The AP-1 adaptor complex is essential for intracellular trafficking of the ORF2 capsid protein and assembly of Hepatitis E virus.

Although the Hepatitis E virus (HEV) is an emerging global health burden, little is known about its interaction with the host cell. HEV genome encodes three proteins including the ORF2 capsid protein that is produced in different forms, the ORF2i protein which is the structural component of viral particles, and the ORF2g/c proteins which are massively secreted but are not associated with infectious material. We recently demonstrated that the endocytic recycling compartment (ERC) is hijacked by HEV to serve as a viral factory. However, host determinants involved in the subcellular shuttling of viral proteins to viral factories are unknown. Here, we demonstrate that the AP-1 adaptor complex plays a pivotal role in the targeting of ORF2i protein to viral factories. This complex belongs to the family of adaptor proteins that are involved in vesicular transport between the trans-Golgi network and early/recycling endosomes. An interplay between the AP-1 complex and viral protein(s) has been described for several viral lifecycles. In the present study, we demonstrated that the ORF2i protein colocalizes and interacts with the AP-1 adaptor complex in HEV-producing or infected cells. We showed that silencing or drug-inhibition of the AP-1 complex prevents ORF2i protein localization in viral factories and reduces viral production in hepatocytes. Modeling of the ORF2i/AP-1 complex also revealed that the S domain of ORF2i likely interacts with the σ1 subunit of AP-1 complex. Hence, our study identified for the first time a host factor involved in addressing HEV proteins (i.e. ORF2i protein) to viral factories.

An Arginine-Rich Motif in the ORF2 capsid protein regulates the hepatitis E virus lifecycle and interactions with the host cell.

Hepatitis E virus (HEV) infection is the most common cause of acute viral hepatitis worldwide. Hepatitis E is usually asymptomatic and self-limiting but it can become chronic in immunocompromised patients and is associated with increased fulminant hepatic failure and mortality rates in pregnant women. HEV genome encodes three proteins including the ORF2 protein that is the viral capsid protein. Interestingly, HEV produces 3 isoforms of the ORF2 capsid protein which are partitioned in different subcellular compartments and perform distinct functions in the HEV lifecycle. Notably, the infectious ORF2 (ORF2i) protein is the structural component of virions, whereas the genome-free secreted and glycosylated ORF2 proteins likely act as a humoral immune decoy. Here, by using a series of ORF2 capsid protein mutants expressed in the infectious genotype 3 p6 HEV strain as well as chimeras between ORF2 and the CD4 glycoprotein, we demonstrated how an Arginine-Rich Motif (ARM) located in the ORF2 N-terminal region controls the fate and functions of ORF2 isoforms. We showed that the ARM controls ORF2 nuclear translocation likely to promote regulation of host antiviral responses. This motif also regulates the dual topology and functionality of ORF2 signal peptide, leading to the production of either cytosolic infectious ORF2i or reticular non-infectious glycosylated ORF2 forms. It serves as maturation site of glycosylated ORF2 by furin, and promotes ORF2-host cell membrane interactions. The identification of ORF2 ARM as a unique central regulator of the HEV lifecycle uncovers how viruses settle strategies to condense their genetic information and hijack cellular processes.

Lactose malabsorption and colonic fermentations alter host metabolism in rats.

