RESUMEN

Programmed death-1 (PD-1) has a pivotal role in the attenuation of adaptive immune responses and peripheral tolerance. Here we describe the identification of the Pekin duck programmed death-1 orthologue (duPD-1). The duPD-1 cDNA encodes a 283-amino acid polypeptide that has an amino acid identity of 70%, 32% and 31% with chicken, murine and human PD-1, respectively. The duck PD-1 gene shares five conserved exons with chicken, murine and human PD-1 genes. A cluster of putative regulatory elements within the conserved region B (CR-B) of the basal promotor is conserved. Homology modeling was most compatible with the two ß-sheet IgV domain structure of murine PD-1. Contact residues, shown to be critical for binding of the respective human and murine PD-1 ligands are mostly conserved between avian and mammalian species, whereas residues that define the cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM) are highly conserved across higher vertebrates and frog. Constitutive expression of duPD-1 transcripts was predominantly found in lymphocyte-rich tissues, and mitogen-stimulation of duck peripheral blood mononuclear cells transiently increased duPD-1 mRNA expression. A soluble duPD-1 protein was expressed and shown to engage the identified duck PD-1 ligands. Our observations show considerable evolutionary conservation between mammalian and avian PD-1 orthologues. This work will facilitate further investigation of the role of PD-1 signaling in adaptive immunity in the Pekin duck, a non-mammalian vertebrate and pathogen host with relevance for human and animal health.

Asunto(s)

Proteínas Aviares/química , Proteínas Aviares/genética , Receptor de Muerte Celular Programada 1/química , Receptor de Muerte Celular Programada 1/genética , Animales , Proteínas Aviares/clasificación , Mapeo Cromosómico , Clonación Molecular , Patos , Expresión Génica , Ligandos , Modelos Moleculares , Filogenia , Receptor de Muerte Celular Programada 1/clasificación , Receptor de Muerte Celular Programada 1/metabolismo , Dominios Proteicos , Estructura Secundaria de Proteína , ARN Mensajero/metabolismo , Alineación de Secuencia , Análisis de Secuencia de Proteína , Distribución Tisular

RESUMEN

Programmed death-1 (PD-1), upon engagement by its ligands, programmed death ligand-1 (PD-L1) and programmed death ligand-2 (PD-L2), provides signals that attenuate adaptive immune responses. Here we describe the identification of the Pekin duck PD-L2 (duPD-L2) and its gene structure. The duPD-L2 cDNA encodes a 321 amino acid protein that has an amino acid identity of 76% and 35% with chicken and human PD-L2, respectively. Mapping of the duPD-L2 cDNA with duck genomic sequences revealed an exonic structure similar to that of the human Pdcd1lg2 gene. Homology modelling of the duPD-L2 protein was compatible with the murine PD-L2 ectodomain structure. Residues known to be important for PD-1 receptor binding of murine PD-L2 were mostly conserved in duPD-L2 within sheets A and G and partially conserved within sheets C and F. DuPD-L2 mRNA was constitutively expressed in all tissues examined with highest expression levels in lung, spleen, cloaca, bursa, cecal tonsil, duodenum and very low levels of expression in muscle, kidney and brain. Lipopolysaccharide treatment of adherent duck PBMC upregulated duPD-L2 mRNA expression. Our work shows evolutionary conservation of the PD-L2 ectodomain structure and residues important for PD-1 binding in vertebrates including fish. The information provided will be useful for further investigation of the role of duPD-L2 in the regulation of duck adaptive immunity and exploration of PD-1-targeted immunotherapies in the duck hepatitis B infection model.

