The endocytic recycling compartment serves as a viral factory for hepatitis E virus.

Although hepatitis E virus (HEV) is the major leading cause of enterically transmitted viral hepatitis worldwide, many gaps remain in the understanding of the HEV lifecycle. Notably, viral factories induced by HEV have not been documented yet, and it is currently unknown whether HEV infection leads to cellular membrane modeling as many positive-strand RNA viruses. HEV genome encodes the ORF1 replicase, the ORF2 capsid protein and the ORF3 protein involved in virion egress. Previously, we demonstrated that HEV produces different ORF2 isoforms including the virion-associated ORF2i form. Here, we generated monoclonal antibodies that specifically recognize the ORF2i form and antibodies that recognize the different ORF2 isoforms. One antibody, named P1H1 and targeting the ORF2i N-terminus, recognized delipidated HEV particles from cell culture and patient sera. Importantly, AlphaFold2 modeling demonstrated that the P1H1 epitope is exposed on HEV particles. Next, antibodies were used to probe viral factories in HEV-producing/infected cells. By confocal microscopy, we identified subcellular nugget-like structures enriched in ORF1, ORF2 and ORF3 proteins and viral RNA. Electron microscopy analyses revealed an unprecedented HEV-induced membrane network containing tubular and vesicular structures. We showed that these structures are dependent on ORF2i capsid protein assembly and ORF3 expression. An extensive colocalization study of viral proteins with subcellular markers, and silencing experiments demonstrated that these structures are derived from the endocytic recycling compartment (ERC) for which Rab11 is a central player. Hence, HEV hijacks the ERC and forms a membrane network of vesicular and tubular structures that might be the hallmark of HEV infection.

National early warning score to predict intensive care unit transfer and mortality in COVID-19 in a French cohort.

BACKGROUND: No risk stratification tool has been validated in hospitalised patients with coronavirus disease 2019 (COVID-19), despite a high rate of intensive care requirement and in-hospital mortality. We aimed to determine whether the National Early Warning Score (NEWS) at admission can accurately predict in-hospital mortality and ICU transfer. METHODS: This was a retrospective cohort study from January 24 to April 16, 2020, at Lille University Hospital. All consecutive adult patients with laboratory-confirmed COVID-19 who were initially admitted to non-ICU wards were included. The primary outcome was a composite criterion consisting of ICU transfer or in-hospital mortality. We evaluated the prognostic performance of NEWS by calculating the area under (AUC) the receiver operating characteristic curve, the optimal threshold value of NEWS, and its association with the primary outcome. RESULTS: Of the 202 COVID-19 patients, the median age was 65 (interquartile range 52-78), 38.6% were women and 136 had at least one comorbidity. The median NEWS was 4 (2-6). A total of 65 patients were transferred to the ICU or died in the hospital. Compared with patients with favourable outcome, these patients were significantly older, had more comorbidities and higher NEWS. The AUC for NEWS was 0.68 (0.60-0.77) and the best cutoff value was 6. Adjusted odds ratio for NEWS ≥ 6 as an independent predictor was 3.78 (1.94-7.09). CONCLUSIONS: In hospitalised COVID-19 patients, NEWS was an independent predictor of ICU transfer and in-hospital death. In daily practice, NEWS ≥ 6 at admission may help to identify patients who are at risk to deteriorate.

The C-terminal domain of the MERS coronavirus M protein contains a trans-Golgi network localization signal.