Lactose malabsorption is associated with rapid production of high levels of osmotic compounds, such as organic acids and SCFA in the colon, suspected to contribute to the onset of lactose intolerance. Adult rats are lactase deficient and the present study was conducted to evaluate in vivo the metabolic consequences of acute lactose ingestion, including host-microbiota interactions. Rats received diets of 25% sucrose (S25 control group) or 25% lactose (L25 experimental group). SCFA and lactic acid were quantified in intestinal contents and portal blood. Expression of SCFA transporter genes was quantified in the colonic mucosa. Carbohydrate oxidation (Cox) and lipid oxidation (Lox) were computed by indirect calorimetry. Measurements were performed over a maximum of 13 h. Time, diet and time × diet variables had significant effects on SCFA concentration in the caecum (P<0·001, P=0·004 and P=0·007, respectively) and the portal blood (P<0·001, P=0·04 and P<0·001, respectively). Concomitantly, expression of sodium monocarboxylate significantly increased in the colonic mucosa of the L25 group (P=0·003 at t = 6 h and P<0·05 at t = 8 h). During 5 h after the meal, the L25 group's changes in metabolic parameters (Cox, Lox) were significantly lower than those of the S25 group (P=0·02). However, after 5 h, L25 Cox became greater than S25 (P=0·004). Thus, enhanced production and absorption of SCFA support the metabolic changes observed in calorimetry. These results underline the consequences of acute lactose malabsorption and measured compensations occurring in the host's metabolism, presumably through the microbiota fermentations and microbiota-host interactions.

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.

Processing and Subcellular Localization of the Hepatitis E Virus Replicase: Identification of Candidate Viral Factories.

Hepatitis E virus (HEV) is the major cause of acute hepatitis worldwide. HEV is a positive-sense RNA virus expressing three open reading frames (ORFs). ORF1 encodes the ORF1 non-structural polyprotein, the viral replicase which transcribes the full-length genome and a subgenomic RNA that encodes the structural ORF2 and ORF3 proteins. The present study is focused on the replication step with the aim to determine whether the ORF1 polyprotein is processed during the HEV lifecycle and to identify where the replication takes place inside the host cell. As no commercial antibody recognizes ORF1 in HEV-replicating cells, we aimed at inserting epitope tags within the ORF1 protein without impacting the virus replication efficacy. Two insertion sites located in the hypervariable region were thus selected to tolerate the V5 epitope while preserving HEV replication efficacy. Once integrated into the infectious full-length Kernow C-1 p6 strain, the V5 epitopes did neither impact the replication of genomic nor the production of subgenomic RNA. Also, the V5-tagged viral particles remained as infectious as the wildtype particles to Huh-7.5 cells. Next, the expression pattern of the V5-tagged ORF1 was compared in heterologous expression and replicative HEV systems. A high molecular weight protein (180 kDa) that was expressed in all three systems and that likely corresponds to the unprocessed form of ORF1 was detected up to 25 days after electroporation in the p6 cell culture system. Additionally, less abundant products of lower molecular weights were detected in both in cytoplasmic and nuclear compartments. Concurrently, the V5-tagged ORF1 was localized by confocal microscopy inside the cell nucleus but also as compact perinuclear substructures in which ORF2 and ORF3 proteins were detected. Importantly, using in situ hybridization (RNAScope ®), positive and negative-strand HEV RNAs were localized in the perinuclear substructures of HEV-producing cells. Finally, by simultaneous detection of HEV genomic RNAs and viral proteins in these substructures, we identified candidate HEV factories.

Persistent low body weight in humans is associated with higher mitochondrial activity in white adipose tissue.

BACKGROUND: Constitutional thinness (CT) is a state of low but stable body weight (BMI ≤18 kg/m2). CT subjects have normal-range hormonal profiles and food intake but exhibit resistance to weight gain despite living in the modern world's obesogenic environment. OBJECTIVE: The goal of this study is to identify molecular mechanisms underlying this protective phenotype against weight gain. METHODS: We conducted a clinical overfeeding study on 30 CT subjects and 30 controls (BMI 20-25 kg/m2) matched for age and sex. We performed clinical and integrative molecular and transcriptomic analyses on white adipose and muscle tissues. RESULTS: Our results demonstrate that adipocytes were markedly smaller in CT individuals (mean ± SEM: 2174 ± 142 µm 2) compared with controls (3586 ± 216 µm2) (P < 0.01). The mitochondrial respiratory capacity was higher in CT adipose tissue, particularly at the level of complex II of the electron transport chain (2.2-fold increase; P < 0.01). This higher activity was paralleled by an increase in mitochondrial number (CT compared with control: 784 ± 27 compared with 675 ± 30 mitochondrial DNA molecules per cell; P < 0.05). No evidence for uncoupled respiration or "browning" of the white adipose tissue was found. In accordance with the mitochondrial differences, CT subjects had a distinct adipose transcriptomic profile [62 differentially expressed genes (false discovery rate of 0.1 and log fold change >0.75)], with many differentially expressed genes associating with positive metabolic outcomes. Pathway analyses revealed an increase in fatty acid oxidation ( P = 3 × 10-04) but also triglyceride biosynthesis (P = 3.6 × 10-04). No differential response to the overfeeding was observed in the 2 groups. CONCLUSIONS: The distinct molecular signature of the adipose tissue in CT individuals suggests the presence of augm ented futile lipid cycling, rather than mitochondrial uncoupling, as a way to increase energy expenditure in CT individuals. We propose that increased mitochondrial function in adipose tissue is an important mediator in sustaining the low body weight in CT individuals. This knowledge could ultimately allow more targeted approaches for weight management treatment strategies. This trial was registered at clinicaltrials.gov as NCT02004821.

