Recombinant Genomes Research Articles

Genomic distinctiveness and recombination in tomato leaf curl New Delhi virus (ToLCNDV-BG) isolates infecting bitter gourd.

The online version contains supplementary material available at 10.1007/s13205-024-04009-3.

Resolving the 22q11.2 deletion using CTLR-Seq reveals chromosomal rearrangement mechanisms and individual variance in breakpoints

We developed a generally applicable method, CRISPR/Cas9-targeted long-read sequencing (CTLR-Seq), to resolve, haplotype-specifically, the large and complex regions in the human genome that had been previously impenetrable to sequencing analysis, such as large segmental duplications (SegDups) and their associated genome rearrangements. CTLR-Seq combines in vitro Cas9-mediated cutting of the genome and pulse-field gel electrophoresis to isolate intact large (i.e., up to 2,000 kb) genomic regions that encompass previously unresolvable genomic sequences. These targets are then sequenced (amplification-free) at high on-target coverage using long-read sequencing, allowing for their complete sequence assembly. We applied CTLR-Seq to the SegDup-mediated rearrangements that constitute the boundaries of, and give rise to, the 22q11.2 Deletion Syndrome (22q11DS), the most common human microdeletion disorder. We then performed de novo assembly to resolve, at base-pair resolution, the full sequence rearrangements and exact chromosomal breakpoints of 22q11.2DS (including all common subtypes). Across multiple patients, we found a high degree of variability for both the rearranged SegDup sequences and the exact chromosomal breakpoint locations, which coincide with various transposons within the 22q11.2 SegDups, suggesting that 22q11DS can be driven by transposon-mediated genome recombination. Guided by CTLR-Seq results from two 22q11DS patients, we performed three-dimensional chromosomal folding analysis for the 22q11.2 SegDups from patient-derived neurons and astrocytes and found chromosome interactions anchored within the SegDups to be both cell type-specific and patient-specific. Lastly, we demonstrated that CTLR-Seq enables cell-type specific analysis of DNA methylation patterns within the deletion haplotype of 22q11DS.

Complete mitochondrial genome of Melia azedarach L., reveals two conformations generated by the repeat sequence mediated recombination

Melia azedarach is a species of enormous value of pharmaceutical industries. Although the chloroplast genome of M. azedarach has been explored, the information of mitochondrial genome (Mt genome) remains surprisingly limited. In this study, we used a hybrid assembly strategy of BGI short-reads and Nanopore long-reads to assemble the Mt genome of M. azedarach. The Mt genome of M. azedarach is characterized by two circular chromosomes with 350,142 bp and 290,387 bp in length, respectively, which encodes 35 protein-coding genes (PCGs), 23 tRNA genes, and 3 rRNA genes. A pair of direct repeats (R1 and R2) were associated with genome recombination, resulting in two conformations based on the Sanger sequencing and Oxford Nanopore sequencing. Comparative analysis identified 19 homologous fragments between Mt and chloroplast genome, with the longest fragment of 12,142 bp. The phylogenetic analysis based on PCGs were consist with the latest classification of the Angiosperm Phylogeny Group. Notably, a total of 356 potential RNA editing sites were predicted based on 35 PCGs, and the editing events lead to the formation of the stop codon in the rps10 gene and the start codons in the nad4L and atp9 genes, which were verified by PCR amplification and Sanger sequencing. Taken together, the exploration of M. azedarach gap-free Mt genome provides a new insight into the evolution research and complex mitogenome architecture.

RNA 2'-O-methylation promotes persistent R-loop formation and AID-mediated IgH class switch recombination

