have posed significant threats to human life and health. The mortality rate of patients infected with the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is 35 %. The main protease (Mpro) plays a crucial role in the MERS-CoV life cycle, and Mpro exhibited a high degree of conservation among different coronaviruses. Therefore inhibition of Mpro has become an effective strategy for the development of broad-spectrum anti-coronaviral drugs. The inhibition of SARS-CoV-2 Mpro by the anti-tumor drug carmofur has been revealed, but structural studies of carmofur in complex with Mpro from other types of coronavirus have not been reported. Hence, we revealed the structure of the MERS-CoV Mpro-carmofur complex, analysed the structural basis for the binding of carmofur to MERS-CoV Mpro in detail, and compared the binding patterns of carmofur to Mpros of two different coronaviruses, MERS-CoV and SARS-CoV-2. Considering the importance of Mpros for coronavirus therapy, structural understanding of Mpro inhibition by carmofur could contribute to the design and development of novel antiviral drugs with safe and broad-spectrum efficacy.

Antibody-dependent enhancement (ADE) of SARS-CoV-2 in patients exposed to MERS-CoV and SARS-CoV-2 antigens.

This study evaluated the potential for antibody-dependent enhancement (ADE) in serum samples from patients exposed to Middle East respiratory syndrome coronavirus (MERS-CoV). Furthermore, we evaluated the effect of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination on ADE in individuals with a MERS infection history. We performed ADE assay in sera from MERS recovered and SARS-CoV-2-vaccinated individuals using BHK cells expressing FcgRIIa, SARS-CoV-2, and MERS-CoV pseudoviruses (PVs). Further, we analyzed the association of ADE to serum IgG levels and neutralization. Out of 16 MERS patients, nine demonstrated ADE against SARS-CoV-2 PV, however, none of the samples demonstrated ADE against MERS-CoV PV. Furthermore, out of the seven patients exposed to SARS-CoV-2 vaccination after MERS-CoV infection, only one patient (acutely infected with MERS-CoV) showed ADE for SARS-CoV-2 PV. Further analysis indicated that IgG1, IgG2, and IgG3 against SARS-CoV-2 S1 and RBD subunits, IgG1 and IgG2 against the MERS-CoV S1 subunit, and serum neutralizing activity were low in ADE-positive samples. In summary, samples from MERS-CoV-infected patients exhibited ADE against SARS-CoV-2 and was significantly associated with low levels of neutralizing antibodies. Subsequent exposure to SARS-CoV-2 vaccination resulted in diminished ADE activity while the PV neutralization assay demonstrated a broadly reactive antibody response in some patient samples.

Recombination analysis on the receptor switching event of MERS-CoV and its close relatives: implications for the emergence of MERS-CoV.

BACKGROUND: PlMERS-CoV is a coronavirus known to cause severe disease in humans, taxonomically classified under the subgenus Merbecovirus. Recent findings showed that the close relatives of MERS-CoV infecting vespertillionid bats (family Vespertillionidae), named NeoCoV and PDF-2180, use their hosts' ACE2 as their entry receptor, unlike the DPP4 receptor usage of MERS-CoV. Previous research suggests that this difference in receptor usage between these related viruses is a result of recombination. However, the precise location of the recombination breakpoints and the details of the recombination event leading to the change of receptor usage remain unclear. METHODS: We used maximum likelihood-based phylogenetics and genetic similarity comparisons to characterise the evolutionary history of all complete Merbecovirus genome sequences. Recombination events were detected by multiple computational methods implemented in the recombination detection program. To verify the influence of recombination, we inferred the phylogenetic relation of the merbecovirus genomes excluding recombinant segments and that of the viruses' receptor binding domains and examined the level of congruency between the phylogenies. Finally, the geographic distribution of the genomes was inspected to identify the possible location where the recombination event occurred. RESULTS: Similarity plot analysis and the recombination-partitioned phylogenetic inference showed that MERS-CoV is highly similar to NeoCoV (and PDF-2180) across its whole genome except for the spike-encoding region. This is confirmed to be due to recombination by confidently detecting a recombination event between the proximal ancestor of MERS-CoV and a currently unsampled merbecovirus clade. Notably, the upstream recombination breakpoint was detected in the N-terminal domain and the downstream breakpoint at the S2 subunit of spike, indicating that the acquired recombined fragment includes the receptor-binding domain. A tanglegram comparison further confirmed that the receptor binding domain-encoding region of MERS-CoV was acquired via recombination. Geographic mapping analysis on sampling sites suggests the possibility that the recombination event occurred in Africa. CONCLUSION: Together, our results suggest that recombination can lead to receptor switching of merbecoviruses during circulation in bats. These results are useful for future epidemiological assessments and surveillance to understand the spillover risk of bat coronaviruses to the human population.

