Context The human endonuclease Artemis, required for immunoglobulin rearrangements of V(D)J segments in B cells and thereby contributing to the adaptive immune response, belongs to the metallo-β-lactamase enzyme superfamily. β-lactam antibiotics, including ceftriaxone, are widely used in clinical practice and display strong affinity for β-lactamase catalytic sites. In vitro, ceftriaxone has been shown to block the activity of Artemis. Based on these structural similarities, the hypothesis according to which certain β-lactams could inhibit Artemis and therefore the immune response was previously proposed by our teams. This study aimed to determine in vivo whether ceftriaxone exposure alters vaccine-induced humoral immunity. Methods To explore the hypothesis put forward above, we developed an animal model to evaluate the antibody immune response to tetanus toxin induced by a diphtheria–tetanus–poliomyelelitis (DTP) vaccine in the presence or absence of ceftriaxone. Male BALB/c mice were vaccinated either before or after a five-day intraperitoneal ceftriaxone treatment; control groups received isotonic saline. The mice immune response to vaccine was quantified by measuring interactions with specific antigens using immunochromatographic tests, enabling longitudinal assessment of anti-tetanus toxoid antibodies. Results We report the first experimental evidence of a specific weakening of vaccine-induced immune response in animals receiving ceftriaxone, with significantly reduced anti-tetanus antibody levels when vaccination followed antibiotic exposure. The response remained efficient in animals receiving isotonic saline treatment as control, and in mice vaccinated prior to ceftriaxone administration. Conclusions These results confirm our hypothesis that the immune response to tetanus toxin is impaired and that a refractory period for humoral immunity may be caused by the inhibition of Artemis by ceftriaxone. This work highlights a clinically relevant interaction between β-lactam antibiotics and adaptive immune responses.

Theories of evolution seeded by Lamarck and Darwin two centuries ago put forward inherited modifications of genomes while ignoring interactions between organisms living in the same ecosystem, and rearrangements (with recombinations and reassortments), introgressive evolution, transposable elements, and sequence losses within genomes. Here we summarized data that currently indicate a different, optimized, and broadly applicable vision of genome evolution. In fact, two paradigms coexist in evolutionary biology: one is that of linear, gradual evolution linked to mutations with selection in a given environment, as drawn by Darwin, Lamarck, and the Red Queen theory. Another is that of unpredictable, chaotic, leap-making evolution, as illustrated by the Court Jester theory. Key drivers of genome expansion comprise gains of sequences, among which invasions by either endogenous or exogenous transposable elements that resemble epidemics and determine defense countermeasures, as well as genome duplications and polyploidizations. Sympatric conditions with biological melting pots promote sequence gains and increase genome mosaicism. Sequence loss, which can involve degradation and non-repair, is another major player in evolution in reduced ecosystems, with allopatric conditions promoting specializations. “Fertile selfish sequences” are transmitted between biological entities and can multiply within genomes, while junk DNA consists of remnants of genes progressively degraded. Besides, coding density within genomes tends to decrease as organism complexity increases, regardless of genome size, and therefore the proportion of genomes that is functional is mostly unknown. Overall, current genomic data indicate that genetic evolution is chaotic and permanently creative and innovative, and that representing genomes as single sequences is not realistic, just as phylogenetic trees are an inaccurate representation of sequence evolution, which is more realistically represented by rhizomes or networks, as genomes are not unique but multiple, plastic, and mosaic. Beyond, chaotic intentionality and random purpose should be considered, and the question of the finality of evolution cannot truly be resolved. Finally, genomic data support an anthropocenic rather than anthropocentric paradigm of evolution.

Human cellular APOBEC enzymes were largely reported as involved in innate antiviral defenses. We previously highlighted that in SARS-CoV-2 genomes obtained in our center, nearly half of 'hyperfertile' or 'fertile' mutations while 23% of neutral/weakly deleterious mutations had APOBEC signatures. Here we determined that 29% of mutations we named 'lethal' as detected in quasispecies but not in consensus genomes have APOBEC signatures. Overall, these results do not suggest that human APOBEC acts as defense agents against SARS-CoV-2 but as 'Court Jesters', being neither friends nor foes but only enzymes whose activity can either favor or hamper viral fitness according to the Kimura theory of neutral evolution.

