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Hepatitis C virus (HCV) exhibits high genetic diversity, characterized by regional variations in genotype prevalence. This poses a challenge to the improved development of vaccines and pan-genotypic treatments, which require the consideration of global trends in HCV genotype prevalence. Here we provide the first comprehensive survey of these trends. To approximate national HCV genotype prevalence, studies published between 1989 and 2013 reporting HCV genotypes are reviewed and combined with overall HCV prevalence estimates from the Global Burden of Disease (GBD) project. We also generate regional and global genotype prevalence estimates, inferring data for countries lacking genotype information. We include 1,217 studies in our analysis, representing 117 countries and 90% of the global population. We calculate that HCV genotype 1 is the most prevalent worldwide, comprising 83.4 million cases (46.2% of all HCV cases), approximately one-third of which are in East Asia. Genotype 3 is the next most prevalent globally (54.3 million, 30.1%); genotypes 2, 4, and 6 are responsible for a total 22.8% of all cases; genotype 5 comprises the remaining

Inherited alleles account for most of the genetic risk for schizophrenia. However, new (de novo) mutations, in the form of large chromosomal copy number changes, occur in a small fraction of cases and disproportionally disrupt genes encoding postsynaptic proteins. Here we show that small de novo mutations, affecting one or a few nucleotides, are overrepresented among glutamatergic postsynaptic proteins comprising activity-regulated cytoskeleton-associated protein (ARC) and N-methyl-d-aspartate receptor (NMDAR) complexes. Mutations are additionally enriched in proteins that interact with these complexes to modulate synaptic strength, namely proteins regulating actin filament dynamics and those whose messenger RNAs are targets of fragile X mental retardation protein (FMRP). Genes affected by mutations in schizophrenia overlap those mutated in autism and intellectual disability, as do mutation-enriched synaptic pathways. Aligning our findings with a parallel case-control study, we demonstrate reproducible insights into aetiological mechanisms for schizophrenia and reveal pathophysiology shared with other neurodevelopmental disorders.

The hepatitis C virus (HCV), which currently infects an estimated 3% of people worldwide, has been present in some human populations for several centuries, notably HCV genotypes 1 and 2 in West Africa and genotype 6 in Southeast Asia. Here we use newly developed methods of sequence analysis to conduct the first comprehensive investigation of the epidemic and evolutionary history of HCV in Asia. Our analysis includes new HCV core ( n = 16) and NS5B ( n = 14) gene sequences, obtained from serum samples of jaundiced patients from Laos. These exceptionally diverse isolates were analyzed in conjunction with all available reference strains using phylogenetic and Bayesian coalescent methods. We performed statistical tests of phylogeographic structure and applied a recently developed “relaxed molecular clock” approach to HCV for the first time, which indicated an unexpectedly high degree of rate variation. Our results reveal a >1,000-year-long development of genotype 6 in Asia, characterized by substantial phylogeographic structure and two distinct phases of epidemic history, before and during the 20th century. We conclude that HCV lineages representing preexisting and spatially restricted strains were involved in multiple, independent local epidemics during the 20th century. Our analysis explains the generation and maintenance of HCV diversity in Asia and could provide a template for further investigations of HCV spread in other regions.

Background. The effect that high-dose interferon (IFN)-alpha induction therapy for hepatitis C virus (HCV) infection has on cellular immune responses is currently unknown. Methods. Thirty-one treatment-naive patients with chronic HCV infection received amantadine and ribavirin, combined with 6 weeks of high-dose IFN-alpha-2b induction therapy followed by weekly pegylated IFN-alpha-2b, for 24 or 48 weeks. Using IFN-gamma and interleukin (IL)-2 enzyme-linked immunospot (ELISpot) assays, we analyzed the pattern of cytokine secretion by structural and nonstructural HCV- and cytomegalovirus (CMV)-specific T cells before, during, and after therapy. Results. HCV-specific T cell responses, which were predominantly IFN-gamma secreting and which correlated with alanine transaminase levels ([Formula: see text]; [Formula: see text]), were found before treatment in 10 of 15 patients with a sustained virological response (SVR) and in 11 of 16 in the non-SVR group. There was a striking loss of IFN-gamma and IL-2 HCV-specific T cells during therapy, predominantly in the SVR group. This response recovered after cessation of therapy, regardless of outcome. Suppression of CMV-specific T cell responses, in addition to total lymphocyte counts, was also observed. Conclusions. High-dose IFN-alpha induction therapy leads to a profound decline in IL-2- and IFN-gamma-secreting HCV- and CMV-specific T cells. These data indicate that restoration of T cell responses is unlikely to be causally linked to an early response or SVR to therapy.