Quantitative assay to analyze neutralization and inhibition of authentic Middle East respiratory syndrome coronavirus.

To date, there is no licensed vaccine for Middle East respiratory syndrome coronavirus (MERS-CoV). Therefore, MERS-CoV is one of the diseases targeted by the Coalition for Epidemic Preparedness Innovations (CEPI) vaccine development programs and has been classified as a priority disease by the World Health Organization (WHO). An important measure of vaccine immunogenicity and antibody functionality is the detection of virus-neutralizing antibodies. We have developed and optimized a microneutralization assay (MNA) using authentic MERS-CoV and standardized automatic counting of virus foci. Compared to our standard virus neutralization assay, the MNA showed improved sensitivity when analyzing 30 human sera with good correlation of results (Spearman's correlation coefficient r = 0.8917, p value < 0.0001). It is important to use standardized materials, such as the WHO international standard (IS) for anti-MERS-CoV immunoglobulin G, to compare the results from clinical trials worldwide. Therefore, in addition to the neutralizing titers (NT50 = 1384, NT80 = 384), we determined the IC50 and IC80 of WHO IS in our MNA to be 0.67 IU/ml and 2.6 IU/ml, respectively. Overall, the established MNA is well suited to reliably quantify vaccine-induced neutralizing antibodies with high sensitivity.

Design of MERS-CoV entry inhibitory short peptides based on helix-stabilizing strategies.

Interaction between Middle East respiratory syndrome coronavirus (MERS-CoV) spike (S) protein heptad repeat-1 domain (HR1) and heptad repeat-2 domain (HR2) is critical for the MERS-CoV fusion process. This interaction is mediated by the α-helical region from HR2 and the hydrophobic groove in a central HR1 trimeric coiled coil. We sought to develop a short peptidomimetic to act as a MERS-CoV fusion inhibitor by reproducing the key recognition features of HR2 helix. This was achieved by the use of helix-stabilizing strategies, including substitution with unnatural helix-favoring amino acids, introduction of ion pair interactions, and conjugation of palmitic acid. The resulting 23-mer lipopeptide, termed AEEA-C16, inhibits MERS-CoV S protein-mediated cell-cell fusion at a low micromolar level comparable to that of the 36-mer HR2 peptide HR2P-M2. Collectively, our studies provide new insights into developing short peptide-based antiviral agents to treat MERS-CoV infection.

Therapeutic nanobodies against SARS-CoV-2 and other pathogenic human coronaviruses.

Nanobodies, single-domain antibodies derived from variable domain of camelid or shark heavy-chain antibodies, have unique properties with small size, strong binding affinity, easy construction in versatile formats, high neutralizing activity, protective efficacy, and manufactural capacity on a large-scale. Nanobodies have been arisen as an effective research tool for development of nanobiotechnologies with a variety of applications. Three highly pathogenic coronaviruses (CoVs), SARS-CoV-2, SARS-CoV, and MERS-CoV, have caused serious outbreaks or a global pandemic, and continue to post a threat to public health worldwide. The viral spike (S) protein and its cognate receptor-binding domain (RBD), which initiate viral entry and play a critical role in virus pathogenesis, are important therapeutic targets. This review describes pathogenic human CoVs, including viral structures and proteins, and S protein-mediated viral entry process. It also summarizes recent advances in development of nanobodies targeting these CoVs, focusing on those targeting the S protein and RBD. Finally, we discuss potential strategies to improve the efficacy of nanobodies against emerging SARS-CoV-2 variants and other CoVs with pandemic potential. It will provide important information for rational design and evaluation of therapeutic agents against emerging and reemerging pathogens.