We studied the fate of a gene called ORF8 in different coronaviruses species during their passage to humans. We showed that this gene harbored stop codons in many epidemic SARS-CoV-2 variants in humans and in up to 86% of SARS-CoV-2 circulating in 2023 and therefore could be a non-virulence gene whose disappearance helps the epidemics spread. We questioned whether this is a general phenomenon following coronavirus passage to humans from another species, most often bats. We studied SARS-CoV, SARS-CoV-2, MERS-CoV, and the four coronaviruses endemic in humans for several years or decades and observed gene degradation after species jumps from animals to humans. For SARS-CoV-2, we observed the progressive disappearance of its ORF8 with one or several stop codons and partial or even complete deletions, which is reminiscent of the 'Cheshire cat phenomenon' described in the L. Caroll's Alice's Adventures in Wonderland novel, where a cat gradually disappears to leave only a smile. In viruses that emerged earlier in humans and adapted to this host, only footprints of a few bases remain, but which are significantly associated with the original gene.

Coxiella burnetii is responsible for Q fever, a zoonosis that mainly manifests as a mild acute infection and may persist in specific individuals causing severe complications, including endocarditis and vascular infections. Transmission mainly occurs through the inhalation of aerosols, underlining bacterial resistance to environmental stress. C. burnetii is an intracellular gram-negative pathogen that can infect and survive in monocytes and macrophages and replicate within acidic compartments. C. burnetii expresses several virulence factors, including lipopolysaccharide and a type IV secretion system, which are involved in hijacking several functions of the immune system and controlling cell death. This review summarizes research on C. burnetii infection from clinical presentation of Q fever to bacterial and host factors of virulence. It compiles immunological data associated with bacterial cure or persistence and discusses a potential link between C. burnetii-macrophage interaction and immune dysregulation of Q fever.

RNA hairpins may constitute a foundation of genetic evolution both in viruses and other organisms. Stem-loops theoretically comprise a stable part, the double-stranded stem, and a single-stranded loop allowing evolution. Here we tested for SARS-CoV-2 if "fertile" mutations were in loops while mutations in stems were poorly tolerated and rarely found in consensus genomes. We combined information on the frequencies of mutations, either "fertile" (present in ≥ 50 genomes) or "non-fertile" (neutral, weakly deleterious or lethal) in 61,397 SARS-CoV-2 genomes, and on whether these mutations occurred at positions where nucleotides were predicted to be either paired or unpaired. The proportion of positions harboring "fertile" mutations was significantly higher in loops than in stems for the whole genome (11.6% vs. 7.6%; p < 0.001, Yates-corrected chi-square test). This was also the case in the RNA-dependent RNA polymerase gene (10.0% vs. 4.9%; p = 0.0003) or in the spike gene (12.3% vs. 8.9%; p = 0.0049). All four most frequent mutations in our set of genomes were located in loops. Thus, apart from some observations in "accessory" genes, evolution in SARS-CoV-2 predominantly occurred in loops while mutations in stems were relatively "non-fertile." These stems could be potential antiviral targets, possibly through their disruption by RNA interference.

SARS-CoV-2 can infect different organs, including the intestine. In an in vitro model of Caco-2 intestinal cell line, we previously found that SARS-CoV-2 modulates the ACE2 receptor expression and affects the expression of molecules involved in intercellular junctions. To further explore the possibility that the intestinal epithelium can serve as an alternative infection route for SARS-CoV-2, we used a model of polarized monolayers of Caco-2 cells (or co-cultures of two intestinal cell lines: Caco-2 and HT29) grown on the polycarbonate membrane of Transwell inserts, inoculated with the virus either in the upper or lower chamber of culture to determine the tropism of the virus for the apical or basolateral pole of these cells. In both polarized Caco-2 cell monolayers and co-culture Caco-2/HT29 cell monolayer, apical SARS-CoV-2 inoculation was found to be much more effective in establishing infection than basolateral inoculation. In addition, apical SARS-CoV-2 infection triggers monolayer degeneration, as shown by histological examination, measurement of trans-epithelial electrical resistance, and cell adhesion molecule expression. During apical infection, the infectious viruses reach the lower chamber, suggesting either a transcytosis mechanism from the apical side to the basolateral side of cells, a paracellular trafficking of the virus after damage to intercellular junctions in the epithelial barrier, or both. Taken together, these data indicate a preferential tropism of SARS-CoV-2 for the apical pole of the human intestinal tract and suggest that infection via the intestinal lumen leads to a systemic infection.