V(D)J genomic recombination joins single gene segments to encode an extensive repertoire of antigen receptor specificities in T and B lymphocytes. This process initiates with double-stranded breaks adjacent to conserved recombination signal sequences that contain either 12- or 23-nucleotide spacer regions. Only recombination between signal sequences with unequal spacers results in productive coding genes, a phenomenon known as the “12/23 rule.” Here we present two novel genomic tools that allow the capture and analysis of immune locus rearrangements from whole thymic and splenic tissues using second-generation sequencing. Further, we provide strong evidence that the 12/23 rule of genomic recombination is frequently violated under physiological conditions, resulting in unanticipated hybrid recombinations in ∼10% of Tcra excision circles. Hence, we demonstrate that strict adherence to the 12/23 rule is intrinsic neither to recombination signal sequences nor to the catalytic process of recombination and propose that nonclassical excision circles are liberated during the formation of antigen receptor diversity.

European Community Recent years have seen considerable progress in epidemiological and molecular genetic research into environmental and genetic factors in schizophrenia, but methodological uncertainties remain with regard to validating environmental exposures, and the population risk conferred by individual molecular genetic variants is small. There are now also a limited number of studies that have investigated molecular genetic candidate gene-environment interactions (G x E), however, so far, thorough replication of findings is rare and G x E research still faces several conceptual and methodological challenges. in this article, we aim to review these recent developments and illustrate how integrated, large-scale investigations may overcome contemporary challenges in G x E research, drawing on the example of a large, international, multi-center study into the identification and translational application of G x E in schizophrenia. While such investigations are now well underway, new challenges emerge for G x E research from late-breaking evidence that genetic variation and environmental exposures are, to a significant degree, shared across a range of psychiatric disorders, with potential overlap in phenotype. Maastricht Univ, Med Ctr, Dept Psychiat & Neuropsychol, Sch Mental Hlth & Neurosci,South Limburg Mental H, NL-6200 MD Maastricht, Netherlands Kings Coll London, Inst Psychiat, Dept Psychosis Studies, London WC2R 2LS, England Mondriaan Mental Hlth Trust, Maastricht, Heerlen, Netherlands Univ Groningen, Univ Med Ctr Groningen, Rob Giel Clin Res, Univ Ctr Psychiat, Groningen, Netherlands Kings Coll London, Inst Psychiat, Dept Hlth Serv & Populat Res, London WC2R 2LS, England Kings Coll London, Inst Psychiat, Dept Psychol, London WC2R 2LS, England Cardiff Univ, MRC, Ctr Neuropsychiat Genet, Cardiff CF10 3AX, S Glam, Wales Cent Inst Mental Hlth, Dept Psychiat & Psychotherapy, Mannheim, Germany Dokuz Eylul Univ, Sch Med, Dept Psychiat, Konak, Turkey Istanbul Univ, Istanbul Fac Med, Dept Psychiat, Psychot Disorders Res Unit, Istanbul, Turkey Ankara Univ, Sch Med, Dept Psychiat, Cebeci Hosp, TR-06100 Ankara, Turkey Ankara Univ, Brain Res Ctr, TR-06100 Ankara, Turkey Yale Univ, Sch Med, Dept Psychiat, New Haven, CT USA Middle E Tech Univ, Dept Psychol, TR-06531 Ankara, Turkey Diskapi YB Res & Training Hosp, Ankara, Turkey Turkish Federat Schizophrenia Assoc, Ankara, Turkey Ataturk Training & Res Hosp, Psychiat Clin, Ankara, Turkey Manisa Mental Hlth Hosp, Manisa, Turkey Istanbul Univ, Expt Med Res Inst, Dept Adv Neurol Sci, Istanbul Fac Med, Istanbul, Turkey Univ Complutense, IiSGM CIBERSAM, Dept Child & Adolescent Psychiat, Hosp Gen Univ Gregorio Maranon,Sch Med, E-28040 Madrid, Spain Univ Barcelona, Dept Psychiat, Hosp Clin, IDIBAPS,Ctr Invest Biomed Red Salud Mental CIBERS, Barcelona, Spain Univ Valencia, Sch Med, Dept Psychiat, Ctr Invest Biomed Red Salud Mental CIBERSAM, Valencia, Spain Univ Oviedo, Sch Med, Dept Med,Psychiat Area, Ctr Invest Biomed Red Salud Mental CIBERSAM, Oviedo, Spain Univ Santiago de Compostela, Dept Mental Hlth & Drug Addit Assistance, Hlth Serv Galicia,Psychiat Genet Grp IDIS, Hosp Clin,Ctr