RESUMEN
Development of curative therapies for chronic hepatitis B virus (HBV) infection will likely require new animal models. Here, we evaluate HBV infection in squirrel monkeys based on the high-sequence homology of the HBV receptor, Na+/taurocholate co-transporting peptide (NTCP), between humans and squirrel monkeys. HBV PreS1 peptide was examined for binding human and squirrel monkey NTCP. Immunodeficient Fah -/- , NOD, Rag1 -/- , Il2Rg null (FNRG) mice engrafted with human or squirrel monkey hepatocytes were challenged with HBV or Woolly Monkey HBV (WMHBV). In addition, adult squirrel monkeys were inoculated with HBV, WMHBV, adeno-associated virus containing an infectious genome of HBV (AAV-HBV), and AAV-WMHBV. Finally, neonate squirrel monkeys were assessed for the potential of chronic infection with WMHBV. PreS1 peptide efficiently bound to human and squirrel monkey NTCP but not to mouse or capuchin NTCP. FNRG mice engrafted with squirrel monkey hepatocytes were susceptible to infection by WMHBV but not human HBV. Similarly, adult squirrel monkeys could be infected with WMHBV but not human HBV, whereas chimeric mice engrafted with human hepatocytes were susceptible to HBV but not WMHBV. Infection of squirrel monkeys with AAV-WMHBV yielded maximum viremia of 108 genomes/mL with detectable virus for up to 8 months. Notably, covalently closed circular DNA was detected in the liver of these animals. Infection of neonates with WMHBV led to detectable viremia for up to 6 months. Conclusions: Adult and neonate squirrel monkeys exhibited prolonged WMHBV viremia lasting 6-8 months. This is greater than twice the duration of viremia achieved in other nonhuman primates and suggests that squirrel monkeys may be a suitable model for testing HBV therapeutics.
RESUMEN
Simarteriviruses (Arteriviridae: Simarterivirinae) are commonly found at high titers in the blood of African monkeys but do not cause overt disease in these hosts. In contrast, simarteriviruses cause severe disease in Asian macaques upon accidental or experimental transmission. Here, we sought to better understand the host-dependent drivers of simarterivirus pathogenesis by infecting olive baboons (n = 4) and rhesus monkeys (n = 4) with the simarterivirus Southwest baboon virus 1 (SWBV-1). Surprisingly, none of the animals in our study showed signs of disease following SWBV-1 inoculation. Three animals (two rhesus monkeys and one olive baboon) became infected and sustained high levels of SWBV-1 viremia for the duration of the study. The course of SWBV-1 infection was highly predictable: plasma viremia peaked between 1 × 107 and 1 × 108 vRNA copies/mL at 3â»10 days post-inoculation, which was followed by a relative nadir and then establishment of a stable set-point between 1 × 106 and 1 × 107 vRNA copies/mL for the remainder of the study (56 days). We characterized cellular and antibody responses to SWBV-1 infection in these animals, demonstrating that macaques and baboons mount similar responses to SWBV-1 infection, yet these responses are ineffective at clearing SWBV-1 infection. SWBV-1 sequencing revealed the accumulation of non-synonymous mutations in a region of the genome that corresponds to an immunodominant epitope in the simarterivirus major envelope glycoprotein GP5, which likely contribute to viral persistence by enabling escape from host antibodies.
Asunto(s)
Arteriviridae/patogenicidad , Infecciones Asintomáticas , Macaca mulatta/virología , Papio/virología , Infecciones por Virus ARN/veterinaria , Animales , Anticuerpos Antivirales/sangre , Genoma Viral , Inmunidad Celular , Masculino , Mutación , Infecciones por Virus ARN/inmunología , Proteínas del Envoltorio Viral/inmunología , Carga Viral , Viremia , Replicación ViralRESUMEN
The virus-host relationship in simian immunodeficiency virus (SIV) infected chimpanzees is thought to be different from that found in other SIV infected African primates. However, studies of captive SIVcpz infected chimpanzees are limited. Previously, the natural SIVcpz infection of one chimpanzee, and the experimental infection of six chimpanzees was reported, with limited follow-up. Here, we present a long-term study of these seven animals, with a retrospective re-examination of the early stages of infection. The only clinical signs consistent with AIDS or AIDS associated disease was thrombocytopenia in two cases, associated with the development of anti-platelet antibodies. However, compared to uninfected and HIV-1 infected animals, SIVcpz infected animals had significantly lower levels of peripheral blood CD4+ T-cells. Despite this, levels of T-cell activation in chronic infection were not significantly elevated. In addition, while plasma levels of ß2 microglobulin, neopterin and soluble TNF-related apoptosis inducing ligand (sTRAIL) were elevated in acute infection, these markers returned to near-normal levels in chronic infection, reminiscent of immune activation patterns in 'natural host' species. Furthermore, plasma soluble CD14 was not elevated in chronic infection. However, examination of the secondary lymphoid environment revealed persistent changes to the lymphoid structure, including follicular hyperplasia in SIVcpz infected animals. In addition, both SIV and HIV-1 infected chimpanzees showed increased levels of deposition of collagen and increased levels of Mx1 expression in the T-cell zones of the lymph node. The outcome of SIVcpz infection of captive chimpanzees therefore shares features of both non-pathogenic and pathogenic lentivirus infections.
