Molecular Characterization and Genomic Diversity of SARS‐CoV‐2 Spike Gene Variants Circulating in Iraq: Mutational Impact on ACE2 Affinity, RBD Immune Escape, and Viral Transmission

Surveillance for SARS-CoV-2 variants of concern in the Australian context.

The H9N2 influenza viruses that are enzootic in terrestrial poultry in China pose a persistent pandemic threat to humans. To investigate whether the continuous circulation and adaptation of these viruses in terrestrial poultry increased their infectivity to pigs, we conducted a serological survey in pig herds with H9N2 viruses selected from the aquatic avian gene pool (Y439 lineage) and the enzootic terrestrial poultry viruses (G1 and Y280 lineages). We also compared the infectivity and transmissibility of these viruses in pigs. It was found that more than 15% of the pigs sampled from 2010 to 2012 in southern China were seropositive to either G1 or Y280 lineage viruses, but none of the sera were positive to the H9 viruses from the Y439 lineage. Viruses of the G1 and Y280 lineages were able to infect experimental pigs, with detectable nasal shedding of the viruses and seroconversion, whereas viruses of the Y439 lineage did not cause a productive infection in pigs. Thus, adaptation and prevalence in terrestrial poultry could lead to interspecies transmission of H9N2 viruses from birds to pigs. Although H9N2 viruses do not appear to be continuously transmissible among pigs, repeated introductions of H9 viruses to pigs naturally increase the risk of generating mammalian-adapted or reassorted variants that are potentially infectious to humans. This study highlights the importance of monitoring the activity of H9N2 viruses in terrestrial poultry and pigs. H9N2 subtype of influenza viruses has repeatedly been introduced into mammalian hosts, including humans and pigs, so awareness of their activity and evolution is important for influenza pandemic preparedness. However, since H9N2 viruses usually cause mild or even asymptomatic infections in mammalian hosts, they may be overlooked in influenza surveillance. Here, we found that the H9N2 viruses established in terrestrial poultry had higher infectivity in pigs than those from aquatic birds, which suggests that adaptation of the H9N2 viruses in terrestrial poultry might have increased the infectivity of the virus to mammals. Therefore, monitoring the prevalence and evolution of H9 viruses prevalent in terrestrial birds and conducting risk assessment of their threat to mammals are critical for evaluating the pandemic potential of this virus.

During the COVID-19 pandemic, SARS-CoV-2 variants drove large waves of infections, fueled by increased transmissibility and immune escape. Current models focus on changes in variant frequencies without linking them to underlying transmission mechanisms of intrinsic transmissibility and immune escape. We introduce a framework connecting variant dynamics to these mechanisms, showing how host population immunity interacts with viral transmissibility and immune escape to determine relative variant fitness. We advance a selective pressure metric that provides an early signal of epidemic growth using genetic data alone, crucial with current underreporting of cases. Additionally, we show that a latent immunity space model approximates immunological distances, offering insights into population susceptibility and immune evasion. These insights refine real-time forecasting and lay the groundwork for research into the interplay between viral genetics, immunity, and epidemic growth.

The latest wave of SARS-CoV-2 Omicron variants displayed a growth advantage and increased viral fitness through convergent evolution of functional hotspots that work synchronously to balance fitness requirements for productive receptor binding and efficient immune evasion. In this study, we combined AlphaFold2-based structural modeling approaches with atomistic simulations and mutational profiling of binding energetics and stability for prediction and comprehensive analysis of the structure, dynamics, and binding of the SARS-CoV-2 Omicron BA.2.86 spike variant with ACE2 host receptor and distinct classes of antibodies. We adapted several AlphaFold2 approaches to predict both the structure and conformational ensembles of the Omicron BA.2.86 spike protein in the complex with the host receptor. The results showed that the AlphaFold2-predicted structural ensemble of the BA.2.86 spike protein complex with ACE2 can accurately capture the main conformational states of the Omicron variant. Complementary to AlphaFold2 structural predictions, microsecond molecular dynamics simulations reveal the details of the conformational landscape and produced equilibrium ensembles of the BA.2.86 structures that are used to perform mutational scanning of spike residues and characterize structural stability and binding energy hotspots. The ensemble-based mutational profiling of the receptor binding domain residues in the BA.2 and BA.2.86 spike complexes with ACE2 revealed a group of conserved hydrophobic hotspots and critical variant-specific contributions of the BA.2.86 convergent mutational hotspots R403K, F486P, and R493Q. To examine the immune evasion properties of BA.2.86 in atomistic detail, we performed structure-based mutational profiling of the spike protein binding interfaces with distinct classes of antibodies that displayed significantly reduced neutralization against the BA.2.86 variant. The results revealed the molecular basis of compensatory functional effects of the binding hotspots, showing that BA.2.86 lineage may have evolved to outcompete other Omicron subvariants by improving immune evasion while preserving binding affinity with ACE2 via through a compensatory effect of R493Q and F486P convergent mutational hotspots. This study demonstrated that an integrative approach combining AlphaFold2 predictions with complementary atomistic molecular dynamics simulations and robust ensemble-based mutational profiling of spike residues can enable accurate and comprehensive characterization of structure, dynamics, and binding mechanisms of newly emerging Omicron variants.

