Spatiotemporal Scan and Age-Period-Cohort Analysis of Hepatitis C Virus in Henan,China: 2005–2012

Background

Studies have shown that hepatitis C virus (HCV) infection increased during the past decades in China. However, little evidence is available on when, where, and who were infected with HCV. There are gaps in knowledge on the epidemiological burden and evolution of the HCV epidemic in China.

Methods

Data on HCV cases were collected by the disease surveillance system from 2005 to 2012 to explore the epidemic in Henan province. Spatiotemporal scan statistics and age-period-cohort (APC) model were used to examine the effects of age, period, birth cohort, and spatiotemporal clustering.

Results

177,171 HCV cases were reported in Henan province between 2005 and 2012. APC modelling showed that the HCV reported rates significantly increased in people aged > 50 years. A moderate increase in HCV reported rates was observed for females aged about 25 years. HCV reported rates increased over the study period. Infection rates were greatest among people born between 1960 and 1980. People born around 1970 had the highest relative risk of HCV infection. Women born between 1960 and 1980 had a five-fold increase in HCV infection rates compared to men, for the same birth cohort. Spatiotemporal mapping showed major clustering of cases in northern Henan, which probably evolved much earlier than other areas in the province.

Conclusions

Spatiotemporal mapping and APC methods are useful to help delineate the evolution of the HCV epidemic. Birth cohort should be part of the criteria screening programmes for HCV in order to identify those at highest risk of infection and unaware of their status. As Henan is unique in the transmission route for HCV, these methods should be used in other high burden provinces to help identify subpopulations at risk.     

Analysis of Functional Differences between Hepatitis C Virus NS5A of Genotypes 1–7 in Infectious Cell Culture Systems

Hepatitis C virus (HCV) is an important cause of chronic liver disease. Several highly diverse HCV genotypes exist with potential key functional differences. The HCV NS5A protein was associated with response to interferon (IFN)-α based therapy, and is a primary target of currently developed directly-acting antiviral compounds. NS5A is important for replication and virus production, but has not been studied for most HCV genotypes. We studied the function of NS5A using infectious NS5A genotype 1–7 cell culture systems, and through reverse genetics demonstrated a universal importance of the amphipathic alpha-helix, domain I and II and the low-complexity sequence (LCS) I for HCV replication; the replicon-enhancing LCSI mutation S225P attenuated all genotypes. Mutation of conserved prolines in LCSII led to minor reductions in virus production for the JFH1(genotype 2a) NS5A recombinant, but had greater effects on other isolates; replication was highly attenuated for ED43(4a) and QC69(7a) recombinants. Deletion of the conserved residues 414-428 in domain III reduced virus production for most recombinants but not JFH1(2a). Reduced virus production was linked to attenuated replication in all cases, but ED43(4a) and SA13(5a) also displayed impaired particle assembly. Compared to the original H77C(1a) NS5A recombinant, the changes in LCSII and domain III reduced the amounts of NS5A present. For H77C(1a) and TN(1a) NS5A recombinants, we observed a genetic linkage between NS5A and p7, since introduced changes in NS5A led to changes in p7 and vice versa. Finally, NS5A function depended on genotype-specific residues in domain I, as changing genotype 2a-specific residues to genotype 1a sequence and vice versa led to highly attenuated mutants. In conclusion, this study identified NS5A genetic elements essential for all major HCV genotypes in infectious cell culture systems. Genotype- or isolate- specific NS5A functional differences were identified, which will be important for understanding of HCV NS5A function and therapeutic targeting.     

