Role of SARS-CoV-2 mutations in the evolution of the COVID-19 pandemic.

OBJECTIVES: The 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. METHODS: We 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. RESULTS: A 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 1000 nucleotides per gene. They were 3.7-times more frequent in accessory than informational genes (13.8 versus 3.7/1000 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). CONCLUSIONS: most 'hyperfertile' mutations did not predict emergence of a new epidemic, and some were stop codons indicating the existence of so-named 'non-virulence' genes.

Incidental diagnosis of mpox virus infection in patients undergoing sexually transmitted infection screening-findings from a study in France.

OBJECTIVES: This study aimed to investigate the prevalence of mpox virus (MPXV) infections in the general population consulting for routine sexually transmitted infections (STIs) in our Marseille public hospital. In fact, the recent worldwide MPXV outbreak mainly impacted men who have sex with men and the prevalence of MPXV infections in the general population remains poorly defined. METHODS: All samples addressed routinely to our microbiological laboratory for STIs between July 1 and October 15, 2022 were screened with MPXV-specific quantitative polymerase chain reaction. RESULTS: A total of 2688 samples from 1896 patients suspected of having STIs were tested and eight (0.4%) patients were incidentally diagnosed with MPXV infection, including six men and two women. MPXV was detected in rectal swabs (n = 2), urine (n = 2), vaginal swabs (n = 2), a urethral swab (n = 1), and a skin swab (n = 1). CONCLUSIONS: This study suggests that some MPXV infections are likely to be underdiagnosed because of their non-specific clinical presentation and/or insufficient clinical knowledge of the disease. Our data showed that systematic screening was particularly useful for detecting MPXV in patients without classic lesions or cases of asymptomatic carriage in patients reporting recent high-risk exposure and in patients presenting no obvious risk factor.

Influenza at the 2021 Grand Magal of Touba and possible spread to rural villages in South Senegal - a genomic epidemiological study.

OBJECTIVES: Influenza is frequent among pilgrims participating in the Grand Magal de Touba (GMT), in Senegal, with a potential to spread to contacts when they return home. METHODS: Ill pilgrims consulting at a health care center in Mbacké city close to Touba during the 2021 GMT, pilgrims returning to Dielmo and Ndiop villages, and patients who did not travel to Touba and consulted at health care centers in these two villages in 2021 were tested for the influenza virus by polymerase chain reaction on nasopharyngeal samples. Next-generation sequencing and comparative and phylogenetic analyses of influenza A virus genomes were performed. RESULTS: A total of 62 of 685 patients tested positive for influenza A virus, including 34 of 53 who were consulted in Mbacké in late September, six of 129 pilgrims who returned home in early October, and 20 of 42 villagers from October 3 to 29. A total of 27 genomes were obtained. Four clusters were observed based on the phylogenetic analyses, suggesting that Mbacké patients and returned pilgrims may have shared closely related viral strains with patients inhabiting the villages who did not participate in the GMT. CONCLUSIONS: Villagers in Ndiop and Dielmo may have been infected with viral strains originating from the GMT and possibly imported by pilgrims who returned from the GMT.

Resistance-associated mutations to the anti-SARS-CoV-2 agent nirmatrelvir: Selection not induction.

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.

From viral democratic genomes to viral wild bunch of quasispecies.

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.

The return of the "Mistigri" (virus adaptative gain by gene loss) through the SARS-CoV-2 XBB.1.5 chimera that predominated in 2023.

Severe acute respiratory syndrome coronavirus 2 XBB.1.5 is the first recombinant lineage to predominate at the country and global scales. Very interestingly, like the Marseille-4B subvariant (or B.1.160) and the pandemic variant B.1.1.7 (or Alpha) previously, it has its ORF8 gene inactivated by a stop codon. We aimed here to study the distribution of stop codons in ORF8 of XBB.1.5 and non-XBB.1.5 genomes. We identified that a stop codon was present at 89 (74%) ORF8 codons in ≥1 of 15 222 404 genomes available in GISAID. The mean proportion of genomes with a stop codon per codon was 0.11% (range, 0%-7.8%). In addition, a stop codon was detected at 15 (12%) codons in at least 1000 genomes. These 15 codons are notably located on seven stem-loop hairpin regions and in the signal peptide region for the case of the XBB.1.5 lineage (codon 8). Thus, it is very likely that stop codons in ORF8 gene contributed on at least three occasions and independently during the pandemic to the evolutionary success of a lineage that became transiently predominant. Such association of gene loss with evolutionary success, which suits the recently described Mistigri rule, is an important biological phenomenon very unknown in virology while largely described in cellular organisms.

