Identification of interferon-stimulated genes with modulated expression during hepatitis E virus infection in pig liver tissues and human HepaRG cells.

Introduction: Hepatitis E virus (HEV) is a common cause of enterically transmitted acute hepatitis worldwide. The virus is transmitted by the fecal-oral route via the consumption of contaminated water supplies and is also a zoonotic foodborne pathogen. Swine are the main reservoir of zoonotic HEV. In humans, HEV infection is usually asymptomatic or causes acute hepatitis that is self-limited. However, fulminant hepatic failure and chronic cases of HEV infection can occur in some patients. In contrast, HEV infection in pigs remains asymptomatic, although the virus replicates efficiently, suggesting that swine are able to control the virus pathogenesis. Upon viral infection, IFN is secreted and activates cellular pathways leading to the expression of many IFN-stimulated genes (ISGs). ISGs can restrict the replication of specific viruses and establish an antiviral state within infected and neighboring cells. Methods: In this study, we used PCR arrays to determine the expression level of up to 168 ISGs and other IFN-related genes in the liver tissues of pigs infected with zoonotic HEV-3c and HEV-3f and in human bipotent liver HepaRG cells persistently infected with HEV-3f. Results and discussion: The expression of 12 and 25 ISGs was found to be up-regulated in infected swine livers and HepaRG cells, respectively. The expression of CXCL10, IFIT2, MX2, OASL and OAS2 was up-regulated in both species. Increased expression of IFI16 mRNA was also found in swine liver tissues. This study contributes to the identification of potential ISGs that could play a role in the control or persistence of HEV infection.

Low prevalence of hepatitis E virus in the liver of Corsican pigs slaughtered after 12 months despite high antibody seroprevalence.

Hepatitis E virus (HEV) infection can be acute and benign or evolve to chronic hepatitis with rapid progression toward cirrhosis or liver failure in humans. Hence, hepatitis E (HE) disease is a major public health concern. In countries where pig populations are highly contaminated with HEV, human cases of HE are mainly foodborne, occurring frequently after consumption of raw or undercooked pork products or liver. Among factors associated to the presence of HEV in pork livers from intensive rearing systems, early slaughter (≤6 months) seems to be major. In Corsica, local pigs are raised in extensive farming systems and slaughtered after 12 months. To evaluate if slaughter of pigs over 12 months reduces the risk of HEV presence in livers, 1197 liver samples were randomly collected in 2 Corsican slaughterhouses. Presence of HEV RNA was detected in liver and HEV seroprevalence was determined in paired serum. The sampling included 1083 livers from animals between 12 and 48 months and 114 livers from animals <12 months. The samples were predominantly from semi-extensive and extensive farms (n = 1154). Estimated HEV seroprevalence was high, that is, >88%, and HEV RNA prevalence in adult pig livers (>12 months old) was low, that is, 0.18%. However, in livers from younger animals (<12 months), including piglets below 6 months old, 5.3% (6/114) of the samples were positive for HEV RNA. Sequences recovered from positive livers belonged to HEV genotype 3c and 3f. The presence of infectious HEV was confirmed in two livers by the detection of HEV replication in HepaRG cell cultures. Thus, this study demonstrates the low prevalence of HEV in livers of pigs over 12 months, even in farms with high HEV circulation. This observation may open new perspectives on the preferential use of livers from animals older than 12 months in raw pork liver products.

Characterization of a Cell Culture System of Persistent Hepatitis E Virus Infection in the Human HepaRG Hepatic Cell Line.