BackgroundRNA–DNA hybrids or R-loops are associated with deleterious genomic instability and protective immunoglobulin class switch recombination (CSR). However, the underlying phenomenon regulating the two contrasting functions of R-loops is unknown. Notably, the underlying mechanism that protects R-loops from classic RNase H-mediated digestion thereby promoting persistence of CSR-associated R-loops during CSR remains elusive.ResultsHere, we report that during CSR, R-loops formed at the immunoglobulin heavy (IgH) chain are modified by ribose 2′-O-methylation (2′-OMe). Moreover, we find that 2′-O-methyltransferase fibrillarin (FBL) interacts with activation-induced cytidine deaminase (AID) associated snoRNA aSNORD1C to facilitate the 2′-OMe. Moreover, deleting AID C-terminal tail impairs its association with aSNORD1C and FBL. Disrupting FBL, AID or aSNORD1C expression severely impairs 2′-OMe, R-loop stability and CSR. Surprisingly, FBL, AID’s interaction partner and aSNORD1C promoted AID targeting to the IgH locus.ConclusionTaken together, our results suggest that 2′-OMe stabilizes IgH-associated R-loops to enable productive CSR. These results would shed light on AID-mediated CSR and explain the mechanism of R-loop-associated genomic instability.

HIV-1 subtype A1, D, and recombinant proviral genome landscapes during long-term suppressive therapy

The primary obstacle to curing HIV-1 is a reservoir of CD4+ cells that contain stably integrated provirus. Previous studies characterizing the proviral landscape, which have been predominantly conducted in males in the United States and Europe living with HIV-1 subtype B, have revealed that most proviruses that persist during antiretroviral therapy (ART) are defective. In contrast, less is known about proviral landscapes in females with non-B subtypes, which represents the largest group of individuals living with HIV-1. Here, we analyze genomic DNA from resting CD4+ T-cells from 16 female and seven male Ugandans with HIV-1 receiving suppressive ART (n = 23). We perform near-full-length proviral sequencing at limiting dilution to examine the proviral genetic landscape, yielding 607 HIV-1 subtype A1, D, and recombinant proviral sequences (mean 26/person). We observe that intact genomes are relatively rare and clonal expansion occurs in both intact and defective genomes. Our modification of the primers and probes of the Intact Proviral DNA Assay (IPDA), developed for subtype B, rescues intact provirus detection in Ugandan samples for which the original IPDA fails. This work will facilitate research on HIV-1 persistence and cure strategies in Africa, where the burden of HIV-1 is heaviest.

De Novo Hybrid Assembly Unveils Multi-Chromosomal Mitochondrial Genomes in Ludwigia Species, Highlighting Genomic Recombination, Gene Transfer, and RNA Editing Events.

Biological invasions have been identified as the fifth cause of biodiversity loss, and their subsequent dispersal represents a major ecological challenge. The aquatic invasive species Ludwigia grandiflora subsp. hexapetala (Lgh) and Ludwigia peploides subsp. montevidensis (Lpm) are largely distributed in aquatic environments in North America and in Europe. However, they also present worrying terrestrial forms that are able to colonize wet meadows. To comprehend the mechanisms of the terrestrial adaptation of Lgh and Lpm, it is necessary to develop their genomic resources, which are currently poorly documented. We performed de novo assembly of the mitogenomes of Lgh and Lpm through hybrid assemblies, combining short reads (SR) and/or long reads (LR) before annotating both mitogenomes. We successfully assembled the mitogenomes of Lgh and Lpm into two circular molecules each, resulting in a combined total length of 711,578 bp and 722,518 bp, respectively. Notably, both the Lgh and Lpm molecules contained plastome-origin sequences, comprising 7.8% of the mitochondrial genome length. Additionally, we identified recombinations that were mediated by large repeats, suggesting the presence of multiple alternative conformations. In conclusion, our study presents the first high-quality mitogenomes of Lpm and Lgh, which are the only ones in the Myrtales order found as two circular molecules.

Exploring the Accuracy and Limits of Algorithms for Localizing Recombination Breakpoints.

