RESUMO
Entry of hepatitis C virus (HCV) into hepatocytes is a complex process that involves numerous cellular factors, including the scavenger receptor class B type 1 (SR-B1), the tetraspanin CD81, and the tight junction (TJ) proteins claudin-1 (CLDN1) and occludin (OCLN). Despite expression of all known HCV-entry factors, in vitro models based on hepatoma cell lines do not fully reproduce the in vivo susceptibility of liver cells to primary HCV isolates, implying the existence of additional host factors which are critical for HCV entry and/or replication. Likewise, HCV replication is severely impaired within hepatocellular carcinoma (HCC) tissue in vivo, but the mechanisms responsible for this restriction are presently unknown. Here, we identify tumor-associated calcium signal transducer 2 (TACSTD2), one of the most downregulated genes in primary HCC tissue, as a host factor that interacts with CLDN1 and OCLN and regulates their cellular localization. TACSTD2 gene silencing disrupts the typical linear distribution of CLDN1 and OCLN along the cellular membrane in both hepatoma cells and primary human hepatocytes, recapitulating the pattern observed in vivo in primary HCC tissue. Mechanistic studies suggest that TACSTD2 is involved in the phosphorylation of CLDN1 and OCLN, which is required for their proper cellular localization. Silencing of TACSTD2 dramatically inhibits HCV infection with a pan-genotype effect that occurs at the level of viral entry. Our study identifies TACSTD2 as a novel regulator of two major HCV-entry factors, CLDN1 and OCLN, which is strongly downregulated in malignant hepatocytes. These results provide new insights into the complex process of HCV entry into hepatocytes and may assist in the development of more efficient cellular systems for HCV propagation in vitro.
Assuntos
Antígenos de Neoplasias/metabolismo , Carcinoma Hepatocelular/virologia , Moléculas de Adesão Celular/metabolismo , Claudina-1/metabolismo , Hepacivirus/patogenicidade , Hepatite C/virologia , Neoplasias Hepáticas/virologia , Ocludina/metabolismo , Antígenos de Neoplasias/genética , Carcinoma Hepatocelular/epidemiologia , Carcinoma Hepatocelular/metabolismo , Moléculas de Adesão Celular/genética , Claudina-1/genética , Regulação para Baixo , Hepatite C/complicações , Hepatite C/metabolismo , Hepatócitos/metabolismo , Hepatócitos/patologia , Hepatócitos/virologia , Humanos , Neoplasias Hepáticas/epidemiologia , Neoplasias Hepáticas/metabolismo , Ocludina/genética , Internalização do Vírus , Replicação ViralRESUMO
Hepatitis E virus (HEV) is a common cause of acute hepatitis that results in high mortality in pregnant women and may establish chronic infections in immunocompromised patients. We demonstrate for the first time that alpha interferon (IFN-α) and ribavirin inhibit in vitro HEV replication in both a subgenomic replicon and an infectious culture system based on a genotype 3 strain. IFN-α showed a moderate but significant synergism with ribavirin. These findings corroborate the reported clinical effectiveness of both drugs. In addition, the antiviral activity of ribavirin against wild-type genotype 1, 2, and 3 strains was confirmed by immunofluorescence staining. Furthermore, the in vitro activity of ribavirin depends on depletion of intracellular GTP pools, which is evident from the facts that (i) other GTP-depleting agents (5-ethynyl-1-ß-d-ribofuranosylimidazole-4-carboxamide [EICAR] and mycophenolic acid) inhibit viral replication, (ii) exogenously added guanosine reverses the antiviral effects, and (iii) a strong correlation (R(2) = 0.9998) exists between the antiviral activity and GTP depletion of ribavirin and other GTP-depleting agents.