Myocarditis has been recognized as a possible rare complication of COVID-19 mRNA vaccination. It concerns between one and five vaccinated people per 100,000 in the general population, with increased incidence in adolescent and young adult men. Most often, cases of myocarditis have been reported in the days following the second dose of vaccine mainly in younger male patients. This rare complication of vaccination usually resolves within days or weeks. However, the pathophysiological events responsible for the increase in frequency of myocarditis after COVID-19 vaccination remain unclear. Several recent reports have highlighted that free spike proteins circulating in the blood of patients at high levels appear to play a major role in myocarditis. Here, we review the most relevant data that partly lift the veil on the molecular mechanisms of the induction of myocarditis following mRNA-based COVID-19 vaccination. We hypothesize that a mechanism of molecular mimicry of the viral spike triggers transient dysregulation of angiotensin-converting enzyme 2, leading to increased soluble angiotensin II binding to the transmembrane receptor angiotensin II type I receptor, similar to what is observed during SARS-CoV-2 infection. We suggest to standardize management of suspected cases of mRNA-based COVID-19 vaccine-induced myocarditis, including angiotensin II and spike antigenemia monitoring.

The polioviruses (PVs) are mainly transmitted by direct contact with an infected person through the fecal-oral route and respiratory secretions (or more rarely via contaminated water or food) and have a primary tropism for the gut. After their replication in the gut, in rare cases (far less than 1% of the infected individuals), PVs can spread to the central nervous system leading to flaccid paralysis, which can result in respiratory paralysis and death. By the middle of the 20th century, every year the wild polioviruses (WPVs) are supposed to have killed or paralyzed over half a million people. The introduction of the oral poliovirus vaccines (OPVs) through mass vaccination campaigns (combined with better application of hygiene measures), was a success story which enabled the World Health Organization (WHO) to set the global eradication of poliomyelitis as an objective. However this strategy of viral eradication has its limits as the majority of poliomyelitis cases today arise in individuals infected with circulating vaccine-derived polioviruses (cVDPVs) which regain pathogenicity following reversion or recombination. In recent years (between January 2018 and May 2023), the WHO recorded 8.8 times more cases of polio which were linked to the attenuated OPV vaccines (3,442 polio cases after reversion or recombination events) than cases linked to a WPV (390 cases). Recent knowledge of the evolution of RNA viruses and the exchange of genetic material among biological entities of the intestinal microbiota, call for a reassessment of the polio eradication vaccine strategies.

An HFMD outbreak spread over the city of Hải Phòng from summer 2011 to autumn 2012. This epidemic was chosen because it was the very first HFMD epidemic in North Vietnam, eliminating thus interferences with previous outbreaks. This epidemic displayed three separate waves. A complete dataset was collected for more than 9500 patients during this period, which enabled us to analyze this epidemic at different scales. Access to the healthcare system was crucial during this period, which was possible due to a reorganization of the system in February–March 2012. An analysis at the commune level enabled us to track the epidemic along certain communication routes. The three-waves structure reveals a wide disparity at the district level. We developed a mathematical model showing high accuracy at the adjustment of data for both the total number of cases and for the number of cases per week. As a consequence, the model was able to accurately determine the dates of the beginning and end of each wave and to show that they overlapped. Using mathematical functions associated with this model, it was possible to calculate the probability for a patient to belong to a specific wave.

Mutations associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) resistance to antiprotease nirmatrelvir were reported. We aimed to detect them in SARS-CoV-2 genomes and quasispecies retrieved in our institute before drug availability in January 2022 and to analyze the impact of mutations on protease (3CLpro) structure. We sought for 38 3CLpro nirmatrelvir resistance mutations in a set of 62 673 SARS-CoV-2 genomes obtained in our institute from respiratory samples collected between 2020 and 2023 and for these mutations in SARS-CoV-2 quasispecies for 90 samples collected in 2020, using Python. SARS-CoV-2 protease with major mutation E166V was generated with Swiss Pdb Viewer and Molegro Molecular Viewer. We detected 22 (58%) of the resistance-associated mutations in 417 (0.67%) of the genomes analyzed; 325 (78%) of these genomes had been obtained from samples collected in 2020-2021. APOBEC signatures were found for 12/22 mutations. We also detected among viral quasispecies from 90 samples some minority reads harboring any of 15 nirmatrelvir resistance mutations, including E166V. Also, we predicted that E166V has a very limited effect on 3CLpro structure but may prevent drug attachment. Thus, we evidenced that mutations associated with nirmatrelvir resistance pre-existed in SARS-CoV-2 before drug availability. These findings further warrant SARS-CoV-2 genomic surveillance and SARS-CoV-2 quasispecies characterization.