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Enfermedades del Simio Antropoideo/virología , VIH-1/fisiología , Infecciones por Lentivirus/veterinaria , Lentivirus de los Primates/fisiología , Pan troglodytes , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/fisiología , Animales , Enfermedades del Simio Antropoideo/inmunología , Enfermedades del Simio Antropoideo/patología , Enfermedades del Simio Antropoideo/fisiopatología , Enfermedades Autoinmunes/etiología , Enfermedades Autoinmunes/veterinaria , Biomarcadores/sangre , Recuento de Linfocito CD4 , Femenino , VIH-1/inmunología , VIH-1/aislamiento & purificación , Hiperplasia , Infecciones por Lentivirus/inmunología , Infecciones por Lentivirus/fisiopatología , Infecciones por Lentivirus/virología , Lentivirus de los Primates/inmunología , Lentivirus de los Primates/aislamiento & purificación , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/patología , Ganglios Linfáticos/virología , Masculino , Proteínas de Resistencia a Mixovirus/metabolismo , Neopterin/sangre , Fragmentos de Péptidos/sangre , Fragmentos de Péptidos/química , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/sangre , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/química , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/patología , Síndrome de Inmunodeficiencia Adquirida del Simio/fisiopatología , Virus de la Inmunodeficiencia de los Simios/inmunología , Virus de la Inmunodeficiencia de los Simios/aislamiento & purificación , Trombocitopenia/etiología , Trombocitopenia/veterinaria , Carga Viral , Microglobulina beta-2/sangreRESUMEN
The marmoset is an important nonhuman primate model for regenerative medicine. For experimental autologous cell therapy based on induced pluripotent (iPS) cells in the marmoset, cells must be able to undergo robust and reliable directed differentiation that will not require customization for each specific iPS cell clone. When marmoset iPS cells were aggregated in a hanging drop format for 3 days, followed by exposure to dual SMAD inhibitors and retinoic acid in monolayer culture for 3 days, we found substantial variability in the response of different iPS cell clones. However, when clones were pretreated with 0.05-2% dimethyl sulfoxide (DMSO) for 24 hours, all clones showed a very similar maximal response to the directed differentiation scheme. Peak responses were observed at 0.5% DMSO in two clones and at 1% DMSO in a third clone. When patterns of gene expression were examined by microarray analysis, hierarchical clustering showed very similar responses in all 3 clones when they were pretreated with optimal DMSO concentrations. The change in phenotype following exposure to DMSO and the 6 day hanging drop/monolayer treatment was confirmed by immunocytochemistry. Analysis of DNA content in DMSO-exposed cells indicated that it is unlikely that DMSO acts by causing cells to exit from the cell cycle. This approach should be generally valuable in the directed neural differentiation of pluripotent cells for experimental cell therapy.