Biomarker Discovery for Early Detection of Hepatocellular Carcinoma in Hepatitis C–infected Patients

Chronic hepatic disease damages the liver, and the resulting wound-healing process leads to liver fibrosis and the subsequent development of cirrhosis. The leading cause of hepatic fibrosis and cirrhosis is infection with hepatitis C virus (HCV), and of the patients with HCV-induced cirrhosis, 2% to 5% develop hepatocellular carcinoma (HCC), with a survival rate of 7%. HCC is one of the leading causes of cancer-related death worldwide, and the poor survival rate is largely due to late-stage diagnosis, which makes successful intervention difficult, if not impossible. The lack of sensitive and specific diagnostic tools and the urgent need for early-stage diagnosis prompted us to discover new candidate biomarkers for HCV and HCC. We used aptamer-based fractionation technology to reduce serum complexity, differentially labeled samples (six HCV and six HCC) with fluorescent dyes, and resolved proteins in pairwise two-dimensional difference gel electrophoresis. DeCyder software was used to identify differentially expressed proteins and spots picked, and MALDI-MS/MS was used to determine that ApoA1 was down-regulated by 22% (p < 0.004) in HCC relative to HCV. Differential expression quantified via two-dimensional difference gel electrophoresis was confirmed by means of ¹⁸O/¹⁶O stable isotope differential labeling with LC-MS/MS zoom scans. Technically independent confirmation was demonstrated by triple quadrupole LC-MS/MS selected reaction monitoring (SRM) assays with three peptides specific to human ApoA1 (DLATVYVDVLK, WQEEMELYR, and VSFLSALEEYTK) using ¹⁸O/¹⁶O-labeled samples and further verified with AQUA peptides as internal standards for quantification. In 50 patient samples (24 HCV and 26 HCC), all three SRM assays yielded highly similar differential expression of ApoA1 in HCC and HCV patients. These results validated the SRM assays, which were independently confirmed by Western blotting. Thus, ApoA1 is a candidate member of an SRM biomarker panel for early diagnosis, prognosis, and monitoring of HCC. Future multiplexing of SRM assays for other candidate biomarkers is envisioned to develop a biomarker panel for subsequent verification and validation studies.Hepatocellular carcinoma (HCC)¹ is the most common type of primary liver cancer and ranks third among cancers as a cause of death worldwide. With a five-year survival rate of less than 7% (1), it is responsible for more than a million deaths annually (2). Hepatitis C virus (HCV) is a major risk factor for the development of HCC, and an estimated 3 to 4 million people are infected with HCV annually (3).The projected rise in new HCC cases in the United States is due mainly to latent HCV infections (4) in the general population, with the onset of HCC coming several decades after initial infection. The poor prognosis associated with HCC is primarily due to the disease being diagnosed at a late stage, making successful therapeutic intervention difficult, if not impossible. Early diagnosis is important for successful treatment by means of ablation, resection, and/or transplant. Although α-fetoprotein (AFP) is routinely used for screening, it is often normal or indeterminate in early cancer cases. AFP is a low-sensitivity biomarker that is normal in up to 40% of patients with HCC, particularly during the early stages (5). AFP is also a low-specificity biomarker, as it is seen in patients with cirrhosis or exacerbations of chronic hepatitis and pancreatitis (6, 7). These limitations generate anxiety for patients and physicians alike. Other screening modalities for early HCC detection are variously inaccurate, expensive (computed tomography or MRI), or potentially dangerous (biopsy). These concerns present the urgent need for a sensitive, specific, and facile screening modality for early detection, diagnosis, and monitoring of HCC that would provide significant clinical benefit. Thus, there is a critical unmet medical need to discover and validate novel specific biomarkers for the early detection of HCC.Progression from chronic infection to cirrhosis and then to HCC usually results in changes in proteins found in hepatic tissues and peripheral blood (8). Accordingly, the exploration of serum to discover clinically useful protein and peptide biomarkers is promising. Serum provides a rich sample for diagnostic analyses because of the expression and release of proteins (potential biomarkers) into the bloodstream in response to specific physiological states. The proteomic characterization of human serum for the identification of disease-specific biomarkers promises to be a powerful diagnostic tool for defining the onset, progression, and prognosis of human diseases. Indeed, much of our current understanding of the serum proteome has come from the use of techniques such as two-dimensional PAGE and LC/MS, which have proven to be exceptionally useful for separating and identifying individual protein and peptide constituents in serum. Although the easily obtainable nature and high protein content of serum make it a valuable specimen for biomarker determination (9), there are still numerous hurdles to overcome when analyzing human serum, one of the most complex proteomes known. Serum contains ∼20 highly abundant proteins, which account for greater than 95% of the total protein mass, and a large number of medium- and low-abundance proteins that span some 12 orders of magnitude in concentration and represent an extremely formidable analytical challenge (10, 11). Thus the reliable proteomic characterization of serum and identification of biomarkers would be dramatically improved if the complexity of the serum proteome were reduced. Many fractionation techniques have been used to address this challenge; we used aptamer-based technology to reduce abundant proteins, thereby enriching lower abundance proteins with minimal or no loss of serum proteome information.In this study, several analytical approaches were used to discover and confirm ApoA1 as a candidate serum-based biomarker for early HCC detection. We applied two-dimensional difference gel electrophoresis (DIGE) together with nanoflow liquid chromatography–tandem mass spectrometry (nano-LC-MS/MS) to detect proteins that are differentially expressed between HCV and HCC. Although many serum biomarker candidates have been discovered, verification and validation of these candidates are the rate-limiting steps in a biomarker pipeline (12) because conventional methods using antibodies (e.g. Western blotting, enzyme-linked immunosorbent assay) are not suitable for large-scale analysis because of their poor throughput and antibody availability (13). To overcome these challenges, we used selective reaction monitoring (SRM) to quantitatively verify and validate ApoA1 as a candidate biomarker in HCC and HCV.     

