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.

COVID-19 Is a Confounder of Increased Candida Airway Colonisation.

An increased incidence of invasive fungal infection was reported in SARS-CoV-2-infected patients hospitalised in the intensive care unit. However, the impact of COVID-19 on Candida airway colonisation has not yet been assessed. This study aimed to test the impact of several factors on Candida airway colonisation, including SARS-CoV-2 infection. We conducted a two-pronged monocentric retrospective study. First, we analysed the prevalence of positive yeast culture in respiratory samples obtained from 23 departments of the University Hospital of Marseille between 1 January 2018 and 31 March 2022. We then conducted a case-control study, comparing patients with documented Candida airway colonisation to two control groups. We observed an increase in the prevalence of yeast isolation over the study period. The case-control study included 300 patients. In the multivariate logistic regression, diabetes, mechanical ventilation, length of stay in the hospital, invasive fungal disease, and the use of antibacterials were independently associated with Candida airway colonisation. The association of SARS-CoV-2 infection with an increased risk of Candida airway colonisation is likely to be a consequence of confounding factors. Nevertheless, we found the length of stay in the hospital, mechanical ventilation, diabetes, and the use of antibacterials to be statistically significant independent risk factors of Candida airway colonisation.

The emergence, spread and vanishing of a French SARS-CoV-2 variant exemplifies the fate of RNA virus epidemics and obeys the Mistigri rule.

The nature and dynamics of mutations associated with the emergence, spread, and vanishing of SARS-CoV-2 variants causing successive waves are complex. We determined the kinetics of the most common French variant ("Marseille-4") for 10 months since its onset in July 2020. Here, we analyzed and classified into subvariants and lineages 7453 genomes obtained by next-generation sequencing. We identified two subvariants, Marseille-4A, which contains 22 different lineages of at least 50 genomes, and Marseille-4B. Their average lifetime was 4.1 ± 1.4 months, during which 4.1 ± 2.6 mutations accumulated. Growth rate was 0.079 ± 0.045, varying from 0.010 to 0.173. Most of the lineages exhibited a bell-shaped distribution. Several beneficial mutations at unpredicted sites initiated a new outbreak, while the accumulation of other mutations resulted in more viral heterogenicity, increased diversity and vanishing of the lineages. Marseille-4B emerged when the other Marseille-4 lineages vanished. Its ORF8 gene was knocked out by a stop codon, as reported in SARS-CoV-2 of mink and in the Alpha variant. This subvariant was associated with increased hospitalization and death rates, suggesting that ORF8 is a nonvirulence gene. We speculate that the observed heterogenicity of a lineage may predict the end of the outbreak.

Investigation of a COVID-19 outbreak on the Charles de Gaulle aircraft carrier, March to April 2020: a retrospective cohort study.

BackgroundSARS-CoV-2 emergence was a threat for armed forces. A COVID-19 outbreak occurred on the French aircraft carrier Charles de Gaulle from mid-March to mid-April 2020.AimTo understand how the virus was introduced, circulated then stopped circulation, risk factors for infection and severity, and effectiveness of preventive measures.MethodsWe considered the entire crew as a cohort and collected personal, clinical, biological, and epidemiological data. We performed viral genome sequencing and searched for SARS-CoV-2 in the environment.ResultsThe attack rate was 65% (1,148/1,767); 1,568 (89%) were included. The male:female ratio was 6.9, and median age was 29 years (IQR: 24-36). We examined four clinical profiles: asymptomatic (13.0%), non-specific symptomatic (8.1%), specific symptomatic (76.3%), and severe (i.e. requiring oxygen therapy, 2.6%). Active smoking was not associated with severe COVID-19; age and obesity were risk factors. The instantaneous reproduction rate (Rt) and viral sequencing suggested several introductions of the virus with 4 of 5 introduced strains from within France, with an acceleration of Rt when lifting preventive measures. Physical distancing prevented infection (adjusted OR: 0.55; 95% CI: 0.40-0.76). Transmission may have stopped when the proportion of infected personnel was large enough to prevent circulation (65%; 95% CI: 62-68).ConclusionNon-specific clinical pictures of COVID-19 delayed detection of the outbreak. The lack of an isolation ward made it difficult to manage transmission; the outbreak spread until a protective threshold was reached. Physical distancing was effective when applied. Early surveillance with adapted prevention measures should prevent such an outbreak.