Phylogenetic methods are widely used to reconstruct the evolutionary relationships among species and individuals. However, recombination can obscure ancestral relationships as individuals may inherit different regions of their genome from different ancestors. It is, therefore, often necessary to detect recombination events, locate recombination breakpoints, and select recombination-free alignments prior to reconstructing phylogenetic trees. While many earlier studies have examined the power of different methods to detect recombination, very few have examined the ability of these methods to accurately locate recombination breakpoints. In this study, we simulated genome sequences based on ancestral recombination graphs and explored the accuracy of three popular recombination detection methods: MaxChi, 3SEQ, and Genetic Algorithm Recombination Detection. The accuracy of inferred breakpoint locations was evaluated along with the key factors contributing to variation in accuracy across datasets. While many different genomic features contribute to the variation in performance across methods, the number of informative sites consistent with the pattern of inheritance between parent and recombinant child sequences always has the greatest contribution to accuracy. While partitioning sequence alignments based on identified recombination breakpoints can greatly decrease phylogenetic error, the quality of phylogenetic reconstructions depends very little on how breakpoints are chosen to partition the alignment. Our work sheds light on how different features of recombinant genomes affect the performance of recombination detection methods and suggests best practices for reconstructing phylogenies based on recombination-free alignments.

Probing recombinant AAV capsid integrity and genome release after thermal stress by mass photometry

Adeno-associated viruses (AAVs) are gaining traction as delivery vehicles for gene therapy although the molecular understanding of AAV-transgene release is still limited. Typically, the process of viral uncoating is investigated (in vitro) through thermal stress, revealing capsid disintegration at elevated temperatures. To assess the (in)stability of different empty and filled AAV preparations, we used the light-scattering-based interferometric microscopy technique of mass photometry (MP) that, on a single-particle basis, determines the molecular weight of AAVs. By introducing a heat-stable DNA plasmid as an internal standard, we quantitatively probed the impact of heat on AAVs. Generally, empty AAVs exhibited greater heat resistance than genome-filled particles. Our data also indicate that upon DNA release, the capsids do not transform into empty AAVs, but seem to aggregate or disintegrate. Strikingly, some AAVs exhibited an intermediate state with disrupted capsids but preserved bound genome, a feature that experimentally only emerged following incubation with a nuclease. Our data demonstrate that the thermal uncoating process is highly AAV specific (i.e., can be influenced by serotype, genome, host system). We argue that nuclease treatment in combination with MP can be used as an additional analytical tool for assessing structural integrity of recombinant and/or clinical AAV vectors.

Completed genome and emergence scenario of the multidrug-resistant nosocomial pathogen Staphylococcus epidermidis ST215

BackgroundA multidrug-resistant lineage of Staphylococcus epidermidis named ST215 is a common cause of prosthetic joint infections and other deep surgical site infections in Northern Europe, but is not present elsewhere. The increasing resistance among S. epidermidis strains is a global concern. We used whole-genome sequencing to characterize ST215 from healthcare settings.ResultsWe completed the genome of a ST215 isolate from a Swedish hospital using short and long reads, resulting in a circular 2,676,787 bp chromosome and a 2,326 bp plasmid. The new ST215 genome was placed in phylogenetic context using 1,361 finished public S. epidermidis reference genomes. We generated 10 additional short-read ST215 genomes and 11 short-read genomes of ST2, which is another common multidrug-resistant lineage at the same hospital. We studied recombination’s role in the evolution of ST2 and ST215, and found multiple recombination events averaging 30–50 kb. By comparing the results of antimicrobial susceptibility testing for 31 antimicrobial drugs with the genome content encoding antimicrobial resistance in the ST215 and ST2 isolates, we found highly similar resistance traits between the isolates, with 22 resistance genes being shared between all the ST215 and ST2 genomes. The ST215 genome contained 29 genes that were historically identified as virulence genes of S. epidermidis ST2. We established that in the nucleotide sequence stretches identified as recombination events, virulence genes were overrepresented in ST215, while antibiotic resistance genes were overrepresented in ST2.ConclusionsThis study features the extensive antibiotic resistance and virulence gene content in ST215 genomes. ST215 and ST2 lineages have similarly evolved, acquiring resistance and virulence through genomic recombination. The results highlight the threat of new multidrug-resistant S. epidermidis lineages emerging in healthcare settings.

