Hepatitis E virus (HEV) is considered an emerging pathogen in industrialized countries. The occurrence of HEV genotypes in samples of faeces from asymptomatic migrants arriving on the coasts of South Italy and environmental samples was investigated. Analyses of sequences were used to compare human and environmental genotypes. A total of 40 stool specimens, 12 samples of untreated urban sewage, 12 samples of treated urban sewage and 12 samples of surface water were analysed. Viruses were concentrated from water samples by the tangential flow ultrafiltration technique. The presence of HEV RNA was detected by nested RT-PCR. Viral isolates were sequenced and phylogenetically characterized. Two (5%) of the 40 faecal samples tested were found to be positive for HEV RNA (G1 and G3 genotypes). The virus was detected in 25% (3/12) of the untreated sewage samples and 25% (3/12) of the surface water samples: all isolates belonged to G3 genotype. None of the treated sewage samples were found to be HEV RNA positive. The virus was detected in the faeces of two asymptomatic subjects, suggesting a potential role for symptom-free HEV carriers as a human reservoir. G3 HEV strains were detected in the untreated sewage, as observed in similar studies conducted in other European countries but differing from another study conducted in Italy recently. Moreover, our results show the first case of HEV isolated from fresh surface waters.

Peroxisome proliferator-activated receptor gamma (PPAR gamma) is a nuclear receptor that regulates gene expression of inflammatory mediators in liver injury. Hepatitis B virus (HBV) suppresses the PPAR gamma-mediated transactivation in liver cancerous cell lines. However, the role of PPAR gamma in patients with chronic HBV infection has not fully demonstrated. Our present study was firstly to determine the clinical relevance of peripheral PPAR gamma mRNA levels in chronic hepatitis B (CHB) patients, and then, the DNA methylation of PPAR gamma promoter was investigated. Peripheral blood mononuclear cells (PBMCs) were isolated from 91 CHB patients and 18 healthy controls. The mRNA level of PPAR gamma was determined by quantitative real-time PCR; meanwhile, the CpG island methylation was assessed by methylation-specific PCR. CHB patients showed significantly lower mRNA level of PPAR gamma than healthy controls (P = 0.005). The mRNA level was decreased in HBV-DNA-positive group than HBV-DNA-negative group (P = 0.041). Interaction analysis demonstrated that the DNA methylation pattern was responsible for the suppression of peripheral PPAR gamma transcription in CHB patients (P = 0.003). Furthermore, the hypermethylation of PPAR gamma gene promoter was significantly associated with liver inflammation and fibrosis in CHB. In conclusion, DNA methylation patterns were responsible for the decreased mRNA level of peripheral PPAR gamma in CHB patients. Liver inflammation and fibrosis were found to be associated with hypermethylation of PPAR gamma promoter.

Quantification of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) and their change model during treatment are emerging as a useful tool for assessing the outcome of hepatitis B virus (HBV) infection and predicting the efficacy of antiviral therapy. The aim of this study was to compare the performance of the Elecsys and Architect assays for HBsAg and HBeAg quantification. Quantification of HBsAg and HBeAg, determined by these two assays, were assessed in 1292 sera from patients with chronic hepatitis B(CHB). HBeAg quantification in serum was performed by calibrating the results through HBeAg Paul-Ehrlich international (PEI) reference standard. The HBV genotype was determined by direct sequencing and phylogenetic analysis. Of 1292 samples, the distribution of genotype was 514 (39.78%) genotype B, 776 (60.06%) genotype C, 2 (0.16%) genotype D. The results of HBsAg and HBeAg quantification between the Architect and Elecsys assays were significantly correlated (HBsAg: r = 0.939; HBeAg: r = 0.987), independent of HBV genotype and treatment phase. The mean differences between the two methods (the log10 [Elecsys] - the log10 [Architect]) were 0.075 log10  IU/mL and -0.149 log10 PE IU/mL in quantifying HBsAg and HBeAg, respectively. This study demonstrates a high correlation between the Elecsys and the Architect assays in quantifying HBsAg and HBeAg, regardless of HBV genotype. Both the two assays can be used to monitor the HBsAg and HBeAg levels in patients with chronic hepatitis B.