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Diferenciación Celular/efectos de los fármacos , Dimetilsulfóxido/farmacología , Células Madre Pluripotentes Inducidas/citología , Neuronas/citología , Animales , Callithrix , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Diferenciación Celular/genética , Línea Celular , Inmunohistoquímica , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los ResultadosRESUMEN
Persistent infections with hepatitis B virus (HBV) or hepatitis C virus (HCV) account for the majority of cases of hepatic cirrhosis and hepatocellular carcinoma (HCC) worldwide. Small, non-coding RNAs play important roles in virus-host interactions. We used high throughput sequencing to conduct an unbiased profiling of small (14-40 nts) RNAs in liver from Japanese subjects with advanced hepatitis B or C and hepatocellular carcinoma (HCC). Small RNAs derived from tRNAs, specifically 30-35 nucleotide-long 5' tRNA-halves (5' tRHs), were abundant in non-malignant liver and significantly increased in humans and chimpanzees with chronic viral hepatitis. 5' tRH abundance exceeded microRNA abundance in most infected non-cancerous tissues. In contrast, in matched cancer tissue, 5' tRH abundance was reduced, and relative abundance of individual 5' tRHs was altered. In hepatitis B-associated HCC, 5' tRH abundance correlated with expression of the tRNA-cleaving ribonuclease, angiogenin. These results demonstrate that tRHs are the most abundant small RNAs in chronically infected liver and that their abundance is altered in liver cancer.
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Hepatitis B Crónica/genética , Hepatitis C Crónica/genética , ARN de Transferencia/metabolismo , Animales , Secuencia de Bases , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Hepatitis B Crónica/patología , Hepatitis C Crónica/patología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inmunoprecipitación , Hígado/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , MicroARNs/química , MicroARNs/metabolismo , Datos de Secuencia Molecular , Pan troglodytes/metabolismo , ARN de Transferencia/química , Reacción en Cadena en Tiempo Real de la Polimerasa , Ribonucleasa Pancreática/genética , Análisis de Secuencia de ARNRESUMEN
UNLABELLED: Persistent infection is a key feature of hepatitis C virus (HCV). However, chimpanzee infections with cell culture-derived viruses (JFH1 or related chimeric viruses that replicate efficiently in cell culture) have been limited to acute-transient infections with no pathogenicity. Here, we report persistent infection with chronic hepatitis in a chimpanzee challenged with cell culture-derived genotype 1a virus (H77S.2) containing 6 cell culture-adaptive mutations. Following acute-transient infection with a chimeric H77/JFH1 virus (HJ3-5), intravenous (i.v.) challenge with 10(6) FFU H77S.2 virus resulted in immediate seroconversion and, following an unusual 4- to 6-week delay, persistent viremia accompanied by alanine aminotransferase (ALT) elevation, intrahepatic innate immune responses, and diffuse hepatopathy. This first persistent infection with cell culture-produced HCV provided a unique opportunity to assess evolution of cell culture-adapted virus in vivo. Synonymous and nonsynonymous nucleotide substitution rates were greatest during the first 8 weeks of infection. Of 6 cell culture-adaptive mutations in H77S.2, Q1067R (NS3) had reverted to Q1067 and S2204I (NS5A) was replaced by T2204 within 8 weeks of infection. By 62 weeks, 4 of 6 mutations had reverted to the wild-type sequence, and all reverted to the wild-type sequence by 194 weeks. The data suggest H77S.2 virus has greater potential for persistence and pathogenicity than JFH1 and demonstrate both the capacity of a nonfit virus to persist for weeks in the liver in the absence of detectable viremia as well as strong selective pressure against cell culture-adaptive mutations in vivo. IMPORTANCE: This study shows that mutations promoting the production of infectious genotype 1a HCV in cell culture have the opposite effect and attenuate replication in the liver of the only fully permissive animal species other than humans. It provides the only example to date of persistent infection in a chimpanzee challenged with cell culture-produced virus and provides novel insight into the forces shaping molecular evolution of that virus during 5 years of persistent infection. It demonstrates that a poorly fit virus can replicate for weeks within the liver in the absence of detectable viremia, an observation that expands current concepts of HCV pathogenesis and that is relevant to relapses observed with direct-acting antiviral therapies.
