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1.
Virology ; 512: 48-55, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28915405

RESUMO

Despite drug advances for Hepatitis C virus (HCV), re-infections remain prevalent in high-risk populations. Unfortunately, the role of preexisting viral immunity and how it modulates re-infection is unclear. GBV-B infection of common marmosets is a useful model to study tissue immune responses in hepacivirus infections, and in this study we re-challenged 4 animals after clearance of primary viremia. Although only low-to-absent viremia was observed following re-challenge, GBV-B viral RNA was detectable in liver, confirming re-infection. Microscopic hepatic lesions indicated severe-to-mild lymphocyte infiltration and fibrosis in 3 out of 4 animals. Further, GBV-B-specific T cells were elevated in animals with moderate-to-severe hepatopathology, and up to 3-fold increases in myeloid dendritic and activated natural killer cells were observed after infection. Our data indicate that occult hepacivirus re-infections occur and that new liver pathology is possible even in the presence of anti-hepacivirus T cells and in the absence of high viremia.


Assuntos
Infecções por Flaviviridae/imunologia , Vírus GB B/fisiologia , Hepatite Viral Animal/imunologia , Animais , Callithrix , Infecções por Flaviviridae/patologia , Hepatite Viral Animal/patologia , Imunidade Inata/fisiologia , Fígado/patologia , Fígado/virologia , Linfócitos T/fisiologia , Carga Viral , Viremia
2.
J Virol ; 90(20): 9153-62, 2016 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-27489267

RESUMO

UNLABELLED: Despite its importance in shaping adaptive immune responses, viral clearance, and immune-based inflammation, tissue-specific innate immunity remains poorly characterized for hepatitis C virus (HCV) infection due to the lack of access to acutely infected tissues. In this study, we evaluated the impact of natural killer (NK) cells and myeloid (mDCs) and plasmacytoid (pDCs) dendritic cells on control of virus replication and virus-induced pathology caused by another, more rapidly resolving hepacivirus, GB virus B (GBV-B), in infections of common marmosets. High plasma and liver viral loads and robust hepatitis characterized acute GBV-B infection, and while viremia was generally cleared by 2 to 3 months postinfection, hepatitis and liver fibrosis persisted after clearance. Coinciding with peak viral loads and liver pathology, the levels of NK cells, mDCs, and pDCs in the liver increased up to 3-fold. Although no obvious numerical changes in peripheral innate cells occurred, circulating NK cells exhibited increased perforin and Ki67 expression levels and increased surface expression of CXCR3. These data suggested that increased NK cell arming and proliferation as well as tissue trafficking may be associated with influx into the liver during acute infection. Indeed, NK cell frequencies in the liver positively correlated with plasma (R = 0.698; P = 0.015) and liver (R = 0.567; P = 0.057) viral loads. Finally, soluble factors associated with NK cells and DCs, including gamma interferon (IFN-γ) and RANTES, were increased in acute infection and also were associated with viral loads and hepatitis. Collectively, the findings showed that mobilization of local and circulating innate immune responses was linked to acute virus-induced hepatitis, and potentially to resolution of GBV-B infection, and our results may provide insight into similar mechanisms in HCV infection. IMPORTANCE: Hepatitis C virus (HCV) infection has created a global health crisis, and despite new effective antivirals, it is still a leading cause of liver disease and death worldwide. Recent evidence suggests that innate immunity may be a potential therapeutic target for HCV, but it may also be a correlate of increased disease. Due to a lack of access to human tissues with acute HCV infection, in this study we evaluated the role of innate immunity in resolving infection with a hepacivirus, GBV-B, in common marmosets. Collectively, our data suggest that NK cell and DC mobilization in acute hepacivirus infection can dampen virus replication but also regulate acute and chronic liver damage. How these two opposing effects on the host may be modulated in future therapeutic and vaccine approaches warrants further study.


Assuntos
Células Dendríticas/imunologia , Vírus GB B/imunologia , Hepatite Viral Animal/imunologia , Hepatite Viral Animal/patologia , Imunidade Inata , Células Matadoras Naturais/imunologia , Animais , Callithrix , Citocinas/metabolismo , Vírus GB B/patogenicidade , Fatores Imunológicos/metabolismo , Fígado/patologia , Fígado/virologia , Carga Viral
3.
J Virol ; 90(18): 8198-211, 2016 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-27384651