Randomized clinical trial: efficacy of a food supplement, TRANSITECH, on healthy individuals with mild intermittent constipation.

BACKGROUND: Constipation is a common disorder in the general population and can be observed in healthy individuals. A natural product leading to an increase in bowel movements and decrease in colonic transit time (CTT), without bloating, could be useful for the patient's care. OBJECTIVES: To investigate the effects of TRANSITECH, a food supplement composed of plants and lactic ferments, on bowel movements, CTT and bloating. METHODS: A total of 100 healthy participants, presenting two to five stools per week, were selected and followed over a 6-day baseline period. They were randomly assigned to receive daily two tablets of TRANSITECH or placebo during 10 days. They were then followed up over 28 days after intervention. Participants daily recorded in a home questionnaire the characteristics of stools (frequency and consistency), and the importance of bloating during the preintervention period (from D-6 to D0), the intervention period (from D0 to D10) and the postintervention period (from D10 to D38). Their CTTs were also evaluated by following the propagation of radiopaque markers at D0 and D10. RESULTS: At D10, the food supplement group showed, compared with the placebo group, higher daily stool emission (0.95±0.50, 0.70±0.20, P<0.001), softer stool consistency (2.5±0.6 vs. 3.0±0.8, P<0.001) and lower CTT (33.8±28.2 vs. 56.4±36.2 h, P=0.01). The active group also showed a sustained increase in daily stool emissions observed at D38 compared with D0 (P=0.03). CONCLUSION: TRANSITECH is an efficient natural solution for the treatment of constipation. It increases the number of bowel movements, decreases the oroanal and segmental CTT, is well tolerated, and presents sustained effects after treatment completion.

Rational and design of an overfeeding protocol in constitutional thinness: Understanding the physiology, metabolism and genetic background of resistance to weight gain.

BACKGROUND: Constitutional thinness (CT) is a natural state of underweight (13-17.5kg/m2) without the presence of any eating disorders and abnormal hormonal profile, and with preserved menses in women. We previously conducted a four-week fat overfeeding study showing weight gain resistance in CT women and one of our main results was the identification of an energy gap: a positive energy balance (higher energy intake than energy expenditure). OBJECTIVE: This new overfeeding study is designed to confirm the energy gap and propose mechanistic hypothesis. DESIGN: A 2-week overfeeding (daily consumption of one bottle of Renutryl® Booster (600kcal, 30g protein, 72g carbohydrate, 21g fat) on top of the dietary intake) is performed to compare 15 women and men in each CT group (Body Mass Index [BMI]<18.5kg/m2) to their controls (BMI 20-25kg/m2). Bodyweight, food intake, energy expenditure (canopy, calorimetric chamber and Actiheart), body composition (DXA), appetite regulatory hormone profiles after a test meal, proteomics, metabolomics, urinary metabolic profiles, stool microbiome and lipids, fat and muscle transcriptomics are monitored before and after overfeeding. RESULTS AND CONCLUSIONS: Data inter-linking will be able to be established with results of this study. The findings could possibly open to therapeutic approaches to help CT patients to gain weight as well as provide a better understanding of energy regulation with regard to treat obesity (resistance to weight loss), a mirror image of CT (resistance to weight gain).