Identification of a new HIV-1 circulating recombinant form (CRF159_01103) derived from CRF103_01B and CRF01_AE in Hebei Province, China

Recombinant HIV-1 genomes identified in three or more epidemiological unrelated individuals are defined as circulating recombinant forms (CRFs). CRFs can further recombine with other pure subtypes or recombinants to produce secondary recombinants. In this study, a new HIV-1 intersubtype CRF, designated CRF159_01103, isolated from three men who have sex with men with no epidemiological linkage, was identified in Baoding city, Hebei Province, China. CRF159_01103 was derived from CRF103_01B and CRF01_AE. Bayesian molecular clock analysis was performed on the CRF01-AE and CRF103_01B regions of CRF159_01103. The time of origin of CRF159_01103 was predicted to be 2018–2019, indicating that it is a recent recombinant virus. The emergence of CRF159_01103 has increased the complexity of the HIV-1 epidemic in Hebei Province.

Genomic Gigantism is not Associated with Reduced Selection Efficiency in Neotropical Salamanders

Genome size variation in eukaryotes has myriad effects on organismal biology from the genomic to whole-organism level. Large genome size may be associated with lower selection efficiency because lower effective population sizes allow fixation of deleterious mutations via genetic drift, increasing genome size and decreasing selection efficiency. Because of a hypothesized negative relationship between genome size and recombination rate per base pair, increased genome size could also increase the effect of linked selection in the genome, decreasing the efficiency with which natural selection can fix or remove mutations. We used a transcriptomic dataset of 15 and a subset of six Neotropical salamander species ranging in genome size from 12 to 87 pg to study the relationship between genome size and efficiency of selection. We estimated dN/dS of salamanders with small and large genomes and tested for relaxation of selection in the larger genomes. Contrary to our expectations, we did not find a significant relationship between genome size and selection efficiency or strong evidence for higher dN/dS values in species with larger genomes for either species set. We also found little evidence for relaxation of selection in species with larger genomes. A positive correlation between genome size and range size (a proxy of population size) in this group disagrees with predictions of stronger drift in species with larger genomes. Our results highlight the complex interactions between the many forces shaping genomic variation in organisms with genomic gigantism.

Comprehensive analysis of the complete mitochondrial genome of Lilium tsingtauense reveals a novel multichromosome structure.

Lilium tsingtauense mitogenome comprises 27 independent chromosome molecules, it undergoes frequent genomic recombination, and the rate of recombination and mutation between different repetitive sequences affects the formation of multichromosomal structures. Given the extremely large genome of Lily, which likely harbors additional genetic resources, it serves as an ideal material for studying the phylogenetic evolution of organisms. Although the Lilium chloroplast genome has been documented, the sequence of its mitochondrial genome (mitogenome) remains uncharted. Using BGI short reads and Nanopore long reads, we sequenced, assembled, and annotated the mitogenome of Lilium tsingtauense. This effort culminated in the characterization of Lilium's first complete mitogenome. Comparative analysis with other angiosperms revealed the unique multichromosomal structure of the L. tsingtauense mitogenome, spanning 1,125,108 bp and comprising 27 independent circular chromosomes. It contains 36 protein-coding genes, 12 tRNA genes, and 3 rRNA genes, with a GC content of 44.90%. Notably, three chromosomes in the L. tsingtauense mitogenome lack identifiable genes, hinting at the potential existence of novel genes and noncoding elements. The high degree of observed genome fragmentation implies frequent reorganization, with recombination and mutation rates among diverse repetitive sequences likely driving the formation of multichromosomal structures. Our comprehensive analysis, covering genome size, coding genes, structure, RNA editing, repetitive sequences, and sequence migration, sheds light on the evolutionary and molecular biology of multichromosomal mitochondria in Lilium. This high-quality mitogenome of L. tsingtauense not only enriches our understanding of multichromosomal mitogenomes but also establishes a solid foundation for future genome breeding and germplasm innovation in Lilium.

Simultaneous construction strategy using two types of fluorescent markers for HVT vector vaccine against infectious bursal disease and H9N2 avian influenza virus by NHEJ-CRISPR/Cas9.