Coronavirus M proteins represent the major protein component of the viral envelope. They play an essential role during viral assembly by interacting with all of the other structural proteins. Coronaviruses bud into the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC), but the mechanisms by which M proteins are transported from their site of synthesis, the ER, to the budding site remain poorly understood. Here, we investigated the intracellular trafficking of the Middle East respiratory syndrome coronavirus (MERS-CoV) M protein. Subcellular localization analyses revealed that the MERS-CoV M protein is retained intracellularly in the trans-Golgi network (TGN), and we identified two motifs in the distal part of the C-terminal domain as being important for this specific localization. We identified the first motif as a functional diacidic DxE ER export signal, because substituting Asp-211 and Glu-213 with alanine induced retention of the MERS-CoV M in the ER. The second motif, 199KxGxYR204, was responsible for retaining the M protein in the TGN. Substitution of this motif resulted in MERS-CoV M leakage toward the plasma membrane. We further confirmed the role of 199KxGxYR204 as a TGN retention signal by using chimeras between MERS-CoV M and the M protein of infectious bronchitis virus (IBV). Our results indicated that the C-terminal domains of both proteins determine their specific localization, namely TGN and ERGIC/cis-Golgi for MERS-M and IBV-M, respectively. Our findings indicate that MERS-CoV M protein localizes to the TGN because of the combined presence of an ER export signal and a TGN retention motif.

Hepatitis E Virus Lifecycle and Identification of 3 Forms of the ORF2 Capsid Protein.

BACKGROUND & AIMS: Hepatitis E virus (HEV) infection is a major cause of acute hepatitis worldwide. Approximately 2 billion people live in areas endemic for HEV and are at risk of infection. The HEV genome encodes 3 proteins, including the ORF2 capsid protein. Detailed analyses of the HEV life cycle has been hampered by the lack of an efficient viral culture system. METHODS: We performed studies with gt3 HEV cell culture-produced particles and patient blood and stool samples. Samples were fractionated on iodixanol gradients and cushions. Infectivity assays were performed in vitro and in human liver chimeric mice. Proteins were analyzed by biochemical and proteomic approaches. Infectious particles were analyzed by transmission electron microscopy. HEV antigen levels were measured with the Wantaï enzyme-linked immunosorbent assay. RESULTS: We developed an efficient cell culture system and isolated HEV particles that were infectious in vitro and in vivo. Using transmission electron microscopy, we defined the ultrastructure of HEV cell culture-produced particles and particles from patient sera and stool samples. We also identified the precise sequence of the infectious particle-associated ORF2 capsid protein. In cultured cells and in samples from patients, HEV produced 3 forms of the ORF2 capsid protein: infectious/intracellular ORF2 (ORF2i), glycosylated ORF2 (ORF2g), and cleaved ORF2 (ORF2c). The ORF2i protein associated with infectious particles, whereas the ORF2g and ORF2c proteins were massively secreted glycoproteins not associated with infectious particles. ORF2g and ORF2c were the most abundant antigens detected in sera from patients. CONCLUSIONS: We developed a cell culture system and characterized HEV particles; we identified 3 ORF2 capsid proteins (ORF2i, ORF2g, and ORFc). These findings will advance our understanding of the HEV life cycle and improve diagnosis.

HCoV-229E spike protein fusion activation by trypsin-like serine proteases is mediated by proteolytic processing in the S2' region.

Human coronavirus 229E (HCoV-229E) is responsible for common colds. Like other coronaviruses, HCoV-229E exploits cellular proteases to activate fusion mediated by the spike protein. We analysed the proteolytic processing of the HCoV-229E spike protein by trypsin-like serine proteases leading to activation of the fusion process. Unlike in other coronaviruses, HCoV-229E fusion activation appears to be a one-step process. Indeed, cleavage of the S1/S2 interface does not seem to be a prerequisite, and the fusion activation is highly reliant on the S2' region, with arginine residue 683 acting as the recognition site.

Clinical features and viral diagnosis of two cases of infection with Middle East Respiratory Syndrome coronavirus: a report of nosocomial transmission.