Agreement between indirect calorimetry and traditional tests of lactose malabsorption.

BACKGROUND: Lactose malabsorption occurs frequently and the variable consequent intolerance may seriously impair quality of life. No reliable and convenient test method is in routine clinical practice. A recent animal study showed that the respiratory quotient changed significantly after ingestion of sucrose and lactose in naturally lactase-deficient rats. AIMS: This exploratory study evaluated the relevance of monitoring the respiratory quotient after lactose ingestion to detect malabsorption. METHODS: Healthy volunteers were identified and classified lactose absorbers and malabsorbers by a lactose tolerance test (25 g). After an overnight fast, a second lactose challenge was performed to monitor hydrogen excretion and respiratory quotient kinetics over 4h. Participants also completed questionnaires to score and localise their gastrointestinal symptoms. RESULTS: 20 subjects were enrolled (10 per group, 60% males, mean age 34 ± 4 years). Respiratory quotient kinetics were different between absorbers and malabsorbers during the first 100 min after lactose ingestion (p<0.01) and during the initial 30-50 min period. Respiratory quotient was significantly, positively correlated to peak glycaemia (R=0.74) and negatively correlated to hydrogen excretion (R=-0.51) and symptoms score (R=-0.46). CONCLUSIONS: Indirect calorimetry could improve the reliability of lactose malabsorption diagnosis. Studies on larger populations are needed to confirm the validity of this test and propose a simplified measurement.

Assessment of the removal and inactivation of influenza viruses H5N1 and H1N1 by drinking water treatment.

Since 2003, there has been significant concern about the possibility of an outbreak of avian influenza virus subtype H5N1. Moreover, in the last few months, a pandemic of a novel swine-origin influenza A virus, namely A(H1N1), has already caused hundreds of thousands of human cases of illness and thousands of deaths. As those viruses could possibly contaminate water resources through wild birds excreta or through sewage, the aim of our work was to find out whether the treatment processes in use in the drinking water industry are suitable for eradicating them. The effectiveness of physical treatments (coagulation-flocculation-settling, membrane ultrafiltration and ultraviolet) was assessed on H5N1, and that of disinfectants (monochloramine, chlorine dioxide, chlorine, and ozone) was established for both the H5N1 and H1N1 viruses. Natural water samples were spiked with human H5N1/H1N1 viruses. For the coagulation-settling experiments, raw surface water was treated in jar-test pilots with 3 different coagulating agents (aluminum sulfate, ferric chloride, aluminum polychorosulfate). Membrane performance was quantified using a hollow-fiber ultrafiltration system. Ultraviolet irradiation experiments were conducted with a collimated beam that made it possible to assess the effectiveness of various UV doses (25-60 mJ/cm2). In the case of ozone, 0.5 mg/L and 1 mg/L residual concentrations were tested with a contact time of 10 min. Finally, for chlorine, chlorine dioxide and monochloramine treatments, several residual oxidant target levels were tested (from 0.3 to 3 mg/L) with contact times of 5-120 min. The infectivity of the H5N1 and H1N1 viruses in water samples was quantified in cell culture using a microtiter endpoint titration. The impact of coagulation-settling on the H5N1 subtype was quite low and variable. In contrast, ultrafiltration achieved more than a 3-log reduction (and more than a 4-log removal in most cases), and UV treatment was readily effective on its inactivation (more than a 5-log inactivation with a UV dose of 25 mJ/cm2). Of the chemical disinfection treatments, ozone, chlorine and chlorine dioxide were all very effective in inactivating H5N1 and H1N1, whereas monochloramine treatment required higher doses and longer contact times to achieve significant reductions. Our findings suggest that the water treatment strategies that are currently used for surface water treatment are entirely suitable for removing and/or inactivating influenza A viruses. Appropriate preventive actions can be defined for single disinfection treatment plants.