RESUMEN

Interferons (IFNs) are the first line of defense against viral infections in vertebrates. Type III interferon (IFN-λ) is recognized for its key role in innate immunity of tissues of epithelial origin. Here we describe the identification of the Pekin duck IFN-λ ortholog (duIFN-λ). The predicted duIFN-λ protein has an amino acid identity of 63%, 38%, 37% and 33% with chicken IFN-λ and human IFN-λ3, IFN-λ2 and IFN-λ1, respectively. The duck genome contains a single IFN-λ gene that is comprised of five exons and four introns. Recombinant duIFN-λ up-regulated OASL and Mx-1 mRNA in primary duck hepatocytes. Our observations suggest evolutionary conservation of genomic organization and structural features implicated in receptor binding and antiviral activity. The identification and expression of duIFN-λ will facilitate further study of the role of type III IFN in antiviral defense and inflammatory responses of the Pekin duck, a non-mammalian vertebrate and pathogen host with relevance for human and animal health.

Asunto(s)

Patos/genética , Interferones/genética , Interferones/metabolismo , 2',5'-Oligoadenilato Sintetasa/genética , 2',5'-Oligoadenilato Sintetasa/metabolismo , Secuencia de Aminoácidos , Animales , Pollos/genética , Clonación Molecular , Exones , Regulación de la Expresión Génica , Hepatocitos/fisiología , Interferones/química , Interleucinas/genética , Intrones , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas de Resistencia a Mixovirus/genética , Filogenia , Conformación Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido

RESUMEN

BACKGROUND: Feedback mechanisms between interferons α and λ (IFNs) may be affected by single nucleotide polymorphisms (SNP) in interleukin 28B (IL-28B; IFN-λ3) promoter region and may influence cytomegalovirus (CMV) replication. METHODS: We associated IL-28B SNPs with the risk of CMV replication after transplantation. Next, we examined the effect of IL-28B genotypes on IL-28B, and IFN-stimulated gene (ISG) expression, and CMV replication in human foreskin fibroblast (HFF) and peripheral blood mononuclear cells (PBMCs). RESULTS: Transplant recipients with an IL-28B SNP (rs8099917) had significantly less CMV replication (P = .036). Both HFF-cells and PBMCs with a SNP showed lower IL-28B expression during infection with CMV, but higher "antiviral" ISG expression (eg, OAS1). Fibroblasts with a SNP had a 3-log reduction of CMV replication at day 4 (P = .004). IL-28B pretreatment induced ISG expression in noninfected fibroblasts, but a relative decrease of ISG expression could be observed in CMV-infected fibroblasts. The inhibitory effects of IL-28B could be abolished by siRNA or antagonistic peptides against the IL-28 receptor. In fibroblasts, inhibition of IL-28 signaling resulted in an increase of ISG expression and 3-log reduction of CMV-replication (P = .01). CONCLUSIONS: We postulate that IL-28B may act as a key regulator of ISG expression during primary CMV infection. IL-28B SNPs may be associated with higher antiviral ISG expression, which results in better replication control.

Asunto(s)

Infecciones por Citomegalovirus/genética , Infecciones por Citomegalovirus/inmunología , Citomegalovirus/inmunología , Interleucinas/genética , Interleucinas/inmunología , Adulto , Anciano , Células Cultivadas , Citomegalovirus/fisiología , Femenino , Fibroblastos/virología , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Interferones , Leucocitos Mononucleares/virología , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Trasplante , Replicación Viral

RESUMEN

Here we describe the cloning and expression of Pekin duck IL-10 (duIL-10) and a six exon-5 intron structure of an IL-10 gene. Two transcripts encoding duIL-10 with an alternatively spliced 3'UTR, and a transcript lacking exon 5 with a novel coding sequence for its C-terminus (duIL-10ΔE5) were isolated from splenocytes. The duIL-10 protein has an amino acid identity of 79% and 47% with chicken and human IL-10, respectively. The duck IL-10 gene shares a similar structure of the respective exons 1-5 with the IL-10 genes of other vertebrates but has an alternative exon. The duIL-10 3D structure by homology modeling was similar to that of the human IL-10 monomer, whereas the predicted duIL-10ΔE5 protein lacks helix F. DuIL-10 and duIL-10ΔE5 transcripts were most abundant in primary and secondary immune organs and lung. Recombinant duIL-10 suppressed duck IL-2 transcripts in mitogen-activated PBMCs. Our observation suggests evolutionary conservation of structure and function of the duIL-10 protein but the roles of the novel IL-10 splice variants in the regulation of duck immune responses and evolution of vertebrate immunity remain to be elucidated.

