Limited permissibility of ENL-R and Mv-1-Lu mink cell lines to SARS-CoV-2.

The SARS-CoV-2 pandemic started in the end of 2019 in Wuhan, China, which highlighted the scenario of frequent cross-species transmission events. From the outbreak possibly initiated by viral spill-over into humans from an animal reservoir, now we face the human host moving globally while interacting with domesticated and peridomestic animals. The emergence of a new virus into the ecosystem leads to selecting forces and species-specific adaptations. The adaptation of SARS-CoV-2 to other animals represents a risk to controlling the dissemination of this coronavirus and the emergence of new variants. Since 2020, several mink farms in Europe and the United States have had SARS-CoV-2 outbreaks with human-mink and mink-human transmission, where the mink-selected variants possibly hold evolutionary concerning advantages. Here we investigated the permissibility of mink lung-derived cells using two cell lines, Mv-1-Lu and ENL-R, against several lineages of SARS-CoV-2, including some classified as variants of concern. The viral release rate and the infectious titers indicate that these cells support infections by different SARS-CoV-2 lineages. The viral production occurs in the first few days after infection with the low viral release by these mink cells, which is often absent for the omicron variant for lung cells. The electron microscopy reveals that during the viral replication cycle, the endomembrane system of the mink-host cell undergoes typical changes while the viral particles are produced, especially in the first days of infection. Therefore, even if limited, mink lung cells may represent a selecting source for SARS-CoV-2 variants, impacting their transmissibility and pathogenicity and making it difficult to control this new coronavirus.

Screening and Whole Genome Sequencing of SARS-CoV-2 Circulating During the First Three Waves of the COVID-19 Pandemic in Libreville and the Haut-Ogooué Province in Gabon.

Since the onset of the COVID-19 pandemic, the SARS-CoV-2 viral dynamics in Africa have been less documented than on other continents. In Gabon, a Central African country, a total number of 37,511 cases of COVID-19 and 281 deaths have been reported as of December 8, 2021. After the first COVID-19 case was reported on March 12, 2020, in the capital Libreville, the country experienced two successive waves. The first one, occurred in March 2020 to August 2020, and the second one in January 2021 to May 2021. The third wave began in September 2021 and ended in November 2021. In order to reduce the data gap regarding the dynamics of SARS-CoV-2 in Central Africa, we performed a retrospective genotyping study using 1,006 samples collected from COVID-19 patients in Gabon from 2020 to 2021. Using SARS-CoV-2 variant screening by Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) and whole genome sequencing (WGS), we genotyped 809 SARS-CoV-2 samples through qRT-PCR and identified to generated 291 new genomes. It allowed us to describe specific mutations and changes in the SARS-CoV-2 variants in Gabon. The qRT-PCR screening of 809 positive samples from March 2020 to September 2021 showed that 119 SARS-CoV-2 samples (14.7%) were classified as VOC Alpha (Pangolin lineage B.1.1.7), one (0.1%) was a VOC Beta (B.1.351), and 198 (24.5 %) were VOC Delta (B.1.617.2), while 491 samples (60.7%) remained negative for the variants sought. The B1.1 variant was predominant during the first wave while the VOC Alpha dominated the second wave. The B1.617.2 Delta variant is currently the dominant variant of the third wave. Similarly, the analysis of the 291 genome sequences indicated that the dominant variant during the first wave was lineage B.1.1, while the dominant variants of the second wave were lineages B.1.1.7 (50.6%) and B.1.1.318 (36.4%). The third wave started with the circulation of the Delta variant (B.1.617). Finally, we compared these results to the SARS-CoV-2 sequences reported in other African, European, American and Asian countries. Sequences of Gabonese SARS-CoV-2 strains presented the highest similarities with those of France, Belgium and neighboring countries of Central Africa, as well as West Africa.

Evidence of antibodies against SARS-CoV-2 in wild mustelids from Brittany (France).