The human population is currently facing the third and possibly the worst pandemic caused by human coronaviruses (CoVs). The virus was first reported in Wuhan, China, on 31 December 2019 and spread within a short time to almost all countries of the world. Genome analysis of the early virus isolates has revealed high similarity with SARS-CoV and hence the new virus was officially named SARS-CoV-2. Since CoVs have the largest genome among all RNA viruses, they can adapt to many point mutation and recombination events; particularly in the spike gene, which enable these viruses to rapidly change and evolve in nature. CoVs are known to cross the species boundaries by using different cellular receptors. Both animal reservoir and intermediate host for SARS-CoV-2 are still unresolved and necessitate further investigation. In the current review, different aspects of SARS-CoV-2 biology and pathogenicity are discussed, including virus genetics and evolution, spike protein and its role in evolution and adaptation to novel hosts, and virus transmission and persistence in nature. In addition, the immune response developed during SARS-CoV-2 infection is demonstrated with special reference to the interplay between immune cells and their role in disease progression. We believe that the SARS-CoV-2 outbreak will not be the last and spillover of CoVs from bats will continue. Therefore, establishing intervention approaches to reduce the likelihood of future CoVs spillover from natural reservoirs is a priority.

Adaptive mutations in the H5N1 polymerase complex

The H1N1 Eurasian avian-like swine (EAsw) influenza viruses originated from an avian H1N1 virus. To characterize potential changes in the membrane fusion activity of the hemagglutinin (HA) during avian-to-swine adaptation of the virus, we studied EAsw viruses isolated in the first years of their circulation in pigs and closely related contemporary H1N1 viruses of wild aquatic birds. Compared to the avian viruses, the swine viruses were less sensitive to neutralization by lysosomotropic agent NH4Cl in MDCK cells, had a higher pH optimum of hemolytic activity, and were less stable at acidic pH. Eight amino acid substitutions in the HA were found to separate the EAsw viruses from their putative avian precursor; four substitutions-T492S, N722D, R752K, and S1132F-were located in the structural regions of the HA2 subunit known to play a role in acid-induced conformational transition of the HA. We also studied low-pH-induced syncytium formation by cell-expressed HA proteins and found that the HAs of the 1918, 1957, 1968, and 2009 pandemic viruses required a lower pH for fusion induction than did the HA of a representative EAsw virus. Our data show that transmission of an avian H1N1 virus to pigs was accompanied by changes in conformational stability and fusion promotion activity of the HA. We conclude that distinctive host-determined fusion characteristics of the HA may represent a barrier for avian-to-swine and swine-to-human transmission of influenza viruses. Continuing cases of human infections with zoonotic influenza viruses highlight the necessity to understand which viral properties contribute to interspecies transmission. Efficient binding of the HA to cellular receptors in a new host species is known to be essential for the transmission. Less is known about required adaptive changes in the membrane fusion activity of the HA. Here we show that adaptation of an avian influenza virus to pigs in Europe in 1980s was accompanied by mutations in the HA, which decreased its conformational stability and increased pH optimum of membrane fusion activity. This finding represents the first formal evidence of alteration of the HA fusion activity/stability during interspecies transmission of influenza viruses under natural settings.