BACKGROUND: Human infection with a novel coronavirus named Middle East Respiratory Syndrome coronavirus (MERS-CoV) was first identified in Saudi Arabia and the Middle East in September, 2012, with 44 laboratory-confirmed cases as of May 23, 2013. We report detailed clinical and virological data for two related cases of MERS-CoV disease, after nosocomial transmission of the virus from one patient to another in a French hospital. METHODS: Patient 1 visited Dubai in April, 2013; patient 2 lives in France and did not travel abroad. Both patients had underlying immunosuppressive disorders. We tested specimens from the upper (nasopharyngeal swabs) or the lower (bronchoalveolar lavage, sputum) respiratory tract and whole blood, plasma, and serum specimens for MERS-CoV by real-time RT-PCR targeting the upE and Orf1A genes of MERS-CoV. FINDINGS: Initial clinical presentation included fever, chills, and myalgia in both patients, and for patient 1, diarrhoea. Respiratory symptoms rapidly became predominant with acute respiratory failure leading to mechanical ventilation and extracorporeal membrane oxygenation (ECMO). Both patients developed acute renal failure. MERS-CoV was detected in lower respiratory tract specimens with high viral load (eg, cycle threshold [Ct] values of 22·9 for upE and 24 for Orf1a for a bronchoalveolar lavage sample from patient 1; Ct values of 22·5 for upE and 23·9 for Orf1a for an induced sputum sample from patient 2), whereas nasopharyngeal specimens were weakly positive or inconclusive. The two patients shared the same room for 3 days. The incubation period was estimated at 9-12 days for the second case. No secondary transmission was documented in hospital staff despite the absence of specific protective measures before the diagnosis of MERS-CoV was suspected. Patient 1 died on May 28, due to refractory multiple organ failure. INTERPRETATION: Patients with respiratory symptoms returning from the Middle East or exposed to a confirmed case should be isolated and investigated for MERS-CoV with lower respiratory tract sample analysis and an assumed incubation period of 12 days. Immunosuppression should also be taken into account as a risk factor. FUNDING: French Institute for Public Health Surveillance, ANR grant Labex Integrative Biology of Emerging Infectious Diseases, and the European Community's Seventh Framework Programme projects EMPERIE and PREDEMICS.

Effect of High Hydrostatic Pressure Processing and Holder Pasteurization of Human Milk on Inactivation of Human Coronavirus 229E and Hepatitis E Virus.

In preterm infants, sterilized donor milk (DM) is frequently used for feeding when breast milk is lacking. Most human milk banks use the Holder pasteurization method (HoP) to ensure the microbiological safety of DM. However, this method degrades many bioactive factors and hormones. Recently, high hydrostatic pressure (HHP) processing, which preserves bioactive factors in human milk, has been proposed as an alternative method to ensure the safety of DM. Although HHP treatment has been shown to be effective for viral inactivation, the effect of HHP on viruses that may be present in the complex nutritional matrix of human milk has not yet been defined. In the present study, we compared the efficacy of two HHP protocols (4 cycles at 350 MPa at 38 °C designated as 4xHP350 treatment, and 1 cycle at 600 MPa at 20 °C designated as 1xHP600 treatment) with the HoP method on artificially virus-infected DM. For this purpose, we used human coronavirus 229E (HCoV-229E) and hepatitis E virus (HEV) as surrogate models for enveloped and non-enveloped viruses. Our results showed that HCoV-229E is inactivated by HHP and HoP treatment. In particular, the 4xHP350 protocol is highly effective in inactivating HCoV-229E. However, our results demonstrated a matrix effect of human milk on HCoV-229E inactivation. Furthermore, we demonstrated that HEV is stable to moderate pressure HHP treatment, but the milk matrix does not protect it from inactivation by the high-pressure HHP treatment of 600 MPa. Importantly, the complex nutritional matrix of human milk protects HEV from inactivation by HoP treatment. In conclusion, we demonstrated that HHP and HoP treatments do not lead to complete inactivation of both surrogate virus models, indicating that these treatments cannot guarantee total viral safety of donor milk.

Serum neutralization of SARS coronavirus 2 Omicron sublineages BA.1 and BA.2 and cellular immune responses 3 months after booster vaccination.