Asunto(s)

Patos/genética , Patos/inmunología , Interleucina-10/genética , Empalme Alternativo , Animales , Secuencia de Bases , Humanos , Interleucina-10/química , Interleucina-10/inmunología , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Alineación de Secuencia

RESUMEN

The potential of CD154 (CD40L) as a powerful immunological adjuvant has been shown in various strategies. In this study we examine the immunogenicity and protective efficacy of a CD40-targeting avian influenza hemagglutinin (HA) subunit DNA vaccine in ducks. DNA constructs encoded the ectodomain of the HA protein of LPAI A/mallard/BC/373/2005 (H5N2) with or without fusion to the ectodomain of duck CD154. CD40-targeting significantly accelerated and enhanced humoral responses to the vector-encoded HA protein. In viral challenge experiments with A/chicken/Vietnam/14/2005 (H5N1), DNA immunization conferred partial protection against the genetically distant HPAI. The observed improved kinetics and magnitude of immune induction suggest that CD40-targeting holds promise for influenza A vaccine development.

Asunto(s)

Ligando de CD40/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Vacunas contra la Influenza/inmunología , Gripe Aviar/prevención & control , Vacunas de ADN/inmunología , Estructuras Animales/patología , Animales , Anticuerpos Antivirales/sangre , Ligando de CD40/genética , Protección Cruzada , Patos , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Histocitoquímica , Vacunas contra la Influenza/genética , Gripe Aviar/patología , Microscopía , Pruebas de Neutralización , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Vacunas de ADN/genética , Vacunas de Subunidad/genética , Vacunas de Subunidad/inmunología , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología

RESUMEN

Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4, CD152) is an inhibitory T cell receptor predominately expressed on activated T cells. The duck CTLA-4 (DuCTLA-4) cDNA and a transcript lacking the predicted transmembrane encoding region (DuCTLA-4DeltaTM) were isolated from splenocytes using RT-PCR. The predicted DuCTLA-4 protein showed an identity of 92%, 49% and 47% with chicken, human and mouse homologues, respectively. Sequence comparison revealed conservation of residues implicated in the B7 ligand binding, disulfide linkages, glycosylation and intracellular signaling. DuCTLA-4 mRNA was predominately expressed in primary and secondary immune organs. DuCTLA-4 and DuCTLA-4DeltaTM transcripts were differentially regulated in PBMCs. Flow cytometric analysis showed constitutive expression of DuCTLA-4 protein on freshly isolated PBMCs and a modest increase upon mitogen stimulation. Our observations suggest that DuCTLA-4 and its isoform DuCTLA-4DeltaTM evolved before the divergence of birds and mammals. Both DuCTLA-4 isoforms have significant structural homology to mammalian CTLA-4 proteins but their individual roles in the regulation of duck immune responses remains to be elucidated.

Asunto(s)

Antígenos CD/aislamiento & purificación , Patos/inmunología , Inmunidad Adaptativa/inmunología , Secuencia de Aminoácidos , Animales , Antígenos CD/genética , Antígenos CD/inmunología , Secuencia de Bases , Antígeno CTLA-4 , Clonación Molecular , Patos/genética , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Filogenia , Isoformas de Proteínas , ARN Mensajero/química , ARN Mensajero/genética , Técnica del ADN Polimorfo Amplificado Aleatorio , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Alineación de Secuencia , Análisis de Secuencia de ADN