Assuntos
Antivirais/farmacologia , Guanosina Trifosfato/metabolismo , Vírus da Hepatite E/efeitos dos fármacos , Vírus da Hepatite E/metabolismo , Interferon-alfa/farmacologia , Ribavirina/farmacologia , Replicação Viral/efeitos dos fármacos , Linhagem Celular , HumanosRESUMO
The family Hepeviridae consists of positive-stranded RNA viruses that infect a wide range of mammalian species, as well as chickens and trout. A subset of these viruses infects humans and can cause a self-limiting acute hepatitis that may become chronic in immunosuppressed individuals. Current published descriptions of the taxonomical divisions within the family Hepeviridae are contradictory in relation to the assignment of species and genotypes. Through analysis of existing sequence information, we propose a taxonomic scheme in which the family is divided into the genera Orthohepevirus (all mammalian and avian hepatitis E virus (HEV) isolates) and Piscihepevirus (cutthroat trout virus). Species within the genus Orthohepevirus are designated Orthohepevirus A (isolates from human, pig, wild boar, deer, mongoose, rabbit and camel), Orthohepevirus B (isolates from chicken), Orthohepevirus C (isolates from rat, greater bandicoot, Asian musk shrew, ferret and mink) and Orthohepevirus D (isolates from bat). Proposals are also made for the designation of genotypes within the human and rat HEVs. This hierarchical system is congruent with hepevirus phylogeny, and the three classification levels (genus, species and genotype) are consistent with, and reflect discontinuities in the ranges of pairwise distances between amino acid sequences. Adoption of this system would include the avoidance of host names in taxonomic identifiers and provide a logical framework for the assignment of novel variants.
Assuntos
Hepatite E/veterinária , Hepatite E/virologia , Vírus de RNA/classificação , Vírus de RNA/genética , Animais , Análise por Conglomerados , Humanos , Filogenia , Vírus de RNA/isolamento & purificação , RNA Viral/genéticaRESUMO
BACKGROUND: The true incidence of transfusion-associated hepatitis (TAH) before blood screening is unknown. Our aims were to reevaluate blood recipients receiving unscreened blood and analyze hepatitis viruses circulating more than 45 years ago. STUDY DESIGN AND METHODS: Cryopreserved serum samples from 66 patients undergoing open heart surgery in the 1960s were reevaluated with modern diagnostic tests to determine the incidence of TAH and its virologic causes. RESULTS: In this heavily transfused population receiving a mean of 20 units per patient of predominantly paid-donor blood, 30 of 66 (45%) developed biochemical evidence of hepatitis; of these, 20 (67%) were infected with hepatitis C virus (HCV) alone, four (13%) with hepatitis B virus (HBV) alone, and six (20%) with both viruses. Among the 36 patients who did not develop hepatitis, four (11%) were newly infected with HCV alone, nine (25%) with HBV alone, and one (3%) with both viruses. Overall, 100% of patients with hepatitis and 39% of those without hepatitis were infected with HBV and/or HCV; one patient was also infected with hepatitis E virus. The donor carrier rate for HBV and/or HCV was estimated to be more than 6%; contemporaneously prepared pooled normal human plasma was also contaminated with multiple hepatitis viruses. CONCLUSION: TAH virus infections were a larger problem than perceived 50 years ago and HCV was the predominant agent transmitted. All hepatitis cases could be attributed to HCV and/or HBV and hence there was no evidence to suggest that an additional hepatitis agent existed undetected in the blood supply.
Assuntos
Transfusão de Sangue , Hepacivirus , Vírus da Hepatite B , Hepatite B , Hepatite C , Adulto , Idoso , Feminino , Seguimentos , Hepatite B/epidemiologia , Hepatite B/transmissão , Hepatite C/epidemiologia , Hepatite C/transmissão , Hepatite E/epidemiologia , Hepatite E/transmissão , Vírus da Hepatite E , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Fatores de TempoRESUMO
Hepatitis E virus (HEV) is transmitted via the fecal-oral route and has been recognized as a common source of large waterborne outbreaks involving contaminated water in developing countries. Thus, there is the need to produce experimental data on the disinfection kinetics of HEV by chlorine in water samples with diverse levels of fecal contamination. Here, the inactivation of HEV and human adenovirus C serotype 2 (HAdV2), used as a reference virus, was monitored using immunofluorescence and quantitative reverse transcription polymerase chain reaction (RT-qPCR) assays. HEV has been shown to be susceptible to chlorine disinfection and presented equivalent kinetics to human adenoviruses. The C(t) values observed for a 2-log reduction of HEV were 0.41 in buffered demand-free water and 11.21 mg/L × min in the presence of 1% sewage. The results indicate that the inactivation kinetics of HEV and HAdV2 are equivalent and support the use of chlorine disinfection as an effective strategy to control HEV waterborne transmission.
