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OBJECTIVES We attempted to establish a molecular investigation by Next Generation sequencing of the measles virus (MeV) strains circulating in Marseille-France during the last outbreak that occurred between 2017 and 2019. METHODS The circulating MeV were isolated from clinical samples using cell culture method and whole genomes were sequenced by Illumina Miseq Next Generation. Genotyping and comparative analyses were assessed by phylogenetic reconstructions. Clinical and epidemiological data from cases were also recorded. RESULTS A total of 110 MeV strains were isolated in cell culture. Our analysis based on whole genome sequences of 98 isolates confirmed that 93 strains belonged to the genotype D8 and 5 to the genotype B3. Phylogenetic analyses revealed 4 distinct MeV circulating clones in Marseille. Measles mostly occured in children

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.

The emergence of COVID-19 disease due to SARS-CoV-2 at the end of 2019 was rapidly associated with the isolation of the strain from co-culture onto VERO cells. These isolations quickly made it possible to carry out the first tests for antiviral agents' susceptibility and drug repurposing. However, it seems important to make an inventory of all the cells that can support the growth of this virus and evaluate possible differences between isolates. In the present work, we tested 4 strains of SARS-CoV-2 locally isolated on a panel of 34 cell lines present in our laboratory and commonly used for the isolation of human pathogenic microorganism. After inoculation, cells were observed for cytopathic effects and quantitative real-time polymerase reaction was used to measure the virus replication on the cells. We were able to obtain growth on 7 cell lines, 6 simian, and the human Caco-2. The cytopathogenic effects are variable, ranging from lysis of the cell monolayer in 48-72 h to no cytopathic effect in spite of intense multiplication, as in Caco-2 cells. Interestingly, effect and multiplication varied widely according to the strain tested. In this paper, we explored the species specificity and tissue tropism of SARS-CoV-2 in vitro on a panel of cells available in our laboratory and identified human and animal cell lines susceptible to support SARS-CoV-2 replication. Our work highlights the importance of testing multiple strains when testing antiviral molecules and performing patho-physiological analyzes.

SARS-CoV-2, a novel coronavirus infecting humans, is responsible for the current COVID-19 global pandemic. If several strains could be isolated worldwide, especially forin-vitrodrug susceptibility testing and vaccine development, few laboratories routinely isolate SARS-CoV-2. This is due to the fact that the current co-culture strategy is highly time consuming and requires working in a biosafety level 3 laboratory. In this work, we present a new strategy based on high content screening automated microscopy (HCS) allowing large scale isolation of SARS-CoV-2 from clinical samples in 1 week. A randomized panel of 104 samples, including 72 tested positive by RT-PCR and 32 tested negative, were processed with our HCS procedure and were compared to the classical isolation procedure. Isolation rate was 43 % with both strategies on RT-PCR positive samples, and was correlated with the initial RNA viral load in the samples, where we obtained a positivity threshold of 27 Ct. Co-culture delays were shorter with HCS strategy, where 80 % of the positive samples were recovered by the third day of co-culture, as compared to only 25 % with the classic strategy. Moreover, only the HCS strategy allowed us to recover all the positive elements after 1 week of co-culture. This system allows rapid and automated screening of clinical samples with minimal operator work load, thus reducing the risks of contamination.

Acanthamoeba spp. are predominant free-living amoebae of water and soil. They have been used as tools for the isolation and culture of microbes that resist after their phagocytosis, such as Legionella-like bacteria, and, more recently giant viruses for which differences in permissiveness have been reported. However, problems have been reported regarding their identification at the species level. The present work implemented specific PCR systems for the detection and identification of Acanthamoeba species through comparison of sequences and phylogenetic analyses. Thirty-three Acanthamoeba isolates were studied, including 20 reference strains and 13 isolates retrieved from water, soil or clinical samples. Previous delineation of a core genome encompassing 826 genes based on draft genome sequences from 14 Acanthamoeba species allowed designing PCR systems for one of these core genes that encodes an alanine-tRNA ligase. These primers allowed an efficient and specific screening to detect Acanthamoeba presence. In addition, they identified all 20 reference strains, while partial and complete sequences coding for 18S ribosomal RNA identified only 11 (55%). We found that four isolates may be considered as new Acanthamoeba species. Consistent with previous studies, we demonstrated that some Acanthamoeba isolates were incorrectly assigned to species using the 18S rDNA sequences. Our implemented tool may help determining which Acanthamoeba strains are the most efficient for the isolation of associated microorganisms.