Control of common viral epidemics but not of SARS-CoV-2 through the application of hygiene and distancing measures.

BACKGROUND: We systematically survey respiratory and gastrointestinal infections of viral origin in samples sent to our university hospital institute in Marseille, southern France. Here, we evaluated whether the measures implemented to fight COVID-19 had an effect on the dynamics of viral respiratory or gastrointestinal infections. METHODS: We analysed PCR performed and positive for the diagnoses of viral respiratory and gastrointestinal infections over five years (January 2017-February 2021). Data were collected from our epidemiological surveillance system (MIDaS). Dates and contents of French measures against SARS-CoV-2 were collected from: https://www.gouvernement.fr/info-coronavirus/les-actions-du-gouvernement. RESULTS: Over the 2017-2021 period, 990,364 analyses were carried out for respiratory infections not including SARS-CoV-2, 510,671 for SARS-CoV-2 and 27,719 for gastrointestinal infections. During winter 2020-2021, when the most restrictive lockdown measures were in place in France, a marked decrease of infections with influenza viruses (one case versus 1,839-1,850 cases during 2017-2020 cold seasons) and with the RSV (56 cases versus 988-1,196 cases during 2017-2020 cold seasons) was observed, demonstrating the relative effectiveness of these measures on their occurrence. SARS-CoV-2 incidence seemed far less affected. Rhinoviruses, parainfluenza 3 virus, and the coronavirus NL63 remained at comparable levels. Also, the norovirus winter season positivity rates decreased continuously and significantly over time from 9.3% in 2017-2018 to 2.0% in 2020-2021. CONCLUSION: The measures taken to control COVID-19 were effective against lower respiratory tract infections viruses and gastroenteritis agents, but not on the agents of the common winter cold and SARS-CoV-2. This suggests that more specific measures to prevent COVID-19 and upper respiratory tract infections need to be discovered to limit the spread of this epidemic.

Antilogic, a new supervised machine learning software for the automatic interpretation of antibiotic susceptibility testing in clinical microbiology: proof-of-concept on three frequently isolated bacterial species.

OBJECTIVE: Antibiotic susceptibility testing (AST) is necessary in order to adjust empirical antibiotic treatment, but the interpretation of results requires experience and knowledge. We have developed a machine learning software that is capable of reading AST images without any human intervention and that automatically interprets the AST, based on a database of antibiograms that have been clinically validated with European Committee on Antimicrobial Susceptibility Testing rules. METHODS: We built a database of antibiograms that were labelled by senior microbiologists for three species: Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus. We then developed Antilogic, a Python software based on an original image segmentation module and supervised learning models that we trained against the database. Finally, we blind tested Antilogic against a validation set of 5100 photos of antibiograms. RESULTS: We trained Antilogic against a database of 18072 pictures of antibiograms. Overall agreement against the validation set reached 97% (16 855/17 281) regarding phenotypes. The severity rate of errors was also evaluated: 1.66% (287/17 281) were major errors and 0.80% (136/17 281) were very major errors. After implementation of uncertainty quantifications, the rate of errors decreased to 0.80% (114/13 451) and 0.42% (51/13 451) for major and very major errors respectively. DISCUSSION: Antilogic is the first machine learning software that has been developed for AST interpretation. It is based on a novel approach that differs from the typical diameter measurement and expert system approach. Antilogic is a proof of concept that artificial intelligence can contribute to faster and easier diagnostic methods in the field of clinical microbiology.

Using MALDI-TOF spectra in epidemiological surveillance for the detection of bacterial subgroups with a possible epidemic potential.

