The coronavirus recombination pathway.

Recombination is thought to be a mechanism that facilitates cross-species transmission in coronaviruses, thus acting as a driver of coronavirus spillover and emergence. Despite its significance, the mechanism of recombination is poorly understood, limiting our potential to estimate the risk of novel recombinant coronaviruses emerging in the future. As a tool for understanding recombination, here, we outline a framework of the recombination pathway for coronaviruses. We review existing literature on coronavirus recombination, including comparisons of naturally observed recombinant genomes as well as in vitro experiments, and place the findings into the recombination pathway framework. We highlight gaps in our understanding of coronavirus recombination illustrated by the framework and outline how further experimental research is critical for disentangling the molecular mechanism of recombination from external environmental pressures. Finally, we describe how an increased understanding of the mechanism of recombination can inform pandemic predictive intelligence, with a retrospective emphasis on SARS-CoV-2.

Virological characteristics of the SARS-CoV-2 XBB variant derived from recombination of two Omicron subvariants.

In late 2022, SARS-CoV-2 Omicron subvariants have become highly diversified, and XBB is spreading rapidly around the world. Our phylogenetic analyses suggested that XBB emerged through the recombination of two cocirculating BA.2 lineages, BJ.1 and BM.1.1.1 (a progeny of BA.2.75), during the summer of 2022. XBB.1 is the variant most profoundly resistant to BA.2/5 breakthrough infection sera to date and is more fusogenic than BA.2.75. The recombination breakpoint is located in the receptor-binding domain of spike, and each region of the recombinant spike confers immune evasion and increases fusogenicity. We further provide the structural basis for the interaction between XBB.1 spike and human ACE2. Finally, the intrinsic pathogenicity of XBB.1 in male hamsters is comparable to or even lower than that of BA.2.75. Our multiscale investigation provides evidence suggesting that XBB is the first observed SARS-CoV-2 variant to increase its fitness through recombination rather than substitutions.

Evolutionary adaptation of bovine coronavirus (BCoV): Screening of natural recombinations across the complete genomes.

Bovine coronavirus (BCoV) is a member of pathogenic Betacoronaviruses that has been circulating for several decades in multiple host species. Given the similarity between BCoV and human coronaviruses, the current study aimed to review the complete genomes of 107 BCoV strains available on the GenBank database, collected between 1983 and 2017 from different countries. The maximum-likelihood based phylogenetic analysis revealed three main BCoV genogroups: GI, GII, and GIII. GI is further divided into nine subgenogroups: GI-a to GI-i. The GI-a to GI-d are restricted to Japan, and GI-e to GI-i to the USA. The evolutionary relationships were also inferred using phylogenetic network analysis, revealing two major distinct networks dominated by viruses identified in the USA and Japan, respectively. The USA strains-dominated Network Cluster includes two sub-branches: France/Germany and Japan/China in addition to the United States, while Japan strains-dominated Network Cluster is limited to Japan. Twelve recombination events were determined, including 11 intragenogroup (GI) and one intergenogroup (GII vs. GI-g). The breakpoints of the recombination events were mainly located in ORF1ab and the spike glycoprotein ORF. Interestingly, 10 of 12 recombination events occurred between Japan strains, one between the USA strains, and one from intercontinental recombination (Japan vs. USA). These findings suggest that geographical characteristics, and population density with closer contact, might significantly impact the BCoV infection and co-infection and boost the emergence of more complex virus lineages.

Whole-Genome Characterization of SARS-CoV-2 Reveals Simultaneous Circulation of Three Variants and a Putative Recombination (20B/20H) in Pets, Brazzaville, Republic of the Congo.

Following the emergence of SARS-CoV-2, cases of pets infected with variants circulating among humans were reported. In order to evaluate the occurrence of SARS-CoV-2 circulation among pets in the Republic of the Congo, we conducted a ten-month study of dogs and cats living in COVID-19-positive households in Brazzaville and neighboring localities. Real-time PCR and the Luminex platform were used to detect SARS-CoV-2 RNA and antibodies to SARS-CoV-2 RBD and S proteins, respectively. Our results show for the first time the simultaneous circulation of several variants of SARS-CoV-2, including viruses from clades 20A and 20H and a putative recombinant variant between viruses from clades 20B and 20H. We found a high seroprevalence of 38.6%, with 14% of tested pets positive for SARS-CoV-2 RNA. Thirty-four percent of infected pets developed mild clinical signs, including respiratory and digestive signs, and shed the virus for about one day to two weeks. These results highlight the potential risk of SARS-CoV-2 interspecies transmission and the benefits of a "One Health" approach that includes SARS-CoV-2 diagnosis and surveillance of viral diversity in pets. This approach aims to prevent transmission to surrounding wildlife as well as spillback to humans.