RESUMEN

BACKGROUND: Severe Combined Immune Deficient (SCID)/Urokinase-type Plasminogen Activator (uPA) mice undergo liver failure and are useful hosts for the propagation of transplanted human hepatocytes (HH) which must compete with recipient-derived hepatocytes for replacement of the diseased liver parenchyma. While partial replacement by HH has proven useful for studies with Hepatitis C virus, complete replacement of SCID/uPA mouse liver by HH has never been achieved and limits the broader application of these mice for other areas of biomedical research. The herpes simplex virus type-1 thymidine kinase (HSVtk)/ganciclovir (GCV) system is a powerful tool for cell-specific ablation in transgenic animals. The aim of this study was to selectively eliminate murine-derived parenchymal liver cells from humanized SCID/uPA mouse liver in order to achieve mice with completely humanized liver parenchyma. Thus, we reproduced the HSVtk (vTK)/GCV system of hepatic failure in SCID/uPA mice. METHODOLOGY/PRINCIPAL FINDINGS: In vitro experiments demonstrated efficient killing of vTK expressing hepatoma cells after GCV treatment. For in vivo experiments, expression of vTK was targeted to the livers of FVB/N and SCID/uPA mice. Hepatic sensitivity to GCV was first established in FVB/N mice since these mice do not undergo liver failure inherent to SCID/uPA mice. Hepatic vTK expression was found to be an integral component of GCV-induced pathologic and biochemical alterations and caused death due to liver dysfunction in vTK transgenic FVB/N and non-transplanted SCID/uPA mice. In SCID/uPA mice with humanized liver, vTK/GCV caused death despite extensive replacement of the mouse liver parenchyma with HH (ranging from 32-87%). Surprisingly, vTK/GCV-dependent apoptosis and mitochondrial aberrations were also localized to bystander vTK-negative HH. CONCLUSIONS/SIGNIFICANCE: Extensive replacement of mouse liver parenchyma by HH does not provide a secure therapeutic advantage against vTK/GCV-induced cytotoxicity targeted to residual mouse hepatocytes. Functional support by engrafted HH may be secured by strategies aimed at limiting this bystander effect.

Asunto(s)

Trasplante de Células/métodos , Hepatocitos/trasplante , Hepatopatías/cirugía , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Ganciclovir/farmacología , Ganciclovir/toxicidad , Hepatocitos/citología , Herpesvirus Humano 1/enzimología , Humanos , Immunoblotting , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/ultraestructura , Hepatopatías/etiología , Hepatopatías/genética , Masculino , Ratones , Ratones SCID , Ratones Transgénicos , Microscopía Electrónica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Timidina Quinasa/genética , Timidina Quinasa/metabolismo , Transfección , Trasplante Heterólogo , Activador de Plasminógeno de Tipo Uroquinasa/genética

RESUMEN

Duck hepatitis B virus (DHBV) is a model virus for human hepatitis B virus (HBV), which infects approximately 360 million individuals worldwide. Nucleoside analogs can decrease virus production by inhibiting the viral polymerase; however, complete clearance by these drugs is not common because of the persistence of the HBV episome. HBV DNA is present in the nucleus as a covalently closed circular (cccDNA) form, where it drives viral transcription and progeny virus production. cccDNA is not the direct target of antiviral nucleoside analogs and is the source of HBV reemergence when antiviral therapy is stopped. To target cccDNA, six different zinc finger proteins (ZFP) were designed to bind DNA sequences in the DHBV enhancer region. After the binding kinetics were assessed by using electrophoretic mobility shift assays and surface plasmon resonance, two candidates with dissociation constants of 12.3 and 40.2 nM were focused on for further study. The ZFPs were cloned into a eukaryotic expression vector and cotransfected into longhorn male hepatoma cells with the plasmid pDHBV1.3, which replicates the DHBV life cycle. In the presence of each ZFP, viral RNA was significantly reduced, and protein levels were dramatically decreased. As a result, intracellular viral particle production was also significantly decreased. In summary, designed ZFPs are able to bind to the DHBV enhancer and interfere with viral transcription, resulting in decreased production of viral products and progeny virus genomes.