Invest Biomedica Red Salud Mental C, Santiago de Compostela 15706, Spain Hosp Virgen de la Luz, Serv Psiquiat, Dept Psychiat, Cuenca, Spain Hosp Univ Infanta Leonor Hosp Virgen Torre, Villa de Vallecas Mental Hlth Ctr, Villa de Vallecas Mental Hlth Dept, Madrid, Spain Hosp Univ Infanta Leonor Hosp Virgen Torre, Puente de Vallecas Mental Hlth Dept, Ctr Salud Mental Puente Vallecas, Madrid, Spain Hosp Clin Univ, Fdn Publ Galega Med Xenomica, Santiago de Compostela, Spain Univ Complutense, Sch Med, Hosp Gen Univ Gregorio Maranon, Dept Psychiat, E-28040 Madrid, Spain Hosp Psiquiatr Conxo, Santiago de Compostela, Spain Univ Amsterdam, Acad Med Ctr, Early Psychosis Sect, Dept Psychiat, NL-1105 AZ Amsterdam, Netherlands Vrije Univ Amsterdam, Dept Clin Psychol, Amsterdam, Netherlands EMGO Inst Hlth & Care Res, Amsterdam, Netherlands Parnassia Psychiat Inst, Dept Psychosis Res, the Hague, Netherlands Rivierduinen Psychiat Inst, Leiden, Netherlands Grp Hosp Mondor, AP HP, Creteil, France Hop Henri Mondor, INSERM, U955, Equipe 15, F-94010 Creteil, France Univ Paris Est, Fac Med, Creteil, France Fdn Fondamental, Creteil, France CMP B CHU, F-63003 Clermont Ferrand 1, France Univ Auvergne, EA 7280, Clermont Ferrand, France EPS Maison Blanche, Paris, France UPC KU Leuven, Dept Neurosci, UPC, Kortenberg, Belgium UPC, Dept Neurosci, Res Grp Psychiat, Leuven, Belgium Katholieke Univ Leuven, Univ Hosp Gasthuisberg, Dept Human Genet, Leuven, Belgium Assoc Sci Res Multiple Births, Ghent, Belgium Univ Ghent, Dept Neurol, Ghent Univ Hosp, B-9000 Ghent, Belgium Univ Athens, Sch Med, Eginit Hosp, Athens 11528, Greece Med Univ Vienna, Dept Psychiat & Psychotherapy, Vienna, Austria Psychiat Univ Clin Basel, Ctr Gender Res & Early Detect, Basel, Switzerland Psychiat Univ Clin Basel, Diagnost & Crisis Intervent Ctr, Basel, Switzerland Univ Cologne, Dept Psychiat & Psychotherapy, D-50931 Cologne, Germany Univ Hong Kong, Li Ka Shing Fac Med, Ctr Genom Sci, State Key Lab Brain & Cognit Sci, Hong Kong, Hong Kong, Peoples R China Univ Hong Kong, Li Ka Shing Fac Med, Dept Psychiat, Hong Kong, Hong Kong, Peoples R China Univ Hong Kong, Queen Mary Hosp, Li Ka Shing Fac Med, State Key Lab Brain & Cognit Sci, Hong Kong, Hong Kong, Peoples R China Univ Hong Kong, Queen Mary Hosp, Li Ka Shing Fac Med, Dept Psychiat, Hong Kong, Hong Kong, Peoples R China Univ Basque Country, Univ Deusto, Interuniv Chair Law & Human Genome Prov Govt Bisk, Bilbao, Bizkaia, Spain Univ Zaragoza, Zaragoza, Spain Univ Cambridge, Dept Psychiat, Cambridge, England UCL, Div Psychiat, London, England Royal Coll Surgeons Ireland, Beaumont Hosp, Educ & Res Ctr, Dept Psychiat, Dublin 9, Ireland Univ Munich, Dept Psychiat, Div Mol & Clin Neurobiol, Munich, Germany Univ Bologna, Alma Mater Studiorium, Psychiat Unit, Dept Med & Surg Sci, Bologna, Italy Univ Bologna, Alma Mater Studiorium, Genet Unit, Dept Med & Surg Sci, Bologna, Italy Local Hlth Trust, Dept Mental Hlth & Pathol Addict, Bologna, Italy Univ Palermo, Sect Psychiat, Dept Expt Biomed & Clin Neurosci, Palermo, Italy P Giaccone Gen Hosp, Unit Psychiat, Palermo, Italy Univ Melbourne, Ctr Youth Mental Hlth, Parkville, Vic 3052, Australia Univ Melbourne, Melbourne Neuropsychiat Ctr, Carlton, Vic, Australia Univ São Paulo, Fac Med, Dept Med Prevent, BR-01246903 São Paulo, Brazil Univ São Paulo, Nucleo Pesquina Saude Mental Populac, São Paulo, Brazil Univ São Paulo, Fac Med Ribeirao Preto, Dept Neurociencias & Ciencias Comportamento, BR-14049 Ribeirao Preto, Brazil Univ Verona, Sect Psychiat, Dept Publ Hlth & Community Med, I-37100 Verona, Italy Copenhagen Univ Hosp, Res Unit, Mental Hlth Ctr Copenhagen, Copenhagen, Denmark Univ Paris 05, Fac Med, Serv Hosp Univ, Hop St Anne, Paris, France Univ Autonoma Barcelona, Dept Psicol Clin & Salut, E-08193 Barcelona, Spain St Pere Claver Fundacio Sanitaria, Dept Salut Mental, Barcelona, Spain Univ N Carolina, Dept Psychol, Greensboro, NC 27412 USA CIBERSAM, Spanish Mental Hlth Res Network, Barcelona, Spain Universidade Federal de São Paulo, Dept Psychiat, PRISMA Early Intervent Program, São Paulo, Brazil Univ Belgrade, Sch Med, Beograd, Serbia Universidade Federal de São Paulo, Dept Psychiat, PRISMA Early Intervent Program, São Paulo, Brazil European Community: HEALTH-F2-2009-241909 Web of Science