Of variants and vaccines

The persistent evolution of SARS-CoV-2 is driven by mutations in the spike protein that modulate receptor binding, immune evasion, and structural stability. In this study, we deciphered the complex host-virus protein-protein interactions using an integrated molecular dynamics (MD) approach to assess the biophysical impacts of key spike mutations, including T478K, T478A, T478E, E484K, G496S, F490S, Q493E, and Y369C. Our findings reveal that viral adaptation hinges on trade-offs between transmissibility and immune escape. For instance, T478K enhances ACE2 binding through structural rigidification and salt bridge formation (e.g., K478-D30), favoring Omicron’s increased transmissibility. In contrast, T478A introduces polarity loss and interface relaxation, while T478E leads to electrostatic repulsion and weakened binding, both of which compromise interface stability. E484K balances antibody evasion (e.g., against LY-CoV555) with receptor stabilization via compensatory interactions (e.g., K484-D38). In vivo studies support these findings, showing that T478K and E484K enhance viral fitness and immune evasion in animal models. Mutations like G496S and F490S act as stealth adaptations, subtly destabilizing ACE2 or introducing metastability without fully disrupting binding. The high-risk Y369C mutation collapses the N-terminal domain supersite, enhancing immune evasion but requiring compensatory mutations (e.g., G142D) to maintain viability. Evolutionary strategies favor co-mutations (e.g., T478K + Q498R) that distribute fitness costs across residues. Notably, functionally conserved energetic hotspots such as T430, L390, V382, K386, F486, Q493 (RBD), and Q102, R192 (ACE2) consistently contributed to ACE2 engagement across all variants, representing potential targets for broad-spectrum therapeutics. Our work provides the importance of real-time surveillance for mutations that exploit conformational flexibility or compensatory networks, informing the design of durable vaccines and multi-specific antibodies. These insights bridge molecular mechanisms with evolutionary dynamics, offering a framework to anticipate and counter emerging variants.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-15979-6.

Virus transmission is essential for spreading viral infections and is a highly coordinated process which occurs by cell-free transmission or cell–cell contact. The transmission of Bovine Foamy Virus (BFV) is highly cell-associated, with undetectable cell-free transmission. However, BFV particle budding can be induced by overexpression of wild-type (wt) BFV Gag and Env or artificial retargeting of Gag to the plasma membrane via myristoylation membrane targeting signals, closely resembling observations in other foamy viruses. Thus, the particle release machinery of wt BFV appears to be an excellent model system to study viral adaption to cell-free transmission by in vitro selection and evolution. Using selection for BFV variants with high cell-free infectivity in bovine and non-bovine cells, infectivity dramatically increased from almost no infectious units to about 105–106 FFU (fluorescent focus forming units)/mL in both cell types. Importantly, the selected BFV variants with high titer (HT) cell-free infectivity could still transmit via cell-cell contacts and were neutralized by serum from naturally infected cows. These selected HT–BFV variants will shed light into virus transmission and potential routes of intervention in the spread of viral infections. It will also allow the improvement or development of new promising approaches for antiretroviral therapies.

Background/aim In January 2020, the ARTIC Network designed 98 pairs of primers divided into two pools for targeting amplification of the severe acute respiratory syndrome coronavirus 2 genome by multiplex PCR. However, in using this protocol, several users, including our team at Emerging Pathogens Institute at University of Florida, noticed a systematic dropout of reads covering amplicon 18, which overlaps the gene for nonstructural protein 3 (nsp3) in ORF1a, and amplicon 76, targeting the spike (S) gene. The aim of this work was to verify the design of primers for the dropout region of amplicon 76 in the V3 ARTIC protocol covering the severe acute respiratory syndrome coronavirus 2 spike gene, to evaluate correlation of dropout to viral load and determine if there is a correlation between dropouts and viral load and viral lineages and as well as to further explore potential mutations in the primer regions. Materials and methods A total of 544 samples presenting dropout at amplicon 76 from the V3 ARTIC primer set were collected over time from December 2020 to December 2021 in Alachua County, Florida. RNA was extracted, followed by cDNA synthesis and PCR amplification, which targeted the dropout region within the spike. PCR amplification was performed with forward and reverse primers designed in-house. Sequencing was performed to detect potential mutations at primer sites. Viral load was performed for 96 samples showing a dropout of the spike genome at amplicon 76. The results were compared and correlated to lineage/variant of concern. Results A total of 544 dropout samples were amplified with the primers designed in-house. Overall, 381 (70%) of these samples showed bands visible by gel electrophoresis. This result, along with Sanger sequencing, was enough to verify the efficacy of our designed primers in amplifying the dropout region. Sequencing revealed that primer regions were conserved; no mutations were observed at the site corresponding to the Illumina primers for amplicon 76. The mean Ct value of dropouts was 32.53, and the mean Ct value of nondropouts was 26.11. Welch test on Ct values of dropouts versus nondropout showed significant difference (P<10−6). The 96 dropout samples tested for viral load included 17 different lineages, with delta lineage (B.1.617.2-like) being the most common (21.8%), followed by B.1.2 (10.4%), alpha (B.1.1.7-like) (9.4%), B.1.234 (9.4%), iota (B.1.526-like) (5.2%), and gamma (P.1-like) (4.2%). Unexpectedly, when determining the effect the lineage on the chance of a sample being a dropout, the delta lineage had an odds ratio of less than 1, implying that a sequence belonging to delta lineage was less likely to produce a dropout. Conclusions Our study showed that dropouts are not due to mutation at the primer sites. Viral loads likely affect the odds of a sample presenting a dropout of amplicon 76 but was not the only cause of dropout. Several dropout samples could not be classified because of undefined genomic segments, which may have skewed the resulting odds of lineage on sequencing. It is possible, as previously suggested, that dropouts may be due to interaction and dimer formation between primers of the multiplexed PCR reaction.