BACKGROUND: For the purpose of epidemiological surveillance, the Hospital University Institute Méditerranée infection has implemented since 2013 a system named MIDaS, based on the systematic collection of routine activity materials, including MALDI-TOF spectra, and results. The objective of this paper is to present the pipeline we use for processing MALDI-TOF spectra during epidemiological surveillance in order to disclose proteinic cues that may suggest the existence of epidemic processes in complement of incidence surveillance. It is illustrated by the analysis of an alarm observed for Streptococcus pneumoniae. METHODS: The MALDI-TOF spectra analysis process looks for the existence of clusters of spectra characterized by a double time and proteinic close proximity. This process relies on several specific methods aiming at contrasting and clustering the spectra, presenting graphically the results for an easy epidemiological interpretation, and for determining the discriminating spectra peaks with their possible identification using reference databases. RESULTS: The use of this pipeline in the case of an alarm issued for Streptococcus pneumoniae has made it possible to reveal a cluster of spectra with close proteinic and temporal distances, characterized by the presence of three discriminant peaks (5228.8, 5917.8, and 8974.3 m/z) and the absence of peak 4996.9 m/z. A further investigation on UniProt KB showed that peak 5228.8 is possibly an OxaA protein and that the absent peak may be a transposase. CONCLUSION: This example shows this pipeline may support a quasi-real time identification and characterization of clusters that provide essential information on a potentially epidemic situation. It brings valuable information for epidemiological sensemaking and for deciding on the continuation of the epidemiological investigation, in particular the involving of additional costly resources to confirm or invalidate the alarm. CLINICAL TRIALS REGISTRATION: NCT03626987.

Early combination therapy with hydroxychloroquine and azithromycin reduces mortality in 10,429 COVID-19 outpatients.

We evaluated the age-specific mortality of unselected adult outpatients infected with SARS-CoV-2 treated early in a dedicated COVID-19 day hospital and we assessed whether the use of hydroxychloroquine (HCQ) + azithromycin (AZ) was associated with improved survival in this cohort. A retrospective monocentric cohort study was conducted in the day hospital of our center from March to December 2020 in adults with PCR-proven infection who were treated as outpatients with a standardized protocol. The primary endpoint was 6-week mortality, and secondary endpoints were transfer to the intensive care unit and hospitalization rate. Among 10,429 patients (median age, 45 [IQR 32-57] years; 5597 [53.7%] women), 16 died (0.15%). The infection fatality rate was 0.06% among the 8315 patients treated with HCQ+AZ. No deaths occurred among the 8414 patients younger than 60 years. Older age and male sex were associated with a higher risk of death, ICU transfer, and hospitalization. Treatment with HCQ+AZ (0.17 [0.06-0.48]) was associated with a lower risk of death, independently of age, sex and epidemic period. Meta-analysis evidenced consistency with 4 previous outpatient studies (32,124 patients-Odds ratio 0.31 [0.20-0.47], I2 = 0%). Early ambulatory treatment of COVID-19 with HCQ+AZ as a standard of care is associated with very low mortality, and HCQ+AZ improve COVID-19 survival compared to other regimens.

Consequences of the COVID-19 Outbreak Lockdown on Non-Viral Infectious Agents as Reported by a Laboratory-Based Surveillance System at the IHU Méditerranée Infection, Marseille, France.

The objective of this paper is to describe the surveillance system MIDaS and to show how this system has been used for evaluating the consequences of the French COVID-19 lockdown on the bacterial mix of AP-HM and the antibiotic resistance. MIDas is a kind of surveillance activity hub, allowing the automatic construction of surveillance control boards. We investigated the diversity and resistance of bacterial agents from respiratory, blood, and urine samples during the lockdown period (from week 12 to 35 of 2020), using the same period of years from 2017 to 2019 as control. Taking into account the drop in patient recruitment, several species have exhibited significant changes in their relative abundance (either increasing or decreasing) with changes up to 9%. The changes were more important for respiratory and urine samples than for blood samples. The relative abundance in respiratory samples for the whole studied period was higher during the lockdown. A significant increase in the percentage of wild phenotypes during the lockdown was observed for several species. The use of the MIDaS syndromic collection and surveillance system made it possible to efficiently detect, analyze, and follow changes of the microbiological population as during the lockdown period.