Interaction of L-SIGN with Hepatitis C Virus Envelope Protein E2 Up-Regulates Raf–MEK–ERK Pathway

Liver/lymph node-specific intercellular adhesion molecule-3-grabbing integrin (L-SIGN) facilitates hepatitis C virus (HCV) infection through interaction with HCV envelope protein E2. Signaling events triggered by the E2 via L-SIGN are poorly understood. Here, kinase cascades of Raf–MEK–ERK pathway were defined upon the E2 treatment in NIH3T3 cells with stable expression of L-SIGN. The E2 bound to the cells through interaction with L-SIGN and such binding subsequently resulted in phosphorylation and activation of Raf, MEK, and ERK. Blockage of L-SIGN with antibody against L-SIGN reduced the E2-induced phosphorylation of Raf, MEK, and ERK. In the cells infected with cell culture-derived HCV, phosphorylation of these kinases was enhanced by the E2. Up-regulation of Raf–MEK–ERK pathway by HCV E2 via L-SIGN provides new insights into signaling cascade of L-SIGN, and might be a potential target for control and prevention of HCV infection.     

Combined Liquid Chromatography–Tandem Mass Spectrometry Analysis of Progesterone Metabolites

Progesterone has a number of important functions throughout the human body. While the roles of progesterone are well known, the possible actions and implications of progesterone metabolites in different tissues remain to be determined. There is a growing body of evidence that these metabolites are not inactive, but can have significant biological effects, as anesthetics, anxiolytics and anticonvulsants. Furthermore, they can facilitate synthesis of myelin components in the peripheral nervous system, have effects on human pregnancy and onset of labour, and have a neuroprotective role. For a better understanding of the functions of progesterone metabolites, improved analytical methods are essential. We have developed a combined liquid chromatography—tandem mass spectrometry (LC-MS/MS) method for detection and quantification of progesterone and 16 progesterone metabolites that has femtomolar sensitivity and good reproducibility in a single chromatographic run. MS/MS analyses were performed in positive mode and under constant electrospray ionization conditions. To increase the sensitivity, all of the transitions were recorded using the Scheduled MRM algorithm. This LC-MS/MS method requires small sample volumes and minimal sample preparation, and there is no need for derivatization. Here, we show the application of this method for evaluation of progesterone metabolism in the HES endometrial cell line. In HES cells, the metabolism of progesterone proceeds mainly to (20S)-20-hydroxy-pregn-4-ene-3-one, (20S)-20-hydroxy-5α-pregnane-3-one and (20S)-5α-pregnane-3α,20-diol. The investigation of possible biological effects of these metabolites on the endometrium is currently undergoing.     