Enhanced recombination among Omicron subvariants of SARS-CoV-2 contributes to viral immune escape.

Genetic recombination is an important driver of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolution, which requires the coinfection of a single host cell with different SARS-CoV-2 strains. To understand the emergence and prevalence of recombinant SARS-CoV-2 lineages through time and space, we analyzed SARS-CoV-2 genome sequences collected from November 2019 to July 2022. We observed an extraordinary increase in the emergence of SARS-CoV-2 recombinant lineages during the Omicron wave, particularly in Northern America and Europe. This phenomenon was independent of the sequencing frequency or genetic diversity of circulating SARS-CoV-2 strains. The recombination breakpoints were more prevalent in the 3'-untranslated region of the viral genome. Importantly, we noted the enrichment of certain amino acids in the Spike protein of recombinant lineages, which have been reported to confer immune escape from neutralizing antibodies and increase angiotensin-converting enzyme 2 receptor binding in some cases. We also observed I42V amino acid change genetically fixated in the NSP14 of the Omicron lineage, which needs further characterization for its potential role in enhanced recombination. Overall, we report the important and timely observation of accelerated recombination in the currently circulating SARS-CoV-2 Omicron variants and explore their potential contribution to viral fitness, particularly immune escape.

VirusRecom: an information-theory-based method for recombination detection of viral lineages and its application on SARS-CoV-2.

Genomic recombination is an important driving force for viral evolution, and recombination events have been reported for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the Coronavirus Disease 2019 pandemic, which significantly alter viral infectivity and transmissibility. However, it is difficult to identify viral recombination, especially for low-divergence viruses such as SARS-CoV-2, since it is hard to distinguish recombination from in situ mutation. Herein, we applied information theory to viral recombination analysis and developed VirusRecom, a program for efficiently screening recombination events on viral genome. In principle, we considered a recombination event as a transmission process of ``information'' and introduced weighted information content (WIC) to quantify the contribution of recombination to a certain region on viral genome; then, we identified the recombination regions by comparing WICs of different regions. In the benchmark using simulated data, VirusRecom showed a good balance between precision and recall compared to two competing tools, RDP5 and 3SEQ. In the detection of SARS-CoV-2 XE, XD and XF recombinants, VirusRecom providing more accurate positions of recombination regions than RDP5 and 3SEQ. In addition, we encapsulated the VirusRecom program into a command-line-interface software for convenient operation by users. In summary, we developed a novel approach based on information theory to identify viral recombination within highly similar sequences, providing a useful tool for monitoring viral evolution and epidemic control.

Heterologous RNA Recombination in the Cystoviruses φ6 and φ8: A Mechanism of Viral Variation and Genome Repair.

Recombination and mutation of viral genomes represent major mechanisms for viral evolution and, in many cases, moderate pathogenicity. Segmented genome viruses frequently undergo reassortment of the genome via multiple infection of host organisms, with influenza and reoviruses being well-known examples. Specifically, major genomic shifts mediated by reassortment are responsible for radical changes in the influenza antigenic determinants that can result in pandemics requiring rapid preventative responses by vaccine modifications. In contrast, smaller mutational changes brought about by the error-prone viral RNA polymerases that, for the most part, lack a replication base mispairing editing function produce small mutational changes in the RNA genome during replication. Referring again to the influenza example, the accumulated mutations-known as drift-require yearly vaccine updating and rapid worldwide distribution of each new formulation. Coronaviruses with a large positive-sense RNA genome have long been known to undergo intramolecular recombination likely mediated by copy choice of the RNA template by the viral RNA polymerase in addition to the polymerase-based mutations. The current SARS-CoV-2 origin debate underscores the importance of understanding the plasticity of viral genomes, particularly the mechanisms responsible for intramolecular recombination. This review describes the use of the cystovirus bacteriophage as an experimental model for recombination studies in a controlled manner, resulting in the development of a model for intramolecular RNA genome alterations. The review relates the sequence of experimental studies from the laboratory of Leonard Mindich, PhD at the Public Health Research Institute-then in New York City-and covers a period of approximately 12 years. Hence, this is a historical scientific review of research that has the greatest relevance to current studies of emerging RNA virus pathogens.

Origin of New Lineages by Recombination and Mutation in Avian Infectious Bronchitis Virus from South America.