Utilizing sinapic acid as an inhibitory antiviral agent against MERS-CoV PLpro.

Concerns about the social and economic collapse, high mortality rates, and stress on the healthcare system are developing due to the coronavirus onslaught in the form of various species and their variants. In the recent past, infections brought on by coronaviruses severe acute respiratory syndrome coronaviruses (SARS-CoV and SARS-CoV-2) as well as middle east respiratory syndrome coronavirus (MERS-CoV) have been reported. There is a severe lack of medications to treat various coronavirus types including MERS-CoV which is hazard to public health due to its ability for pandemic spread by human-to-human transmission. Here, we utilized sinapic acid (SA) against papain-like protease (PLpro), a crucial enzyme involved in MERS-CoV replication, because phytomedicine derived from nature has less well-known negative effects. The thermal shift assay (TSA) was used in the current study to determine whether the drug interact with the recombinant MERS-CoV PLpro. Also, inhibition assay was conducted as the hydrolysis of fluorogenic peptide from the Z-RLRGG-AMC-peptide bond in the presence of SA to determine the level of inhibition of the MERS-CoV PLpro. To study the structural binding efficiency Autodock Vina was used to dock SA to the MERS-CoV PLpro and results were analyzed using PyMOL and Maestro Schrödinger programs. Our results show a convincing interaction between SA and the MERS protease, as SA reduced MERS-CoV PLpro in a dose-dependent way IC50 values of 68.58 µM (of SA). The TSA showed SA raised temperature of melting to 54.61 °C near IC50 and at approximately 2X IC50 concentration (111.5 µM) the Tm for SA + MERS-CoV PLpro was 59.72 °C. SA was docked to MERS-CoV PLpro to identify the binding site. SA bound to the blocking loop (BL2) region of MERS-CoV PLpro interacts with F268, E272, V275, and P249 residues of MERS-CoV PLpro. The effectiveness of protease inhibitors against MERS-CoV has been established and SA is already known for broad range biological activity including antiviral properties; it can be a suitable candidate for anti-MERS-CoV treatment.

A Multifaceted Computational Approach to Understanding the MERS-CoV Main Protease and Brown Algae Compounds' Interaction.

Middle East Respiratory Syndrome (MERS) is a viral respiratory disease caused b a special type of coronavirus called MERS-CoV. In the search for effective substances against the MERS-CoV main protease, we looked into compounds from brown algae, known for their medicinal benefits. From a set of 1212 such compounds, our computer-based screening highlighted four-CMNPD27819, CMNPD1843, CMNPD4184, and CMNPD3156. These showed good potential in how they might attach to the MERS-CoV protease, comparable to a known inhibitor. We confirmed these results with multiple computer tests. Studies on the dynamics and steadiness of these compounds with the MERS-CoV protease were performed using molecular dynamics (MD) simulations. Metrics like RMSD and RMSF showed their stability. We also studied how these compounds and the protease interact in detail. An analysis technique, PCA, showed changes in atomic positions over time. Overall, our computer studies suggest brown algae compounds could be valuable in fighting MERS. However, experimental validation is needed to prove their real-world effectiveness.

Development and preliminary validation of a MERS-CoV ELISA for serological testing of camels and alpacas.

This study describes the development and preliminary validation of a new serological assay using MERS-CoV S1 protein in an indirect enzyme-linked immunosorbent assay (ELISA) format. This assay has the advantage of being able to test MERS-CoV serum samples in a PC2 laboratory without the need for a high-level biocontainment laboratory (PC3 or PC4), which requires highly trained and skilled staff and a high level of resources and equipment. Furthermore, this MERS-CoV S1 ELISA enables a larger number of samples to be tested quickly, with results obtained in approximately five hours. The MERS-CoV S1 ELISA demonstrated high analytical specificity, with no cross-reactivity observed in serum of animals infected with other viruses, including different coronaviruses. We tested 166 positive and 40 negative camel serum samples and have estimated the diagnostic sensitivity (DSe) to be 99.4% (95% CI: 96.7 - 100.0%) and diagnostic specificity (DSp) to be 100% (95% CI: 97.2%-100.0%) relative to the assigned serology results (ppNT and VNT) using a S/P ratio cut-off value of >0.58. The findings of this study showed that our MERS-CoV S1 ELISA was more sensitive than the commercial EUROIMMUN ELISA (Se 99.4% vs 84.9%) and comparable to the ppNT assay, and therefore could be used as a diagnostic aid in countries in the Middle East where MERS-CoV is endemic in dromedary camels. The assay reagents and protocol were easily adapted and transferred from an Australian laboratory to a laboratory in the University of Hong Kong. Thus, the results described here show that the MERS-CoV S1 ELISA represents a cheap, rapid, robust, and reliable assay to support surveillance of MERS-CoV in camels in endemic regions.