Several new direct-acting antiviral (DAA) drugs are in development for chronic hepatitis C viral (HCV) infection, and NS3-NS4A serine protease and the NS5B RNA-dependent RNA polymerase have been the major targets. HCV variants displaying drug-resistant phenotypes have been observed both in vitro and during clinical trials. Our aim was to characterize amino acid changes at positions previously associated with resistance in protease (NS3) and polymerase (NS5B) regions from treatment-naïve HCV patients infected with genotypes 1a, 1b and 3a. All 1383 NS3 protease sequences (genotype 1a = 680, 1b = 498 and 3a = 205) and 806 NS5B polymerase sequences (genotypes 1a = 471, 1b = 329, 3a = 6) were collected from Los Alamos databank. Genotype 3a protease sequences showed the typical low-level resistance mutation V36L. NS3 sequences from other genotypes presented mutations on positions 36, 39, 41, 43, 54, 80, 109, 155 and 168 in a frequency lower than 2%, except for the mutation Q80R found in 35% of genotype 1a isolates. Polymerase sequences from genotype 3a patients showed five typical mutations: L419I, I424V, I482L, V499A and S556G. Two positions presented high polymorphism in the NS5B region from genotype 1a (V499A) and genotype 1b (C316N) subjects. Our results demonstrated a natural profile of genotype 3a that can be associated with the pre-existence of HCV variants resistant to first-generation protease inhibitors and to non-nucleoside polymerase inhibitors. Likewise, genotype 1b isolates and genotype 1a sequences exhibited pre-existing mutations associated with resistance to Palm II and Thumb I polymerase inhibitors, respectively.

Chemokines and cytokines play a vital role in directing and regulating immune responses to viral infections. Persistent hepatitis C virus (HCV) infection is characterized by the loss of anti-HCV cellular immune responses, while control of HCV infection is associated with maintenance of anti-HCV cellular immune responses. To determine whether plasma concentrations of 19 chemokines and cytokines controlling T-cell trafficking and function differed based on infection outcome, we compared them in at-risk subjects followed prospectively for HCV infection. Levels were compared over time in subjects who controlled HCV infection (Clearance) and subjects who developed persistent HCV infection (Persistence) at two time points during acute infection: (i) first viraemic sample (initial viraemia) and (ii) last viraemic sample in Clearance subjects and time-matched samples in Persistence subjects. At initial viraemia, increased pro-inflammatory tumour necrosis factor α (TNFα) plasma concentrations were observed in the Clearance group, while the plasma levels of anti-inflammatory interleukin (IL)-2, IL-10 and IL-13 were higher in the Persistence group. IL-13 was positively correlated with IL-2 and IL-10 at initial viraemia in the Persistence group. At the time of last viraemia, plasma levels of eotaxin, macrophage chemoattractant protein-4 (MCP-4), IL-5 and IL-10 were higher in the Persistence group and IL-10 and IL-5 levels were positively correlated. Collectively, these results suggest that the development of persistent infection is associated with an anti-inflammatory and pro-fibrogenic chemokine and cytokine profile that is evident at the onset of infection and maintained throughout acute infection.

Study C209 evaluated the activity of telaprevir in treatment-naïve patients with genotypes 2 or 3 (G2, G3) hepatitis C virus (HCV) infection. Telaprevir monotherapy showed potent activity against HCV G2, but limited activity against G3. This analysis was performed to characterize HCV viral variants emerging during telaprevir-based treatment of G2/G3 HCV-infected patients. Patients were randomized to receive 2 weeks of treatment with telaprevir (telaprevir monotherapy), telaprevir plus peginterferon alfa-2a and ribavirin (triple therapy), or placebo plus peginterferon alfa-2a and ribavirin (control), followed by 22-24 weeks of peginterferon/ribavirin alone. Viral breakthrough was defined as an increase >1 log10 in HCV RNA from nadir, or HCV RNA >100 IU/mL in patients previously reaching <25 IU/mL. Twenty-three patients (47%) had G2 and 26 (53%) had G3 HCV. Viral breakthrough occurred during the initial 2-week treatment phase in six G2 patients (66.7%; subtypes 2, 2a and 2b) and three G3 patients (37.5%; all subtype 3a), all in the telaprevir monotherapy arm. Four breakthrough patients (three G2, one G3) subsequently achieved sustained virologic response (SVR). In all patients with breakthrough and available sequence data, mutations associated with reduced susceptibility to telaprevir in genotype 1 (G1) HCV were observed. No novel G2/G3-specific mutations were associated with telaprevir resistance. The telaprevir resistance profile appeared consistent across HCV genotypes 1, 2 and 3. Although viral breakthrough with resistance occurred in patients receiving telaprevir monotherapy, half of these patients achieved an SVR upon addition of peginterferon/ribavirin highlighting the importance of combination therapy.