The gammacoronavirus avian infectious bronchitis virus (IBV) is a highly contagious respiratory pathogen of primary economic importance to the global poultry industry. Two IBV lineages (GI-11 and GI-16) have been widely circulating for decades in South America. GI-11 is endemic to South America, and the GI-16 is globally distributed. We obtained full-length IBV genomes from Argentine and Uruguayan farms using Illumina sequencing. Genomes of the GI-11 and GI-16 lineages from Argentina and Uruguay differ in part of the spike coding region. The remaining genome regions are similar to the Chinese and Italian strains of the GI-16 lineage that emerged in Asia or Europe in the 1970s. Our findings support that the indigenous GI-11 strains recombine extensively with the invasive GI-16 strains. During the recombination process, GI-11 acquired most of the sequences of the GI-16, retaining the original S1 sequence. GI-11 strains with recombinant genomes are circulating forms that underwent further local evolution. The current IBV scenario in South America includes the GI-16 lineage, recombinant GI-11 strains sharing high similarity with GI-16 outside S1, and Brazilian GI-11 strains with a divergent genomic background. There is also sporadic recombinant in the GI-11 and GI-16 lineages among vaccine and field strains. Our findings exemplified the ability of IBV to generate emergent lineage by using the S gene in different genomic backgrounds. This unique example of recombinational microevolution underscores the genomic plasticity of IBV in South America.

Pandemic-scale phylogenomics reveals the SARS-CoV-2 recombination landscape.

Accurate and timely detection of recombinant lineages is crucial for interpreting genetic variation, reconstructing epidemic spread, identifying selection and variants of interest, and accurately performing phylogenetic analyses1-4. During the SARS-CoV-2 pandemic, genomic data generation has exceeded the capacities of existing analysis platforms, thereby crippling real-time analysis of viral evolution5. Here, we use a new phylogenomic method to search a nearly comprehensive SARS-CoV-2 phylogeny for recombinant lineages. In a 1.6 million sample tree from May 2021, we identify 589 recombination events, which indicate that around 2.7% of sequenced SARS-CoV-2 genomes have detectable recombinant ancestry. Recombination breakpoints are inferred to occur disproportionately in the 3' portion of the genome that contains the spike protein. Our results highlight the need for timely analyses of recombination for pinpointing the emergence of recombinant lineages with the potential to increase transmissibility or virulence of the virus. We anticipate that this approach will empower comprehensive real-time tracking of viral recombination during the SARS-CoV-2 pandemic and beyond.

Potential intervariant and intravariant recombination of Delta and Omicron variants.

Among numerous severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concerns, Omicron is more infectious and immune-escaping, while Delta is more pathogenic. Here, we provide evidence for both intervariant and intravariant recombination of the rapidly evolving new SARS-CoV-2 genomes, including XD/XE/XF and BA.3, raising concerns of potential more infectious, immune-escaping, and disease-causing Omicron and Delta-Omicron variants.

Proteolytic Processing of the Coronavirus Replicase Nonstructural Protein 14 Exonuclease Is Not Required for Virus Replication but Alters RNA Synthesis and Viral Fitness.

Coronaviruses (CoVs) initiate replication by translation of the positive-sense RNA genome into the replicase polyproteins connecting 16 nonstructural protein domains (nsp1-16), which are subsequently processed by viral proteases to yield mature nsp. For the betacoronavirus murine hepatitis virus (MHV), total inhibition of translation or proteolytic processing of replicase polyproteins results in rapid cessation of RNA synthesis. The nsp5-3CLpro (Mpro) processes nsps7-16, which assemble into functional replication-transcription complexes (RTCs), including the enzymatic nsp12-RdRp and nsp14-exoribonuclease (ExoN)/N7-methyltransferase. The nsp14-ExoN activity mediates RNA-dependent RNA proofreading, high-fidelity RNA synthesis, and replication. To date, the solved partial RTC structures, biochemistry, and models use or assume completely processed, mature nsp. Here, we demonstrate that in MHV, engineered deletion of the cleavage sites between nsp13-14 and nsp14-15 allowed recovery of replication-competent virus. Compared to wild-type (WT) MHV, the nsp13-14 and nsp14-15 cleavage deletion mutants demonstrated delayed replication kinetics, impaired genome production, altered abundance and patterns of recombination, and impaired competitive fitness. Further, the nsp13-14 and nsp14-15 mutant viruses demonstrated mutation frequencies that were significantly higher than with the WT. The results demonstrate that cleavage of nsp13-14 or nsp14-15 is not required for MHV viability and that functions of the RTC/nsp14-ExoN are impaired when assembled with noncleaved intermediates. These data will inform future genetic, structural, biochemical, and modeling studies of coronavirus RTCs and nsp 13, 14, and 15 and may reveal new approaches for inhibition or attenuation of CoV infection. IMPORTANCE Coronavirus replication requires proteolytic maturation of the nonstructural replicase proteins to form the replication-transcription complex. Coronavirus replication-transcription complex models assume mature subunits; however, mechanisms of coronavirus maturation and replicase complex formation have yet to be defined. Here, we show that for the coronavirus murine hepatitis virus, cleavage between the nonstructural replicase proteins nsp13-14 and nsp14-15 is not required for replication but does alter RNA synthesis and recombination. These results shed new light on the requirements for coronavirus maturation and replication-transcription complex assembly, and they may reveal novel therapeutic targets and strategies for attenuation.