In the French region of Brittany, mainly in the department of the Côtes d'Armor, during the first half of 2021, seropositivity for SARS-CoV-2 was detected in five wild mustelids out of 33 animals tested (15.6%). Anti-SARS-CoV-2 IgG was detected against at least four out of five recombinant viral proteins (S1 receptor binding domain, nucleocapsid, S1 subunit, S2 subunit and spike) in three pine martens (Martes martes) and in two badgers (Meles meles) using the automated western blot technique. An ELISA test also identified seropositive cases, although these did not align with western blot results. Although the 171 qPCRs carried out on samples from the 33 mustelids were all negative, these preliminary results from this observational study nevertheless bear witness to infections of unknown origin. The epidemiological surveillance of Covid-19 in wildlife must continue, in particular with effective serology tools.

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.

Seroprevalence of SARS-CoV-2 among high-density communities and hyper-endemicity of COVID-19 in Vietnam.

OBJECTIVE: To assess the magnitude of active and recovering COVID-19 patients among at-risk communities and to identify the factors associated with positive serology. METHODS: Four hundred and eighty-three close contacts of COVID-19 patients residing in Ho Chi Minh City, Vietnam, during the fourth wave of the COVID-19 epidemic (September and October 2021) were included. Five weeks after exposure to a COVID-19 patient, they underwent a serology test using the BIOSYNEX COVID-19 BSS kit. RESULTS: The median age of participants was 37 years. A total of 34.6% individuals presented at least one clinical symptom between the time of contact with the COVID-19 patient and inclusion in study. A total of 1.7% unvaccinated individuals tested positive for SARS-CoV-2 using real-time PCR, and 9.5% had evidence of recent infection (positive PCR and/or IgM). A further 26.7% unvaccinated individuals presented evidence of a past infection (positive IgG only). Socio-demographic characteristics, vaccination status and clinical symptoms were not associated with a positive IgM test. CONCLUSION: This is the first serosurvey conducted during the fourth wave of the epidemic in Vietnam. It revealed a seropositivity rate higher than in previous studies and confirmed the hyperendemicity of SARS-CoV-2. Testing using rapid serological tests proved to be a reliable, easy-to-use method and enabled a rapid estimation of the burden of COVID-19.

Emergence in southern France of a new SARS-CoV-2 variant harbouring both N501Y and E484K substitutions in the spike protein.

SARS-CoV-2 variants have become a major virological, epidemiological, and clinical concern, particularly with regard to the risk of escape from vaccine-induced immunity. Here, we describe the emergence of a new variant, with the index case returning from travel in Cameroon. For 13 SARS-CoV-2-positive patients living in the same geographical area of southeastern France, a qPCR test for screening variant-associated mutations showed an atypical combination. The genome sequences were obtained by next-generation sequencing with Oxford Nanopore Technologies on GridION instruments within about 8 h. Analysis revealed 46 nucleotide substitutions and 37 deletions, resulting in 30 amino acid substitutions and 12 deletions. Fourteen of the amino acid substitutions, including N501Y and E484K, and nine deletions are located in the spike protein. This genotype pattern led to the establishment of a new Pangolin lineage, named B.1.640.2, that is a phylogenetic sister group to the old B.1.640 lineage, which has now been renamed B.1.640.1. The lineages differ by 25 nucleotide substitutions and 33 deletions. The combination of mutations in these isolates and their phylogenetic position indicate, based on our previous definition, that they represent a new variant, which we have named "IHU". These data are a further example of the unpredictability of the emergence of SARS-CoV-2 variants, and of their possible introduction into a given geographical area from abroad.

SARS-CoV-2 Vaccination and Protection Against Clinical Disease: A Retrospective Study, Bouches-du-Rhône District, Southern France, 2021.