International audience; The presence of the giant virus of amoeba Marseillevirus has been identified at many different sites on the human body, including in the bloodstream of asymptomatic subjects, in the lymph nodes of a child with adenitis, in one adult with Hodgkin's disease, and in the pharynx of an adult. A high seroprevalence of the Marseillevirus has been recorded in the general population. Whether Marseillevirus can disseminate and persist within a mammal after entry remains unproven. We aimed to assess the ability of the virus to disseminate and persist into healthy organisms, especially in the lymphoid organs. Parenteral inoculations were performed by intraperitoneal injection (in rats and mice) or intravenous injection (in rats). Airway inoculation was performed by aerosolization (in mice). Dissemination and persistence were assessed by using PCR and amebal co-culture. Serologies were performed by immunofluorescent assay. Pathological examination was conducted after standard and immunohistochemistry staining. After intraperitoneal inoculation in mice and rats, Marseillevirus was detected in the bloodstream during the first 24 h. Persistence was noted until the end of the experiment, i.e., at 14 days in rats. After intravenous inoculation in rats, the virus was first detected in the blood until 48 h and then in deep organs with infectious virus detected until 14 and 21 days in the liver and the spleen, respectively. Its DNA was detected for up to 30 days in the liver and the spleen. After aerosolization in mice, infectious Marseillevirus was present in the lungs and nasal associated lymphoid tissue until 30 days post inoculation but less frequently and at a lower viral load in the lung than in the nasal associated lymphoid tissue. No other site of dissemination was found after aerosol exposure. Despite no evidence of disease being observed, the 30-day long persistence of Marseillevirus in rats and mice, regardless of the route of inoculation, supports the hypothesis of an infective potential of the virus in certain conditions. Its constant and long-term detection in nasal associated lymphoid tissue in mice after an aerosol exposure suggests the involvement of naso-pharyngeal associated lymphoid tissues in protecting the host against environmental Marseillevirus.

BACKGROUND: Necrotizing enterocolitis (NEC) is the most common and serious gastrointestinal disorder among preterm neonates. We aimed to assess a specific gut microbiota profile associated with NEC. METHODS: Stool samples and clinical data were collected from 4 geographically independent neonatal intensive care units, over a 48-month period. Thirty stool samples from preterm neonates with NEC (n = 15) and controls (n = 15) were analyzed by 16S ribosomal RNA pyrosequencing and culture-based methods. The results led us to develop a specific quantitative polymerase chain reaction (qPCR) assay for Clostridium butyricum, and we tested stool samples from preterm neonates with NEC (n = 93) and controls (n = 270). We sequenced the whole genome of 16 C. butyricum strains, analyzed their phylogenetic relatedness, tested their culture supernatants for cytotoxic activity, and searched for secreted toxins. RESULTS: Clostridium butyricum was specifically associated with NEC using molecular and culture-based methods (15/15 vs 2/15; P < .0001) or qPCR (odds ratio, 45.4 [95% confidence interval, 26.2-78.6]; P < .0001). Culture supernatants of C. butyricum strains from preterm neonates with NEC (n = 14) exhibited significant cytotoxic activity (P = .008), and we identified in all a homologue of the β-hemolysin toxin gene shared by Brachyspira hyodysenteriae, the etiologic agent of swine dysentery. The corresponding protein was secreted by a NEC-associated C. butyricum strain. CONCLUSIONS: NEC was associated with C. butyricum strains and dysbiosis with an oxidized, acid, and poorly diversified gut microbiota. Our findings highlight the plausible toxigenic mechanism involved in the pathogenesis of NEC.

To the Editor: Circular replication initiation protein (Rep)–encoding single-stranded DNA (ssDNA) (CRESS-DNA) genomes are found in diverse group II virus families, which all possess a conserved Rep-encoding gene and a nonenveloped icosahedral capsid, except geminiviruses, which have twinned particles (1). Gemycircularvirus (GcV) were initially discovered in fungi, but a growing number of new species has been characterized by metagenomics in air, sewage, insects, and feces from a broad range of vertebrates (1–5). GcVs have also been found in the brain and serum of humans with multiple sclerosis; in the cerebrospinal fluid of a patient with encephalitis; and in several blood samples, including those from an HIV-positive blood donor (6–8). We report the presence of 2 divergent GcVs and a novel CRESS-DNA virus (CV) in 2 pericardial fluid samples from a patient with idiopathic recurrent pericarditis.

International audience; A Gram-stain-negative, bacilli-shaped bacterial strain, LS-1(T), was isolated from a sewage water sample collected in Jeddah, Saudi Arabia. The taxonomic position of strain LS-1(T) was investigated using a polyphasic taxonomic approach. Phylogenetic analysis based on 16S rRNA gene sequences and those of four other genes indicated that strain LS-1(T) belongs to the genus Legionella in the family Legionellaceae. Regarding the 16S rRNA gene, the most closely related species are Legionella rowbothamii LLAP-6(T) (98.6 %) and Legionella lytica L2(T) (98.5 %). The mip gene sequence of strain LS-1(T) showed 94% sequence similarity with that of L. lytica L2(T) and 93% similarity with that of L. rowbothamii LLAP-6(T). Strain LS-1(T) grew optimally at a temperature of 32 degrees C on a buffered charcoal yeast extract (BCYE) agar plate in a 5% CO2 atmosphere and had a flagellum. The combined phylogenetic, phenotypic and genomic sequence data suggest that strain LS-1(T) represents a novel species of the genus Legionella, for which the name Legionella saoudiensis sp. nov. is proposed. The type strain is LS-1(T) (= DSM 101682(T) = CSUR P2101(T)).