Human leukocyte antigen B27 (HLA-B27) is associated with protection in human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infection. This protective role is linked to single immunodominant HLA-B27-restricted CD8+ T-cell epitopes in both infections. In order to define the relative contribution of a specific HLA-B27-restricted epitope to the natural course of HCV infection, we compared the biological impact of the highly conserved HCV genotype 1 epitope, for which the protective role has been described, with the corresponding region in genotype 3 that differs in its sequence by three amino acid residues. The genotype 3a peptide was not recognized by CD8+ T cells specific for the genotype 1 peptide. Furthermore, patients with acute or chronic infection with HCV genotype 3a did not mount T-cell responses to this epitope region, and their autologous viral sequences showed no evidence of T-cell pressure. Finally, we found a significantly higher frequency of HLA-B27 positivity in patients with chronic HCV genotype 3a infection compared to genotype 1 infection, indicating that there is no protection by HLA-B27 in HCV genotype 3 infection. Conclusion: Our data indicate that the protective effect of HLA-B27 is limited to HCV genotype 1 infection and does not expand to other genotypes such as genotype 3a. This can most likely be explained by intergenotype sequence diversity leading to the loss of the immunodominant HLA-B27 epitope in viral strains other than genotype 1. Our results underline the central role of a single HLA-B27-restricted epitope-specific CD8+ T-cell response in mediating protection in HCV genotype 1 infection. (HEPATOLOGY 2010;51:54–62.)