From January 18th to August 13th, 2021, 13,804 unvaccinated and 1,156 patients who had received at least one COVID-19 vaccine dose were tested qPCR-positive for SARS-CoV-2 in our center. Among vaccinated patients, 949, 205 and 2 had received a single, two or three vaccine doses, respectively. Most patients (80.3%) had received the Pfizer-BioNTech vaccine. The SARS-CoV-2 variants infecting vaccinated patients varied over time, reflecting those circulating in the Marseille area, with a predominance of the Marseille-4/20A.EU2 variant from weeks 3 to 6, of the Alpha/20I variant from weeks 7 to 25, and of the Delta/21A variant from week 26. SARS-CoV-2 infection was significantly more likely to occur in the first 13 days post-vaccine injection in those who received a single dose (48.9%) than two doses (27.4%, p< 10-3). Among 161 patients considered as fully vaccinated, i.e., >14 days after the completion of the vaccinal scheme (one dose for Johnson and Johnson and two doses for Pfizer/BioNTech, Moderna and Sputnik vaccines), 10 (6.2%) required hospitalization and four (2.5%) died. Risks of complications increased with age in a nonlinear pattern, with a first breakpoint at 54, 33, and 53 years for death, transfer to ICU, and hospitalization, respectively. Among patients infected by the Delta/21A or Alpha/20I variants, partial or complete vaccination exhibited a protective effect with a risk divided by 3.1 for mortality in patients ≥ 55 years, by 2.8 for ICU transfer in patients ≥ 34 years, and by 1.8 for hospitalization in patients ≥ 54 years. Compared to partial vaccination, complete vaccination provided an even stronger protective effect, confirming effectiveness to prevent severe forms of COVID-19.

Contagion Management at the Méditerranée Infection University Hospital Institute.

The Méditerranée Infection University Hospital Institute (IHU) is located in a recent building, which includes experts on a wide range of infectious disease. The IHU strategy is to develop innovative tools, including epidemiological monitoring, point-of-care laboratories, and the ability to mass screen the population. In this study, we review the strategy and guidelines proposed by the IHU and its application to the COVID-19 pandemic and summarise the various challenges it raises. Early diagnosis enables contagious patients to be isolated and treatment to be initiated at an early stage to reduce the microbial load and contagiousness. In the context of the COVID-19 pandemic, we had to deal with a shortage of personal protective equipment and reagents and a massive influx of patients. Between 27 January 2020 and 5 January 2021, 434,925 nasopharyngeal samples were tested for the presence of SARS-CoV-2. Of them, 12,055 patients with COVID-19 were followed up in our out-patient clinic, and 1888 patients were hospitalised in the Institute. By constantly adapting our strategy to the ongoing situation, the IHU has succeeded in expanding and upgrading its equipment and improving circuits and flows to better manage infected patients.

Emergence and outcomes of the SARS-CoV-2 'Marseille-4' variant.

BACKGROUND: In Marseille, France, following a first severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak in March-May 2020, a second epidemic phase occurred from June, involving 10 new variants. The Marseille-4 variant caused an epidemic that started in August and is still ongoing. METHODS: The 1038 SARS-CoV-2 whole genome sequences obtained in our laboratory by next-generation sequencing with Illumina technology were analysed using Nextclade and nextstrain/ncov pipelines and IQ-TREE. A Marseille-4-specific qPCR assay was implemented. Demographic and clinical features were compared between patients with the Marseille-4 variant and those with earlier strains. RESULTS: Marseille-4 harbours 13 hallmark mutations. One leads to an S477N substitution in the receptor binding domain of the spike protein targeted by current vaccines. Using a specific qPCR, it was observed that Marseille-4 caused 12-100% of SARS-CoV-2 infections in Marseille from September 2020, being involved in 2106 diagnoses. This variant was more frequently associated with hypoxemia than were clade 20A strains before May 2020. It caused a re-infection in 11 patients diagnosed with different SARS-CoV-2 strains before June 2020, suggesting either short-term protective immunity or a lack of cross-immunity. CONCLUSIONS: Marseille-4 should be considered as a major SARS-CoV-2 variant. Its sudden appearance points towards an animal reservoir, possibly mink. The protective role of past exposure and current vaccines against this variant should be evaluated.