Genome Recombination between the Delta and Alpha Variants of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

Prominent genomic recombination has been observed between the Delta and Alpha variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), isolated from clinical specimens in Japan. Interestingly, the recombination variant detected in this study carries a spike protein identical to that in the domestic Delta variant, thereby suggesting that further risks would not be associated with infectivity and immune escape. The recombinant was classified as an XC lineage in the PANGOLIN database. It is necessary to intensively study such marked genetic variations and characterize emerging variants after careful verification of their lineage and clade assignment.

A Bayesian approach to infer recombination patterns in coronaviruses.

As shown during the SARS-CoV-2 pandemic, phylogenetic and phylodynamic methods are essential tools to study the spread and evolution of pathogens. One of the central assumptions of these methods is that the shared history of pathogens isolated from different hosts can be described by a branching phylogenetic tree. Recombination breaks this assumption. This makes it problematic to apply phylogenetic methods to study recombining pathogens, including, for example, coronaviruses. Here, we introduce a Markov chain Monte Carlo approach that allows inference of recombination networks from genetic sequence data under a template switching model of recombination. Using this method, we first show that recombination is extremely common in the evolutionary history of SARS-like coronaviruses. We then show how recombination rates across the genome of the human seasonal coronaviruses 229E, OC43 and NL63 vary with rates of adaptation. This suggests that recombination could be beneficial to fitness of human seasonal coronaviruses. Additionally, this work sets the stage for Bayesian phylogenetic tracking of the spread and evolution of SARS-CoV-2 in the future, even as recombinant viruses become prevalent.

Viral Nucleases from Herpesviruses and Coronavirus in Recombination and Proofreading: Potential Targets for Antiviral Drug Discovery.

In this review, we explore recombination in two very different virus families that have become major threats to human health. The Herpesviridae are a large family of pathogenic double-stranded DNA viruses involved in a range of diseases affecting both people and animals. Coronaviridae are positive-strand RNA viruses (CoVs) that have also become major threats to global health and economic stability, especially in the last two decades. Despite many differences, such as the make-up of their genetic material (DNA vs. RNA) and overall mechanisms of genome replication, both human herpes viruses (HHVs) and CoVs have evolved to rely heavily on recombination for viral genome replication, adaptation to new hosts and evasion of host immune regulation. In this review, we will focus on the roles of three viral exonucleases: two HHV exonucleases (alkaline nuclease and PolExo) and one CoV exonuclease (ExoN). We will review the roles of these three nucleases in their respective life cycles and discuss the state of drug discovery efforts against these targets.

Prevention and Control of Porcine Epidemic Diarrhea: The Development of Recombination-Resistant Live Attenuated Vaccines.

Porcine epidemic diarrhea (PED), causing up to 100% mortality in neonatal pigs, is a highly contagious enteric disease caused by PED virus (PEDV). The highly virulent genogroup 2 (G2) PEDV emerged in 2010 and has caused huge economic losses to the pork industry globally. It was first reported in the US in 2013, caused country-wide outbreaks, and posed tremendous hardship for many pork producers in 2013-2014. Vaccination of pregnant sows/gilts with live attenuated vaccines (LAVs) is the most effective strategy to induce lactogenic immunity in the sows/gilts and provide a passive protection via the colostrum and milk to suckling piglets against PED. However, there are still no safe and effective vaccines available after about one decade of endeavor. One of the biggest concerns is the potential reversion to virulence of an LAV in the field. In this review, we summarize the status and the major obstacles in PEDV LAV development. We also discuss the function of the transcriptional regulatory sequences in PEDV transcription, contributing to recombination, and possible strategies to prevent the reversion of LAVs. This article provides insights into the rational design of a promising LAV without safety issues.