Asunto(s)

ADN Circular/genética , Virus de la Hepatitis B del Pato/metabolismo , Ingeniería de Proteínas/métodos , ARN Viral/metabolismo , Transcripción Genética , Dedos de Zinc/fisiología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Núcleo Celular/virología , Pollos , Elementos de Facilitación Genéticos , Cinética , Datos de Secuencia Molecular , Resonancia por Plasmón de Superficie

RESUMEN

Binding of CD154, a member of the TNF ligand superfamily, to its receptor CD40 is essential for the development and regulation of adaptive immune responses in mammals. The duck CD154 (DuCD154) encoding gene was isolated from activated splenocytes using RT-PCR. Sequence analysis of the cloned DuCD154 gene revealed an open reading frame of 819 base pairs encoding a 272 amino acid protein. The extracellular domain of DuCD154 was identified and expressed for characterization and generation of antibodies. DuCD154 mRNA was predominantly expressed in spleen, thymus and duodenum. DuCD154 protein generated in cell culture was secreted and formed dimers. DuCD154 markedly enhanced proliferative responses in duck splenocytes when used alone or in conjunction with LPS or PHA. These observations suggest that DuCD154 has functional equivalence with mammalian CD154 and that the central role of CD154 as an immunoregulatory protein had already evolved before the divergence of birds and mammals.

Asunto(s)

Ligando de CD40/genética , Ligando de CD40/inmunología , Patos/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Ligando de CD40/metabolismo , Células Cultivadas , Patos/genética , Evolución Molecular , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Bazo/citología , Bazo/inmunología

RESUMEN

Engagement of CD154 on activated T cells with CD40 on antigen-presenting cells (APCs) potentiates adaptive immune responses in mammals. Soluble multimeric forms of CD154 have been used as an adjuvant or in immunotargeting strategies to enhance vaccine responses. The objective of our study was to examine the ability of duck CD154 (DuCD154) to enhance DNA vaccine responses in the duck hepatitis B model. Constructs were generated to express the functional domain of DuCD154 (tCD154), truncated duck hepatitis B virus (DHBV) core antigen (tcore) and chimera of tcore fused to tCD154 (tcore-tCD154). Expression in LMH cells demonstrated that all proteins were secreted and that tCD154 and tcore-tCD154 formed multimers. Ducks immunized with the plasmid ptcore-tCD154 developed accelerated and enhanced core-specific antibody responses compared to ducks immunized with ptcore or ptcore plus ptCD154. Antibody responses were better sustained in both ptcore-tCD154- and ptcore plus ptCD154-immunized ducks. Core-specific proliferative responses of duck peripheral blood mononuclear cells were enhanced in ducks immunized with ptcore-tCD154 or ptcore alone. This study suggests that the role of CD154 in the regulation of adaptive immune responses had already evolved before the divergence of birds and mammals. Thus, targeting of antigens to APCs with CD154 is an effective strategy to enhance DNA vaccine responses not only in mammalian species but also in avian species.

Asunto(s)

Adyuvantes Inmunológicos/uso terapéutico , Antígenos CD40/inmunología , Ligando de CD40/inmunología , Patos/inmunología , Hepatitis B/inmunología , Vacunas de ADN/inmunología , Animales , Anticuerpos Monoclonales/sangre , Ligando de CD40/genética , Modelos Animales de Enfermedad , Escherichia coli/genética , Vectores Genéticos , Hepatitis B/sangre , Hepatitis B/prevención & control , Esquemas de Inmunización , Leucocitos Mononucleares/inmunología , Ligandos , Datos de Secuencia Molecular , Plásmidos , Vacunas de ADN/uso terapéutico