RESUMO

UNLABELLED: A lack of immunocompetent-small-primate models has been an obstacle for developing hepatitis C virus (HCV) vaccines and affordable antiviral drugs. In this study, HCV/GB virus B (GBV-B) chimeric virus carrying the major nonstructural proteins NS2 to NS4A (HCV NS2 to -4A chimera) was produced and used to infect common marmosets, since HCV NS2 to NS4A proteins are critical proteases and major antigens. Seven marmosets were inoculated intrahepatically with HCV NS2 to -4A chimera RNA for primary infection or intravenously injected with chimera-containing serum for passage infection. Three animals used as controls were injected with phosphate-buffered saline (PBS) or GBV-B, respectively. Six of seven HCV NS2 to -4A chimera-infected marmosets exhibited consistent viremia and one showed transient viremia during the course of follow-up detection. All six infected animals with persistent circulating viremia presented characteristics typical of viral hepatitis, including viral RNA and proteins in hepatocytes and histopathological changes in liver tissue. Viremia was consistently detected for 5 to 54 weeks of follow-up. FK506 immunosuppression facilitated the establishment of persistent chimera infection in marmosets. An animal with chimera infection spontaneously cleared the virus in blood 7 weeks following the first inoculation, but viral-RNA persistence, low-level viral protein, and mild necroinflammation remained in liver tissue. The specific antibody and T-cell response to HCV NS3 in this viremia-resolved marmoset was boosted by rechallenging, but no viremia was detected during 57 weeks of follow-up. The chimera-infected marmosets described can be used as a suitable small-primate animal model for studying novel antiviral drugs and T-cell-based vaccines against HCV infection. IMPORTANCE: HCV infection causes approximately 70% of chronic hepatitis and is frequently associated with primary liver cancer globally. Chimpanzees have been used as a reliable primate model for HCV infection, but ethical considerations have restricted their utility in biomedical research. GB virus B (GBV-B) is a flavivirus related to HCV. It can infect common marmosets, a New World small primate, and induces viral hepatitis similar to HCV infection in humans. To minimize differences between GBV-B and HCV, we generated HCV NS2 to -4A/GBV-B chimeric viruses and established a chimera-infected marmoset model. HCV NS2 to -4A chimera-infected marmosets provide a small-animal model for evaluating novel antiviral drugs targeting HCV NS3-NS4A protease and T-cell-based HCV vaccines.


Assuntos
Infecções por Flaviviridae/virologia , Vírus GB B/crescimento & desenvolvimento , Hepatite Viral Animal/virologia , Recombinação Genética , Proteínas não Estruturais Virais/genética , Animais , Callithrix , Infecções por Flaviviridae/patologia , Vírus GB B/genética , Anticorpos Anti-Hepatite C/sangue , Hepatite Viral Animal/patologia , Hepatócitos/virologia , Fígado/patologia , Fígado/virologia , Linfócitos T/imunologia , Viremia
4.
Microbiol Immunol ; 60(1): 26-34, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26634303

RESUMO

The development of effective hepatitis C virus (HCV) vaccines is essential for the prevention of further HCV dissemination, especially in developing countries. Therefore the aim of this study is to establish a feasible and immunocompetent surrogate animal model of HCV infection that will help in evaluation of the protective efficacy of newly developing HCV vaccine candidates. To circumvent the narrow host range of HCV, an HCV genotype 1b-based chimeric clone carrying E1, E2 and p6 regions from GB virus B (GBV-B), which is closely related to HCV, was generated. The chimera between HCV and GBV-B, named HCV/G, replicated more efficiently as compared with the HCV clone in primary marmoset hepatocytes. Furthermore, it was found that the chimera persistently replicated in a tamarin for more than 2 years after intrahepatic inoculation of the chimeric RNA. Although relatively low (<200 copies/mL), the viral RNA loads in plasma were detectable intermittently during the observation period. Of note, the chimeric RNA was found in the pellet fraction obtained by ultracentrifugation of the plasma at 73 weeks, indicating production of the chimeric virus. Our results will help establish a novel non-human primate model for HCV infection on the basis of the HCV/G chimera in the major framework of the HCV genome.


Assuntos
Vírus GB B/fisiologia , Hepatite Viral Animal/virologia , Doenças dos Macacos/virologia , Platirrinos/virologia , Replicação Viral/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Quimera/genética , Quimera/virologia , Modelos Animais de Doenças , Infecções por Flaviviridae/virologia , Vírus GB B/genética , Vírus GB B/imunologia , Humanos , Dados de Sequência Molecular , RNA Viral/genética , Vacinas contra Hepatite Viral/imunologia , Proteínas não Estruturais Virais
5.
J Virol ; 89(23): 12131-44, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26401036