The Main Protease of Middle East Respiratory Syndrome Coronavirus Induces Cleavage of Mitochondrial Antiviral Signaling Protein to Antagonize the Innate Immune Response.

Mitochondrial antiviral signaling protein (MAVS) is a crucial signaling adaptor in the sensing of positive-sense RNA viruses and the subsequent induction of the innate immune response. Coronaviruses have evolved multiple mechanisms to evade this response, amongst others, through their main protease (Mpro), which is responsible for the proteolytic cleavage of the largest part of the viral replicase polyproteins pp1a and pp1ab. Additionally, it can cleave cellular substrates, such as innate immune signaling factors, to dampen the immune response. Here, we show that MAVS is cleaved in cells infected with Middle East respiratory syndrome coronavirus (MERS-CoV), but not in cells infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This cleavage was independent of cellular negative feedback mechanisms that regulate MAVS activation. Furthermore, MERS-CoV Mpro expression induced MAVS cleavage upon overexpression and suppressed the activation of the interferon-ß (IFN-ß) and nuclear factor-κB (NF-κB) response. We conclude that we have uncovered a novel mechanism by which MERS-CoV downregulates the innate immune response, which is not observed among other highly pathogenic coronaviruses.

Identification of potential antiviral compounds from Egyptian sea stars against MERS-CoV with the in vitro and in silico experiments.

Recently, the world faced many epidemics which were caused by viral respiratory pathogens. Marine creatures including Asteroidea class have been one of the recent research topics due to their diverse and complex secondary metabolites. Some of these constituents exhibit antiviral activities. The present study aimed to extract and identify the potential antiviral compounds from Pentaceraster cumingi, Astropecten polyacanthus and Pentaceraster mammillatus. The results showed that promising activity of the methanolic extract of P. cumingi with 50% inhibitory concentration (IC50) of 3.21 mg/ml against MERS-CoV with a selective index (SI) of 13.975. The biochemical components of the extracts were identified by GC/MS analysis. The Molecular docking study highlighted the virtual mechanism of binding the identified compounds towards three PDB codes of MERS-CoV non-structural protein 10/16. Interestingly, 2-mono Linolein showed promising binding energy of -14.75 Kcal/mol with the second PDB code (5YNI) and -15.22 Kcal/mol with the third PDB code (5YNQ).

Inactivated Split MERS-CoV Antigen Prevents Lethal Middle East Respiratory Syndrome Coronavirus Infections in Mice.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes fatal infections, with about 36% mortality in humans, and is endemic to the Middle East. MERS-CoV uses human dipeptidyl peptidase 4 (hDPP4) as a receptor for infection. Despite continued research efforts, no licensed vaccine is available for protection against this disease in humans. Therefore, this study sought to develop an inactivated fragmented MERS-CoV vaccine grown in Vero cells in an hDPP4-transgenic mouse model. Two-dose immunisation in mice with 15, 20, or 25 µg of spike proteins of inactivated split MERS-CoV antigens induced neutralising antibodies, with titres ranging from NT 80 to 1280. In addition, all immunised mice were completely protected, with no virus detection in tissues, weight loss, or mortality. The immunised splenocytes produced more cytokines that stimulate immune response (IFN-γ and TNF-α) than those that regulate it (IL-4 and IL-10). Taken together, the inactivated fragmented MERS-CoV vaccine is effective for the protection of mice against lethal MERS-CoV. Thus, the inactivated fragmented MERS-CoV vaccine warrants further testing in other hosts.