International audience; The number of novel giant viruses identified and characterized from the recently proposed order Megavirales has increased in recent years and new questions have been raised regarding viral diversity and evolution. Here, we describe the isolation and characterization of Saudi moumouvirus (SDMV), a new giant virus belonging to Mimivirus lineage B, isolated from a sewage sample taken from the King Abdulaziz University hospital in Jeddah, Saudi Arabia. SDMV presented 500 nm icosahedral particles with a 1,046,087 bp genome, which is larger than moumouvirus-like genomes which have been described in the past. The SDMV genome was predicted to encode 868 ORFs, ranging in size from 54 to 2,914 amino acids, with a mean size of 349 aa. Furthermore, this genome was predicted to encode 40 new genes (ORFans) without similarity with other sequences (ORFan L850 transcript was detected by qPCR in infected amoeba), in addition to 42 hypothetical proteins (pseudo-ORFs) with less than 100 aa, which matched other sequences in the NCBI nr database. Phylogenetic analysis showed that SDMV clustered together with mimiviruses from lineage B, including moumouvirus-like strains. It is, therefore, the third Mimivirus to be isolated in Asia and the first of group B.

Faustovirus, a new Asfarviridae-related giant virus, was recently isolated in Vermamoeba vermiformis, a protist found in sewage water in various geographical locations and occasionally reported in human eye infection cases. As part of a global metagenomic analysis of viral communities existing in biting midges, we report here for the first time the identification and isolation of a Faustovirus-like virus in hematophagous arthropods and its detection in their animal hosts. The DNA virome analysis of three pools of Culicoides sp., engorged female Culicoides imicola and non-engorged male/female C. imicola biting midges collected in Senegal, revealed the presence of amoeba-infecting giant viruses and, among them, a majority of sequences related to Faustovirus. Phylogenetic analyses conducted on several structural genes of Faustovirus confirmed the clustering of the arthropod-borne Faustovirus with sewage-borne Faustoviruses, with a distinct geographical clustering of Senegalese Faustovirus strains. Transmission electron microscopy identified viral particles with morphologies and diameters which were compatible with Faustovirus. The presence of infectious arthropod-borne Faustovirus was finally confirmed by successful isolation on V. vermiformis amoeba. Global proteomic analysis of biting midges identified that arthropods' blood meal originating from cattle, rodents and humans. Further screening of cattle sera and rodent tissue resulted in prevalence of Faustovirus being estimated at 38% in rodents and 14% in cattle, suggesting a possible origin of Faustovirus presence in arthropods via the ingestion of contaminated blood meal. Viral loads were the highest in rodents' urine and kidney samples, suggesting a possible excretion of viral particles into the environment. Faustovirus DNA polymerase-related sequences were also detected in more than 9 and 11% of febrile patients and healthy Senegalese human sera, respectively. Our study thus, highlights the need to investigate the role of arthropods, wildlife, and domestic animals in the lifecycle of amoeba-infecting giant viruses and, in particular, the environmental cycle of Faustovirus.

A distinct class of infectious agents, the virophages that infect giant viruses of the Mimiviridae family, has been recently described. Here we report the simultaneous discovery of a giant virus of Acanthamoeba polyphaga (Lentille virus) that contains an integrated genome of a virophage (Sputnik 2), and a member of a previously unknown class of mobile genetic elements, the transpovirons. The transpovirons are linear DNA elements of ∼7 kb that encompass six to eight protein-coding genes, two of which are homologous to virophage genes. Fluorescence in situ hybridization showed that the free form of the transpoviron replicates within the giant virus factory and accumulates in high copy numbers inside giant virus particles, Sputnik 2 particles, and amoeba cytoplasm. Analysis of deep-sequencing data showed that the virophage and the transpoviron can integrate in nearly any place in the chromosome of the giant virus host and that, although less frequently, the transpoviron can also be linked to the virophage chromosome. In addition, integrated fragments of transpoviron DNA were detected in several giant virus and Sputnik genomes. Analysis of 19 Mimivirus strains revealed three distinct transpovirons associated with three subgroups of Mimiviruses. The virophage, the transpoviron, and the previously identified self-splicing introns and inteins constitute the complex, interconnected mobilome of the giant viruses and are likely to substantially contribute to interviral gene transfer.