Evaluation of Strategies to Fight COVID-19: The French Paradigm.

(1) Background: We collected COVID-19 mortality data and the age distribution of the deceased in France and other European countries, as well as specifically in the cities of Paris and Marseille, and compared them. (2) Methods: Data on mortality related to COVID-19 and the associated age distribution were collected from government institutions in various European countries. In France, data were obtained from INSEE and Santé Publique France. All-cause mortality was also examined in order to study potential excess mortality using EuroMOMO. The Marseille data came from the epidemiological surveillance system. (3) Results: France is one of the European countries most impacted by COVID-19. Its proportion of deaths in people under 60 years of age is higher (6.5%) than that of Italy (4.6%) or Spain (4.7%). Excess mortality (5% more deaths) was also observed. Ile-de-France and the Grand-Est are the two French regions with the highest mortality. The proportion of deaths in the under-60 age group was considerable in Ile-de-France (9.9% vs. 4.5% in the Southern region). There are significantly higher numbers of patients hospitalized, in intensive care and deceased in Paris than in Marseille. (4) Conclusions: No patient management, i.e., from screening to diagnosis, including biological assessment and clinical examination, likely explains the high mortality associated with COVID-19.

Viral RNA in City Wastewater as a Key Indicator of COVID-19 Recrudescence and Containment Measures Effectiveness.

In recent years, and more specifically at the beginning of the COVID-19 crisis, wastewater surveillance has been proposed as a tool to monitor the epidemiology of human viral infections. In the present work, from July to December 2020, the number of copies of SARS-CoV-2 RNA in Marseille's wastewater was correlated with the number of new positive cases diagnosed in our Institute of Infectious Disease, which tested about 20% of the city's population. Number of positive cases and number of copies of SARS-CoV-2 RNA in wastewater were significantly correlated (p = 0.013). During the great epidemic peak, from October to December 2020, the curves of virus in the sewers and the curves of positive diagnoses were perfectly superposed. During the summer period, the superposition of curves was less evident as subject to many confounding factors that were discussed. We also tried to correlate the effect of viral circulation in wastewater with containment measures, probably the most unbiased correlation on their potential inflection effect of epidemic curves. Not only is this correlation not obvious, but it also clearly appears that the drop in cases as well as the drop in the viral load in the sewers occur before the containment measures. In fact, this suggests that there are factors that initiate the end of the epidemic peak independently of the containment measure. These factors will therefore need to be explored more deeply in the future.

Clinical outcomes in COVID-19 patients infected with different SARS-CoV-2 variants in Marseille, France.

OBJECTIVES: To compare the clinical and epidemiological aspects associated with different predominant lineages circulating in Marseille from March 2020 to January 2021. METHODS: In this single-centre retrospective cohort study, characteristics of patients infected with four different severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants were documented from medical files. The outcome was the occurrence of clinical failure, defined as hospitalization (for outpatients), transfer to the intensive care unit (inpatients) and death (all). RESULTS: A total of 254 patients were infected with clade 20A (20AS), 85 with Marseille-1 (M1V), 190 with Marseille-4 (M4V) and 211 with N501Y (N501YV) variants. 20AS presented a bell-shaped epidemiological curve and nearly disappeared around May 2020. M1V reached a very weak peak, then disappeared after six weeks. M4V appeared in July presented an atypical wave form for 7 months. N501YV has only recently appeared. Compared with 20AS, patients infected with M1V were less likely to report dyspnoea (adjusted odds ratio (OR) 0.50, p 0.04), rhinitis (aOR 0.57, p 0.04) and to be hospitalized (aOR 0.22, p 0.002). Patients infected with M4V were more likely to report fever than those with 20AS and M1V (aOR 2.49, p < 0.0001 and aOR 2.30, p 0.007, respectively) and to be hospitalized than those with M1V (aOR 4.81, p 0.003). Patients infected with N501YV reported lower rate of rhinitis (aOR 0.50, p 0.001) and anosmia (aOR 0.57, p 0.02), compared with those infected with 20AS. A lower rate of hospitalization was associated with N501YV infection compared with 20AS and M4V (aOR 0.33, p < 0.0001 and aOR 0.27, p < 0.0001, respectively). CONCLUSIONS: The four lineages have presentations that differ from one another, epidemiologically and clinically. This supports SARS-CoV-2 genomic surveillance through next-generation sequencing.