How to distinguish mpox from its mimickers: An observational retrospective cohort study.

During the current global outbreak of mpox (formerly monkeypox), atypical features were frequently described outside endemic areas, raising concerns around differential diagnosis. In this study, we included 372 adult patients who had clinical signs consistent with mpox and who were screened using non-variola orthopoxvirus specific quantitative polymerase chain reaction (PCR) between 15 May and 15 November 2022 at the University Hospital Institute Méditerranée Infection, Marseille, France. At least one clinical sample was positive for 143 (38.4%) of these patients and 229 (61.6%) were negative. Clinically, patients who had mpox presented more frequently with systemic signs (69.9% vs. 31.0%, p < 10-6 ) including fever (51.0% vs. 30.1%, p < 10-3 ), myalgia (33.5% vs. 17.9%, p = 0.002), and lymphadenopathy (38.5% vs. 13.1%, p < 10-6 ). Among the patients who were negative for the non-variola orthopoxvirus, an alternative diagnosis was identified in 58 of them (25.3%), including chickenpox (n = 30, 13.1%), syphilis (n = 9, 4%), bacterial skin infection (n = 8, 3.5%), gonococcus (n = 5, 2.2%), HSV infection (n = 5, 2.2%), and histoplasmosis (n = 1, 0.4%). Overall, in the current outbreak, we show that mpox has a poorly specific clinical presentation. This reinforces the importance of microbiological confirmation. In symptomatic patients who are negative for the monkeypox virus by PCR, a broad differential diagnosis should be maintained.

Scurvy complicated with Capnocytophaga sputigena sepsis as a possible cause of death of king Saint-Louis of France (1270 AD).

The cause of death of Saint-Louis is not known, but recent findings indicated that he presented scurvy and inflammatory jaw disease, which has been associated with infection by oral commensals. Here, we have the exceptional opportunity to analyze the relics of the viscera of King Saint-Louis. A 4.3 g sample from the viscera relics of King Saint-Louis conserved in Versailles' cathedral was subjected to radiocarbon dating, electronic and optic microscopy, and elementary, palynological, molecular, proteomics and microbiological analyses including specific PCR and v3v4 16 S rRNA gene amplification prior to large-scale sequencing using an Illumina MiSeq instrument. The measured radiocarbon age was Cal 1290 CE-1400, which was compatible with that of the viscera of St Louis viscera, considering the addition of lime, incense and vegetables within the human organs. Elemental and palynological analyses confirmed a medieval embalming process. Proteomics analysis identified mainly human muscle and blood proteins. Specific PCR for plague, amoebiasis, shigellosis and typhoid fever was negative. C. sputigena was identified as the main pathogenic species representing 10.8 % of all microbial sequences. In contrast, C. sputigena was found in only 0.001 % of samples sequenced in our center, and the 23 positive human samples showed a dramatically lower abundance (0.02-2.6 %). In the literature, human infections with C. sputigena included odontitis, dental abscess, sinusitis, thoracic infections and bacteremia, particularly in immunocompromised patients with oral and dental diseases consistent with recent analysis of King Saint-Louis' jaw. C. sputigena, a commensal of the mouth that is potentially pathogenic and responsible for fatal bacteremia, may have been the cause of the king's death.

Emergence of a second SARS-CoV-2 variant with a tremendous genetic leap from its ancestors.