ObjectivesThe SARS-CoV-2 pandemic and large-scale genomic surveillance provided an exceptional opportunity to analyze mutations that appeared over three years in viral genomes. Here we studied mutations and their epidemic consequences for SARS-CoV-2 genomes from our center.MethodsWe analyzed 61,397 SARS-CoV-2 genomes we sequenced from respiratory samples for genomic surveillance. Mutations frequencies were calculated using Nextclade, Microsoft Excel, and an in-house Python script.ResultsA total of 22,225 nucleotide mutations were identified, 220 (1.0%) being each at the root of ≥836 genomes, classifying mutations as ‘hyperfertile’. Two seeded the European pandemic: P323L in RNA polymerase, associated with an increased mutation rate, and D614G in spike that improved fitness. Most ‘hyperfertile’ mutations occurred in areas not predicted with increased virulence. Their mean number was 8±6 (0-22) per 1,000 nucleotides per gene. They were 3.7-times more frequent in accessory than informational genes (13.9 versus 3.7/1,000 nucleotides). Particularly, they were 4.1-times more frequent in ORF8 than in the RNA polymerase gene. Interestingly, stop codons were present in 97 positions, almost only in accessory genes, including ORF8 (21/100 codons).Conclusionsmost ‘hyperfertile’ mutations did not predict emergence of a new epidemic, and some were stop codons indicating the existence of so-named ‘non-virulence’ genes.

SARS-CoV-2 can infect different organs, including the intestine. In Caco-2 intestinal cell line, SARS-CoV-2 modulates the ACE2 receptor expression and affects the expression of molecules involved in intercellular junctions. To further explore the possibility that the intestinal epithelium serves as an alternative infection route for SARS-CoV-2, we used a model of polarised intestinal cell monolayers grown on the polycarbonate membrane of Transwell inserts, inoculated with the virus either in the upper or lower chamber of culture. In both polarised Caco-2 cell monolayers and co-culture Caco-2/HT29 cell monolayer, apical SARS-CoV-2 inoculation was found to be much more effective in establishing infection than basolateral inoculation. In addition, apical SARS-CoV-2 infection triggers monolayer degeneration, as shown by histological examination, measurement of trans-epithelial electronic resistance, and cell adhesion molecule expression. During this process, the infectious viruses reach the lower chamber, suggesting either a transcytosis mechanism from the apical side to the basolateral side of cells, a paracellular trafficking of the virus after damage to intercellular junctions in the epithelial barrier, or both. Taken together, these data highlight a preferential tropism of SARS-CoV-2 for the apical side of the human intestinal tract and suggests that infection via the intestinal lumen leads to a systemic infection.### Competing Interest StatementThe authors have declared no competing interest.

The evolution of SARS-CoV-2, the agent of COVID-19, has been remarkable for its high mutation potential, leading to the appearance of variants. Some mutations have never appeared in the published genomes, which represent consensus, or bona fide genomes. Here we tested the hypothesis that mutations that did not appear in consensus genomes were, in fact, as frequent as the mutations that appeared during the various epidemic episodes, but were not expressed because lethal. To identify these mutations, we analysed the genomes of 90 nasopharyngeal samples and the quasispecies determined by next-generation sequencing. Mutations observed in the quasispecies and not in the consensus genomes were considered to be lethal, what we called “outlaw” mutations. Among these mutations, we analysed the 21 most frequent. Eight of these “outlaws” were in the RNA polymerase and we were able to use a structural biology model and molecular dynamics simulations to demonstrate the functional incapacity of these mutated RNA polymerases. Three other mutations affected the spike, a major protein involved in the pathogenesis of COVID-19. Overall, by analysing the SARS-CoV-2 quasispecies obtained during sequencing, this method made it possible to identify “outlaws,” showing areas that could potentially become the target of treatments.

From 2011 to 2012, Northern Vietnam suffered its first large-scale hand, foot, and mouth disease (HFMD) epidemic. Two sets of official guidelines were issued during the outbreak to handle the HFMD crisis. The city of Hai Phong was used as a model to analyze the impact of the released guidelines. A total of 9621 HFMD cases were reported in Hai Phong city from April 2011 to December 2012. Three distinct waves of HFMD occurred. Enterovirus A71 and Coxsackievirus A16 were successively associated with the epidemics. Two periods, before and after the guidelines’ release, could be distinguished and characterized by different patient patterns. The time to admission and severity changed notably. Guideline publications help the health system refocus on the 0.5–3 years age group with the highest incidence of the disease. The three waves showed different special distribution, but the main routes of infection were rivers and local secondary roads, most likely through local trade and occupational movements of people.