Hepatitis E virus (HEV) is considered as an emerging global health problem. In most cases, hepatitis E is a self-limiting disease and the virus is cleared spontaneously without the need of antiviral therapy. However, immunocompromised individuals can develop chronic infection and liver fibrosis that can progress rapidly to cirrhosis and liver failure. The lack of efficient and relevant cell culture system and animal models has limited our understanding of the biology of HEV and the development of effective drugs for chronic cases. In the present study, we developed a model of persistent HEV infection in human hepatocytes in which HEV replicates efficiently. This HEV cell culture system is based on differentiated HepaRG cells infected with an isolate of HEV-3 derived from a patient suffering from acute hepatitis E. Efficient replication was maintained for several weeks to several months as well as after seven successive passages on HepaRG naïve cells. Moreover, after six passages onto HepaRG, we found that the virus was still infectious after oral inoculation into pigs. We also showed that ribavirin had an inhibitory effect on HEV replication in HepaRG. In conclusion, this system represents a relevant and efficient in vitro model of HEV replication that could be useful to study HEV biology and identify effective antiviral drugs against chronic HEV infection.

Impact of porcine circovirus type 2 (PCV2) infection on hepatitis E virus (HEV) infection and transmission under experimental conditions.

Hepatitis E virus is a zoonotic pathogen for which pigs have been identified as the main reservoir in industrialised countries. HEV infection dynamics in pig herds and pigs are influenced by several factors, including herd practices and possibly co-infection with immunomodulating viruses. This study therefore investigates the impact of porcine circovirus type 2 (PCV2) on HEV infection and transmission through experimental HEV/PCV2 co-infection of specific-pathogen-free pigs. No statistical difference between HEV-only and HEV/PCV2-infected animals was found for either the infectious period or the quantity of HEV shed in faeces. The HEV latency period was shorter for HEV/PCV2 co-infected pigs than for HEV-only infected pigs (11.6 versus 12.3 days). Its direct transmission rate was three times higher in cases of HEV/PCV2 co-infection than in cases of HEV-only infection (0.12 versus 0.04). On the other hand, the HEV transmission rate through environmental accumulation was lower in cases of HEV/PCV2 co-infection (4.3·10-6 versus 1.5·10-5 g/RNA copies/day for HEV-only infected pigs). The time prior to HEV seroconversion was 1.9 times longer in HEV/PCV2 co-infected pigs (49.4 versus 25.6 days for HEV-only infected pigs). In conclusion, our study shows that PCV2 affects HEV infection and transmission in pigs under experimental conditions.

The Amino-Terminal Region of Hepatitis E Virus ORF1 Containing a Methyltransferase (Met) and a Papain-Like Cysteine Protease (PCP) Domain Counteracts Type I Interferon Response.

Hepatitis E virus (HEV) is responsible for large waterborne epidemics of hepatitis in endemic countries and is an emerging zoonotic pathogen worldwide. In endemic regions, HEV-1 or HEV-2 genotypes are frequently associated with fulminant hepatitis in pregnant women, while with zoonotic HEV (HEV-3 and HEV-4), chronic cases of hepatitis and severe neurological disorders are reported. Hence, it is important to characterize the interactions between HEV and its host. Here, we investigated the ability of the nonstructural polyprotein encoded by the first open reading frame (ORF1) of HEV to modulate the host early antiviral response and, in particular, the type I interferon (IFN-I) system. We found that the amino-terminal region of HEV-3 ORF1 (MetYPCP), containing a putative methyltransferase (Met) and a papain-like cysteine protease (PCP) functional domain, inhibited IFN-stimulated response element (ISRE) promoter activation and the expression of several IFN-stimulated genes (ISGs) in response to IFN-I. We showed that the MetYPCP domain interfered with the Janus kinase (JAK)/signal transducer and activator of the transcription protein (STAT) signalling pathway by inhibiting STAT1 nuclear translocation and phosphorylation after IFN-I treatment. In contrast, MetYPCP had no effect on STAT2 phosphorylation and a limited impact on the activation of the JAK/STAT pathway after IFN-II stimulation. This inhibitory function seemed to be genotype-dependent, as MetYPCP from HEV-1 had no significant effect on the JAK/STAT pathway. Overall, this study provides evidence that the predicted MetYPCP domain of HEV ORF1 antagonises STAT1 activation to modulate the IFN response.