UNLABELLED: Many hepatitis C virus (HCV) infections worldwide are with the genotype 1 and 3 strains of the virus. Cellular immune responses are known to be important in the containment of HCV genotype 1 infection, and many genotype 1 T cell targets (epitopes) that are presented by host human leukocyte antigens (HLAs) have been identified. In contrast, there is almost no information known about the equivalent responses to genotype 3. Immune escape mechanisms used by HCV include the evolution of viral polymorphisms (adaptations) that abrogate this host-viral interaction. Evidence of HCV adaptation to HLA-restricted immune pressure on HCV can be observed at the population level as viral polymorphisms associated with specific HLA types. To evaluate the escape patterns of HCV genotypes 1 and 3, we assessed the associations between viral polymorphisms and specific HLA types from 187 individuals with genotype 1a and 136 individuals with genotype 3a infection. We identified 51 HLA-associated viral polymorphisms (32 for genotype 1a and 19 for genotype 3a). Of these putative viral adaptation sites, six fell within previously published epitopes. Only two HLA-associated viral polymorphisms were common to both genotypes. In the remaining sites with HLA-associated polymorphisms, there was either complete conservation or no significant HLA association with viral polymorphism in the alternative genotype. This study also highlights the diverse mechanisms by which viral evasion of immune responses may be achieved and the role of genotype variation in these processes. CONCLUSION: There is little overlap in HLA-associated polymorphisms in the nonstructural proteins of HCV for the two genotypes, implying differences in the cellular immune pressures acting on these viruses and different escape profiles. These findings have implications for future therapeutic strategies to combat HCV infection, including vaccine design.

UNLABELLED: The efficacy of specifically targeted anti-viral therapy for hepatitis C virus (HCV) (STAT-C), including HCV protease and polymerase inhibitors, is limited by the presence of drug-specific viral resistance mutations within the targeted proteins. Genetic diversity within these viral proteins also evolves under selective pressures provided by host human leukocyte antigen (HLA)-restricted immune responses, which may therefore influence STAT-C treatment response. Here, the prevalence of drug resistance mutations relevant to 27 developmental STAT-C drugs, and the potential for drug and immune selective pressures to intersect at sites along the HCV genome, is explored. HCV nonstructural (NS) 3 protease or NS5B polymerase sequences and HLA assignment were obtained from study populations from Australia, Switzerland, and the United Kingdom. Four hundred five treatment-naïve individuals with chronic HCV infection were considered (259 genotype 1, 146 genotype 3), of which 38.5% were coinfected with human immunodeficiency virus (HIV). We identified preexisting STAT-C drug resistance mutations in sequences from this large cohort. The frequency of the variations varied according to individual STAT-C drug and HCV genotype/subtype. Of individuals infected with subtype 1a, 21.5% exhibited genetic variation at a known drug resistance site. Furthermore, we identified areas in HCV protease and polymerase that are under both potential HLA-driven pressure and therapy selection and identified six HLA-associated polymorphisms (P