RESUMO

UNLABELLED: Hepatitis C virus (HCV) only infects humans and chimpanzees, while GB virus B (GBV-B), another hepatotropic hepacivirus, infects small New World primates (tamarins and marmosets). In an effort to develop an immunocompetent small primate model for HCV infection to study HCV pathogenesis and vaccine approaches, we investigated the HCV life cycle step(s) that may be restricted in small primate hepatocytes. First, we found that replication-competent, genome-length chimeric HCV RNAs encoding GBV-B structural proteins in place of equivalent HCV sequences designed to allow entry into simian hepatocytes failed to induce viremia in tamarins following intrahepatic inoculation, nor did they lead to progeny virus in permissive, transfected human Huh7.5 hepatoma cells upon serial passage. This likely reflected the disruption of interactions between distantly related structural and nonstructural proteins that are essential for virion production, whereas such cross talk could be restored in similarly designed HCV intergenotypic recombinants via adaptive mutations in NS3 protease or helicase domains. Next, HCV entry into small primate hepatocytes was examined directly using HCV-pseudotyped retroviral particles (HCV-pp). HCV-pp efficiently infected tamarin hepatic cell lines and primary marmoset hepatocyte cultures through the use of the simian CD81 ortholog as a coreceptor, indicating that HCV entry is not restricted in small New World primate hepatocytes. Furthermore, we observed genomic replication and modest virus secretion following infection of primary marmoset hepatocyte cultures with a highly cell culture-adapted HCV strain. Thus, HCV can successfully complete its life cycle in primary simian hepatocytes, suggesting the possibility of adapting some HCV strains to small primate hosts. IMPORTANCE: Hepatitis C virus (HCV) is an important human pathogen that infects over 150 million individuals worldwide and leads to chronic liver disease. The lack of a small animal model for this infection impedes the development of a preventive vaccine and pathogenesis studies. In seeking to establish a small primate model for HCV, we first attempted to generate recombinants between HCV and GB virus B (GBV-B), a hepacivirus that infects small New World primates (tamarins and marmosets). This approach revealed that the genetic distance between these hepaciviruses likely prevented virus morphogenesis. We next showed that HCV pseudoparticles were able to infect tamarin or marmoset hepatocytes efficiently, demonstrating that there was no restriction in HCV entry into these simian cells. Furthermore, we found that a highly cell culture-adapted HCV strain was able to achieve a complete viral cycle in primary marmoset hepatocyte cultures, providing a promising basis for further HCV adaptation to small primate hosts.


Assuntos
Vírus GB B/fisiologia , Hepacivirus/fisiologia , Estágios do Ciclo de Vida/fisiologia , Modelos Animais , Primatas/virologia , Internalização do Vírus , Animais , Sequência de Bases , Primers do DNA/genética , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Células HEK293 , Hepacivirus/genética , Hepatócitos/virologia , Especificidade de Hospedeiro , Humanos , Immunoblotting , Dados de Sequência Molecular , Plasmídeos/genética , Análise de Sequência de DNA , Viremia
6.
J Virol ; 88(13): 7426-44, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24741107

RESUMO

UNLABELLED: GB virus B (GBV-B), which is hepatotropic in experimentally infected small New World primates, is a member of the Hepacivirus genus but phylogenetically relatively distant from hepatitis C virus (HCV). To gain insights into the role and specificity of hepaciviral nonstructural protein 2 (NS2), which is required for HCV polyprotein processing and particle morphogenesis, we investigated whether NS2 structural and functional features are conserved between HCV and GBV-B. We found that GBV-B NS2, like HCV NS2, has cysteine protease activity responsible for cleavage at the NS2/NS3 junction, and we experimentally confirmed the location of this junction within the viral polyprotein. A model for GBV-B NS2 membrane topology was experimentally established by determining the membrane association properties of NS2 segments fused to green fluorescent protein (GFP) and their nuclear magnetic resonance structures using synthetic peptides as well as by applying an N-glycosylation scanning approach. Similar glycosylation studies confirmed the HCV NS2 organization. Together, our data show that despite limited amino acid sequence similarity, GBV-B and HCV NS2 proteins share a membrane topology with 3 N-terminal transmembrane segments, which is also predicted to apply to other recently discovered hepaciviruses. Based on these data and using trans-complementation systems, we found that intragenotypic hybrid NS2 proteins with heterologous N-terminal membrane segments were able to efficiently trans-complement an assembly-deficient HCV mutant with a point mutation in the NS2 C-terminal domain, while GBV-B/HCV or intergenotypic NS2 chimeras were not. These studies indicate that virus- and genotype-specific intramolecular interactions between N- and C-terminal domains of NS2 are critically involved in HCV morphogenesis. IMPORTANCE: Nonstructural protein 2 (NS2) of hepatitis C virus (HCV) is a multifunctional protein critically involved in polyprotein processing and virion morphogenesis. To gain insights into NS2 mechanisms of action, we investigated whether NS2 structural and functional features are conserved between HCV and GB virus B (GBV-B), a phylogenetically relatively distant primate hepacivirus. We showed that GBV-B NS2, like HCV NS2, carries cysteine protease activity. We experimentally established a model for GBV-B NS2 membrane topology and demonstrated that despite limited sequence similarity, GBV-B and HCV NS2 share an organization with three N-terminal transmembrane segments. We found that the role of HCV NS2 in particle assembly is genotype specific and relies on critical interactions between its N- and C-terminal domains. This first comparative analysis of NS2 proteins from two hepaciviruses and our structural predictions of NS2 from other newly identified mammal hepaciviruses highlight conserved key features of the hepaciviral life cycle.