Amphipathic α-Helices in Apolipoproteins Are Crucial to the Formation of Infectious Hepatitis C Virus Particles

Apolipoprotein B (ApoB) and ApoE have been shown to participate in the particle formation and the tissue tropism of hepatitis C virus (HCV), but their precise roles remain uncertain. Here we show that amphipathic α-helices in the apolipoproteins participate in the HCV particle formation by using zinc finger nucleases-mediated apolipoprotein B (ApoB) and/or ApoE gene knockout Huh7 cells. Although Huh7 cells deficient in either ApoB or ApoE gene exhibited slight reduction of particles formation, knockout of both ApoB and ApoE genes in Huh7 (DKO) cells severely impaired the formation of infectious HCV particles, suggesting that ApoB and ApoE have redundant roles in the formation of infectious HCV particles. cDNA microarray analyses revealed that ApoB and ApoE are dominantly expressed in Huh7 cells, in contrast to the high level expression of all of the exchangeable apolipoproteins, including ApoA1, ApoA2, ApoC1, ApoC2 and ApoC3 in human liver tissues. The exogenous expression of not only ApoE, but also other exchangeable apolipoproteins rescued the infectious particle formation of HCV in DKO cells. In addition, expression of these apolipoproteins facilitated the formation of infectious particles of genotype 1b and 3a chimeric viruses. Furthermore, expression of amphipathic α-helices in the exchangeable apolipoproteins facilitated the particle formation in DKO cells through an interaction with viral particles. These results suggest that amphipathic α-helices in the exchangeable apolipoproteins play crucial roles in the infectious particle formation of HCV and provide clues to the understanding of life cycle of HCV and the development of novel anti-HCV therapeutics targeting for viral assembly.     

Identification of Human Plasma Metabolites Exhibiting Time-of-Day Variation Using an Untargeted Liquid Chromatography–Mass Spectrometry Metabolomic Approach

Although daily rhythms regulate multiple aspects of human physiology, rhythmic control of the metabolome remains poorly understood. The primary objective of this proof-of-concept study was identification of metabolites in human plasma that exhibit significant 24-h variation. This was assessed via an untargeted metabolomic approach using liquid chromatography–mass spectrometry (LC-MS). Eight lean, healthy, and unmedicated men, mean age 53.6 (SD?±?6.0) yrs, maintained a fixed sleep/wake schedule and dietary regime for 1 wk at home prior to an adaptation night and followed by a 25-h experimental session in the laboratory where the light/dark cycle, sleep/wake, posture, and calorific intake were strictly controlled. Plasma samples from each individual at selected time points were prepared using liquid-phase extraction followed by reverse-phase LC coupled to quadrupole time-of-flight MS analysis in positive ionization mode. Time-of-day variation in the metabolites was screened for using orthogonal partial least square discrimination between selected time points of 10:00 vs. 22:00?h, 16:00 vs. 04:00?h, and 07:00 (d 1) vs. 16:00?h, as well as repeated-measures analysis of variance with time as an independent variable. Subsequently, cosinor analysis was performed on all the sampled time points across the 24-h day to assess for significant daily variation. In this study, analytical variability, assessed using known internal standards, was low with coefficients of variation <10%. A total of 1069 metabolite features were detected and 203 (19%) showed significant time-of-day variation. Of these, 34 metabolites were identified using a combination of accurate mass, tandem MS, and online database searches. These metabolites include corticosteroids, bilirubin, amino acids, acylcarnitines, and phospholipids; of note, the magnitude of the 24-h variation of these identified metabolites was large, with the mean ratio of oscillation range over MESOR (24-h time series mean) of 65% (95% confidence interval [CI]: 49–81%). Importantly, several of these human plasma metabolites, including specific acylcarnitines and phospholipids, were hitherto not known to be 24-h variant. These findings represent an important baseline and will be useful in guiding the design and interpretation of future metabolite-based studies. (Author correspondence: Jooern.Ang@icr.ac.uk or Florence.Raynaud@icr.ac.uk)     

Dimerization of Hepatitis E Virus Capsid Protein E2s Domain Is Essential for Virus–Host Interaction