OBJECTIVES: We investigated serum neutralizing activity against BA.1 and BA.2 Omicron sublineages and T cell response before and 3 months after administration of the booster vaccine in healthcare workers (HCWs). METHODS: HCWs aged 18-65 years who were vaccinated and received booster doses of the BNT162b2 vaccine were included. Anti-SARS coronavirus 2 IgG levels and cellular response (through interferon γ ELISpot assay) were evaluated in all participants, and neutralizing antibodies against Delta, BA.1, and BA.2 were evaluated in participants with at least one follow-up visit 1 or 3 months after the administration of the booster dose. RESULTS: Among 118 HCWs who received the booster dose, 102 and 84 participants attended the 1-month and 3-month visits, respectively. Before the booster vaccine dose, a low serum neutralizing activity against Delta, BA.1, and BA.2 was detectable in only 39/102 (38.2%), 8/102 (7.8%), and 12/102 (11.8%) participants, respectively. At 3 months, neutralizing antibodies against Delta, BA.1, and BA.2 were detected in 84/84 (100%), 79/84 (94%), and 77/84 (92%) participants, respectively. Geometric mean titres of neutralizing antibodies against BA.1 and BA.2 were 2.2-fold and 2.8-fold reduced compared with those for Delta. From 1 to 3 months after the administration of the booster dose, participants with a recent history of SARS coronavirus 2 infection (n = 21/84) had persistent levels of S1 reactive specific T cells and neutralizing antibodies against Delta and BA.2 and 2.2-fold increase in neutralizing antibodies against BA.1 (p 0.014). Conversely, neutralizing antibody titres against Delta (2.5-fold decrease, p < 0.0001), BA.1 (1.5-fold, p 0.02), and BA.2 (2-fold, p < 0.0001) declined from 1 to 3 months after the administration of the booster dose in individuals without any recent infection. DISCUSSION: The booster vaccine dose provided significant and similar response against BA.1 and BA.2 Omicron sublineages; however, the immune response declined in the absence of recent infection.

Immunogenicity of BNT162b2 vaccine booster against SARS-CoV-2 Delta and Omicron variants in nursing home residents: A prospective observational study in older adults aged from 68 to 98 years.

Background: The present study aimed to evaluate the persistent immunogenicity offered by a third dose of BNT162b2 against Delta and Omicron variants, in nursing home (NH) residents. Methods: In this monocenter prospective observational study, anti-spike IgG levels, S1 domain reactive T cell counts, serum neutralizing antibody titers against Delta and Omicron variants were compared before and up to three months after the BNT162b2 booster dose, in NH residents without COVID-19 (COVID-19 naive) or with COVID-19 prior to initial vaccination (COVID-19 recovered). Findings: 106 NH residents (median [interquartile range] age: 86·5 [81;91] years) were included. The booster dose induced a high increase of anti-spike antibody levels in all subjects (p < 0.0001) and a mild transient increase of specific T cells. Before the booster dose, Delta neutralization was detected in 19% (n = 8/43) and 88% (n = 37/42) of COVID-19 naive and COVID-19 recovered subjects, respectively. Three months after the booster dose, all NH residents developed and maintained a higher Delta neutralization (p < 0·0001). Before the booster dose, Omicron neutralization was detected in 5% (n = 2/43) and 55% (n = 23/42) of COVID-19 naive and COVID-19 recovered subjects, respectively, and three months after, in 84% and 95%, respectively. Neutralizing titers to Omicron were lower than to Delta in both groups with a 35-fold reduction compared to Delta. Interpretation: The booster dose restores high neutralization titers against Delta in all NH residents, and at a lower level against Omicron in a large majority of participants. Future studies are warranted to assess if repeated BNT162b2 booster doses or new specific vaccines might be considered for protecting such fragile patients against Omicron and/or future SARS-CoV-2 variants. Funding: French government through the Programme Investissement d'Avenir (I-SITE ULNE/ANR-16-IDEX-0004 ULNE) and the Label of COVID-19 National Research Priority (National Steering Committee on Therapeutic Trials and Other COVID-19 Research, CAPNET).

Enteroviruses and type 1 diabetes: towards a better understanding of the relationship.