Assuntos
Membrana Celular/metabolismo , Infecções por Flaviviridae/metabolismo , Hepatite C/metabolismo , Hepatite Viral Humana/metabolismo , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Infecções por Flaviviridae/virologia , Imunofluorescência , Vírus GB B/fisiologia , Hepacivirus/fisiologia , Hepatite C/virologia , Hepatite Viral Humana/virologia , Humanos , Immunoblotting , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de Aminoácidos , Proteínas não Estruturais Virais/química , Replicação Viral
7.
Virus Res ; 179: 93-101, 2014 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-24246306

RESUMO

Flaviviruses related to hepatitis C virus (HCV) in suitable animal models may provide further insight into the role that cellular immunity contributes to spontaneous clearance of HCV. We characterised changes in lymphocyte populations in tamarins with an acute GBV-B infection, a hepatitis virus of the flaviviridae. Major immune cell populations were monitored in peripheral and intra-hepatic lymphocytes at high viraemia or following a period when peripheral virus was no longer detected. Limited changes in major lymphocyte populations were apparent during high viraemia; however, the proportions of CD3(+) lymphocytes decreased and CD20(+) lymphocytes increased once peripheral viraemia became undetectable. Intrahepatic lymphocyte populations increased at both time points post-infection. Distinct expression patterns of PD-1, a marker of T-cell activation, were observed on peripheral and hepatic lymphocytes; notably there was elevated PD-1 expression on hepatic CD4(+) T-cells during high viraemia, suggesting an activated phenotype, which decreased following clearance of peripheral viraemia. At times when peripheral vRNA was not detected, suggesting viral clearance, we were able to readily detect GBV-B RNA in the liver, indicative of long-term virus replication. This study is the first description of changes in lymphocyte populations during GBV-B infection of tamarins and provides a foundation for more detailed investigations of the responses that contribute to the control of GBV-B infection.


Assuntos
Modelos Animais de Doenças , Infecções por Flaviviridae/virologia , Vírus GB B/fisiologia , Hepatite Viral Humana/virologia , Fígado/imunologia , Saguinus , Animais , Infecções por Flaviviridae/imunologia , Vírus GB B/imunologia , Hepatite Viral Humana/imunologia , Humanos , Fígado/virologia , Ativação Linfocitária , Saguinus/imunologia , Saguinus/virologia , Linfócitos T/imunologia , Viremia/imunologia , Viremia/virologia , Replicação Viral
8.
J Virol ; 87(16): 8971-81, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23740998

RESUMO

GB virus B (GBV-B; family Flaviviridae, genus Hepacivirus) has been studied in New World primates as a model for human hepatitis C virus infection, but the distribution of GBV-B and its relatives in nature has remained obscure. Here, we report the discovery of a novel and highly divergent GBV-B-like virus in an Old World monkey, the black-and-white colobus (Colobus guereza), in Uganda. The new virus, guereza hepacivirus (GHV), clusters phylogenetically with GBV-B and recently described hepaciviruses infecting African bats and North American rodents, and it shows evidence of ancient recombination with these other hepaciviruses. Direct sequencing of reverse-transcribed RNA from blood plasma from three of nine colobus monkeys yielded near-complete GHV genomes, comprising two distinct viral variants. The viruses contain an exceptionally long nonstructural 5A (NS5A) gene, approximately half of which codes for a protein with no discernible homology to known proteins. Computational structure-based analyses indicate that the amino terminus of the GHV NS5A protein may serve a zinc-binding function, similar to the NS5A of other viruses within the family Flaviviridae. However, the 521-amino-acid carboxy terminus is intrinsically disordered, reflecting an unusual degree of structural plasticity and polyfunctionality. These findings shed new light on the natural history and evolution of the hepaciviruses and on the extent of structural variation within the Flaviviridae.


Assuntos
Vírus GB B/genética , Vírus GB B/isolamento & purificação , Hepatite C/veterinária , Doenças dos Primatas/virologia , Proteínas não Estruturais Virais/genética , Animais , Análise por Conglomerados , Colobus , Simulação por Computador , Vírus GB B/química , Genoma Viral , Hepatite C/virologia , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Conformação Proteica , RNA Viral/genética , Análise de Sequência de DNA , Uganda , Proteínas não Estruturais Virais/química
9.
J Virol ; 87(13): 7338-47, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23616647