Assuntos
Adenovírus Humanos/efeitos dos fármacos , Cloro/farmacologia , Desinfetantes/farmacologia , Desinfecção/métodos , Vírus da Hepatite E/efeitos dos fármacos , Purificação da Água/métodos , Adenovírus Humanos/fisiologia , Imunofluorescência , Vírus da Hepatite E/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esgotos/virologia , Inativação de VírusRESUMO
The RNA virus, hepatitis E virus (HEV) is the most or second-most important cause of acute clinical hepatitis in adults throughout much of Asia, the Middle East, and Africa. In these regions it is an important cause of acute liver failure, especially in pregnant women who have a mortality rate of 20-30%. Until recently, hepatitis E was rarely identified in industrialized countries, but Hepatitis E now is reported increasingly throughout Western Europe, some Eastern European countries, and Japan. Most of these cases are caused by genotype 3, which is endemic in swine, and these cases are thought to be zoonotically acquired. However, transmission routes are not well understood. HEV that infect humans are divided into nonzoonotic (types 1, 2) and zoonotic (types 3, 4) genotypes. HEV cell culture is inefficient and limited, and thus far HEV has been cultured only in human cell lines. The HEV strain Kernow-C1 (genotype 3) isolated from a chronically infected patient was used to identify human, pig, and deer cell lines permissive for infection. Cross-species infections by genotypes 1 and 3 were studied with this set of cultures. Adaptation of the Kernow-C1 strain to growth in human hepatoma cells selected for a rare virus recombinant that contained an insertion of 174 ribonucleotides (58 amino acids) of a human ribosomal protein gene.
Assuntos
Vírus da Hepatite E/genética , Hepatite E/genética , Mutagênese Insercional , Recombinação Genética , Proteínas Ribossômicas/genética , Animais , Sequência de Bases , Células CACO-2 , Cervos/virologia , Feminino , Genótipo , Hepatite E/mortalidade , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Gravidez , Complicações Infecciosas na Gravidez/genética , Complicações Infecciosas na Gravidez/mortalidade , Especificidade da Espécie , Suínos/virologia , Doenças dos Suínos/genética , Doenças dos Suínos/mortalidadeRESUMO
Hepatitis E virus (genus Hepevirus, family Hepeviridae) is one of the most important causes of acute hepatitis in adults, particularly among pregnant women, throughout Asia and Africa where mortality rates can be 20-30â%. Hepatitis E virus has a single-stranded positive-sense RNA genome that contains three translated ORFs. The two 3' ORFs are translated from a subgenomic RNA. Functional RNA elements have been identified in and adjacent to the genomic 5' and 3' UTRs and in and around the intergenic region. Here we describe an additional RNA element that is located in a central region of ORF2. The RNA element is predicted to fold into two highly conserved stem-loop structures, ISL1 and ISL2. Mutations that disrupt the predicted structures, without altering the encoded amino acid sequence, result in a drastic reduction in capsid protein synthesis. This indicates that the RNA element plays an important role in one of the early steps of virus replication. The structures were further investigated using a replicon that expresses Gaussia luciferase in place of the capsid protein. Single mutations in ISL2 severely reduced luciferase expression, but a pair of compensatory mutations that were predicted to restore the ISL2 structure, restored luciferase expression to near-WT levels, thus lending experimental support to the predicted structure. Nonetheless the precise role of the ISL1+ISL2 element remains unknown.