Since the start of the SARS-CoV-2 pandemic, the rapid replacement of one lineage by another has been observed. Indeed, SARS-CoV-2 is evolving through a quasispecies mechanism leading to post-infection mutation selection under positive evolutionary pressure (host-driven viral evolution). These mutations may reduce the effectiveness of the specific neutralizing immune response against the virus. We provide here evidence that apart from the selection of SARS-CoV-2 variants by the immune system, selection by the cellular receptor can just as well select variants which escape neutralization.

A few days after being infected with SARS-CoV-2, a fraction of people remain asymptomatic but suffer from a decrease in arterial oxygen saturation in the absence of apparent dyspnea. In light of our clinical investigation on the modulation of molecules belonging to the renin angiotensin system (RAS) in COVID-19 patients, we propose a model that explains 'silent hypoxia'. The RAS imbalance caused by SARS-CoV-2 results in an accumulation of angiotensin 2 (Ang II), which activates the angiotensin 2 type 1 receptor (AT1R) and triggers a harmful cascade of intracellular signals leading to the nuclear translocation of the hypoxia-inducible factor (HIF)-1α. HIF-1α transactivates many genes including the angiotensin-converting enzyme 1 (ACE1), while at the same time, ACE2 is downregulated. A growing number of cells is maintained in a hypoxic condition that is self-sustained by the presence of the virus and the ACE1/ACE2 ratio imbalance. This is associated with a progressive worsening of the patient's biological parameters including decreased oxygen saturation, without further clinical manifestations. When too many cells activate the Ang II-AT1R-HIF-1α axis, there is a 'hypoxic spillover', which marks the tipping point between 'silent' and symptomatic hypoxia in the patient. Immediate ventilation is required to prevent the 'hypoxic spillover'.

The tremendous majority of RNA genomes from pathogenic viruses analyzed and deposited in databases are consensus or "democratic" genomes. They represent the genomes most frequently found in the clinical samples of patients but do not account for the huge genetic diversity of coexisting genomes, which is better described as quasispecies. A viral quasispecies is defined as the dynamic distribution of nonidentical but closely related mutants, variants, recombinant, or reassortant viral genomes. Viral quasispecies have collective behavior and dynamics and are the subject of internal interactions that comprise interference, complementation, or cooperation. In the setting of SARS-CoV-2 infection, intrahost SARS-CoV-2 genetic diversity was recently notably reported for immunocompromised, chronically infected patients, for patients treated with monoclonal antibodies targeting the viral spike protein, and for different body compartments of a single patient. A question that deserves attention is whether such diversity is generated postinfection from a clonal genome in response to selection pressure or is already present at the time of infection as a quasispecies. In the present review, we summarize the data supporting that hosts are infected by a "wild bunch" of viruses rather than by multiple virions sharing the same genome. Each virion in the "wild bunch" may have different virulence and tissue tropisms. As the number of viruses replicated during host infections is huge, a viral quasispecies at any time of infection is wide and is also influenced by host-specific selection pressure after infection, which accounts for the difficulty in deciphering and predicting the appearance of more fit variants and the evolution of epidemics of novel RNA viruses.

Having previously shown that soluble E-cadherin (sE-cad) is found in sera of Q fever patients and that infection of BeWo cells by C. burnetii leads to modulation of the E-cad/β-cat pathway, our purpose was to identify which sheddase(s) might catalyze the cleavage of E-cad. Here, we searched for a direct mechanism of cleavage initiated by the bacterium itself, assuming the possible synthesis of a sheddase encoded in the genome of C. burnetii or an indirect mechanism based on the activation of a human sheddase. Using a straightforward bioinformatics approach to scan the complete genomes of four laboratory strains of C. burnetii, we demonstrate that C. burnetii encodes a 451 amino acid sheddase (CbHtrA) belonging to the HtrA family that is differently expressed according to the bacterial virulence. An artificial CbHtrA gene (CoxbHtrA) was expressed, and the CoxbHtrA recombinant protein was found to have sheddase activity. We also found evidence that the C. burnetii infection triggers an over-induction of the human HuHtrA gene expression. Finally, we demonstrate that cleavage of E-cad by CoxbHtrA on macrophages-THP-1 cells leads to an M2 polarization of the target cells and the induction of their secretion of IL-10, which "disarms" the target cells and improves C. burnetii replication. Taken together, these results demonstrate that the genome of C. burnetii encodes a functional HtrA sheddase and establishes a link between the HtrA sheddase-induced cleavage of E-cad, the M2 polarization of the target cells and their secretion of IL-10, and the intracellular replication of C. burnetii.