Developments in treatment for middle east respiratory syndrome coronavirus (MERS-CoV).

INTRODUCTION: An important respiratory pathogen that has led to multiple hospital outbreaks both inside and outside of the Arabian Peninsula is the Middle East Respiratory Syndrome Coronavirus (MERS-CoV). Given the elevated case fatality rate, there exists a pressing requirement for efficacious therapeutic agents. AREAS COVERED: This is an updated review of the developments in MERS treatment approaches. Using databases like PubMed, Embase, Cochrane, Scopus, and Google Scholar, a thorough search was carried out utilizing keywords like 'MERS,' 'MERS-CoV,' and 'Middle East respiratory syndrome' in conjunction with 'treatment' or 'therapy' from Jan 2012 to Feb 2024. EXPERT OPINION: MERS-CoV is a highly pathogenic respiratory infection that emerged in 2012 and continues to pose a significant public health threat. Despite ongoing efforts to control the spread of MERS-CoV, there is currently no specific antiviral treatment available. While many agents have been tested both in vivo and in vitro, none of them have been thoroughly examined in extensive clinical trials. Only case reports, case series, or cohort studies have been made available as clinical studies. However, there is a limited number of randomized-controlled trials. Because cases are irregular and sporadic, conducting a large prospective randomized trials for establishing an efficacious treatment might be difficult.

Characterization and immunogenicity assessment of MERS-CoV pre-fusion spike trimeric oligomers as vaccine immunogen.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a lethal beta-coronavirus that emerged in 2012. The virus is part of the WHO blueprint priority list with a concerning fatality rate of 35%. Scientific efforts are ongoing for the development of vaccines, anti-viral and biotherapeutics, which are majorly directed toward the structural spike protein. However, the ongoing effort is challenging due to conformational instability of the spike protein and the evasion strategy posed by the MERS-CoV. In this study, we have expressed and purified the MERS-CoV pre-fusion spike protein in the Expi293F mammalian expression system. The purified protein was extensively characterized for its biochemical and biophysical properties. Thermal stability analysis showed a melting temperature of 58°C and the protein resisted major structural changes at elevated temperature as revealed by fluorescence spectroscopy and circular dichroism. Immunological assessment of the MERS-CoV spike immunogen in BALB/c mice with AddaVaxTM and Imject alum adjuvants showed elicitation of high titer antibody responses but a more balanced Th1/Th2 response with AddaVaxTM squalene like adjuvant. Together, our results suggest the formation of higher-order trimeric pre-fusion MERS-CoV spike proteins, which were able to induce robust immune responses. The comprehensive characterization of MERS-CoV spike protein warrants a better understanding of MERS spike protein and future vaccine development efforts.

Sulfated Glycans Inhibit the Interaction of MERS-CoV Receptor Binding Domain with Heparin.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic virus with high contagion and mortality rates. Heparan sulfate proteoglycans (HSPGs) are ubiquitously expressed on the surface of mammalian cells. Owing to its high negatively charged property, heparan sulfate (HS) on the surface of host cells is used by many viruses as cofactor to facilitate viral attachment and initiate cellular entry. Therefore, inhibition of the interaction between viruses and HS could be a promising target to inhibit viral infection. In the current study, the interaction between the receptor-binding domain (RBD) of MERS-CoV and heparin was exploited to assess the inhibitory activity of various sulfated glycans such as glycosaminoglycans, marine-sourced glycans (sulfated fucans, fucosylated chondroitin sulfates, fucoidans, and rhamnan sulfate), pentosan polysulfate, and mucopolysaccharide using Surface Plasmon Resonance. We believe this study provides valuable insights for the development of sulfated glycan-based inhibitors as potential antiviral agents.

Development and characterization of three novel mouse monoclonal antibodies targeting spike protein S1 subunit of Middle East respiratory syndrome corona virus.