A COVID-19 outbreak on board ship: Analysis of the sociotechnical system of epidemiological management in the French Navy.

In late 2019, an epidemic of SARS-CoV-2 broke out in central China. Within a few months, this new virus had spread right across the globe, officially being classified as a pandemic on 11 March 2020. In France, which was also being affected by the virus, the government applied specific epidemiological management strategies and introduced unprecedented public health measures. This article describes the outbreak management system that was applied within the French military and, more specifically, analyzes an outbreak of COVID-19 that occurred on board a nuclear aircraft carrier. We applied the AcciMap systemic analysis approach to understand the course of events that led to the outbreak and identify the relevant human and organizational failures. Results highlight causal factors at several levels of the outbreak management system. They reveal problems with the benchmarks used for diagnosis and decision-making, and underscore the importance of good communication between different levels. We discuss ways of improving epidemiological management in military context.

Sputum proteomic analysis for distinguishing between pulmonary tuberculosis and non-tuberculosis using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS): preliminary results.

OBJECTIVES: The aim was to evaluate the feasibility and diagnostic contribution of protein profiling using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) applied to sputum to diagnose pulmonary tuberculosis. METHODS: Sputum samples collected from patients suspected of having pulmonary tuberculosis were analysed using MALDI-TOF MS. Using the differentially expressed protein peaks, we compared three groups of patients, including those with confirmed pulmonary tuberculosis (PTB), those without tuberculosis but with a lower respiratory tract infection (non-TB LRTI) and those without tuberculosis and without an LRTI (non-TB controls). RESULTS: A total of 102 patients included 35 PTB, 36 non-TB LRTI and 31 non-TB controls. The model differentiated between the PTB patients and the non-TB controls using the 25 most differentially expressed protein peaks, with a sensitivity of 97%, 95% CI 85-100%, and a specificity of 77%, 95% CI 59-90%. The model distinguished the PTB patients from the non-TB LRTI patients using the ten most differentially expressed protein peaks, with a sensitivity of 80%, 95% CI 63-92%, and a specificity of 89%, 95% CI 74-97%. We observed that the negative predictive value of MALDI-TOF MS sputum analysis was higher (96%, 95% CI 80-100%) than that of direct sputum microscopic examination and sputum culture (78%, 95% CI 62-89%) for non-TB controls. When MALDI-TOF MS sputum analysis and direct microscopic examination were combined, the negative predictive value reached 94%, 95% CI 80-99%, for non-TB LRTI patients. DISCUSSION: These results suggest that MALDI-TOF MS sputum analysis coupled with microscopic examination could be used as a screening tool for diagnosing pulmonary TB.

Introduction into the Marseille geographical area of a mild SARS-CoV-2 variant originating from sub-Saharan Africa: An investigational study.

BACKGROUND: In Marseille, France, the COVID-19 incidence evolved unusually with several successive epidemic phases. The second outbreak started in July, was associated with North Africa, and involved travelers and an outbreak on passenger ships. This suggested the involvement of a new viral variant. METHODS: We sequenced the genomes from 916 SARS-CoV-2 strains from COVID-19 patients in our institute. The patients' demographic and clinical features were compared according to the infecting viral variant. RESULTS: From June 26th to August 14th, we identified a new viral variant (Marseille-1). Based on genome sequences (n = 89) or specific qPCR (n = 53), 142 patients infected with this variant were detected. It is characterized by a combination of 10 mutations located in the nsp2, nsp3, nsp12, S, ORF3a, ORF8 and N/ORF14 genes. We identified Senegal and Gambia, where the virus had been transferred from China and Europe in February-April as the sources of the Marseille-1 variant, which then most likely reached Marseille through Maghreb when French borders reopened. In France, this variant apparently remained almost limited to Marseille. In addition, it was significantly associated with a milder disease compared to clade 20A ancestor strains, in univariate analysis. CONCLUSION: Our results demonstrate that SARS-CoV-2 can genetically diversify rapidly, its variants can diffuse internationally and cause successive outbreaks.