Assuntos
Vírus da Hepatite E/genética , RNA Viral/genética , Proteínas Virais/genética , Replicação Viral , Animais , Sequência de Bases , Capsídeo/metabolismo , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Genoma Viral/genética , Vírus da Hepatite E/metabolismo , Vírus da Hepatite E/fisiologia , Humanos , Dados de Sequência Molecular , Mutação , Proteínas Virais/químicaRESUMO
BACKGROUND & AIMS: The diagnosis of drug-induced liver injury relies on exclusion of other causes, including viral hepatitis A, B, and C. Hepatitis E virus (HEV) infection has been proposed as another cause of suspected drug-induced liver disease. We assessed the frequency of HEV infection among patients with drug-induced liver injury in the United States. METHODS: The Drug-Induced Liver Injury Network (DILIN) is a prospective study of patients with suspected drug-induced liver injury; clinical information and biological samples are collected to investigate pathogenesis and disease progression. We analyzed serum samples, collected from patients enrolled in DILIN, for immunoglobulin (Ig) G and IgM against HEV; selected samples were tested for HEV RNA. RESULTS: Among 318 patients with suspected drug-induced liver injury, 50 (16%) tested positive for anti-HEV IgG and 9 (3%) for anti-HEV IgM. The samples that contained anti-HEV IgM (collected 2 to 24 weeks after onset of symptoms) included 4 that tested positive for HEV RNA genotype 3. Samples from the 6-month follow-up visit were available from 4 patients; they were negative for anti-HEV IgM, but levels of anti-HEV IgG increased with time. Patients who had anti-HEV IgM were mostly older men (89%; mean age, 67 years), and 2 were human immunodeficiency virus positive. Clinical reassessment of the 9 patients with anti-HEV IgM indicated that acute hepatitis E was the most likely diagnosis for 7 and might be the primary diagnosis for 2. CONCLUSIONS: HEV infection contributes to a small but important proportion of cases of acute liver injury that are suspected to be drug induced. Serologic testing for HEV infection should be performed, particularly if clinical features are compatible with acute viral hepatitis.
Assuntos
Lesão Pulmonar Aguda/epidemiologia , Lesão Pulmonar Aguda/etiologia , Doença Hepática Induzida por Substâncias e Drogas/epidemiologia , Hepatite E/complicações , Hepatite E/epidemiologia , Doença Aguda , Lesão Pulmonar Aguda/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Doença Hepática Induzida por Substâncias e Drogas/sangue , Doença Hepática Induzida por Substâncias e Drogas/diagnóstico , Diagnóstico Diferencial , Feminino , Anticorpos Anti-Hepatite/sangue , Hepatite E/diagnóstico , Vírus da Hepatite E/imunologia , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Masculino , Pessoa de Meia-Idade , Prevalência , Estudos Prospectivos , Estudos Retrospectivos , Estados Unidos/epidemiologiaRESUMO
Hepatitis C virus infections proceed to chronicity in the majority of cases. In patients, hepatitis C viruses exist as a dynamic and complex quasispecies. The dominant species at any one time arises in response to host immune pressure and other, incompletely understood factors. It is critical to understand all the mechanisms by which dominance is achieved, but this is difficult to study in vivo. Therefore, it would be useful to develop a cell culture system in which naturally occurring quasispecies could be studied. Hepatitis C virus glycoprotein genes E1 and E2 were PCR amplified as a cassette from the plasma of a chronically infected patient and shotgun cloned into a modified 1a/JFH1 infectious cDNA clone. Following transformation of bacteria, plasmids were batch harvested, transcribed, and transfected into Huh7.5 cells to produce a quasispecies of hypervariable region 1 (HVR1) that mimicked that circulating in vivo. Serial passage of the quasispecies in vitro resulted in replacement of the initially dominant species with a new HVR1 species coexisting with selected growth-enhancing mutations located outside HVR1. Antibody raised against one HVR1 sequence neutralized virus with the homologous HVR1 and cross-neutralized virus with a different sequence. Reciprocal swapping of the HVR1 regions between the two dominating species demonstrated that the HVR1 sequence affects the efficiency of replication and of neutralization by anti-HVR1 but that both processes are strongly influenced by regions outside HVR1.