Hepatitis E virus (HEV), a non-enveloped, positive-stranded RNA virus, is transmitted in a faecal-oral manner, and causes acute liver diseases in humans. The HEV capsid is made up of capsomeres consisting of homodimers of a single structural capsid protein forming the virus shell. These dimers are believed to protrude from the viral surface and to interact with host cells to initiate infection. To date, no structural information is available for any of the HEV proteins. Here, we report for the first time the crystal structure of the HEV capsid protein domain E2s, a protruding domain, together with functional studies to illustrate that this domain forms a tight homodimer and that this dimerization is essential for HEV–host interactions. In addition, we also show that the neutralizing antibody recognition site of HEV is located on the E2s domain. Our study will aid in the development of vaccines and, subsequently, specific inhibitors for HEV.     

Incidence of Hepatitis C Virus (HCV) in a Multicenter Cohort of HIV-Positive Patients in Spain 2004–2011: Increasing Rates of HCV Diagnosis but Not of HCV Seroconversions

Objectives

We aim to describe rates and risk factors of Hepatitis C Virus (HCV) diagnoses, follow-up HCV testing and HCV seroconversion from 2004–2011 in a cohort of HIV-positive persons in Spain.

Methods

CoRIS is a multicentre, open and prospective cohort recruiting adult HIV-positive patients naïve to antiretroviral therapy. We analysed patients with at least one negative and one follow-up HCV serology. Incidence Rates (IR) were calculated and multivariate Poisson regression was used to estimate adjusted Rates Ratios (aIRR).

Results

Of 2112 subjects, 53 HCV diagnoses were observed, IR = 0.93/100py (95%CI: 0.7–1.2). IR increased from 0.88 in 2004–05 to 1.36 in 2010–11 (aIRR = 1.55; 95%CI: 0.37–6.55). In men who have sex with men (MSM) from 0.76 to 1.10 (aIRR = 1.45; 95%CI: 0.31–6.82); in heterosexual (HTX) subjects from 1.19 to 1.28 (aIRR = 1.08; 95%CI: 0.11–10.24). HCV seroconversion rates decreased from 1.77 to 0.65 (aIRR = 0.37; 95%CI: 0.12–1.11); in MSM from 1.06 to 0.49 (aIRR = 0.46; 95%CI: 0.09–2.31); in HTX from 2.55 to 0.59 (aIRR = 0.23; 95%CI: 0.06–0.98). HCV infection risk was higher for injecting drug users (IDU) compared to HTX (aIRR = 9.63;95%CI: 2.9–32.2); among MSM, for subjects aged 40–50 compared to 30 or less (IRR = 3.21; 95%CI: 1.7–6.2); and among HTX, for female sex (aIRR = 2.35; 95%CI: 1.03–5.34) and <200 CD4-count (aIRR = 2.39; 95%CI: 0.83–6.89).

Conclusion

We report increases in HCV diagnoses rates which seem secondary to intensification of HCV follow-up testing but not to rises in HCV infection rates. HCV IR is higher in IDU. In MSM, HCV IR increases with age. Among HTX, HCV IR is higher in women and in subjects with impaired immunological situation.     

Virus–Receptor Interactions: The Key to Cellular Invasion

Virus–receptor interactions play a key regulatory role in viral host range, tissue tropism, and viral pathogenesis. Viruses utilize elegant strategies to attach to one or multiple receptors, overcome the plasma membrane barrier, enter, and access the necessary host cell machinery. The viral attachment protein can be viewed as the “key” that unlocks host cells by interacting with the “lock”—the receptor—on the cell surface, and these lock-and-key interactions are critical for viruses to successfully invade host cells. Many common themes have emerged in virus–receptor utilization within and across virus families demonstrating that viruses often target particular classes of molecules in order to mediate these events. Common viral receptors include sialylated glycans, cell adhesion molecules such as immunoglobulin superfamily members and integrins, and phosphatidylserine receptors. The redundancy in receptor usage suggests that viruses target particular receptors or “common locks” to take advantage of their cellular function and also suggests evolutionary conservation. Due to the importance of initial virus interactions with host cells in viral pathogenesis and the redundancy in viral receptor usage, exploitation of these strategies would be an attractive target for new antiviral therapeutics.     

