A New Model to Produce Infectious Hepatitis C Virus without the Replication Requirement

Numerous constraints significantly hamper the experimental study of hepatitis C virus (HCV). Robust replication in cell culture occurs with only a few strains, and is invariably accompanied by adaptive mutations that impair in vivo infectivity/replication. This problem complicates the production and study of authentic HCV, including the most prevalent and clinically important genotype 1 (subtypes 1a and 1b). Here we describe a novel cell culture approach to generate infectious HCV virions without the HCV replication requirement and the associated cell-adaptive mutations. The system is based on our finding that the intracellular environment generated by a West-Nile virus (WNV) subgenomic replicon rendered a mammalian cell line permissive for assembly and release of infectious HCV particles, wherein the HCV RNA with correct 5′ and 3′ termini was produced in the cytoplasm by a plasmid-driven dual bacteriophage RNA polymerase-based transcription/amplification system. The released particles preferentially contained the HCV-based RNA compared to the WNV subgenomic RNA. Several variations of this system are described with different HCV-based RNAs: (i) HCV bicistronic particles (HCVbp) containing RNA encoding the HCV structural genes upstream of a cell-adapted subgenomic replicon, (ii) HCV reporter particles (HCVrp) containing RNA encoding the bacteriophage SP6 RNA polymerase in place of HCV nonstructural genes, and (iii) HCV wild-type particles (HCVwt) containing unmodified RNA genomes of diverse genotypes (1a, strain H77; 1b, strain Con1; 2a, strain JFH-1). Infectivity was assessed based on the signals generated by the HCV RNA molecules introduced into the cytoplasm of target cells upon virus entry, i.e. HCV RNA replication and protein production for HCVbp in Huh-7.5 cells as well as for HCVwt in HepG2-CD81 cells and human liver slices, and SP6 RNA polymerase-driven firefly luciferase for HCVrp in target cells displaying candidate HCV surface receptors. HCV infectivity was inhibited by pre-incubation of the particles with anti-HCV antibodies and by a treatment of the target cells with leukocyte interferon plus ribavirin. The production of authentic infectious HCV particles of virtually any genotype without the adaptive mutations associated with in vitro HCV replication represents a new paradigm to decipher the requirements for HCV assembly, release, and entry, amenable to analyses of wild type and genetically modified viruses of the most clinically significant HCV genotypes

Pemanfaatan Jembatan Penyeberangan Orang di Kota Makassar

SIMPULAN \ud \ud ??? Ketinggian anak tangga terlalu tinggi utamanya bagi wanita, manusia lanjut usia (manula), anak-anak dan penderita cacat. Lebar tangga dan lebar JPO kurang lebar.\ud ??? Tidak bisa digunakan bagi penderita cacat utamanya pengguna kursi roda karena fasilitas aksesibilitas (ramp) tidak tersedia. \ud ??? Azas kemudahan, azas keamanan, azas kegunaan dan azas kemandiriaan pemanfaatan JPO di Makassar belum terpenuhi.\ud \ud \ud REKOMENDASI \ud \ud ??? Perlu ada sosialisasi pemanfaatan JPO bagi masyarakat.\ud ??? Perlu azas kemudahan, azas keamanan, azas kegunaan dan azas kemandiriaan dikembangkan pemanfaatan JPO di Makassar yang mengacu KepMen PU Nomor 30/PRT/M/2006.Fasilitas jembatan penyeberangan orang (JPO) merupakan sarana yang berintikan memudahkan bagi pejalan kaki untuk mencapai satu titik ke titik yang lain di seberang jalan dengan mengedepankan azas kemudahan, azas keselamatan, azas kemandirian, dan azas kegunaan. Metode penelitian deskriptif eksplanatif, data fisik JPO dianalisis dengan mengunakan parameter yang telah ditetapkan dalam KepMen PU No. 30 tahun 2006 dan standarisasi yang manusiawi dan data presepsi pengunjung sebagai sampel diolah dengan menggunakan microsoft office excel 2007. Hasil penelitian menunjukan bahwa JPO di Makassar: (a) ketinggian anak tangga relatip masih tinggi utamanya bagi manula, wanita utamanya wanita hamil dan anak-anak; (b) tekstur lantai JPO relatip licin utamanya pada saat hujan; (c) lebar tangga dan lebar JPO kurang lebar; (d) JPO tidak dapat digunakan secara optimal bagi penderita cacat karena tak tersediakan fasilitas aksesibilitas utamanya pengguna kursi roda (tuna daksa). Persepsi pengguna JPO di Makassar terhadap azas kemudahan, azas kegunaan, azas keamanan dan azas kemandirian pemanfaatan JPO belum terpenuhi. Perlu ada petugas khusus disetiap JPO dan perlu secara berkala pemeliharaannya agar pemanfaatan JPO lebih nyaman dan aman.\ud Kata Kunci: jembatan penyeberang orang, pemanfaata

Sequence comparison of an Australian duck hepatitis B virus strain with other avian hepadnaviruses