Recently, herpesvirus of turkeys (HVT), which was initially employed as a vaccine against Marek's disease (MD), has been shown to be a highly effective viral vector for producing recombinant vaccines that can simultaneously express the protective antigens of multiple poultry diseases. Prior to the development of commercial HVT-vectored dual-insert vaccines, the majority of HVT-vectored vaccines in use only contained a single foreign gene and were often generated using time-consuming and inefficient traditional recombination methods. The development of multivalent HVT-vectored vaccines that induce simultaneous protection against several avian diseases is of great value. In particular, efficacy interference between individual recombinant HVT vaccines can be avoided. Herein, we demonstrated the use of CRISPR/Cas9 gene editing technology for the insertion of an IBDV (G2d) VP2 expression cassette into the UL45/46 region of the recombinant rHVT-HA viral genome to generate the dual insert rHVT-VP2-HA recombinant vaccine. The efficacy of this recombinant virus was also evaluated in specific pathogen-free (SPF) chickens. PCR and sequencing results showed that the recombinant virus rHVT-VP2-HA was successfully constructed. Vaccination with rHVT-VP2-HA produced high levels of specific antibodies against IBDV (G2d) and H9N2/Y280. rHVT-VP2-HA can provide 100% protection against challenges with IBDV (G2d) and H9N2/Y280. These results demonstrate that rHVT-VP2-HA is a safe and highly efficacious vaccine for the simultaneous control of IBDV (G2d) and H9N2/Y280.

Sequence Note: Characterization of Two HIV-1 Strains with Novel Unique Recombinant Genome in Hebei, China.

In China, the proportion of HIV-1 infections due to men who have sex with men (MSM) has increased rapidly. More and more new subtypes are found among the MSM population besides known CRF01_AE, CRF07_BC, and B. The co-circulation of several HIV subtypes in the same population provides the opportunity to develop a new circulating recombinant form (CRF) and unique recombinant form (URF). Here we reported two new URFs from two HIV-1 positive subjects infected through homosexual contact in Hebei, China. Phylogenetic and recombinant analyses based on the near full-length genome (NFLG) of the two URFs are the second-generation recombinant strains that originated from B, CRF01_AE, and CRF07_BC. The CRF01_AE segments in the genome of two URFs originated from cluster 4 of CRF01_AE strains, while the CRF07_BC segments were clustered with 07BC_N in the phylogenetic tree. The emergence of the novel CRF01_AE/CRF07_BC and CRF01_AE/B recombinant forms indicated the importance of the continuous monitoring of the HIV-1 epidemic and new URFs among the MSM population.

Accounting for nuclear and mito genome in dairy cattle breeding - a simulation study

Mitochondria play a significant role in numerous cellular processes through proteins encoded by both the nuclear genome (nDNA) and mito genome (mDNA), and increasing evidence shows that traits of interest might be affected by mito-nuclear interactions. While the variation in nDNA is influenced by mutations and recombination of parental genomes, the variation in mDNA is solely driven by mutations. In addition, mDNA is inherited in a haploid form, from the dam. Cattle populations show significant variation in mDNA between and within breeds. Past research suggests that variation in mDNA accounts for 1–5% of the phenotypic variation in dairy traits. Here we simulated a dairy cattle breeding program to assess the impact of accounting for mDNA variation in pedigree-based and genome-based genetic evaluations on the accuracy of estimated breeding values for mDNA and nDNA components. We also examined the impact of alternative definitions of breeding values on genetic gain, including nDNA and mDNA components that both impact phenotype expression, but mDNA is inherited only maternally. We found that accounting for mDNA variation increased accuracy between +0.01 and +0.03 for different categories of animals, especially for young bulls (+0.03) and females without genotype data (between +0.01 and +0.03). Different scenarios of modeling and breeding value definition impacted genetic gain. The standard approach of ignoring mDNA variation achieved competitive genetic gain. Modeling, but not selecting on mDNA expectedly reduced genetic gain, while optimal use of mDNA variation recovered the genetic gain.

Preliminary efficacy and safety of YSCH-01 in patients with advanced solid tumors: an investigator-initiated trial

ObjectiveTo evaluate the safety and preliminary efficacy of YSCH-01 (Recombinant L-IFN adenovirus) in subjects with advanced solid tumors.MethodsIn this single-center, open-label, investigator-initiated trial of YSCH-01, 14 patients with advanced solid...