The most recent wave of SARS-CoV-2 Omicron variants descending from BA.2 and BA.2.86 exhibited improved viral growth and fitness due to convergent evolution of functional hotspots. These hotspots operate in tandem to optimize both receptor binding for effective infection and immune evasion efficiency, thereby maintaining overall viral fitness. The lack of molecular details on structure, dynamics and binding energetics of the latest FLiRT and FLuQE variants with the ACE2 receptor and antibodies provides a considerable challenge that is explored in this study. We combined AlphaFold2-based atomistic predictions of structures and conformational ensembles of the SARS-CoV-2 Spike complexes with the host receptor ACE2 for the most dominant Omicron variants JN.1, KP.1, KP.2 and KP.3 to examine the mechanisms underlying the role of convergent evolution hotspots in balancing ACE2 binding and antibody evasion. Using the ensemble-based mutational scanning of the spike protein residues and computations of binding affinities, we identified binding energy hotspots and characterized molecular basis underlying epistatic couplings between convergent mutational hotspots. The results suggested that the existence of epistatic interactions between convergent mutational sites at L455, F456, Q493 positions that enable to protect and restore ACE2 binding affinity while conferring beneficial immune escape. To examine immune escape mechanisms, we performed structure-based mutational profiling of the spike protein binding with several classes of antibodies that displayed impaired neutralization against BA.2.86, JN.1, KP.2 and KP.3. The results confirmed the experimental data that JN.1, KP.2 and KP.3 harboring the L455S and F456L mutations can significantly impair the neutralizing activity of class-1 monoclonal antibodies, while the epistatic effects mediated by F456L can facilitate the subsequent convergence of Q493E changes to rescue ACE2 binding. Structural and energetic analysis provided a rationale to the experimental results showing that BD55-5840 and BD55-5514 antibodies that bind to different binding epitopes can retain neutralizing efficacy against all examined variants BA.2.86, JN.1, KP.2 and KP.3. The results support the notion that evolution of Omicron variants may favor emergence of lineages with beneficial combinations of mutations involving mediators of epistatic couplings that control balance of high ACE2 affinity and immune evasion.

Astragalus polysaccharide hinders cervical cancer immune escape by targeting NR3C2 and activating SLC40A1.

Characterizations of SARS-CoV-2 mutational profile, spike protein stability and viral transmission

The breach of an interspecies barrier by RNA viruses has facilitated the emergence of lethal hCoVs, particularly SARS-CoV-2, resulting in significant socioeconomic setbacks and public health risks globally in recent years. Moreover, the high evolutionary plasticity of hCoVs has led to the continuous emergence of diverse variants, complicating clinical management and public health responses. Studying the evolutionary trajectory of hCoVs, which provides a molecular roadmap for understanding viruses’ adaptation, tissue tropism, spread, virulence, and immune evasion, is crucial for addressing the challenges of zoonotic spillover of viruses. Tracing the evolutionary trajectory of lethal hCoVs provides essential genomic insights required for risk stratification, variant/sub-variant classification, preparedness for outbreaks and pandemics, and the identification of critical viral elements for vaccine and therapeutic development. Therefore, this review examines the evolutionary landscape of the three known lethal hCoVs, presenting a focused narrative on SARS-CoV-2 variants under monitoring (VUMs) as of May 2025. Using advanced bioinformatics approaches and data visualization, the review highlights key spike protein substitutions, particularly within the receptor-binding domain (RBD), which drive transmissibility, immune escape, and potential resistance to therapeutics. The article highlights the importance of real-time genomic surveillance and intervention strategies in mitigating emerging variant/sub-variant risks within the ongoing COVID-19 landscape.