Environmental factors, especially viruses, are involved in the initiation or the acceleration of type 1 diabetes (T1D) pathogenesis. Epidemiological data strongly suggest that enteroviruses, such as coxsackievirus B4 (CV-B4), can be associated with T1D. It has been demonstrated that enterovirus infections were significantly more prevalent in at risk individuals, such as siblings of diabetic patients, when they developed anti-beta-cell autoantibodies or T1D, and in recently diagnosed diabetic patients, compared with control subjects. The isolation of CV-B4 from the pancreas of diabetic patients strengthened the hypothesis of a relationship between the virus and the disease. Studies performed in vitro and in vivo in animal models helped to discover mechanisms of the infection of pancreas and other tissues, potentially able to play a role in the pathogenesis of T1D. Interestingly, it cannot be excluded that enteroviruses behave as half-devil half-angel since experimental studies suggest that, in certain conditions, these agents would be able to protect individuals against the disease. All of the plausible mechanisms by which enterovirus may be related to T1D will be reviewed here.

Impaired Functional T-Cell Response to SARS-CoV-2 After Two Doses of BNT162b2 mRNA Vaccine in Older People.

Long-term care facility (LTCF) older residents display physiological alterations of cellular and humoral immunity that affect vaccine responses. Preliminary reports suggested a low early postvaccination antibody response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The aim of this study was to focus on the specific T-cell response. We quantified S1-specific IgG, neutralizing antibody titers, total specific IFNγ-secreting T cells by ELISpot, and functionality of CD4+- and CD8+-specific T cells by flow cytometry, after two doses of the BNT162b2 vaccine in younger and older people, with and without previous COVID-19 infection (hereafter referred to as COVID-19-recovered and COVID-19-naive subjects, respectively). Frailty, nutritional, and immunosenescence parameters were collected at baseline in COVID-19-naive older people. We analyzed the immune response in 129 young adults (median age 44.0 years) and 105 older residents living in a LCTF (median age 86.5 years), 3 months after the first injection. Humoral and cellular memory responses were dramatically impaired in the COVID-19-naive older (n = 54) compared with the COVID-19-naive younger adults (n = 121). Notably, older participants' neutralizing antibodies were 10 times lower than the younger's antibody titers (p < 0.0001) and LCTF residents also had an impaired functional T-cell response: the frequencies of IFNγ+ and IFNγ+IL-2+TNFα+ cells among specific CD4+ T cells, and the frequency of specific CD8+ T cells were lower in COVID-19-naive older participants than in COVID-19-naive young adults (p < 0.0001 and p = 0.0018, respectively). However, COVID-19-recovered older participants (n = 51) had greater antibody and T-cell responses, including IFNγ+ and IFNγ+IL-2+TNFα+-specific CD4+ T cells (p < 0.0001), as well as TNFα+-specific CD8+ T cells (p < 0.001), than COVID-19-naive older adults. We also observed that "inflammageing" and particularly high plasma levels of TNFα was associated to poor antibody response in the older participants. In conclusion, our results show that the COVID-19-naive older people had low counts and impaired specific CD4+ and CD8+ T cells, in addition to impaired antibody response, and that specific studies are warranted to assess the efficiency of SARS-CoV-2 mRNA-based vaccines, as in other immunocompromised subjects. Our study also shows that, despite their physiological alterations of immunity, vaccination is highly efficient in boosting the prior natural memory response in COVID-19-recovered older people.

Use of whole-genome sequencing in the molecular investigation of care-associated HCoV-OC43 infections in a hematopoietic stem cell transplant unit.

BACKGROUND: While respiratory viral infections are recognized as a frequent cause of illness in hematopoietic stem cell transplantation (HSCT) recipients, HCoV-OC43 infections have rarely been investigated as healthcare-associated infections in this population. OBJECTIVES: In this report, HCoV-OC43 isolates collected from HSCT patients were retrospectively characterized to identify potential clusters of infection that may stand for a hospital transmission. STUDY DESIGN: Whole-genome and S gene sequences were obtained from nasal swabs using next-generation sequencing and phylogenetic trees were constructed. Similar identity matrix and determination of the most common ancestor were used to compare clusters of patient's sequences. Amino acids substitutions were analysed. RESULTS: Genotypes B, E, F and G were identified. Two clusters of patients were defined from chronological data and phylogenetic trees. Analyses of amino acids substitutions of the S protein sequences identified substitutions specific for genotype F strains circulating among European people. CONCLUSIONS: HCoV-OC43 may be implicated in healthcare-associated infections.