The on-going emergence of the Omicron BA.2.86 variant is one of the major events in SARS-CoV-2 genetic evolution that remain enigmatic regarding the overall virus mutation rate, together with the emergence of the initial Omicron variant, BA.1. Indeed, the genomes of the Omicron BA.2.86 lineage, an offspring of the second major Omicron subvariant, BA.2, harbor 39 additional mutations in the spike compared to this ancestor. Here we comment on the phylogeny of BA.2.86, on the positions, and frequencies in other SARS-CoV-2, of mutations in its spike, and on the structural model of this protein that concentrates most of BA.2.86 additional mutations.

Temporal Shift of the Respiratory Syncytial Virus Epidemic Peak for the Years 2020-2023 in Marseille, Southern France.

Respiratory syncytial virus is among the most common causes of respiratory infections. Typically, this viral infection has a seasonality during the cold months but with the SARS-CoV-2 pandemic this has been considerably modified. Here, we studied the epidemiology of this virus in university hospitals of Marseille, South of France, over the period 2020 to 2023. We tested in our laboratory from July 2020 to October 2021 16,516 nasopharyngeal swabs from 16,468 patients for RSV infection using different qPCR assays. We then analyzed data from previous and subsequent winters (from 2018 to 2023) and previous summers (from 2015 to 2021). A total of 676 patients were RSV-positive; their mean age was 3 years and 91 were under 5 years of age. We observed a delay of 4 months of the RSV epidemic's onset compared to other years with an epidemic that peaked in March 2021. We had significantly more RSV-positive cases during summer 2021 compared to previous summers, whereas the incidence of RSV infections was not significantly higher during winter 2022 versus previous winters. Moreover, 494 patients were diagnosed as RSV-positive in the emergency unit and 181 were subsequently hospitalized, and 34 patients were diagnosed RSV-positive while already in the intensive care unit. Over all the study periods, 38 patients diagnosed as RSV-positive died, the majority of whom (23/28) were over 65 years of age. These data show an atypical evolution of the incidence of RSV infections in our city and is another example of the unpredictability of infectious disease epidemiology.

COVID-19 in 16 West African Countries: An Assessment of the Epidemiology and Genetic Diversity of SARS-CoV-2 after Four Epidemic Waves.

West Africa faced the COVID-19 pandemic in early March 2020 and, as of March 31, 2022, had more than 900,000 confirmed cases and more than 12,000 deaths. During this period, SARS-CoV-2 genomes evolved genetically, resulting in the emergence of distinct lineages. This review was conducted to provide the epidemiological profile of COVID-19, the mutational profile of SARS-CoV-2, and the dynamics of its lineages in the 16 west African countries by analyzing data from 33 studies and seven situation reports. For a more complete representation of the epidemiology and genetic diversity of SARS-CoV-2, we used reliable public data in addition to eligible studies. As of March 31, 2022, the 16 west African countries experienced four epidemic waves with variable intensities. Higher mortality was noted during the third wave with a case fatality rate (CFR) of 1.9%. After these four epidemic waves, Liberia recorded the highest CFR (4.0%), whereas Benin had the lowest CFR (0.6%). Through mutational analysis, a high genetic heterogeneity of the genomes was observed, with a predominance of mutations in the spike protein. From this high mutational rate, different lineages emerged. Our analysis of the evolutionary diversity allowed us to count 205 lineages circulating in west Africa. This study has provided a good representation of the mutational profile and the prevalence of SARS CoV-2 lineages beyond the knowledge of the global epidemiology of the 16 African countries.

Origin, Diversity, and Multiple Roles of Enzymes with Metallo-ß-Lactamase Fold from Different Organisms.