Assuntos
Hepacivirus/crescimento & desenvolvimento , Hepacivirus/imunologia , Hepatite C Crônica/virologia , Replicação Viral , Anticorpos Neutralizantes/imunologia , Técnicas de Cultura de Células , Linhagem Celular , Clonagem Molecular , Reações Cruzadas , Hepacivirus/genética , Hepacivirus/isolamento & purificação , Anticorpos Anti-Hepatite C/imunologia , Hepatócitos/virologia , Humanos , Mutação , Recombinação Genética , Inoculações Seriadas , Transfecção , Proteínas do Envelope Viral/genéticaRESUMO
Hepatitis E virus (HEV) is an important but extremely understudied human pathogen, and the mechanisms of HEV replication and pathogenesis are largely unknown. We previously identified an attenuated genotype 3 HEV mutant (pSHEV-1) containing three unique amino acid mutations (F51L, T59A, and S390L) in the capsid protein. To determine the role of each of these mutations, we constructed three HEV single mutants (rF51L, rT59A, and rS390L) which were all found to be replication competent in Huh7 liver cells. To determine the pathogenicities of the mutants, we utilized the specific-pathogen-free (SPF) pig model for HEV and a unique inoculation procedure that bypasses the need for propagating infectious HEV in vitro. A total of 60 pigs were intrahepatically inoculated, via an ultrasound-guided technique, with in vitro-transcribed full-length capped RNA transcripts from the infectious clones of each single mutant, the pSHEV-1 triple mutant, wild-type pSHEV-3, or phosphate-buffered saline (PBS) buffer (n = 10). The results showed that the F51L mutation partially contributed to virus attenuation, whereas the T59A and S390L mutations resulted in more drastic attenuation of HEV in pigs, as evidenced by a significantly lower incidence of viremia, a delayed appearance and shorter duration of fecal virus shedding and viremia, and lower viral loads in liver, bile, and intestinal content collected at three different necropsy times. The results indicate that the three mutations in the capsid protein collectively contribute to HEV attenuation. This study has important implications for developing a modified live-attenuated vaccine against HEV.
Assuntos
Aminoácidos/genética , Proteínas do Capsídeo/metabolismo , Vírus da Hepatite E/patogenicidade , Mutação de Sentido Incorreto , Animais , Sangue/virologia , Proteínas do Capsídeo/genética , Linhagem Celular , Modelos Animais de Doenças , Fezes/virologia , Hepatite E/patologia , Hepatite E/virologia , Vírus da Hepatite E/genética , Hepatócitos/virologia , Humanos , Suínos , Doenças dos Suínos/patologia , Doenças dos Suínos/virologia , Viremia , Virulência , Eliminação de Partículas ViraisRESUMO
Hepatitis E virus (HEV) is a human pathogen that causes acute hepatitis. When an HEV capsid protein containing a 52-amino-acid deletion at the C terminus and a 111-amino-acid deletion at the N terminus is expressed in insect cells, the recombinant HEV capsid protein can self-assemble into a T=1 virus-like particle (VLP) that retains the antigenicity of the native HEV virion. In this study, we used cryoelectron microscopy and image reconstruction to show that anti-HEV monoclonal antibodies bind to the protruding domain of the capsid protein at the lateral side of the spikes. Molecular docking of the HEV VLP crystal structure revealed that Fab224 covered three surface loops of the recombinant truncated second open reading frame (ORF2) protein (PORF2) at the top part of the spike. We also determined the structure of a chimeric HEV VLP and located the inserted B-cell tag, an epitope of 11 amino acids coupled to the C-terminal end of the recombinant ORF2 protein. The binding site of Fab224 appeared to be distinct from the location of the inserted B-cell tag, suggesting that the chimeric VLP could elicit immunity against both HEV and an inserted foreign epitope. Therefore, the T=1 HEV VLP is a novel delivery system for displaying foreign epitopes at the VLP surface in order to induce antibodies against both HEV and the inserted epitope.