Comprehensive study of the dynamic interaction between SO2 and acetaldehyde during alcoholic fermentation.

In this work, we focused on the effect of the initial content of SO2 in synthetic grape juice on yeast metabolism linked to the production of acetaldehyde. Lengthening of the lag phase duration was observed with an increase in the initial SO2 content. Nevertheless, an interesting finding was a threshold value of an initial SO2 content of 30 mg L-1 in the juice led to equilibrium between intracellular SO2 diffusion and SO2 production from the sulfate pool by yeast. The ratios of free and bound acetaldehydes were measured during fermentation, and the maximum accumulation of free acetaldehyde was observed when SO2 concentration equilibrium between diffusion and production was reached in the fermenting juice. Moreover, it was observed that SO2 addition resulted in significant changes in the synthesis of aroma compounds. Production of volatile molecules related to sulfur metabolism (methionol) was changed. But, more surprisingly, synthesis of some volatile carbon compounds (diacetyl, isoamyl alcohol, isobutyl alcohol, phenyl ethanol and their corresponding esters) was also altered because of major disruptions in the NADPH/NADP+ redox equilibrium. Finally, we demonstrated that acetaldehyde bound to SO2 could not be metabolized by the yeast during the time course of fermentation and that only free acetaldehyde could impact metabolism.

Hepatitis delta virus proteins repress hepatitis B virus enhancers and activate the alpha/beta interferon-inducible MxA gene.

Co-infection and superinfection of hepatitis B virus (HBV) with hepatitis delta virus (HDV) leads to suppression of HBV replication both in patients and in animal and cellular models. The mechanisms behind this inhibition have not previously been explored fully. HBV replication is governed by four promoters and two enhancers, Enh1 and Enh2. Repression of these enhancers has been reported to be one of the main mechanisms of HBV inhibition. Moreover, in a previous study, it has been demonstrated that alpha interferon (IFN-alpha)-inducible MxA protein inhibits HBV replication. HDV encodes two proteins, p24 and p27. p27 was shown to activate several heterologous promoters, including HBV promoters. In an attempt to analyse the mechanisms of HBV inhibition by HDV, the question was raised whether HDV proteins could act directly by repressing HBV enhancers, and/or indirectly by activating the MxA gene. This issue was addressed in a co-transfection model in Huh-7 cells, using p24- or p27-expressing plasmids along with Enh1, Enh2, HBV and MxA promoter-luciferase constructs. Enh1 and Enh2 were strongly repressed, by 60 and 80 % and 40 and 60 %, by p24 and p27, respectively. In addition, p27 was responsible for threefold activation of the MxA promoter and potentiation of IFN-alpha on this promoter. MxA mRNA quantification and a virus yield reduction assay confirmed these results. In conclusion, this study shows that HDV proteins inhibit HBV replication by trans-repressing its enhancers and by trans-activating the IFN-alpha-inducible MxA gene.

Detection of enterovirus RNA in postoperative thyroid tissue specimens.

CONTEXT: Autoimmune thyroiditis is a very common disease. A genetic predisposition and environmental factors such as viruses are thought to contribute to the development of autoimmune thyroiditis. Enteroviruses, which are involved in other autoimmune diseases, are attractive candidates. OBJECTIVE: To investigate the presence of enteroviral genome sequences in postoperative thyroid tissues with lymphocytic infiltration, a common histological feature of thyroiditis. SUBJECTS AND METHODS: Postoperative thyroid specimens collected prospectively from 86 patients were blindly frozen at -80 degrees C. The presence of EV genome sequences in the samples was blindly investigated by real-time RT-PCR. Clinical data, histological findings and levels of anti-TPO antibodies were collected. RESULTS: EV-RNA detection was positive (up to 36 cycles) or weakly positive (37-39 cycles) in 22 out of 86 patients (25%). EV-RNA (positive or weakly positive signal) was detected in 5 out of 27 (18.5%) thyroid specimens with lymphocytic infiltration, and in 17 out of 59 (29%) thyroid specimens without lymphocytic infiltration (P = 0.4). No correlation was observed between EV-RNA detection in thyroid and the presence of anti-TPOAb. EV-RNA was detected in 3 out of 11 patients histologically diagnosed as thyroiditis (27.3%) and in 18 out of 74 patients (24.3%) with thyroid tumours (multinodular goitre, adenoma and carcinoma) (P = 0.5) and in one patient with a normal thyroid. CONCLUSION: EV-RNA can be detected in thyroid tissue from patients with various thyroid diseases, but there is no relationship between the presence of EV-RNA and thyroiditis. Further studies are needed to clarify the role of EV in thyroid diseases.