ß-lactamase enzymes have generated significant interest due to their ability to confer resistance to the most commonly used family of antibiotics in human medicine. Among these enzymes, the class B ß-lactamases are members of a superfamily of metallo-ß-lactamase (MßL) fold proteins which are characterised by conserved motifs (i.e., HxHxDH) and are not only limited to bacteria. Indeed, as the result of several barriers, including low sequence similarity, default protein annotation, or untested enzymatic activity, MßL fold proteins have long been unexplored in other organisms. However, thanks to search approaches which are more sensitive compared to classical Blast analysis, such as the use of common ancestors to identify distant homologous sequences, we are now able to highlight their presence in different organisms including Bacteria, Archaea, Nanoarchaeota, Asgard, Humans, Giant viruses, and Candidate Phyla Radiation (CPR). These MßL fold proteins are multifunctional enzymes with diverse enzymatic or non-enzymatic activities of which, at least thirteen activities have been reported such as ß-lactamase, ribonuclease, nuclease, glyoxalase, lactonase, phytase, ascorbic acid degradation, anti-cancer drug degradation, or membrane transport. In this review, we (i) discuss the existence of MßL fold enzymes in the different domains of life, (ii) present more suitable approaches to better investigating their homologous sequences in unsuspected sources, and (iii) report described MßL fold enzymes with demonstrated enzymatic or non-enzymatic activities.

Genomic Epidemiology of SARS-CoV-2 in Urban Settings in Senegal.

We used whole genome sequencing to identify and analyze mutations in SARS-CoV-2 in urban settings during the deadliest wave of the COVID-19 epidemic-from March to April 2021-in Senegal. Nasopharyngeal samples testing positive for SARS-CoV-2 were sequenced on the Illumina NovaSeq 6000 sequencing system using the COVIDSeq protocol. A total of 291 genotypable consensus genome sequences were obtained. Phylogenetic analyses grouped the genomes into 16 distinct PANGOLIN lineages. The major lineage was B.1.1.420, despite circulation of the Alpha variant of concern (VOC). A total of 1125 different SNPs, identified relative to the Wuhan reference genome, were detected. These included 13 SNPs in non-coding regions. An average density of 37.2 SNPs per 1000 nucleotides was found, with the highest density occurring in ORF10. This analysis allowed, for the first time, the detection of a Senegalese SARS-CoV-2 strain belonging to the P.1.14 (GR/20J, Gamma V3) sublineage of the Brazilian P.1 lineage (or Gamma VOC). Overall, our results highlight substantial SARS-CoV-2 diversification in Senegal during the study period.

Sequencing of monkeypox virus from infected patients reveals viral genomes with APOBEC3-like editing, gene inactivation, and bacterial agents of skin superinfection.

A large outbreak of Monkeypox virus (MPXV) infections has arisen in May 2022 in nonendemic countries. Here, we performed DNA metagenomics using next-generation sequencing with Illumina or Nanopore technologies for clinical samples from MPXV-infected patients diagnosed between June and July 2022. Classification of the MPXV genomes and determination of their mutational patterns were performed using Nextclade. Twenty-five samples from 25 patients were studied. A MPXV genome was obtained for 18 patients, essentially from skin lesions and rectal swabbing. All 18 genomes were classified in clade IIb, lineage B.1, and we identified four B.1 sublineages (B.1.1, B.1.10, B.1.12, B.1.14). We detected a high number of mutations (range, 64-73) relatively to a 2018 Nigerian genome (genome GenBank Accession no. NC_063383.1), which were harbored by a large part of a set of 3184 MPXV genomes of lineage B.1 recovered from GenBank and Nextstrain; and we detected 35 mutations relatively to genome ON563414.3 (a B.1 lineage reference genome). Nonsynonymous mutations occurred in genes encoding central proteins, among which transcription factors and core and envelope proteins, and included two mutations that would truncate a RNA polymerase subunit and a phospholipase d-like protein, suggesting an alternative start codon and gene inactivation, respectively. A large majority (94%) of nucleotide substitutions were G > A or C > U, suggesting the action of human APOBEC3 enzymes. Finally, >1000 reads were identified as from Staphylococcus aureus and Streptococcus pyogenes for 3 and 6 samples, respectively. These findings warrant a close genomic monitoring of MPXV to get a better picture of the genetic micro-evolution and mutational patterns of this virus, and a close clinical monitoring of skin bacterial superinfection in monkeypox patients.

Aetiology of Acute Undifferentiated Fever Among Children Under the Age of Five in Vietnam: A Prospective Study.