Detection of SARS-CoV-2 intra-host recombination during superinfection with Alpha and Epsilon variants in New York City.

Recombination is an evolutionary process by which many pathogens generate diversity and acquire novel functions. Although a common occurrence during coronavirus replication, detection of recombination is only feasible when genetically distinct viruses contemporaneously infect the same host. Here, we identify an instance of SARS-CoV-2 superinfection, whereby an individual was infected with two distinct viral variants: Alpha (B.1.1.7) and Epsilon (B.1.429). This superinfection was first noted when an Alpha genome sequence failed to exhibit the classic S gene target failure behavior used to track this variant. Full genome sequencing from four independent extracts reveals that Alpha variant alleles comprise around 75% of the genomes, whereas the Epsilon variant alleles comprise around 20% of the sample. Further investigation reveals the presence of numerous recombinant haplotypes spanning the genome, specifically in the spike, nucleocapsid, and ORF 8 coding regions. These findings support the potential for recombination to reshape SARS-CoV-2 genetic diversity.

Recombination in Coronaviruses, with a Focus on SARS-CoV-2.

Recombination is a common evolutionary tool for RNA viruses, and coronaviruses are no exception. We review here the evidence for recombination in SARS-CoV-2 and reconcile nomenclature for recombinants, discuss their origin and fitness, and speculate how recombinants could make a difference in the future of the COVID-19 pandemics.

Tracking SARS-CoV-2 Omicron diverse spike gene mutations identifies multiple inter-variant recombination events.

The current pandemic of COVID-19 is fueled by more infectious emergent Omicron variants. Ongoing concerns of emergent variants include possible recombinants, as genome recombination is an important evolutionary mechanism for the emergence and re-emergence of human viral pathogens. In this study, we identified diverse recombination events between two Omicron major subvariants (BA.1 and BA.2) and other variants of concern (VOCs) and variants of interest (VOIs), suggesting that co-infection and subsequent genome recombination play important roles in the ongoing evolution of SARS-CoV-2. Through scanning high-quality completed Omicron spike gene sequences, 18 core mutations of BA.1 (frequency >99%) and 27 core mutations of BA.2 (nine more than BA.1) were identified, of which 15 are specific to Omicron. BA.1 subvariants share nine common amino acid mutations (three more than BA.2) in the spike protein with most VOCs, suggesting a possible recombination origin of Omicron from these VOCs. There are three more Alpha-related mutations in BA.1 than BA.2, and BA.1 is phylogenetically closer to Alpha than other variants. Revertant mutations are found in some dominant mutations (frequency >95%) in the BA.1. Most notably, multiple characteristic amino acid mutations in the Delta spike protein have been also identified in the "Deltacron"-like Omicron Variants isolated since November 11, 2021 in South Africa, which implies the recombination events occurring between the Omicron and Delta variants. Monitoring the evolving SARS-CoV-2 genomes especially for recombination is critically important for recognition of abrupt changes to viral attributes including its epitopes which may call for vaccine modifications.

Ubiquitous Micro-Modular Homologies among Genomes from Viruses to Bacteria to Human Mitochondrial DNA: Platforms for Recombination during Evolution?

The emerging Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) and its variants have raised tantalizing questions about evolutionary mechanisms that continue to shape biology today. We have compared the nucleotide sequence of SARS-CoV-2 RNA to that of genomes of many different viruses, of endosymbiotic proteobacterial and bacterial DNAs, and of human mitochondrial DNA. The entire 4,641,652 nt DNA sequence of Escherichia coli K12 has been computer-matched to SARS-CoV-2 RNA. Numerous, very similar micro-modular clusters of 3 to 13 nucleotides lengths were detected with sequence identities of 40 to >50% in specific genome segments between SARS-CoV-2 and the investigated genomes. These clusters were part of patch-type homologies. Control sequence comparisons between 1000 randomly computer-composed sequences of 29.9 kb and with the A, C, G, T base composition of SARS-CoV-2 genome versus the reference Wuhan SARS-CoV-2 sequence showed similar patterns of sequence homologies. The universal A, C, G, T genetic coding mode might have succeeded in evolution due in part to its built-in capacity to select for a substantial reservoir of micro-modular domains and employ them as platforms for integrative recombination. Their role in SARS-CoV-2 interspecies transition and the generation of variants appears likely, but their actual involvement will require detailed investigations.