Contribution of charged and polar residues for the formation of the E1–E2 heterodimer from Hepatitis C Virus

The transmembrane domains of the envelope glycoprotein E1 and E2 have crucial multifunctional roles in the biogenesis of hepatitis C virus. We have performed molecular dynamics simulations to investigate a structural model of the transmembrane segments of the E1–E2 heterodimer. The simulations support the key role of the Lys370–Asp728 ion pair for mediating the E1–E2 heterodimerization. In comparison to these two residues, the simulation results also reveal the differential effect of the conserved Arg730 residue that has been observed in experimental studies. Furthermore, we discovered the formation of inter-helical hydrogen bonds via Asn367 that stabilize dimer formation. Simulations of single and double mutants further demonstrate the importance of the ion-pair and polar interactions between the interacting helix monomers. The conformation of the E1 fragment in the simulation of the E1–E2 heterodimer is in close agreement with an NMR structure of the E1 transmembrane segment. The proposed model of the E1–E2 heterodimer supports the postulated cooperative insertion of both helices by the translocon complex into the bilayer.     

Evaluation of Phosphatidylinositol-4-Kinase IIIα as a Hepatitis C Virus Drug Target

Phosphatidylinositol-4-kinase IIIα (PI4KIIIα) is an essential host cell factor for hepatitis C virus (HCV) replication. An N-terminally truncated 130-kDa form was used to reconstitute an in vitro biochemical lipid kinase assay that was optimized for small-molecule compound screening and identified potent and specific inhibitors. Cell culture studies with PI4KIIIα inhibitors demonstrated that the kinase activity was essential for HCV RNA replication. Two PI4KIIIα inhibitors were used to select cell lines harboring HCV replicon mutants with a 20-fold loss in sensitivity to the compounds. Reverse genetic mapping isolated an NS4B-NS5A segment that rescued HCV RNA replication in PIK4IIIα-deficient cells. HCV RNA replication occurs on specialized membranous webs, and this study with PIK4IIIα inhibitor-resistant mutants provides a genetic link between NS4B/NS5A functions and PI4-phosphate lipid metabolism. A comprehensive assessment of PI4KIIIα as a drug target included its evaluation for pharmacologic intervention in vivo through conditional transgenic murine lines that mimic target-specific inhibition in adult mice. Homozygotes that induce a knockout of the kinase domain or knock in a single amino acid substitution, kinase-defective PI4KIIIα, displayed a lethal phenotype with a fairly widespread mucosal epithelial degeneration of the gastrointestinal tract. This essential host physiologic role raises doubt about the pursuit of PI4KIIIα inhibitors for treatment of chronic HCV infection.     

Genetic Polymorphism of Interferon-γ, Interferon-γ Receptor, and Interferon Regulatory Factor-1 Genes in Patients with Hepatitis B Virus Infection

The natural history of hepatitis B virus (HBV) infection is probably related to host immune factors. Interferon-γ (IFN-γ) plays significant roles in immune defense. This study was undertaken to investigate the association between HBV infection and single nucleotide polymorphisms (SNPs) of IFN-γ, IFN-γ receptor (IFNGR)-1 and 2, and interferon regulatory factor (IRF)-1 genes. Between March 2002 and December 2002, 614 Korean patients were enrolled in two different groups: an HBV clearance group (n = 201), who were hepatitis B surface antigen (HBsAg) negative with antibodies to HBsAg and hepatitis B core antigen, and an HBV persistence group (n = 413), who were repeatedly HBsAg positive. We assessed polymorphisms in the IFN-γ gene at position +874, in the IFNGR-1 gene at positions −56 and +95, in the IFNGR-2 gene at the second position of codon 64 (Gln64Arg), and in the IRF-1 gene promoter (−410, −388), and the genotype distributions of the HBV clearance and persistence groups were compared. On the basis of unconditional logistic regression analysis with adjustment for age and sex, no statistically significant association with susceptibility to persistent HBV infection was observed with the IFN-γ, IFNGR-1 and 2, and IRF-1 gene polymorphisms under the codominant, dominant, and recessive models.