RESUMO

Hepatitis C virus (HCV) RNA forms an unusual interaction with human microRNA-122 (miR-122) that promotes viral RNA accumulation in cultured human liver cells and in the livers of infected chimpanzees. GB virus B (GBV-B) is a hepatotropic virus and close relative of HCV. Thus, GBV-B has been used as a surrogate system to study HCV amplification in cultured cells and in infected tamarins. It was discovered that the 5'-terminal sequences of GBV-B RNA, like HCV RNA, forms an Argonaute 2-mediated complex with two miR-122 molecules that are essential for accumulation of GBV-B subgenomic replicon RNA. However, sequences in miR-122 that anneal to each viral RNA genome were different, suggesting distinct overall structural features in HCV:miR-122 and GBV-B:miR-122 complexes. Surprisingly, a deletion that removed both miR-122 binding sites from the subgenomic GBV-B RNAs rendered viral RNA amplification independent from miR-122 and Argonaute 2. This finding suggests that structural features at the end of the viral genome dictate whether miR-122 is required to aid in maintaining viral RNA abundance.


Assuntos
Proteínas Argonauta/metabolismo , Vírus GB B/genética , Regulação Viral da Expressão Gênica/fisiologia , MicroRNAs/metabolismo , RNA Viral/metabolismo , Northern Blotting , Linhagem Celular Tumoral , Primers do DNA/genética , Vírus GB B/metabolismo , Hepacivirus/genética , Hepacivirus/metabolismo , Humanos , Luciferases , Mutagênese , Plasmídeos/genética , Reação em Cadeia da Polimerase em Tempo Real , Replicon/genética , Transfecção
10.
Immunogenetics ; 63(10): 619-26, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21681586

RESUMO

The infection of red-bellied tamarins (Saguinus labiatus) with GB virus B (GBV-B) is an important surrogate model of hepatitis C virus infection in man. To fully exploit the value of this model, we have characterised MHC class I G and class II DRB alleles in eight tamarins representing a cross-section of a UK breeding colony. The results indicated a high degree of classes I and II DRB allele sharing. Each animal transcribed three to four putative surface-expressed class I alleles and two to four class II DRB alleles. Most animals also transcribed at least one class I allele predicted to result in a C-terminal truncated protein. These results represent the first description of MHC polymorphism in this species and provide a foundation for characterisation of MHC diversity in breeding populations of red-bellied tamarins. The data will facilitate the identification of associations between MHC polymorphism and control of viral infections, and detailed dissection of cellular immune responses against GBV-B.


Assuntos
Infecções por Flaviviridae/imunologia , Vírus GB B , Genes MHC da Classe II , Genes MHC Classe I , Hepatite Viral Animal/imunologia , Hepatite Viral Humana/imunologia , Saguinus/imunologia , Animais , Modelos Animais de Doenças , Infecções por Flaviviridae/genética , Frequência do Gene , Hepatite Viral Animal/genética , Hepatite Viral Humana/genética , Humanos , Imunidade Celular/genética , Polimorfismo Genético , Saguinus/genética , Saguinus/virologia
11.
Virology ; 406(2): 228-40, 2010 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-20701941

RESUMO

The hepatitis C virus (HCV) serine protease (NS3/4A) processes the NS3-NS5B segment of the viral polyprotein and also cleaves host proteins involved in interferon signaling, making it an important target for antiviral drug discovery and suggesting a wide breadth of substrate specificity. We compared substrate specificities of the HCV protease with that of the GB virus B (GBV-B), a distantly related nonhuman primate hepacivirus, by exchanging amino acid sequences at the NS4B/5A and/or NS5A/5B cleavage junctions between these viruses within the backbone of subgenomic replicons. This mutagenesis study demonstrated that the GBV-B protease had a broader substrate tolerance, a feature corroborated by structural homology modeling. However, despite efficient polyprotein processing, GBV-B RNAs containing HCV sequences at the C-terminus of NS4B had a pseudo-lethal replication phenotype. Replication-competent revertants contained second-site substitutions within the NS3 protease or NS4B N-terminus, providing genetic evidence for an essential interaction between NS3 and NS4B during genome replication.


Assuntos
Replicação do DNA , Vírus GB B/enzimologia , Hepacivirus/enzimologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Infecções por Flaviviridae/virologia , Vírus GB B/química , Vírus GB B/genética , Vírus GB B/metabolismo , Hepacivirus/química , Hepacivirus/genética , Hepacivirus/metabolismo , Hepatite C/virologia , Hepatite Viral Humana/virologia , Humanos , Dados de Sequência Molecular , Ligação Proteica , RNA Helicases/química , RNA Helicases/genética , RNA Helicases/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Especificidade por Substrato , Proteínas não Estruturais Virais/genética , Replicação Viral
12.
J Gen Virol ; 91(Pt 3): 727-33, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19906942

RESUMO

GB virus B (GBV-B) causes acute hepatitis in experimentally infected tamarins. We compared evolutionary features in acute resolving and persistent GBV-B infection. We detected no evidence of evolution in four animals with clearance during weeks 9-12, whereas three animals with clearance during weeks 13-26 had several substitutions in their polyprotein sequence. A single tamarin had long-term GBV-B viraemia; analysis of virus recovered at weeks 2, 5, 12, 20, 26, 52 and 104 demonstrated that mutations accumulated over time. Overall, the amino acid substitution rate was 3.5x10(-3) and 1.1x10(-3) substitutions per site year(-1) during weeks 1-52 and 53-104, respectively. Thus, there was a significant decrease in evolution over time, as found for hepatitis C virus. The rate of non-synonymous substitution per non-synonymous site compared with that of synonymous substitution per synonymous site decreased over time, suggesting reduction of positive selective pressure. These data demonstrate that prolonged GBV-B infection is associated with viral evolution.