Different pattern of the second outbreak of COVID-19 in Marseille, France.

OBJECTIVE: To describe the characteristics of COVID-19 patients seen in March-April and June-August 2020 in Marseille, France with the aim to investigate possible changes in the disease between these two time periods. METHODS: Demographics, hospitalization rate, transfer to intensive care unit (ICU), lethality, clinical and biological parameters were investigated. RESULTS: Compared to those seen in March-April, COVID-19 patients seen in June-August were significantly younger (39.2 vs. 45.3 years), more likely to be male (52.9% vs. 45.6%), and less likely to be hospitalized (10.7 vs. 18.0%), to be transferred to ICU (0.9% vs. 1.8%) and to die (0.1% vs. 1.1%). Their mean fibrinogen and D-dimer blood levels were lower (1.0 vs. 1.5 g/L and 0.6 vs. 1.1 µg/mL, respectively). By contrast, their viral load was higher (cycle threshold ≤16 = 5.1% vs. 3.7%). CONCLUSIONS: Patients in the two periods did not present marked age and sex differences, but markers of severity were undoubtedly less prevalent in the summer period, associating with a 10 times decrease in the lethality rate.

Decision-making during nonroutine outbreak management: Toward an exploration of experts' creative decisions.

Decision-making during critical outbreak management may require standard strategies, but also more creative ones. Our goal was to characterize the expert decision processes that take place during critical situations, where rule-based strategies and usual procedures cannot be satisfactorily applied. More specifically, we focused on the strategies experts use to deal with epidemiological problems, depending on the complexity of the situation. To this end, we carried out a simulated outbreak alert, to place two experts in a situation of epidemiological problem management, based on usual practice but also conducive to implementing creative solutions. To analyze the data, we considered not only the relevance of the solutions proposed by the experts, but also the four creativity criteria defined by Torrance (fluency, flexibility, elaboration and originality). Results allowed us to identify similarities but also differences between the solutions proposed by the experts, depending on their level of experience in this area.

Analysis of SARS-CoV-2 Variants From 24,181 Patients Exemplifies the Role of Globalization and Zoonosis in Pandemics.

After the end of the first epidemic episode of SARS-CoV-2 infections, as cases began to rise again during the summer of 2020, we at IHU Méditerranée Infection in Marseille, France, intensified the genomic surveillance of SARS-CoV-2, and described the first viral variants. In this study, we compared the incidence curves of SARS-CoV-2-associated deaths in different countries and reported the classification of SARS-CoV-2 variants detected in our institute, as well as the kinetics and sources of the infections. We used mortality collected from a COVID-19 data repository for 221 countries. Viral variants were defined based on ≥5 hallmark mutations along the whole genome shared by ≥30 genomes. SARS-CoV-2 genotype was determined for 24,181 patients using next-generation genome and gene sequencing (in 47 and 11% of cases, respectively) or variant-specific qPCR (in 42% of cases). Sixteen variants were identified by analyzing viral genomes from 9,788 SARS-CoV-2-diagnosed patients. Our data show that since the first SARS-CoV-2 epidemic episode in Marseille, importation through travel from abroad was documented for seven of the new variants. In addition, for the B.1.160 variant of Pangolin classification (a.k.a. Marseille-4), we suspect transmission from farm minks. In conclusion, we observed that the successive epidemic peaks of SARS-CoV-2 infections are not linked to rebounds of viral genotypes that are already present but to newly introduced variants. We thus suggest that border control is the best mean of combating this type of introduction, and that intensive control of mink farms is also necessary to prevent the emergence of new variants generated in this animal reservoir.