BACKGROUND: Middle East Respiratory Syndrome Coronavirus is a highly pathogenic virus that poses a significant threat to public health. OBJECTIVE: The purpose of this study is to develop and characterize novel mouse monoclonal antibodies targeting the spike protein S1 subunit of the Middle East Respiratory Syndrome Corona Virus (MERS-CoV). METHODS: In this study, three mouse monoclonal antibodies (mAbs) against MERS-CoV were generated and characterized using hybridoma technology. The mAbs were evaluated for their reactivity and neutralization activity. The mAbs were generated through hybridoma technology by the fusion of myeloma cells and spleen cells from MERS-CoV-S1 immunized mice. The resulting hybridomas were screened for antibody production using enzyme-linked immunosorbent assays (ELISA). RESULTS: ELISA results demonstrated that all three mAbs exhibited strong reactivity against the MERS-CoV S1-antigen. Similarly, dot-ELISA revealed their ability to specifically recognize viral components, indicating their potential for diagnostic applications. Under non-denaturing conditions, Western blot showed the mAbs to have robust reactivity against a specific band at 116 KDa, corresponding to a putative MERS-CoV S1-antigen. However, no reactive bands were observed under denaturing conditions, suggesting that the antibodies recognize conformational epitopes. The neutralization assay showed no in vitro reactivity against MERS-CoV. CONCLUSION: This study successfully generated three mouse monoclonal antibodies against MERS-CoV using hybridoma technology. The antibodies exhibited strong reactivity against MERS-CoV antigens using ELISA and dot ELISA assays. Taken together, these findings highlight the significance of these mAbs for potential use as valuable tools for MERS-CoV research and diagnosis (community and field-based surveillance and viral antigen detection).

The Initial COVID-19 Reliable Interactive DNA Methylation Markers and Biological Implications.

Earlier research has established the existence of reliable interactive genomic biomarkers. However, reliable DNA methylation biomarkers, not to mention interactivity, have yet to be identified at the epigenetic level. This study, drawing from 865,859 methylation sites, discovered two miniature sets of Infinium MethylationEPIC sites, each having eight CpG sites (genes) to interact with each other and disease subtypes. They led to the nearly perfect (96.87-100% accuracy) prediction of COVID-19 patients from patients with other diseases or healthy controls. These CpG sites can jointly explain some post-COVID-19-related conditions. These CpG sites and the optimally performing genomic biomarkers reported in the literature become potential druggable targets. Among these CpG sites, cg16785077 (gene MX1), cg25932713 (gene PARP9), and cg22930808 (gene PARP9) at DNA methylation levels indicate that the initial SARS-CoV-2 virus may be better treated as a transcribed viral DNA into RNA virus, i.e., not as an RNA virus that has concerned scientists in the field. Such a discovery can significantly change the scientific thinking and knowledge of viruses.

Genomic Diversity and Recombination Analysis of the Spike Protein Gene from Selected Human Coronaviruses.

Human coronaviruses (HCoVs) are seriously associated with respiratory diseases in humans and animals. The first human pathogenic SARS-CoV emerged in 2002-2003. The second was MERS-CoV, reported from Jeddah, the Kingdom of Saudi Arabia, in 2012, and the third one was SARS-CoV-2, identified from Wuhan City, China, in late December 2019. The HCoV-Spike (S) gene has the highest mutation/insertion/deletion rate and has been the most utilized target for vaccine/antiviral development. In this manuscript, we discuss the genetic diversity, phylogenetic relationships, and recombination patterns of selected HCoVs with emphasis on the S protein gene of MERS-CoV and SARS-CoV-2 to elucidate the possible emergence of new variants/strains of coronavirus in the near future. The findings showed that MERS-CoV and SARS-CoV-2 have significant sequence identity with the selected HCoVs. The phylogenetic tree analysis formed a separate cluster for each HCoV. The recombination pattern analysis showed that the HCoV-NL63-Japan was a probable recombinant. The HCoV-NL63-USA was identified as a major parent while the HCoV-NL63-Netherland was identified as a minor parent. The recombination breakpoints start in the viral genome at the 142 nucleotide position and end at the 1082 nucleotide position with a 99% CI and Bonferroni-corrected p-value of 0.05. The findings of this study provide insightful information about HCoV-S gene diversity, recombination, and evolutionary patterns. Based on these data, it can be concluded that the possible emergence of new strains/variants of HCoV is imminent.