A complete protocol for whole-genome sequencing of virus from clinical samples: Application to coronavirus OC43.

Genome sequencing of virus has become a useful tool for better understanding of virus pathogenicity and epidemiological surveillance. Obtaining virus genome sequence directly from clinical samples is still a challenging task due to the low load of virus genetic material compared to the host DNA, and to the difficulty to get an accurate genome assembly. Here we introduce a complete sequencing and analyzing protocol called V-ASAP for Virus Amplicon Sequencing Assembly Pipeline. Our protocol is able to generate the viral dominant genome sequence starting from clinical samples. It is based on a multiplex PCR amplicon sequencing coupled with a reference-free analytical pipeline. This protocol was applied to 11 clinical samples infected with coronavirus OC43 (HcoV-OC43), and led to seven complete and two nearly complete genome assemblies. The protocol introduced here is shown to be robust, to produce a reliable sequence, and could be applied to other virus.

Development and Evaluation of a Hybrid Course in Clinical Virology at a Faculty of Pharmacy in Lille, France.

BACKGROUND: During their studies, pharmacy students must acquire the specific skills in clinical virology required for their subsequent professional practice. Recent experiments on teaching and learning in higher education have shown that hybrid courses strengthen the students' commitment to learning and enable high-quality knowledge acquisition. OBJECTIVE: This study concerned the design and deployment of a hybrid course that combines face-to-face and Web-based instruction in clinical virology for fourth-year pharmacy students. The study's objectives were to (1) measure the students' level of involvement in the course, (2) gauge their interest in this type of learning, and (3) highlight any associated difficulties. METHODS: The study included 194 fourth-year pharmacy students from the Lille Faculty of Pharmacy (University of Lille, Lille, France) between January and June 2017. The students followed a hybrid course comprising an online learning module and 5 tutorial sessions in which professional situations were simulated. The learning module and 3 online evaluation sessions were delivered via the Moodle learning management system. Each tutorial session ended with an evaluation. The number of Moodle log-ins, the number of views of learning resources, and the evaluation marks were recorded. The coefficient for the correlation between the marks in the online evaluation and those in the tutorials was calculated. The students' opinions and level of satisfaction were evaluated via a course questionnaire. RESULTS: The course's learning resources and Web pages were viewed 21,446 and 3413 times, respectively. Of the 194 students, 188 (96.9%) passed the course (ie, marks of at least 10 out of 20). There was a satisfactory correlation between the marks obtained in the online evaluations and those obtained after the tutorials. The course met the students' expectations in 53.2% of cases, and 57.4% of the students stated that they were able to work at their own pace. Finally, 26.6% of the students stated that they had difficulty organizing their work around this hybrid course. CONCLUSIONS: Our results showed that pharmacy students were strongly in favor of a hybrid course. The levels of attendance and participation were high. However, teachers must be aware that some students will encounter organizational difficulties.

Vicinal diketones and their precursors in wine alcoholic fermentation: Quantification and dynamics of production.