BACKGROUND: To investigate the aetiology of acute undifferentiated fever (AUF) among children under the age of five in Vietnam. METHODS: This prospective study was conducted in the Thai Binh paediatric hospital, between July 2020 and July 2021 among children with AUF at admission. Real-time PCR testing 18 microbial pathogens were done on blood samples. RESULTS: 286 children were included, with median age of 16 months. 64.7% were male. 53.9% were positive for at least one pathogen by PCR. Enterovirus, human herpesvirus 6, adenovirus, and varicella zoster virus PCR were positive for 31.1, 12.6, 1.4, and 1.0% patients, respectively. Other pathogens tested negative by PCR. During the hospital stay, based on clinical criteria 47.2% children secondarily presented with signs of respiratory tract infections, 18.9% had hand, foot and mouth disease, 4.6% had chickenpox. 4.2% presented signs of central nervous system infections, 1.0% had dengue (antigenic test) and 1.0% had signs of gastrointestinal infection. Finally, 23.1% patients presented a fever with or without a rash and no other symptoms and ultimately received a diagnosis of AUF. CONCLUSION: Real-time PCR of blood is useful for detecting pathogens and diagnosing infectious causes of AUF. Further prospective studies with blood and urine culture testing and PCR investigation of not only blood but also cerebrospinal fluid, throat, and skin samples according to symptoms would be of interest to confirm the predominance of viral infections in children with AUF and to guide therapeutic options.

Protection from successive Omicron variants with SARS-CoV-2 vaccine and monoclonal antibodies in kidney transplant recipients.

Introduction: Kidney transplant recipients (KTRs) are at high risk of severe COVID-19, even when they are fully vaccinated. Additional booster vaccinations or passive immunization with prophylactic monoclonal antibodies are recommended to increase their protection against severe COVID-19. Methods: Here, we describe the neutralization of SARS-CoV-2 Delta, Omicron BA.1, BA.2, BA.4, and BA.5 variants, firstly by 39 serum samples from vaccinated KTRs exhibiting anti-spike antibody concentrations ≥264 binding antibody units (BAU)/mL and, secondly, by tixagevimab/cilgavimab. Results: No neutralization was observed for 18% of the KTRs, while serum from only 46% of patients could neutralize the five variants. Cross-neutralization of the Delta and Omicron variants occurred for 65-87% of sera samples. The anti-spike antibody concentration correlated with neutralization activity for all the variants. The neutralization titers against the Delta variant were higher in vaccinated KTRs who had previously presented with COVID-19, compared to those KTRs who had only been vaccinated. Breakthrough infections occurred in 39% of the KTRs after the study. Tixagevimab/cilgavimab poorly neutralizes Omicron variants, particularly BA.5, and does not neutralize BQ.1, which is currently the most prevalent strain. Discussion: As a result, sera from seropositive vaccinated KTRs had poor neutralization of the successive Omicron variants. Several Omicron variants are able to escape tixagevimab/cilgavimab.

Correlation of Redondovirus and Entamoeba gingivalis Detections in the Human Oral Cavity Suggests That This Amoeba Is Possibly the Redondovirus Host.

The virome of the human oral cavity and the relationships between viruses and diseases such as periodontitis are scarcely deciphered. Redondoviruses were reported in the human oral cavity in 2019, including in periodontitis patients. Here, we aimed at detecting redondoviruses and at searching for a potential viral host in human saliva. Non-stimulated saliva was collected between December 2020 and June 2021. These samples were tested using real-time PCR regarding the presence of redondovirus and Entamoeba gingivalis DNA. Similarity searches were performed using BLAST against eukaryotic and prokaryotic sequences from GenBank. The redondovirus DNA was detected in 46% of the 28 human saliva samples. In addition, short fragments of redondovirus genomes were detected in silico within Entamoeba sequences. Finally, Entamoeba gingivalis DNA was detected in 46% of the 28 saliva samples, with a strong correlation between redondovirus DNA and E. gingivalis DNA detections, in 93% of the cases. Regarded together, these findings and previous ones strongly support the presence of redondoviruses in the human oral cavity and their association to E. gingivalis as their likely host.