Asunto(s)
Evolución Molecular , Hepacivirus/genética , Hepacivirus/aislamiento & purificación , Hepatitis C Crónica/virología , Mutación , Cultivo de Virus , Alanina Transaminasa/sangre , Animales , Modelos Animales de Enfermedad , Genotipo , Hepacivirus/clasificación , Hígado/patología , Pan troglodytes , ViremiaRESUMEN
BACKGROUND & AIMS: Direct-acting antiviral agents suppress hepatitis B virus (HBV) load, but they require life-long use. Stimulation of the innate immune system could increase its ability to control the virus and have long-lasting effects after a finite regimen. We investigated the effects of immune activation with GS-9620--a potent and selective orally active small molecule agonist of Toll-like receptor 7--in chimpanzees with chronic HBV infection. METHODS: GS-9620 was administered to chimpanzees every other day (3 times each week) for 4 weeks at 1 mg/kg and, after a 1-week rest, for 4 weeks at 2 mg/kg. We measured viral load in plasma and liver samples, the pharmacokinetics of GS-9620, and the following pharmacodynamics parameters: interferon-stimulated gene expression, cytokine and chemokine levels, lymphocyte and natural killer cell activation, and viral antigen expression. Clinical pathology parameters were monitored to determine the safety and tolerability of GS-9620. RESULTS: Short-term oral administration of GS-9620 provided long-term suppression of serum and liver HBV DNA. The mean maximum reduction of viral DNA was 2.2 logs, which occurred within 1 week of the end of GS-9620 administration; reductions of >1 log persisted for months. Serum levels of HBV surface antigen and HBV e antigen, and numbers of HBV antigen-positive hepatocytes, were reduced as hepatocyte apoptosis increased. GS-9620 administration induced production of interferon-α and other cytokines and chemokines, and activated interferon-stimulated genes, natural killer cells, and lymphocyte subsets. CONCLUSIONS: The small molecule GS-9620 activates Toll-like receptor 7 signaling in immune cells of chimpanzees to induce clearance of HBV-infected cells. This reagent might be developed for treatment of patients with chronic HBV infection.
Asunto(s)
Antivirales/uso terapéutico , Virus de la Hepatitis B/efectos de los fármacos , Hepatitis B Crónica/tratamiento farmacológico , Factores Inmunológicos/uso terapéutico , Pteridinas/uso terapéutico , Receptor Toll-Like 7/agonistas , Carga Viral/efectos de los fármacos , Administración Oral , Animales , Antivirales/farmacocinética , Hepatitis B Crónica/inmunología , Inmunidad Innata , Factores Inmunológicos/farmacocinética , Pan troglodytes , Pteridinas/farmacocinética , Receptor Toll-Like 7/inmunologíaRESUMEN
Hepatitis A virus (HAV) is an hepatotropic human picornavirus that is associated only with acute infection. Its pathogenesis is not well understood because there are few studies in animal models using modern methodologies. We characterized HAV infections in three chimpanzees, quantifying viral RNA by quantitative RT-PCR and examining critical aspects of the innate immune response including intrahepatic IFN-stimulated gene expression. We compared these infection profiles with similar studies of chimpanzees infected with hepatitis C virus (HCV), an hepatotropic flavivirus that frequently causes persistent infection. Surprisingly, HAV-infected animals exhibited very limited induction of type I IFN-stimulated genes in the liver compared with chimpanzees with acute resolving HCV infection, despite similar levels of viremia and 100-fold greater quantities of viral RNA in the liver. Minimal IFN-stimulated gene 15 and IFIT1 responses peaked 1-2 wk after HAV challenge and then subsided despite continuing high hepatic viral RNA. An acute inflammatory response at 3-4 wk correlated with the appearance of virus-specific antibodies and apoptosis and proliferation of hepatocytes. Despite this, HAV RNA persisted in the liver for months, remaining present long after clearance from serum and feces and revealing dramatic differences in the kinetics of clearance in the three compartments. Viral RNA was detected in the liver for significantly longer (35 to >48 wk) than HCV RNA in animals with acute resolving HCV infection (10-20 wk). Collectively, these findings indicate that HAV is far stealthier than HCV early in the course of acute resolving infection. HAV infections represent a distinctly different paradigm in virus-host interactions within the liver.