Assuntos
Evolução Molecular , Vírus GB B/classificação , Vírus GB B/genética , Hepatite Viral Animal/virologia , Doenças dos Macacos/virologia , Substituição de Aminoácidos/genética , Animais , Modelos Animais de Doenças , Produtos do Gene pol/genética , Leontopithecus
13.
Virology ; 390(2): 186-96, 2009 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-19501869

RESUMO

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.


Assuntos
Antivirais/farmacologia , Callithrix/imunologia , Vírus GB B/imunologia , Hepatócitos/virologia , Interferon-alfa/farmacologia , Interferon gama/farmacologia , Leontopithecus/imunologia , Animais , Células Cultivadas , Hepatócitos/imunologia , Humanos , Interferon-alfa/imunologia , Interferon gama/imunologia , Análise de Sequência com Séries de Oligonucleotídeos
14.
J Virol ; 83(16): 8062-75, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19474092

RESUMO

Approximately 3% of the world population is chronically infected with hepatitis C virus (HCV). GB virus B (GBV-B), a surrogate model for HCV, causes hepatitis in tamarins and is the virus phylogenetically most closely related to HCV. Previously we described a chimeric GBV-B containing an HCV insert from the 5' noncoding region (NCR) that was adapted for efficient replication in tamarins (Saguinus species). We have also demonstrated that wild-type (WT) GBV-B rapidly adapts for efficient replication in a closely related species, the common marmoset (Callithrix jacchus). Here, we demonstrate that the chimeric virus failed to adapt during serial passage in marmosets. The chimeric virus was passaged four times through 24 marmosets. During passage, two marmoset phenotypes were observed: susceptible and partially resistant. Although appearing to adapt in a resistant animal during a prolonged and gradual increase in viremia, the chimeric GBV-B failed to replicate efficiently upon passage to a naïve marmoset. The resistance was specific to the chimeric virus, as the chimeric virus-resistant animals were susceptible to marmoset-adapted WT virus during rechallenge studies. Three isolates of the chimeric virus were sequenced, and 20 nucleotide changes were observed, including eight amino acid changes. Three unique changes were observed in the 5' NCR chimeric insert, an area that is highly conserved in HCV. We speculate that the failure of the chimeric virus to adapt in marmosets might be due to a bottleneck that occurs at the time of infection of resistant animals, which may lead to a loss of fitness upon serial passage.


Assuntos
Callithrix , Modelos Animais de Doenças , Infecções por Flaviviridae/virologia , Vírus GB B/fisiologia , Hepacivirus/fisiologia , Hepatite C/virologia , Animais , Sequência de Bases , Feminino , Vírus GB B/química , Vírus GB B/genética , Hepacivirus/genética , Humanos , Masculino , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo , Inoculações Seriadas , Replicação Viral
15.
J Virol ; 83(11): 5806-14, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19279089

RESUMO

Worldwide, approximately 170 million people are chronically infected with hepatitis C virus (HCV), and chronic infection frequently progresses to serious liver disease, including cirrhosis and hepatocellular carcinoma. GB virus B (GBV-B), the virus phylogenetically most closely related to HCV, causes hepatitis in tamarins. We have demonstrated the suitability of the tamarin as a host for GBV-B and as a surrogate nonhuman primate model for HCV infection, and we have initiated studies of GBV-B infection in a closely related species, the common marmoset (Callithrix jacchus). Here, we demonstrate that marmosets exhibit two phenotypes upon infection with GBV-B: the susceptible phenotype and the partially resistant phenotype. In addition, we identify changes that may correlate with adaptation of the virus to the partially resistant host. GBV-B was serially passaged five times through 14 marmosets as one lineage and two times through 6 marmosets as a second lineage. Virus adapted to the marmosets and eventually exhibited robust infections in two separate lineages, lineages 1 and 2. A third lineage was initiated with a molecular clone, and again, susceptible and partially resistant phenotypes were observed. Three isolates were fully sequenced (from lineage 1), and 21 nucleotide changes were observed, with six amino acid changes. We speculate that the marmoset partially resistant phenotype may be due to a polymorphism in the marmoset population that affects critical virus-host interactions and that wild-type GBV-B is capable of rapidly adapting to this altered host.