Assuntos
Antígenos Virais/imunologia , Proteínas do Capsídeo/imunologia , Epitopos/imunologia , Vírus da Hepatite E/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Antígenos Virais/genética , Antígenos Virais/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Microscopia Crioeletrônica , Feminino , Processamento de Imagem Assistida por Computador , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Ligação Proteica , Multimerização Proteica , Deleção de Sequência , Spodoptera , Virossomos/metabolismoRESUMO
UNLABELLED: Liver transplantation (LT) is a unique model to study hepatitis C virus (HCV) entry into hepatocytes. Recent in vitro studies suggest significant changes in the expression of the HCV receptors claudin-1 and occludin after HCV infection. Our aims were: (1) to characterize claudin-1 and occludin expression in grafts from LT recipients and (2) to explore their potential influence on early HCV kinetics and their changes after HCV infection. We included 42 HCV-infected LT recipients and 19 uninfected controls. Claudin-1 and occludin were detected in paraffin-embedded liver biopsies obtained during reperfusion and 3 and 12 months after LT. HCV receptors were characterized by confocal immunofluorescence microscopy; quantification and colocalization studies were performed with dedicated software. Claudin-1 and occludin expression were restricted to the apical pole of hepatocytes. There was a significant correlation between the amount of scavenger receptor B1 at the time of reperfusion and the HCV-RNA decay during the first 24 hours following LT (r = 0.55, P = 0.007). Similarly, there was a significant correlation between the levels of claudin and occludin and the slope of HCV-RNA increase during the first week after LT (r = 0.63, P = 0.005). Occludin and claudin-1 levels increased significantly 12 months after LT (P = 0.03 and P = 0.007, respectively). The expression pattern of both proteins, however, remained unchanged, colocalizing strongly (60%-94%) at the apical membrane of hepatocytes. CONCLUSIONS: HCV receptor levels at the time of LT seem to modulate early HCV kinetics. Hepatitis C recurrence after LT was associated with increased levels of claudin-1 and occludin in the hepatocyte cell membrane, although it did not alter their localization within the tight junctions.
Assuntos
Hepacivirus/fisiologia , Hepatite C/virologia , Transplante de Fígado , Proteínas de Membrana/biossíntese , Receptores Virais/biossíntese , Adulto , Idoso , Claudina-1 , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Ocludina , Estudos RetrospectivosRESUMO
We detected cross-reactive neutralizing antibodies (NtAb) against hepatitis C virus (HCV) in chimpanzees vaccinated with HCV-1 (genotype 1a) recombinant E1/E2 envelope glycoproteins. Five vaccinated chimpanzees, protected following HCV-1 challenge, were initially studied using the heterologous H77 (genotype 1a) HCVpp assay. All animals had developed NtAb after the second vaccination; 4 animals had reciprocal titers of ≥200 at the time of challenge. Using genotypes 1a-6a HCV pseudoparticles (HCVpp) and cell culture-derived HCV (HCVcc) assays, cross-reactive NtAb were detected against 1a, 4a, 5a, and 6a, with limited reactivity against 2a and 3a. Our study provides encouragement for the development of a recombinant envelope-based vaccine against hepatitis C.
Assuntos
Anticorpos Neutralizantes/biossíntese , Hepacivirus/imunologia , Hepatite C Crônica/imunologia , Proteínas do Envelope Viral/imunologia , Vacinas contra Hepatite Viral/imunologia , Animais , Anticorpos Neutralizantes/sangue , Reações Cruzadas , Citometria de Fluxo , Genótipo , Hepacivirus/genética , Hepatite C Crônica/prevenção & controle , Pan troglodytes , Estudos Retrospectivos , Vacinação/métodos , Vacinas Sintéticas/imunologia , Vacinas contra Hepatite Viral/normasRESUMO
Little is known about the presence and role of neutralizing antibodies (NtAbs) in perinatal hepatitis C virus (HCV) infection. Using HCV pseudoparticles, NtAbs were studied longitudinally in 12 HCV-infected children with or without evidence of acute hepatitis during the first year of life. Broadly reactive NtAbs of maternal origin did not prevent vertical HCV transmission or progression to chronicity. NtAbs against homologous genotype or subtype appeared during the chronic phase and were more abundant and sustained in children with acute hepatitis. Cross-reactive NtAbs were present in both groups of children, but their appearance did not correlate with better control of viremia or HCV clearance.