Vicinal diketones produced during wine fermentation influence the organoleptic qualities of wine. Diacetyl and 2,3-pentanedione are well known for their contribution to butter or butterscotch-like flavours. We developed an analysis method to quantify vicinal diketones and their precursors, α-acetolactate and α-acetohydroxybutyrate, under oenological conditions. Five-fold dilution of the sample in a phosphate-citrate buffer (pH7.0) strongly attenuated matrix effects between the beginning and end of alcoholic fermentation and protected the sample from spontaneous precursor decarboxylation. The use of diacetyl-d6 as an internal reference improved precision by eliminating differences in the derivatization and extraction yields between the internal standard and the analytes. We obtained unexpected results for alcoholic fermentation by Saccharomyces cerevisiae using this approach. Indeed, the level of diacetyl and 2,3-pentanedione throughout fermentation were very low. However, we observed a large quantity of both precursors. The production dynamics of α-acetolactate were unconventional and there were two distinct phases of accumulation. The first corresponded to the growth phase, and the second to glucose depletion. There was a rapid decrease of precursor levels at the end of fermentation, but there was still a significant amount of α-acetolactate. The amount of precursor remaining at the end of fermentation constitutes a potential source of diacetyl during wine maturation. α-Acetohydroxybutyrate accumulated during the growth phase followed by a continuous decrease of its concentration during the stationary phase. Residual quantities of α-acetohydroxybutyrate found in wine at the end of fermentation does not constitute a sufficient source of 2,3-pentanedione to affect the aromatic profile.

Comparison of three molecular assays for detection of enteric viruses in stool samples.

Three molecular assays (FTD® Viral GE from Fast-track diagnostics, RIDA®GENE VSP1 from R-Biopharm, and Xpert Norovirus from Cepheid) were compared for virus detection in acute diarrhea samples. RIDA®GENE and FTD® Viral GE showed perfect/almost perfect agreement for Rotavirus, Sapovirus and Norovirus, substantial agreement for Adenovirus, and moderate agreement for Astrovirus.

Impact of initial lipid content and oxygen supply on alcoholic fermentation in champagne-like musts.

Available nitrogen, lipids, or oxygen are nutrients with major impact on the kinetics of winemaking fermentation. Assimilable nitrogen is usually the growth-limiting nutrient which availability determines the fermentation rate and therefore the fermentation duration. In some particular cases, as in Champagne, grape musts have high available nitrogen content and low turbidity, i.e., below 50 Nephelometric Turbidity Unit (NTU). In the case of low turbidity, the availability of lipids, particularly phytosterols, becomes limiting. In this situation, control of oxygenation, which is necessary for lipid synthesis by yeast, is particularly crucial during fermentation. To mimic and understand these situations, a synthetic medium simulating the average composition of a Champagne must was used. This medium contained phytosterol (mainly ß-sitosterol) concentrations ranging from 0 to 8mg/L corresponding to turbidity between 10 and 90 NTU. Population reached during the stationary phase and the maximum fermentation rate are conditioned by the initial phytosterol concentration determining the amount of nitrogen consumption. An early loss of viability was observed when the lipid concentrations were very low. For example, the viability continuously decreased during the stationary phase to a final value of 50% for an initial phytosterol concentration of 1mg/L. In some fermentations, 10mg/L oxygen were added at the end of the growth phase to combine the effects of initial content of phytosterols in the musts and the de novo synthesis of ergosterol and unsaturated fatty acids induced by oxygen addition. Effect of oxygen supply on the fermentation kinetics was particularly significant for media with low phytosterol contents. For example, the maximum fermentation rate was increased by 1.4-fold and the fermentation time was 70h shorter with oxygen addition in the medium containing 2mg/L of phytosterols. As a consequence of the oxygen supply, for the media containing 3, 5 and 8mg/L of phytosterols, the assimilable nitrogen was completely exhausted and the fermentation kinetics, as well as the final populations and viabilities (greater than 90%), were identical for the 3 conditions. The impacts of the lipid content and additional oxygen on acetate, glycerol and succinate synthesis were also studied. The phytosterols decreased the acetate and increased the succinate synthesis, and oxygenation resulted in a decrease in succinate formation. This work highlights the similarities and differences between the effects of lipids and oxygen on fermentation kinetics and yeast metabolism. This research highlights the need for an optimal combined management of lipid content in the must via turbidity and oxygenation, particularly in nitrogen-rich musts.