Asunto(s)
Hepatitis A/inmunología , Hepatitis A/virología , Interferón Tipo I/biosíntesis , ARN Viral/aislamiento & purificación , Enfermedad Aguda , Animales , Secuencia de Bases , Cartilla de ADN/genética , Expresión Génica , Perfilación de la Expresión Génica , Hepacivirus/genética , Hepacivirus/aislamiento & purificación , Hepatitis A/genética , Hepatitis A/patología , Virus de la Hepatitis A/genética , Virus de la Hepatitis A/aislamiento & purificación , Hepatitis C/genética , Hepatitis C/inmunología , Hepatitis C/virología , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunidad Innata/genética , Interferón Tipo I/genética , Hígado/patología , Hígado/virología , Pan troglodytes , ARN Viral/genética , Factores de TiempoRESUMEN
GBV-B induces hepatitis in tamarins and marmosets and is a surrogate model for HCV infections. Here, we cloned and characterized the antiviral activity of tamarin and marmoset interferon (IFN)alpha and IFN gamma. Potent antiviral activity was observed for tamarin and marmoset IFN alpha in primary hepatocyte cultures infected with GBV-B. The antiviral activity was greater in cultures exposed to IFN alpha prior to GBV-B infection, suggesting that either GBV-B was capable of inhibition of the antiviral activity of exogenous IFN alpha or that the preexisting endogenous IFN response to the virus reduced efficacy to exogenous IFN alpha. IFN gamma also exhibited antiviral activity in GBV-B infected hepatocytes. The transcriptional response to IFN alpha in marmoset hepatocytes was characterized using human genome microarrays. Since the GBV-B hepatocyte culture model possesses a functional innate immune response, it will provide opportunities to explore the nature of the antiviral response to a virus closely related to HCV.
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Antivirales/farmacología , Callithrix/inmunología , Virus GB-B/inmunología , Hepatocitos/virología , Interferón-alfa/farmacología , Interferón gamma/farmacología , Leontopithecus/inmunología , Animales , Células Cultivadas , Hepatocitos/inmunología , Humanos , Interferón-alfa/inmunología , Interferón gamma/inmunología , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
UNLABELLED: The mechanism of the interferon-alpha (IFNalpha)-induced antiviral response is not completely understood. We recently examined the transcriptional response to IFNalpha in uninfected chimpanzees. The transcriptional response to IFNalpha in the liver and peripheral blood mononuclear cells (PBMCs) was rapidly induced but was also rapidly down-regulated, with most interferon-alpha-stimulated genes (ISGs) returning to the baseline within 24 hours. We have extended these observations to include chimpanzees chronically infected with hepatitis C virus (HCV). Remarkably, using total genome microarray analysis, we observed almost no induction of ISG transcripts in the livers of chronically infected animals following IFNalpha dosing, whereas the response in PBMCs was similar to that in uninfected animals. In agreement with this finding, no decrease in the viral load occurred with up to 12 weeks of pegylated IFNalpha therapy. The block in the response to exogenous IFNalpha appeared to be HCV-specific because the response in a hepatitis B virus-infected animal was similar to that of uninfected animals. The lack of a response to exogenous IFNalpha may be due to an already maximally induced ISG response because chronically HCV-infected chimpanzees already have a highly up-regulated hepatic ISG response. Alternatively, negative regulation may block the response to exogenous IFNalpha, yet it does not prevent the continued response to endogenous ISG stimuli. The IFNalpha response in chronically HCV-infected chimpanzees may be mechanistically similar to the null response in the human population. CONCLUSION: In chimpanzees infected with HCV, the highly elevated hepatic ISG expression may prevent the further induction of ISGs and antiviral efficacy following an IFNalpha treatment.
Asunto(s)
Antivirales/uso terapéutico , Regulación de la Expresión Génica/efectos de los fármacos , Hepatitis C Crónica/tratamiento farmacológico , Interferón-alfa/uso terapéutico , Hígado/virología , Polietilenglicoles/uso terapéutico , Animales , Quimiocina CXCL10/genética , Quimiocina CXCL10/metabolismo , Mapeo Cromosómico , Relación Dosis-Respuesta a Droga , Hepacivirus/patogenicidad , Interferón alfa-2 , Hígado/efectos de los fármacos , Hígado/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Pan troglodytes , Proteínas Recombinantes , Proteínas Supresoras de la Señalización de Citocinas/genética , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Factores de Tiempo , Resultado del TratamientoRESUMEN
Infections with hepatitis C virus (HCV) genotype 3 exhibit differences in clinical phenotype including an increase in response to interferon therapy and development of steatosis. To initiate studies on genotype 3, we created a chimeric genotype 1b replicon containing a genotype 3a NS5A domain. The chimera was capable of efficient colony formation after the selection of a novel dominant adaptive mutation. Thus, domains from highly different strains can interact to form a functional replicase. A new genotype 1a replicon was constructed as well. Genotype specific influence on interferon sensitivity was examined using genotype 1a, 1b and chimeric 1b-3a replicons. The genotype 3a NS5A domain did not increase the sensitivity of the chimeric replicon to IFNalpha. The results suggest that NS5A is not sufficient to convey the increased IFNalpha response by genotype 3 or the replicon model is not capable of mimicking the events involved in increased sustained viral response.