Methods: HBV-specific T cells were expanded in a 2-dimensional combinatorial expansion assay (67 different conditions from 11 factors) and the frequency of HBV-specific T cells was quantified by intra-cellular cytokine staining. Due to the exponentially increasing technical difficulties associated with analyzing multiple factors of T cell dysfunction, we coupled the expansion assay together with fluorescent cell barcoding to achieve a high throughput flow cytometric analysis. Results: By varying the concentration of an amine reactive dye, we were able to obtain 5 distinct fluorescent barcodes which were used to label different samples. This allows us to pool 5 samples into a single tube, reducing staining antibody consumption and the number of flow cytometer acquisitions to an operationally manageable quantity (from 70 to 14 tubes). The flow cytometric data of each tube was then deconvoluted into the individual samples based on the fluorescent barcodes and analyzed. Interestingly, preliminary results from 4 chronic HBV patients and 4 healthy subjects show that combinations which blocks apoptosis through the action of a pan-caspase inhibitor was able to preferentially enhance the in vitro expansion of exhausted HBV-specific T cells and not functional T cells specific for other viruses (CMV or FLU). Conclusion: The development of this assay makes it possible to simultaneously analyze combinations of multiple strategies to rescue exhausted HBV-specific T cells. Further in-depth analysis is ongoing to determine the ideal combinations to rescue exhausted HBV-specific T cells.

Hepatitis C virus subtype 3a is a highly prevalent and globally distributed strain that is often associated with infection via injection drug use. This subtype exhibits particular phenotypic characteristics. In spite of this, detailed genetic analysis of this subtype has rarely been performed. We performed full-length viral sequence analysis in 18 patients with chronic HCV subtype 3a infection and assessed genomic viral variability in comparison to other HCV subtypes. Two novel regions of intragenotypic hypervariability within the envelope protein E2, of HCV genotype 3a, were identified. We named these regions HVR495 and HVR575. They consisted of flanking conserved hydrophobic amino acids and central variable residues. A 5-amino-acid insertion found only in genotype 3a and a putative glycosylation site is contained within HVR575. Evolutionary analysis of E2 showed that positively selected sites within genotype 3a infection were largely restricted to HVR1, HVR495, and HVR575. Further analysis of clonal viral populations within single hosts showed that viral variation within HVR495 and HVR575 were subject to intrahost positive selecting forces. Longitudinal analysis of four patients with acute HCV subtype 3a infection sampled at multiple time points showed that positively selected mutations within HVR495 and HVR575 arose early during primary infection. HVR495 and HVR575 were not present in HCV subtypes 1a, 1b, 2a, or 6a. Some variability that was not subject to positive selection was present in subtype 4a HVR575. Further defining the functional significance of these regions may have important implications for genotype 3a E2 virus-receptor interactions and for vaccine studies that aim to induce cross-reactive anti-E2 antibodies.

Summary There is little information on the diverse infectious causes of jaundice and hepatitis in the Asiatic tropics. Serology (hepatitis A, B, C and E, leptospirosis, dengue, rickettsia), antigen tests (dengue), PCR assays (hepatitis A, C and E) and blood cultures (septicaemia) were performed on samples from 392 patients admitted with jaundice or raised transaminases (≥ × 3) to Mahosot Hospital, Vientiane, Laos over 3 years. Conservative definitions suggested diagnoses of dengue (8.4%), rickettsioses (7.3%), leptospirosis (6.8%), hepatitis B (4.9%), hepatitis C (4.9%), community-acquired septicaemia (3.3%) and hepatitis E (1.6%). Although anti-hepatitis A virus (HAV) IgM antibody results suggested that 35.8% of patients had acute HAV infections, anti-HAV IgG antibody avidity and HAV PCR suggested that 82% had polyclonal activation and not acute HAV infections. Scrub typhus, murine typhus or leptospirosis were present in 12.8% of patients and were associated with meningism and relatively low AST and ALT elevation. These patients would be expected to respond to empirical doxycycline therapy which, in the absence of virological diagnosis and treatment, may be an appropriate cost-effective intervention in Lao patients with jaundice/hepatitis.