Current Status of Putative Animal Sources of SARS-CoV-2 Infection in Humans: Wildlife, Domestic Animals and Pets.

SARS-CoV-2 is currently considered to have emerged from a bat coronavirus reservoir. However, the real natural cycle of this virus remains to be elucidated. Moreover, the COVID-19 pandemic has led to novel opportunities for SARS-CoV-2 transmission between humans and susceptible animal species. In silico and in vitro evaluation of the interactions between the SARS-CoV-2 spike protein and eucaryotic angiotensin-converting enzyme 2 (ACE2) receptor have tentatively predicted susceptibility to SARS-CoV-2 infection of several animal species. Although useful, these data do not always correlate with in vivo data obtained in experimental models or during natural infections. Other host biological properties may intervene such as the body temperature, level of receptor expression, co-receptor, restriction factors, and genetic background. The spread of SARS-CoV-2 also depends on the extent and duration of viral shedding in the infected host as well as population density and behaviour (group living and grooming). Overall, current data indicate that the most at-risk interactions between humans and animals for COVID-19 infection are those involving certain mustelids (such as minks and ferrets), rodents (such as hamsters), lagomorphs (especially rabbits), and felines (including cats). Therefore, special attention should be paid to the risk of SARS-CoV-2 infection associated with pets.

Mink, SARS-CoV-2, and the Human-Animal Interface.

Mink are small carnivores of the Mustelidae family. The American mink is the most common and was imported to Europe, Asia, and Latin America for breeding, as its fur is very popular. Denmark, the Netherlands, and China are the biggest producers of mink. Mink farms with a high population density in very small areas and a low level of genetic heterogeneity are places conducive to contagion. The mink's receptor for SARS-CoV-2 is very similar to that of humans. Experimental models have shown the susceptibility of the ferret, another mustelid, to become infected with SARS-CoV-2 and to transmit it to other ferrets. On April 23, 2020, for the first time, an outbreak of SARS-CoV-2 in a mink farm was reported in the Netherlands. Since then, COVID-19 has reached numerous mink farms in the Netherlands, Denmark, United States, France, Greece, Italy, Spain, Sweden, Poland, Lithuania, and Canada. Not only do mink become infected from each other, but also they are capable of infecting humans, including with virus variants that have mutated in mink. Human infection with variant mink viruses with spike mutations led to the culling in Denmark of all mink in the country. Several animals can be infected with SARS-CoV-2. However, anthropo-zoonotic outbreaks have only been reported in mink farms. The rapid spread of SARS-CoV-2 in mink farms raises questions regarding their potential role at the onset of the pandemic and the impact of mutants on viral fitness, contagiousness, pathogenicity, re-infections with different mutants, immunotherapy, and vaccine efficacy.

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.

Natural history of COVID-19 and therapeutic options.

Introduction: COVID-19 presents benign forms in young patients who frequently present with anosmia. Infants are rarely infected, while severe forms occur in patients over 65 years of age with comorbidities, including hypertension and diabetes. Lymphopenia, eosinopenia, thrombopenia, increased lactate dehydrogenase, troponin, C-reactive protein, D-dimers and low zinc levels are associated with severity.Areas covered: The authors review the literature and provide an overview of the current state of knowledge regarding the natural history of and therapeutic options for COVID-19. Expert opinion: Diagnosis should rely on PCR and not on clinical presumption. Because of discrepancies between clinical symptoms, oxygen saturation or radiological signs on CT scans, pulse oximetry, and radiological investigation should be systematic. The disease evolves in successive phases: an acute virological phase, and, in some patients, a cytokine storm phase; an uncontrolled coagulopathy; and an acute respiratory distress syndrome. Therapeutic options include antivirals, oxygen therapy, immunomodulators, anticoagulants and prolonged mechanical treatment. Early diagnosis, care, and implementation of an antiviral treatment; the use of immunomodulators at a later stage; and the quality of intensive care are critical regarding mortality rates. The higher mortality observed in Western countries remains unexplained. Pulmonary fibrosis may occur in some patients. Its future is unpredictable.