Asunto(s)
Antivirales/farmacología , Hepacivirus/efectos de los fármacos , Hepacivirus/genética , Interferón-alfa/farmacología , Recombinación Genética , Proteínas no Estructurales Virales/genética , Secuencia de Aminoácidos , Línea Celular , Genotipo , Humanos , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , ARN Viral/biosíntesis , Proteínas Recombinantes de Fusión/efectos de los fármacos , Proteínas Recombinantes de Fusión/genética , Replicón , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas no Estructurales Virales/química , Replicación Viral/efectos de los fármacosRESUMEN
The mechanism of the interferon-alpha (IFN-alpha)-induced antiviral response during hepatitis C virus (HCV) therapy is n o t completely understood. In this study,we examined the transcriptional response to IFN-alpha in uninfected chimpanzees after single doses of chimpanzee, human, or human-pegylated IFN-alpha. Liver and peripheral blood mononuclear cell (PBMC) samples were used for total genome microarray analysis. Most induced genes achieved maximal response within 4 hours, began to decline by 8 hours, and were at baseline levels by 24 hours postinoculation, a time when high levels of circulating pegylated IFN-alpha were still present. The rapid downregulation of the IFN-alpha response may be involved in the transition between the observed phase I and phase II viral kinetics during IFN-alpha therapy in HCV-infected patients. The response to all three forms of IFN-alpha was similar; thus, the reasons for previous failures in antiviral treatment of chimpanzees with human IFN-alpha were not due to species specificity of IFN-alpha. The response to IFN-alpha was partially tissue-specific. A total of 1778 genes were altered in expression by twofold or more by IFN-alpha, with 538 and 950 being unique to the liver or PBMC, respectively. Analysis of the IFN-alpha and IFN-gamma responses in primary chimpanzee and human hepatocytes were compared as well. IFN-alpha and IFN-gamma induced partially overlapping sets of genes in hepatocytes. In conclusion, the response to IFN-alpha is largely tissue-specific, and the response is rapidly downregulated in vivo, which may have a significant influence on the kinetics of antiviral response.
Asunto(s)
Antivirales/farmacología , Regulación hacia Abajo , Genoma Viral/efectos de los fármacos , Hepacivirus/efectos de los fármacos , Hepacivirus/genética , Interferón-alfa/farmacología , Animales , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Interferón gamma/farmacología , Pan troglodytes , Factores de TiempoRESUMEN
Hepatitis B virus (HBV) and woolly monkey hepatitis B virus (WMHBV) are primate hepadnaviruses that display restricted tissue and host tropisms. Hepatitis D virus (HDV) particles pseudotyped with HBV and WMHBV envelopes (HBV-HDV and WM-HDV) preferentially infect human and spider monkey hepatocytes, respectively, thereby confirming host range bias in vitro. The analysis of chimeric HBV and WMHBV large (L) envelope proteins suggests that the pre-S1 domain may comprise two regions that affect infectivity: one within the amino-terminal 40 amino acids of pre-S1 and one downstream of this region. In the present study, we further characterized the role of the amino terminus of pre-S1 in infectivity by examining the ability of synthetic peptides to competitively block HDV infection of primary human and spider monkey hepatocytes. A synthetic peptide representing the first 45 residues of the pre-S1 domain of the HBV L protein blocked infectivity of HBV-HDV and WM-HDV, with a requirement for myristylation of the amino terminal residue. Competition studies with truncated peptides suggested that pre-S1 residues 5 to 20 represent the minimal domain for inhibition of HDV infection and, thus, presumably represent the residues involved in virus-host receptor interaction. Recombinant pre-S1 proteins expressed in insect cells blocked infection with HBV-HDV and WM-HDV at a concentration of 1 nanomolar. The ability of short pre-S1 peptides to efficiently inhibit HDV infection suggests that they represent suitable ligands for identification of the HBV receptor and that a pre-S1 mimetic may represent a rational therapy for the treatment of HBV infection.