Assuntos
Adaptação Biológica/imunologia , Callithrix/imunologia , Infecções por Flaviviridae/imunologia , Vírus GB B/imunologia , Hepatite Viral Animal/imunologia , Animais , Callithrix/virologia , Modelos Animais de Doenças , Infecções por Flaviviridae/virologia , Vírus GB B/genética , Hepatite Viral Animal/virologia , Fenótipo , RNA Viral/genética
16.
PLoS One ; 4(2): e4419, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19204793

RESUMO

GB virus B (GBV-B) is closely related to hepatitis C virus (HCV), infects small non-human primates, and is thus a valuable surrogate for studying HCV. Despite significant differences, the 5' nontranslated RNAs (NTRs) of these viruses fold into four similar structured domains (I-IV), with domains II-III-IV comprising the viral internal ribosomal entry site (IRES). We previously reported the in vivo rescue of a chimeric GBV-B (vGB/III(HC)) containing HCV sequence in domain III, an essential segment of the IRES. We show here that three mutations identified within the vGB/III(HC) genome (within the 3'NTR, upstream of the poly(U) tract, and NS5A coding sequence) are necessary and sufficient for production of this chimeric virus following intrahepatic inoculation of synthetic RNA in tamarins, and thus apparently compensate for the presence of HCV sequence in domain III. To assess the mechanism(s) underlying these compensatory mutations, and to determine whether 5'NTR subdomains participating in genome replication do so in a virus-specific fashion, we constructed and evaluated a series of chimeric subgenomic GBV-B replicons in which various 5'NTR subdomains were substituted with their HCV homologs. Domains I and II of the GBV-B 5'NTR could not be replaced with HCV sequence, indicating that they contain essential, virus-specific RNA replication elements. In contrast, domain III could be swapped with minimal loss of genome replication capacity in cell culture. The 3'NTR and NS5A mutations required for rescue of the related chimeric virus in vivo had no effect on replication of the subgenomic GBneoD/III(HC) RNA in vitro. The data suggest that in vivo fitness of the domain III chimeric virus is dependent on a cooperative interaction between the 5'NTR, 3'NTR and NS5A at a step in the viral life cycle subsequent to genome replication, most likely during particle assembly. Such a mechanism may be common to all hepaciviruses.


Assuntos
Vírus GB B/fisiologia , Hepacivirus/fisiologia , RNA não Traduzido/metabolismo , RNA Viral/metabolismo , Proteínas Virais/metabolismo , Animais , Sequência de Bases , Linhagem Celular Tumoral , Vírus GB B/genética , Vírus GB B/patogenicidade , Genoma Viral/genética , Hepacivirus/genética , Hepacivirus/patogenicidade , Humanos , Dados de Sequência Molecular , Mutação/genética , Conformação de Ácido Nucleico , Biossíntese de Proteínas , RNA não Traduzido/química , RNA não Traduzido/genética , RNA Viral/química , RNA Viral/genética , Replicon , Saguinus/virologia , Análise de Sequência de RNA , Replicação Viral
17.
Microbiol Immunol ; 53(1): 53-7, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19161559

RESUMO

More than 170 million people worldwide are chronically infected by HCV, which is the causative agent of chronic hepatitis C, cirrhosis, and finally liver cancer. Although animal models of viral hepatitis are a prerequisite for the evaluation of antiviral and vaccine efficacy, the restricted host range of HCV has hampered the development of a suitable small animal model of HCV infection. Use of the chimpanzee, the only animal known to be susceptible to HCV infection, is limited by ethical and financial restrictions. In this regard GBV-B, being closely related to HCV, appears to be a promising non-human surrogate model for the study of HCV infection. This review describes the characteristic of GBV-B infection of New World monkeys, and discusses current issues concerning the GBV-B model and its future directions.


Assuntos
Modelos Animais de Doenças , Infecções por Flaviviridae/veterinária , Vírus GB B/fisiologia , Hepacivirus/fisiologia , Hepatite C/virologia , Hepatite Viral Animal/virologia , Platirrinos , Animais , Infecções por Flaviviridae/imunologia , Infecções por Flaviviridae/virologia , Hepatite C/imunologia , Hepatite Viral Animal/imunologia , Humanos
18.
J Hepatol ; 49(6): 908-15, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18845353

RESUMO

BACKGROUND/AIMS: The development of new therapies for hepatitis C virus (HCV) infection has been hampered by the lack of a small animal model. GB virus B (GBV-B), which infects new world monkeys, has been proposed as a surrogate system for HCV replication. Despite their short genetic distance, however, difficulties exist when extrapolating results from GBV-B to the HCV system. One way of addressing this is the creation of chimeric GBV-B containing HCV elements. METHODS: Construction and analysis of GBV-B chimeras in which the p13 ion channel was replaced by its HCV counterpart, p7. RESULTS: Replacing all, or part of, the GBV-B p13 protein with HCV p7 resulted in viable chimeras which replicated at wild-type levels in marmosets following intra-hepatic RNA injection. Serum from one animal injected with chimeric RNA was infectious in three naïve recipients, indicating that chimeras formed fully infectious virions. Amantadine, which blocks the ion channel activity of both HCV and GBV-B proteins in vitro, also inhibited GBV-B replication in primary hepatocytes. CONCLUSIONS: These viruses highlight the potential for chimeric GBV-B in the development of HCV-specific therapies and will provide a means of developing HCV p7 as a therapeutic target.