Development of a Cell Culture Model for Inducible SARS-CoV-2 Replication.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces direct cytopathic effects, complicating the establishment of low-cytotoxicity cell culture models for studying its replication. We initially developed a DNA vector-based replicon system utilizing the CMV promoter to generate a recombinant viral genome bearing reporter genes. However, this system frequently resulted in drug resistance and cytotoxicity, impeding model establishment. Herein, we present a novel cell culture model with SARS-CoV-2 replication induced by Cre/LoxP-mediated DNA recombination. An engineered SARS-CoV-2 transcription unit was subcloned into a bacterial artificial chromosome (BAC) vector. To enhance biosafety, the viral spike protein gene was deleted, and the nucleocapsid gene was replaced with a reporter gene. An exogenous sequence was inserted within NSP1 as a modulatory cassette that is removable after Cre/LoxP-mediated DNA recombination and subsequent RNA splicing. Using the PiggyBac transposon strategy, the transcription unit was integrated into host cell chromatin, yielding a stable cell line capable of inducing recombinant SARS-CoV-2 RNA replication. The model exhibited sensitivity to the potential antivirals forsythoside A and verteporfin. An innovative inducible SARS-CoV-2 replicon cell model was introduced to further explore the replication and pathogenesis of the virus and facilitate screening and assessment of anti-SARS-CoV-2 therapeutics.

Exploration of Genome-Wide Recombination Rate Variation Patterns at Different Scales in Pigs.

Meiotic recombination is a prevalent process in eukaryotic sexual reproduction organisms that plays key roles in genetic diversity, breed selection, and species evolution. However, the recombination events differ across breeds and even within breeds. In this study, we initially computed large-scale population recombination rates of both sexes using approximately 52 K SNP genotypes in a total of 3279 pigs from four different Chinese and Western breeds. We then constructed a high-resolution historical recombination map using approximately 16 million SNPs from a sample of unrelated individuals. Comparative analysis of porcine recombination events from different breeds and at different resolutions revealed the following observations: Firstly, the 1Mb-scale pig recombination maps of the same sex are moderately conserved among different breeds, with the similarity of recombination events between Western pigs and Chinese indigenous pigs being lower than within their respective groups. Secondly, we identified 3861 recombination hotspots in the genome and observed medium- to high-level correlation between historical recombination rates (0.542~0.683) and estimates of meiotic recombination rates. Third, we observed that recombination hotspots are significantly far from the transcription start sites of pig genes, and the silico-predicted PRDM9 zinc finger domain DNA recognition motif is significantly enriched in the regions of recombination hotspots compared to recombination coldspots, highlighting the potential role of PRDM9 in regulating recombination hotspots in pigs. Our study analyzed the variation patterns of the pig recombination map at broad and fine scales, providing a valuable reference for genomic selection breeding and laying a crucial foundation for further understanding the molecular mechanisms of pig genome recombination.

Evolution of homologous recombination rates across bacteria

Bacteria are nonsexual organisms but are capable of exchanging DNA at diverse degrees through homologous recombination. Intriguingly, the rates of recombination vary immensely across lineages where some species have been described as purely clonal and others as "quasi-sexual." However, estimating recombination rates has proven a difficult endeavor and estimates often vary substantially across studies. It is unclear whether these variations reflect natural variations across populations or are due to differences in methodologies. Consequently, the impact of recombination on bacterial evolution has not been extensively evaluated and the evolution of recombination rate-as a trait-remains to be accurately described. Here, we developed an approach based on Approximate Bayesian Computation that integrates multiple signals of recombination to estimate recombination rates. We inferred the rate of recombination of 162 bacterial species and one archaeon and tested the robustness of our approach. Our results confirm that recombination rates vary drastically across bacteria; however, we found that recombination rate-as a trait-is conserved in several lineages but evolves rapidly in others. Although some traits are thought to be associated with recombination rate (e.g., GC-content), we found no clear association between genomic or phenotypic traits and recombination rate. Overall, our results provide an overview of recombination rate, its evolution, and its impact on bacterial evolution.

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.