Asunto(s)
Virus de la Hepatitis B/fisiología , Receptores Virales/análisis , Proteínas del Envoltorio Viral/genética , Secuencia de Aminoácidos , Animales , Cebidae , Células Cultivadas , Hepadnaviridae/genética , Hepadnaviridae/fisiología , Virus de la Hepatitis B/genética , Virus de la Hepatitis Delta/genética , Humanos , Datos de Secuencia Molecular , Huella de Proteína , Estructura Terciaria de Proteína/genética , Receptores Virales/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Especificidad de la Especie , Proteínas del Envoltorio Viral/metabolismo , Replicación ViralRESUMEN
Hepatitis C virus (HCV) infections represent a global health problem and are a major contributor to end-stage liver disease including cirrhosis and hepatocellular carcinoma. An improved understanding of the parameters involved in disease progression is needed to develop better therapies and diagnostic markers of disease manifestation. To better understand the dynamics of host gene expression resulting from persistent virus infection, DNA microarray analyses were conducted on livers from 10 chimpanzees persistently infected with HCV. A total of 162 genes were differentially regulated in chronically infected animals compared to uninfected controls. Many genes exhibited a remarkable consistency in changes in expression in the 10 chronically infected animals. A second method of analysis identified 971 genes altered in expression during chronic infection at a 99% confidence level. As with acute-resolving HCV infections, many interferon (IFN)-stimulated genes (ISGs) were transcriptionally elevated, suggesting an ongoing response to IFN and/or double-stranded RNA which is amplified in downstream ISG expression. Thus, persistent infection with HCV results in a complex and partially predictable pattern of gene expression, although the underlying mechanisms regulating the different pathways are not well defined. A single genotype 3-infected animal was available for analysis, and this animal exhibited reduced levels of ISG expression compared to levels of expression with genotype 1 infections and increased expression of a number of genes potentially involved in steatosis. Gene expression data in concert with other observations from HCV infections permit speculation on the regulation of specific aspects of HCV infection.
Asunto(s)
Perfilación de la Expresión Génica , Hepacivirus/patogenicidad , Hepatitis C Crónica/fisiopatología , Hígado/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica , Hepatitis C Crónica/virología , Hígado/virología , Masculino , Pan troglodytes , Proteínas/genéticaRESUMEN
Hepatitis B virus (HBV) and woolly monkey hepatitis B virus (WMHBV) have natural host ranges that are limited to closely related species. The barrier for infection of primates seems to be at the adsorption and/or entry steps of the viral replication cycle, since a human hepatoma cell line is permissive for HBV and WMHBV replication following transfection of cloned DNA. We hypothesized that the HBV and WMHBV envelope proteins contain the principal viral determinants of host range. As previously shown by using the hepatitis D virus (HDV) system, recombinant HBV-HDV particles were infectious in chimpanzee as well as human hepatocytes. We extended the HDV system to include HDV particles pseudotyped with the WMHBV envelope. In agreement with the natural host ranges of HBV and WMHBV, in vitro infections demonstrated that HBV-HDV and WM-HDV particles preferentially infected human and spider monkey cells, respectively. Previous studies have implicated the pre-S1 region of the large (L) envelope protein in receptor binding and host range; therefore, recombinant HDV particles were pseudotyped with the hepadnaviral envelopes containing chimeric L proteins with the first 40 amino acids from the pre-S1 domain exchanged between HBV and WMHBV. Surprisingly, addition of the human amino terminus to the WMHBV L protein increased infectivity on spider monkey hepatocytes but did not increase infectivity for human hepatocytes. Based upon these data, we discuss the possibility that the L protein may be comprised of two domains that affect infectivity and that sequences downstream of residue 40 may influence host range and receptor binding or entry.
Asunto(s)
Cebidae/virología , Virus de la Hepatitis B/fisiología , Virus de la Hepatitis Delta/fisiología , Hepatocitos/virología , Secuencia de Aminoácidos , Animales , Humanos , Datos de Secuencia Molecular , Fenotipo , Polietilenglicoles/farmacología , ARN Viral/análisis , Especificidad de la Especie , Proteínas del Envoltorio Viral/fisiología , Virión/fisiologíaRESUMEN
Recent studies in humans and chimpanzees suggest that immunity can be induced to diminish the incidence of chronic hepatitis C virus (HCV) infection. However, the immunity that promotes viral recovery is poorly understood, and whether the breadth of this adaptive immunity is sufficient to overcome the substantial intergenotype antigenic diversity represents a final obstacle to demonstrating the feasibility of vaccine development. Here we demonstrate that recovery from a genotype 1 HCV infection protects chimpanzees against infection with representatives of other genotypes that exhibit up to 30% divergence at the amino acid level, including challenges with genotype 4, a mixture of genotypes 2 and 3, and a complex inoculum containing genotypes 1, 2, 3, and 4. In each instance, the level and duration of viremia were markedly reduced in comparison to the primary infection in the same animal. The data indicate that epitopes conserved between genotypes must play an essential role in immunity. The inocula used in the rechallenge studies induced typical primary infection profiles in naïve chimpanzees. Rechallenge infections were associated with rapid increases in the intrahepatic transcripts of interferon-stimulated genes, even in animals exhibiting apparent sterilizing immunity. Protective immunity was often associated with an early increase in gamma interferon transcripts in the liver and increases in intrahepatic transcripts of Mig, a T-cell chemokine that is a gamma interferon response gene. These studies are the first to show that cross-genotype immunity can be induced to HCV, demonstrating the feasibility of developing a vaccine protective against all HCV strains.