Assuntos
Anticorpos Neutralizantes/sangue , Hepacivirus/imunologia , Hepatite C/imunologia , Hepatite C/transmissão , Transmissão Vertical de Doenças Infecciosas , Adolescente , Alanina Transaminase/sangue , Anticorpos Neutralizantes/imunologia , Criança , Pré-Escolar , Feminino , Genótipo , Hepacivirus/genética , Anticorpos Anti-Hepatite C/sangue , Humanos , Lactente , Estudos Longitudinais , Masculino , Estudos Prospectivos , RNA Viral/sangue , Estatísticas não Paramétricas , ViremiaRESUMO
The role of rats in human hepatitis E virus (HEV) infections remains controversial. A genetically distinct HEV was recently isolated from rats in Germany, and its genome was sequenced. We have isolated a genetically similar HEV from urban rats in Los Angeles, California, USA, and characterized its ability to infect laboratory rats and nonhuman primates. Two strains of HEV were isolated from serum samples of 134 wild rats that had a seroprevalence of antibodies against HEV of ≈80%. Virus was transmissible to seronegative Sprague-Dawley rats, but transmission was spotty and magnitude and duration of infection were not robust. Viremia was higher in nude rats. Serologic analysis and reverse transcription PCR were comparably sensitive in detecting infection. The sequence of the Los Angeles virus was virtually identical to that of isolates from Germany. Rat HEV was not transmissible to rhesus monkeys, suggesting that it is not a source of human infection.
Assuntos
Vírus da Hepatite E/isolamento & purificação , Hepatite E/veterinária , Hepatite Viral Animal/virologia , Animais , Animais Selvagens/virologia , Sequência de Bases , Doenças Transmissíveis Emergentes/patologia , Doenças Transmissíveis Emergentes/transmissão , Doenças Transmissíveis Emergentes/veterinária , Doenças Transmissíveis Emergentes/virologia , DNA Viral/genética , Feminino , Hepatite E/patologia , Hepatite E/transmissão , Hepatite E/virologia , Vírus da Hepatite E/genética , Vírus da Hepatite E/patogenicidade , Hepatite Viral Animal/patologia , Hepatite Viral Animal/transmissão , Humanos , Fígado/patologia , Los Angeles , Macaca mulatta , Masculino , Ratos , Ratos Sprague-Dawley , Estudos Soroepidemiológicos , Zoonoses/transmissão , Zoonoses/virologiaRESUMO
Hepatitis E virus genotype 1 strain Sar55 replicated in subcloned Caco-2 intestinal cells and Huh7 hepatoma cells that had been transfected with in vitro transcribed viral genomes, and hepatitis E virions were released into the culture medium of both cell lines. Virus egress from cells depended on open reading frame 3 (ORF3) protein, and a proline-rich sequence in ORF3 was important for egress from cultured cells and for infection of macaques. Both intracellular ORF3 protein accumulation and virus release occurred at the apical membrane of polarized Caco-2 cells. ORF3 protein and lipids were intimately associated with virus particles produced in either cell line; ORF2 epitopes were masked in these particles and could not be immunoprecipitated with anti-ORF2.