RESUMEN

Currently, there is a need for therapeutic vaccines that are effective in inducing robust T helper type 1 (Th1) immune responses capable of mediating viral clearance in chronic hepatitis B infection. Hepatitis B therapeutic vaccines were designed and formulated by loading the hepatitis B core antigen (HBcAg) into poly(D,L-lactic-acid-co-glycolic acid) (PLGA) nanoparticles with or without monophospholipid A (MPLA), a Th1-favoring immunomodulator. These particles were around 300 nm in diameter, spherical in shape and had approximately 50% HBcAg encapsulation efficiency. A single immunization with a vaccine formulation containing (MPLA+HBcAg) coformulated in PLGA nanoparticles induced a stronger Th1 cellular immune response with a predominant interferon-gamma (IFN-gamma) profile than those induced by HBcAg alone, free (HBcAg+MPLA) simple mixture or HBcAg-loaded nanoparticles in a murine model. More importantly, the level of HBcAg-specific IFN-gamma production could be increased further significantly by a booster immunization with the (HBcAg+MPLA)-loaded nanoparticles. In summary, these results demonstrated that codelivery of HBcAg and MPLA in PLGA nanoparticles promoted HBcAg-specific Th1 immune responses with IFN-gamma production. These findings suggest that appropriate design of the vaccine formulation and careful planning of the immunization schedule are important in the successful development of effective HBV therapeutic vaccines.

Asunto(s)

Adyuvantes Inmunológicos/administración & dosificación , Sistemas de Liberación de Medicamentos/métodos , Antígenos del Núcleo de la Hepatitis B/inmunología , Vacunas contra Hepatitis B/inmunología , Ácido Láctico/administración & dosificación , Nanoestructuras , Ácido Poliglicólico/administración & dosificación , Polímeros/administración & dosificación , Células TH1/inmunología , Secuencia de Aminoácidos , Animales , Epítopos de Linfocito T/administración & dosificación , Epítopos de Linfocito T/inmunología , Femenino , Anticuerpos contra la Hepatitis B/biosíntesis , Antígenos del Núcleo de la Hepatitis B/administración & dosificación , Vacunas contra Hepatitis B/administración & dosificación , Virus de la Hepatitis B/inmunología , Virus de la Hepatitis B/ultraestructura , Inmunización Secundaria , Ácido Láctico/inmunología , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Nanoestructuras/ultraestructura , Copolímero de Ácido Poliláctico-Ácido Poliglicólico

RESUMEN

Superinfection exclusion is the phenomenon whereby a virus prevents the subsequent infection of an already infected host cell. The Pekin duck hepatitis B virus (DHBV) model was used to investigate superinfection exclusion in hepadnavirus infections. Superinfection exclusion was shown to occur both in vivo and in vitro with a genetically marked DHBV, DHBV-ClaI, which was unable to establish an infection in either DHBV-infected ducklings or DHBV-infected primary duck hepatocytes (PDHs). In addition, exclusion occurred in vivo even when the second virus had a replicative advantage. Superinfection exclusion appears to be restricted to DHBV, as adenovirus, herpes simplex virus type 1, and vesicular stomatitis virus were all capable of efficiently infecting DHBV-infected PDHs. Exclusion was dependent on gene expression by the original infecting virus, since UV-irradiated DHBV was unable to mediate the exclusion of DHBV-ClaI. Using recombinant adenoviruses expressing DHBV proteins, we determined that the large surface antigen mediated exclusion. The large surface antigen is known to cause down-regulation of a DHBV receptor, carboxypeptidase D (CPD). Receptor down-regulation is a mechanism of superinfection exclusion seen in other viral infections, and so it was investigated as a possible mechanism of DHBV-mediated exclusion. However, a mutant large surface antigen which did not down-regulate CPD was still capable of inhibiting DHBV infection of PDHs. In addition, exclusion of DHBV-ClaI did not correlate with a decrease in CPD levels. Finally, virus binding assays and confocal microscopy analysis of infected PDHs indicated that the block in infection occurs after internalization of the second virus. We suggest that superinfection exclusion may result from the role of the L surface antigen as a regulator of intracellular trafficking.

Asunto(s)

Infecciones por Hepadnaviridae/etiología , Antígenos de Superficie de la Hepatitis B/fisiología , Virus de la Hepatitis B del Pato/inmunología , Animales , Carboxipeptidasas/fisiología , Farmacorresistencia Viral , Patos , Virus de la Hepatitis B del Pato/efectos de los fármacos , Virus de la Hepatitis B del Pato/genética , Hepatocitos/virología , Lamivudine/farmacología , Replicación Viral