Asunto(s)
Quimiocinas CXC/metabolismo , Hepacivirus/clasificación , Hepatitis C/inmunología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Interferón gamma/metabolismo , Hígado/inmunología , Animales , Quimiocina CXCL9 , Quimiocinas CXC/genética , Genotipo , Hepacivirus/genética , Hepacivirus/inmunología , Hepacivirus/patogenicidad , Hepatitis C/virología , Humanos , Inmunidad , Péptidos y Proteínas de Señalización Intercelular/genética , Interferón gamma/genética , Interferón gamma/inmunología , Hígado/virologíaRESUMEN
The recently developed hepatitis C virus (HCV) subgenomic replicon system was utilized to evaluate the efficacy of several known antiviral agents. Cell lines that persistently maintained a genotype 1b replicon were selected. The replicon resident in each cell line had acquired adaptive mutations in the NS5A region that increased colony-forming efficiency, and some replicons had acquired NS3 mutations that alone did not enhance colony-forming efficiency but were synergistic with NS5A mutations. A replicon constructed from the infectious clone of the HCV-1 strain (genotype 1a) was not capable of inducing colony formation even after the introduction of adaptive mutations identified in the genotype 1b replicon. Alpha interferon (IFN-alpha), IFN-gamma, and ribavirin exhibited antiviral activity, while double-stranded RNA (dsRNA) and tumor necrosis factor alpha did not. Analysis of transcript levels for a series of genes stimulated by IFN (ISGs) or dsRNA following treatment with IFN-alpha, IFN-gamma, and dsRNA revealed that both IFNs increased ISG transcript levels, but that some aspect of the dsRNA response pathway was defective in Huh7 cells and replicon cell lines in comparison to primary chimpanzee and tamarin hepatocytes. The colony-forming efficiency of the replicon was reduced or eliminated following replication in the presence of ribavirin, implicating the induction of error-prone replication. The potential role of error-prone replication in the synergy observed between IFN-alpha and ribavirin in attaining sustained viral clearance is discussed. These studies reveal characteristics of Huh7 cells that may contribute to their unique capacity to support HCV RNA synthesis and demonstrate the utility of the replicon system for mechanistic studies on antiviral agents.
Asunto(s)
Antivirales/farmacología , Hepacivirus/efectos de los fármacos , Interferón-alfa/farmacología , Interferón gamma/farmacología , Poli I-C/farmacología , Ribavirina/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Secuencia de Aminoácidos , Línea Celular , Genoma Viral , Genotipo , Hepacivirus/genética , Hepacivirus/fisiología , Datos de Secuencia Molecular , Mutación , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genéticaRESUMEN
A full-length cDNA clone of the hepatitis C virus (HCV) genotype 1 prototype (subtype 1a) sequence was constructed. Synthetic RNA produced from the initial cDNA clone was not infectious following intrahepatic inoculation of a chimpanzee. A consensus clone was prepared by comparison with multiple full-length HCV sequences of genotypes 1, 2 and 3. A total of 11 non-consensus amino acid residues were altered by mutagenesis. Synthetic RNA from the repaired clone initiated a typical, acute-resolving HCV infection following intrahepatic inoculation of a chimpanzee. In addition, at least one of three chimeric cDNA clones constructed between the HCV-1 and H77 genotype 1a strains of HCV was infectious in a chimpanzee. This is the first example of an infectious chimeric HCV clone. An infectious cDNA clone of HCV-1 will be of particular value, since it is the prototype HCV sequence and many commonly used reagents are based on this sequence.