Assuntos
Vírus GB B/genética , Hepacivirus/genética , Hepatite C Crônica/virologia , Proteínas Recombinantes de Fusão/genética , Proteínas Virais/genética , Amantadina/farmacologia , Animais , Antivirais/farmacologia , Callithrix , Linhagem Celular , Modelos Animais de Doenças , Desenho de Fármacos , Genoma Viral , Hepatite C Crônica/tratamento farmacológico , Hepatócitos/citologia , Hepatócitos/virologia , Humanos , Rim/citologia , RNA Viral/sangue , RNA Viral/genética , Transfecção
19.
J Gen Virol ; 89(Pt 8): 1911-1920, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18632962

RESUMO

GB virus B (GBV-B) is the closest relative to hepatitis C virus (HCV) with which it shares a common genome organization, however, unlike HCV in humans, it generally causes an acute resolving hepatitis in New World monkeys. It is important to understand the factors regulating the different disease profiles of the two viruses and in this regard, as well as playing a key role in viral RNA replication, the HCV NS5A non-structural protein modulates a variety of host-cell signalling pathways. We have shown previously that HCV NS5A, expressed either alone, or in the context of the complete polyprotein, inhibits the Ras-extracellular-signal-regulated kinase (Erk) pathway and activates the phosphoinositide 3-kinase (PI3K) pathway. In this report, we investigate whether these functions are shared by GBV-B NS5A. Immunofluorescence analysis revealed that a C-terminally FLAG-tagged GBV-B NS5A exhibited a punctate cytoplasmic distribution. However, unlike HCV NS5A, the GBV-B protein did not partially co-localize with early endosomes. Utilizing a transient luciferase reporter system, we observed that GBV-B NS5A failed to inhibit Ras-Erk signalling, however GBV-B NS5A expression did result in the elevation of beta-catenin-dependent transcription via activation of the PI3K pathway. These effects of GBV-B and HCV NS5A on the PI3K and Ras-Erk pathways were confirmed in cells harbouring subgenomic replicons derived from the two viruses. Based on these data we speculate that the differential effects of the two NS5A proteins on cellular signalling pathways may contribute to the differences in the natural history of the two viruses.


Assuntos
Vírus GB B/patogenicidade , Hepacivirus/patogenicidade , Hepatócitos/virologia , Interações Hospedeiro-Patógeno , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Proteínas não Estruturais Virais/metabolismo , Animais , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Vírus GB B/genética , Vírus GB B/metabolismo , Regulação da Expressão Gênica , Hepacivirus/genética , Hepacivirus/metabolismo , Humanos , Fosfatidilinositol 3-Quinases/genética , Frações Subcelulares/metabolismo , Fator de Transcrição AP-1/antagonistas & inibidores , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Proteínas não Estruturais Virais/genética , beta Catenina/genética , beta Catenina/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismo
20.
Virus Res ; 135(1): 181-6, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18455258

RESUMO

The extent of genetic variability following acute infection of tamarins with GB virus B (GBV-B) is not known. In this study we attempted to define the quasispecies variation of GBV-B 17 days post-infection, by PCR amplification of GBV-B RNA extracted from serum and liver. Cloning followed by sequencing revealed a small number of changes in the three regions studied, namely the 5' untranslated region, E2 and NS3. Moreover, there was no region of high amino acid variability in E2, akin to hypervariable region 1 of hepatitis C virus. This was further confirmed by analysing sequences from two additional animals obtained at a similar time point post-infection. Nevertheless, it was apparent that different variants with one or two amino acid substitutions in the region studied had been selected when comparing the sequences from the three animals. This restricted sequence variation of GBV-B during acute hepatitis may explain the infrequent progression of the infection to a chronic stage.


Assuntos
Infecções por Flaviviridae/virologia , Vírus GB B/genética , Variação Genética , Hepatite Viral Animal/virologia , Regiões 5' não Traduzidas/química , Regiões 5' não Traduzidas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Vírus GB B/química , Leontopithecus , Modelos Animais , Dados de Sequência Molecular , RNA Viral/química , RNA Viral/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Proteínas Virais/química , Proteínas Virais/genética
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