© 2001 Society for General MicrobiologyThe genome of an Australian strain of duck hepatitis B virus (AusDHBV) was cloned from a pool of congenitally DHBV-infected-duck serum, fully sequenced and found by phylogenetic analyses to belong to the ‘Chinese’ DHBV branch of the avian hepadnaviruses. Sequencing of the Pre-S/S gene of four additional AusDHBV clones demonstrated that the original clone (pBL4.8) was representative of the virus present in the pool, and a head-to-tail dimer of the clone was infectious when inoculated into newly hatched ducks. When the published sequences of 20 avian hepadnaviruses were compared, substitutions or deletions in the polymerase (POL) gene were most frequent in the 500 nt segment encoding the ‘spacer’ domain that overlaps with the Pre-S domain of the Pre-S/S gene in a different reading frame. In contrast, substitutions and deletions were rare within the adjacent segment that encodes the reverse transcriptase domain of the POL protein and the S domain of the envelope protein, presumably because they are more often deleterious.Miriam Triyatni, Peter L. Ey, Thien Tran, Marc Le Mire, Ming Qiao, Christopher J. Burrell, and Allison R. Jilberthttp://vir.sgmjournals.org

Monoclonal antibodies with broad specificity for hepatitis C virus hypervariable region 1 variants can recognize viral particles

The hypervariable region 1 (HVR1) of the E2 protein of hepatitis C virus (HCV) is a highly heperogeneous sequence that is promiscuously recognized by human sera via binding to amino acid residues with conserved physicochemical properties. We generated a panel of mAbs from mice immunized with HVR1 surrogate peptides (mimotopes) affinity-selected with sera from HCV-infected patients from a phage display library. A high number of specific clones was obtained after immunization with a pool of nine mimotopes, and the resulting mAbs were shown to recognize several 16- and 27-mer peptides derived form natural HVR1 sequences isolated from patients with acute and chronic HCV infection, suggesting that HVR1 mimotopes were efficient antigenic and immunogenic mimics of naturally occurring HCV variants. Moreover, most mAbs were shown to bind HVR1 in the context of a complete soluble form of the E2 glycoprotein, indicating recognition of correctly folded HVR1. In addition a highly promiscuous mAb was able to specifically capture bona fide viral particles (circulating HCV RNA) as wess al rHCV-like particles assembled in insect cells expressing structural viral polypeptides derived from an HCV 1a isolate. These findings demonstrate that it is possible to induce a broadly cross-reactive clonal Ab response to multiple HCV variants. In consideration of the potentially important role of HVR1 in virus binding to cellular receptor(s), such a mechanism could be exploited for induction of neutralizing Abs specific for a large repertoire of viral variants

Monoclonal antibodies with broad specificity for hepatitis C virus hypervariable region 1 variants can recognize viral particles

The hypervariable region 1 (HVR1) of the E2 protein of hepatitis C virus (HCV) is a highly heterogeneous sequence that is promiscuously recognized by human sera via binding to amino acid residues with conserved physicochemical properties. We generated a panel of mAbs from mice immunized with HVR1 surrogate peptides (mimotopes) affinity-selected with sera from HCV-infected patients from a phage display library. A high number of specific clones was obtained after immunization with a pool of nine mimotopes, and the resulting mAbs were shown to recognize several 16- and 27-mer peptides derived from natural HVR1 sequences isolated from patients with acute and chronic HCV infection, suggesting that HVR1 mimotopes were efficient antigenic and immunogenic mimics of naturally occurring HCV variants. Moreover, most mAbs were shown to bind HVR1 in the context of a complete soluble form of the E2 glycoprotein, indicating recognition of correctly folded HVR1. In addition, a highly promiscuous mAb was able to specifically capture bona fide viral particles (circulating HCV RNA) as well as rHCV-like particles assembled in insect cells expressing structural viral polypeptides derived from an HCV la isolate. These findings demonstrate that it is possible to induce a broadly cross-reactive clonal Ab response to multiple HCV variants. In consideration of the potentially important role of HVR1 in virus binding to cellular receptor(s), such a mechanism could be exploited for induction of neutralizing Abs specific for a large repertoire of viral variants

Intrahepatic Transcriptional Signature Associated with Response to Interferon-α Treatment in the Woodchuck Model of Chronic Hepatitis B.

Recombinant interferon-alpha (IFN-α) is an approved therapy for chronic hepatitis B (CHB), but the molecular basis of treatment response remains to be determined. The woodchuck model of chronic hepatitis B virus (HBV) infection displays many characteristics of human disease and has been extensively used to evaluate antiviral therapeutics. In this study, woodchucks with chronic woodchuck hepatitis virus (WHV) infection were treated with recombinant woodchuck IFN-α (wIFN-α) or placebo (n = 12/group) for 15 weeks. Treatment with wIFN-α strongly reduced viral markers in the serum and liver in a subset of animals, with viral rebound typically being observed following cessation of treatment. To define the intrahepatic cellular and molecular characteristics of the antiviral response to wIFN-α, we characterized the transcriptional profiles of liver biopsies taken from animals (n = 8-12/group) at various times during the study. Unexpectedly, this revealed that the antiviral response to treatment did not correlate with intrahepatic induction of the majority of IFN-stimulated genes (ISGs) by wIFN-α. Instead, treatment response was associated with the induction of an NK/T cell signature in the liver, as well as an intrahepatic IFN-γ transcriptional response and elevation of liver injury biomarkers. Collectively, these data suggest that NK/T cell cytolytic and non-cytolytic mechanisms mediate the antiviral response to wIFN-α treatment. In summary, by studying recombinant IFN-α in a fully immunocompetent animal model of CHB, we determined that the immunomodulatory effects, but not the direct antiviral activity, of this pleiotropic cytokine are most closely correlated with treatment response. This has important implications for the rational design of new therapeutics for the treatment of CHB