Evaluating ELISA, Immunofluorescence, and Lateral Flow Assay for SARS-CoV-2 Serologic Assays.

BACKGROUND: The SARS-CoV-2 outbreak has emerged at the end of 2019. Aside from the detection of viral genome with specific RT-PCR, there is a growing need for reliable determination of the serological status. We aimed at evaluating five SARS-CoV-2 serology assays. METHODS: An in-house immunofluorescence assay (IFA), two ELISA kits (EUROIMMUN® ELISA SARS-CoV-2 IgG and NovaLisa® SARS-CoV-2 IgG and IgM) and two lateral flow assays (T-Tek® SARS-CoV-2 IgG/IgM Antibody Test Kit and Sure Bio-tech® SARS-CoV-2 IgM/IgG Antibody Rapid Test) were compared on 40 serums from RT-PCR-confirmed SARS-CoV-2 infected patients and 10 SARS-CoV-2 RT-PCR negative subjects as controls. RESULTS: Control subjects tested negative for SARS-CoV-2 antibodies with all five systems. Estimated sensitivities varied from 35.5 to 71.0% for IgG detection and from 19.4 to 64.5% for IgM detection. For IgG, in-house IFA, EuroImmun, T-Tek and NovaLisa displayed 50-72.5% agreement with other systems except IFA vs EuroImmun and T-Tek vs NovaLisa. Intermethod agreement for IgM determination was between 30 and 72.5%. DISCUSSION: The overall intermethod agreement was moderate. This inconsistency could be explained by the diversity of assay methods, antigens used and immunoglobulin isotype tested. Estimated sensitivities were low, highlighting the limited value of antibody detection in CoVID-19. CONCLUSION: Comparison of five systems for SARS-CoV-2 IgG and IgM antibodies showed limited sensitivity and overall concordance. The place and indications of serological status assessment with currently available tools in the CoVID-19 pandemic need further evaluations.

Contribution of VitaPCR SARS-CoV-2 to the emergency diagnosis of COVID-19.

BACKGROUND: With the persistent COVID-19 pandemic, there is an urgent need to use rapid and reliable diagnostic tools for highly urgent cases. Antigen tests are disappointing with their lack of sensitivity. Among molecular tools allowing a diagnosis in less than an hour, only one, the Cepheid Xpert Xpress SARS-CoV-2 assay, has exhibited a good sensitivity. However, we are also facing a global shortage of reagents and kits. Thus, it is imperative to evaluate other point-of-care molecular tests. METHODS: We evaluated the VitaPCR™ RT-PCR assay, whose sample analysis time is of approximately 20 min, in nasopharyngeal secretions from 534 patients presenting to our Institute, for the diagnosis of COVID-19, and compared it to our routine RT-PCR assay. We also compared the two assays with tenfold dilutions of a SARS-CoV-2 strain. RESULTS: Compared to our routine RT-PCR and the previous diagnosis of COVID-19, the sensitivity, specificity, positive and negative predictive values of VitaPCR™ can be evaluated to be 99.3 % (155/156), 94.7 % (358/378), 88.6 % (155/175) and 99.7 % (358/359), respectively. Tenfold dilutions of a SARS-CoV-2 strain show that the VitaPCR™ was more sensitive that our routine RT-PCR assay. CONCLUSION: The VitaPCR™ SARS-CoV-2 is an accurate rapid test, suitable for clinical practice that can be performed as part of a point-of-care testing, for the rapid diagnosis of COVID-19.