Preliminary single-dose pharmacokinetics of marbofloxacin in ball pythons (Python regius).

Pharmacokinetics of marbofloxacin in two male and four female adult ball pythons (Python regius) was determined after i.v. and p.o. administration of a single dose. Using a crossover design, each snake was given a single 10 mg/kg dose of marbofloxacin i.v. and p.o. Blood samples were collected prior to and 0.5, 1, 1.5, 3, 6, 12, and 24 hr after marbofloxacin administration. Marbofloxacin was quantitated by use of liquid chromatography-mass spectrometry. Following p.o. administration, marbofloxacin had a peak plasma concentration (Cmax) of 9.40 microg/ml and a time to Cmax (Tmax) of 9.0 hr. Based on the plasma pharmacokinetics generated in this study and pending any further studies to evaluate potential toxicity and multi-dose pharmacokinetics, we suggest a dosage for marbofloxacin in ball pythons of 10 mg/kg p.o. at least every 48 hr, depending on the sensitivity of the pathogen and as a basis for further research.

The Instrumental Activity of Daily Living Profile in Aging: A Feasibility Study.

BACKGROUND: Dysfunctions in complex activities of daily living (ADLs) are a normal part of the aging process. However, differentiating functional decline associated with healthy aging from the subtle decline experienced by individuals with mild cognitive impairment and early dementia constitutes a challenge. Finding an appropriate tool that can capture these subtle but important functional changes represents a priority. OBJECTIVES: The aims of this study were to evaluate the feasibility of using the Instrumental Activities of Daily Living Profile (IADL Profile) with elderly participants and to describe their level of difficulty encountered in each task. METHODS: The tool was administered to a group of 40 elderly participants living in the community. RESULTS: The IADL Profile was found to be feasible to use in older individuals; the tool also showed sensitivity to the difficulties experienced by this population in everyday functioning. CONCLUSION: The IADL Profile is a promising ecological tool to evaluate independence in aging and may help to identify individuals with MCI. This tool may also contribute to the development of tailored interventions to enhance everyday functioning in the older population.

Plasma pharmacokinetics and synovial fluid concentrations after oral administration of single and multiple doses of celecoxib in greyhounds.

OBJECTIVE: To determine the plasma pharmacokinetics and synovial fluid concentrations after oral administration of single and multiple doses of celecoxib in Greyhounds. ANIMALS: 7 adult Greyhounds. PROCEDURES: Dogs received celecoxib (median dose, 11.8 mg/kg [range, 11.5 to 13.6 mg/kg], PO, q 24 h) for 10 days. Blood samples were collected prior to administration of celecoxib and serially for 24 hours after the 1st and 10th doses were administered. A synovial joint catheter was placed into a stifle joint in each dog for collection of synovial fluid samples. Concentrations of celecoxib in plasma and synovial fluid were quantified by use of a validated liquid chromatography/mass spectrometry method. Identification of hydroxy- and carboxyl-celecoxib in plasma and synovial fluid was also performed. Pharmacokinetic parameters were determined by use of noncompartmental analysis. RESULTS: Administration of multiple doses of celecoxib resulted in a significant decrease (40%) in median area under the curve (AUC) values and a corresponding decrease in median maximum concentrations (Cmax; 2,620 to 2,032 ng/mL) between the 1st and 10th doses. Synovial fluid concentrations were less than the corresponding plasma concentrations at all times except 24 hours after administration of the 10th dose of celecoxib. CONCLUSIONS AND CLINICAL RELEVANCE: Celecoxib distributes into the synovial fluid of Greyhounds. Although the exact mechanism for the decreases in AUC and Cmax is not known, results suggested that the plasma pharmacokinetics of celecoxib are different after administration of multiple doses in Greyhounds. These findings warrant further investigation on the absorption, distribution, metabolism, and elimination of celecoxib in Greyhounds and other breeds of dogs.