There is limited information about the role of hepatic stellate cells (HSC) in liver innate immunity against hepatitis C virus (HCV). We thus examined whether HSC can produce antiviral factors that inhibit HCV replication in human hepatocytes. HSC expressed functional Toll-like receptor 3 (TLR-3), which could be activated by its ligand, polyinosine-polycytidylic acid (poly I:C), leading to the induction of interferon-λ (IFN-λ) at both mRNA and protein levels. TLR-3 signalling of HSC also induced the expression of IFN regulatory factor 7 (IRF-7), a key regulator of IFN signalling pathway. When HCV JFH-1-infected Huh7 cells were co-cultured with HSC activated with poly I:C or incubated in media conditioned with supernatant (SN) from poly I:C-activated HSC, HCV replication was significantly suppressed. This HSC SN action on HCV inhibition was mediated through IFN-λ, which was evidenced by the observation that antibody to IFN-λ receptors could neutralize the HSC-mediated anti-HCV effect. The role of IFN-λ in HSC-mediated anti-HCV activity is further supported by the observation that HSC SN treatment induced the expression of IRF-7 and IFN-stimulated genes (ISGs), OAS-1 and MxA in HCV-infected Huh7 cells. These observations indicate that HSC may be a key regulatory bystander, participating in liver innate immunity against HCV infection using an IFN-λ-dependent mechanism.

Studies of IL-28B genotype in patients with hepatitis C virus (HCV) genotype 2/3 infection have yielded conflicting results. The aim of this meta-analysis was to obtain a pooled odds ratio (OR) of the impact of IL-28B genotype on achieving sustained virologic response (SVR) in patients with HCV genotype 2/3 infection treated with pegIFN and ribavirin. A meta-analysis with a random effects model was performed, and study heterogeneity and publication bias were assessed. Forty-three percent of the Caucasians (11 studies) and 86% of Asians (five studies) had the favourable IL-28B genotype. In Caucasians, the pooled OR of SVR with the favourable IL-28B genotype was 1.36 (95%CI: 0.98-1.88, P = 0.07) in all patients and 1.55 (95%CI: 1.10-2.18, P = 0.01) in patients treated with pegIFN and ribavirin for ≥24 weeks. In Asians, the pooled OR of SVR in patients with the favourable IL-28B genotype was 1.99 (95%CI: 0.94-4.25, P = 0.07). The favourable IL-28B genotype was also significantly associated with rapid virologic response (RVR) in both groups (Caucasians: OR: 1.82, 95%CI: 1.12-2.96, P = 0.02; Asians: 2.39, 95%CI: 1.39-4.11, P = 0.002), as well as the likelihood of an SVR in a subgroup of 350 Caucasian patients without an RVR (OR: 3.29, 95%CI: 1.67-6.51, P = 0.001). The favourable IL-28B genotype is a statistically significant predictor of SVR and RVR in Caucasian patients treated with pegIFN and ribavirin for 24 weeks. In contrast, the favourable IL-28B genotype is associated with RVR, but not SVR in Asian HCV genotype 2 patients.

For a long time, the lack of an appropriate cell culture system has hampered the study of neutralizing antibody responses against hepatitis C virus (HCV). However, the last decade has seen the development of several model systems that have significantly advanced our understanding of viral entry and antibody neutralization. Studies of acutely infected patients suggest that a strong and early production of neutralizing antibodies may contribute to control the virus during the acute phase of HCV infection and facilitate viral elimination by cellular immune responses. It also emerges that the early antibody response mainly targets hypervariable region 1 (HVR1) of the envelope glycoprotein E2. This host response can lead to viral escape from neutralization by rapid amino acid changes in this hypervariable region. In contrast, cross-reactive neutralizing antibodies seem to appear later during HCV infection, and several mechanisms contribute to reduce their accessibility to their cognate epitopes. These include the masking of major conserved neutralizing epitopes by HVR1, specific N-linked glycans and the lipid moiety of the viral particle. Other potential mechanisms of evasion from the neutralizing antibody response include a modulation by high-density lipoproteins and interfering antibodies as well as the capacity of the virus to be transferred by cell-to-cell contacts. Finally, the recent identification of several highly conserved neutralizing epitopes provides some opportunities for the design and development of vaccine candidates that elicit a protective humoral immune response.