Assuntos
Células Epiteliais/virologia , Vírus da Hepatite E/fisiologia , Hepatócitos/virologia , Proteínas Virais/fisiologia , Liberação de Vírus , Animais , Linhagem Celular , Hepatite E/virologia , Vírus da Hepatite E/patogenicidade , Humanos , Macaca mulatta , Doenças dos Macacos , Proteínas Virais/genéticaRESUMO
The chimpanzee is the only animal model for investigating the pathogenesis of viral hepatitis types A through E in humans. Studies of the host response, including microarray analyses, have relied on the close relationship between these two primate species: chimpanzee samples are commonly tested with human-based reagents. In this study, the host responses to two dissimilar viruses, hepatitis E virus (HEV) and hepatitis C virus (HCV), were compared in multiple experimentally infected chimpanzees. Affymetrix U133+2.0 human microarray chips were used to assess the entire transcriptome in serial liver biopsies obtained over the course of the infections. Respecting the limitations of microarray probes designed for human target transcripts to effectively assay chimpanzee transcripts, we conducted probe-level analysis of the microarray data in conjunction with a custom mapping of the probe sequences to the most recent human and chimpanzee genome sequences. Time points for statistical comparison were chosen based on independently measured viremia levels. Regardless of the viral infection, the alignment of differentially expressed genes to the human genome sequence resulted in a larger number of genes being identified when compared with alignment to the chimpanzee genome sequence. This probably reflects the lesser refinement of gene annotation for chimpanzees. In general, the two viruses demonstrated very distinct temporal changes in host response genes, although both RNA viruses induced genes that were involved in many of the same biological systems, including interferon-induced genes. The host response to HCV infection was more robust in the magnitude and number of differentially expressed genes compared to HEV infection.
Assuntos
Perfilação da Expressão Gênica , Hepacivirus/patogenicidade , Vírus da Hepatite E/patogenicidade , Interações Hospedeiro-Patógeno/genética , Animais , Genoma , Genoma Humano , Interações Hospedeiro-Patógeno/imunologia , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Pan troglodytes , Alinhamento de Sequência , Fatores de Tempo , ViremiaRESUMO
A recombinant baculovirus(vBacORF2) that expressed the full-length ORF2 capsid protein of a genotype 1 strain of hepatitis E virus(HEV) was constructed. Transduction of S10-3 human hepatoma cells with this baculovirus led to large amounts of ORF2 protein production in ~50% of the cells as determined by immune fluorescence microscopy. The majority of the ORF2 protein detected by Western blot was 72 kDa, the size expected for the full-length protein. To determine if the exogenously-supplied ORF2 protein could transencapsidate viral genomes, S10-3 cell cultures that had been transfected the previous day with an HEV replicon of genotype 1 that contained the gene for green fluorescent protein(GFP), in place of that for ORF2 protein, were transduced with the vBacORF2 virus. Cell lysates were prepared 5 days later and tested for the ability to deliver the GFP gene to HepG2/C3A cells, another human hepatoma cell line. FACS analysis indicated that lysates from cell cultures receiving only the GFP replicon were incapable of introducing the replicon into the HepG2/C3A cells whereas ~2% of the HepG2/C3A cells that received lysate from cultures that had received both the replicon and the baculovirus produced GFP. Therefore, the baculovirus-expressed ORF2 protein was able to trans-encapsidate the viral replicon and form a particle that could infect naïve HepG2/C3A cells. This ex vivo RNA packaging system should be useful for studying many aspects of HEV molecular biology.
Assuntos
Baculoviridae/genética , Carcinoma Hepatocelular/virologia , Expressão Gênica , Vírus da Hepatite E/fisiologia , RNA Viral/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Montagem de Vírus , Baculoviridae/fisiologia , Linhagem Celular Tumoral , Vetores Genéticos/genética , Vetores Genéticos/fisiologia , Vírus da Hepatite E/genética , Humanos , RNA Viral/genéticaRESUMO
The JFH1 strain of hepatitis C virus (HCV) is unique among HCV isolates, in that the wild-type virus can traverse the entire replication cycle in cultured cells. However, without adaptive mutations, only low levels of infectious virus are produced. In the present study, the effects of five mutations that were selected during serial passage in Huh-7.5 cells were studied. Recombinant genomes containing all five mutations produced 3-4 logs more infectious virions than did wild type. Neither a coding mutation in NS5A nor a silent mutation in E2 was adaptive, whereas coding mutations in E2, p7, and NS2 all increased virus production. A single-cycle replication assay in CD81-deficient cells was developed to study more precisely the effect of the adaptive mutations. The E2 mutation had minimal effect on the amount of infectious virus released but probably enhanced entry into cells. In contrast, both the p7 and NS2 mutations independently increased the amount of virus released.
Assuntos
Adaptação Biológica/genética , Hepacivirus/genética , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Humanos , Mutação/genética , Replicação ViralRESUMO
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.