RESUMO
Dengue virus (DENV) is one of the most important arthropod-borne pathogens that cause life-threatening diseases in humans. However, no vaccine or specific antiviral is available for dengue. As seen in other RNA viruses, the innate immune system plays a key role in controlling DENV infection and disease outcome. Although the interferon (IFN) response, which is central to host protective immunity, has been reported to limit DENV replication, the molecular details of how DENV infection is modulated by IFN treatment are elusive. In this study, by employing a gain-of-function screen using a type I IFN-treated cell-derived cDNA library, we identified a previously uncharacterized gene, C19orf66, as an IFN-stimulated gene (ISG) that inhibits DENV replication, which we named Repressor of yield of DENV (RyDEN). Overexpression and gene knockdown experiments revealed that expression of RyDEN confers resistance to all serotypes of DENV in human cells. RyDEN expression also limited the replication of hepatitis C virus, Kunjin virus, Chikungunya virus, herpes simplex virus type 1, and human adenovirus. Importantly, RyDEN was considered to be a crucial effector molecule in the IFN-mediated anti-DENV response. When affinity purification-mass spectrometry analysis was performed, RyDEN was revealed to form a complex with cellular mRNA-binding proteins, poly(A)-binding protein cytoplasmic 1 (PABPC1), and La motif-related protein 1 (LARP1). Interestingly, PABPC1 and LARP1 were found to be positive modulators of DENV replication. Since RyDEN influenced intracellular events on DENV replication and, suppression of protein synthesis from DENV-based reporter construct RNA was also observed in RyDEN-expressing cells, our data suggest that RyDEN is likely to interfere with the translation of DENV via interaction with viral RNA and cellular mRNA-binding proteins, resulting in the inhibition of virus replication in infected cells.
Assuntos
Vírus da Dengue/fisiologia , Dengue/imunologia , Interferons/imunologia , Proteínas Virais/genética , Replicação Viral/imunologia , Linhagem Celular , Vírus da Dengue/crescimento & desenvolvimento , Técnicas de Silenciamento de Genes , Humanos , Immunoblotting , Imunoprecipitação , Espectrometria de Massas , Reação em Cadeia da Polimerase , TransfecçãoRESUMO
Tumor-associated macrophages (TAMs) are known to regulate tumor response to many anti-cancer therapies, including oncolytic virotherapy. Oncolytic virotherapy employing oncolytic paramyxoviruses, such as attenuated measles (MeV) and mumps (MuV) viruses, has demonstrated therapeutic potential against various malignancies. However, the response of TAMs to oncolytic paramyxoviruses and the consequent effect on virotherapeutic efficacy remains to be characterized. Here, we demonstrate that the presence of human monocyte-derived macrophages (MDMs), irrespective of initial polarization state, enhances the virotherapeutic effect of MeV and MuV on breast cancer cells. Notably, our finding contrasts those of several studies involving other oncolytic viruses, which suggest that TAMs negatively impact virotherapeutic efficacy by impeding virus replication and dissemination. We found that the enhanced virotherapeutic effect in the presence of MDMs was due to slightly delayed proliferation and significantly elevated cell death that was not a result of increased virus replication. Instead, we found that the enhanced virotherapeutic effect involved several macrophage-associated anti-tumor mediators, and was associated with the modulation of MDMs towards an anti-tumor phenotype. Our findings present an alternative view on the role of TAMs in oncolytic virotherapy, and highlight the immunotherapeutic potential of oncolytic paramyxoviruses; possibly contributing towards the overall efficacy of oncolytic virotherapy.
Assuntos
Apoptose/fisiologia , Neoplasias da Mama/terapia , Macrófagos/imunologia , Vírus do Sarampo/metabolismo , Vírus da Caxumba/metabolismo , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Microambiente Tumoral , Replicação ViralRESUMO
Background: Muscular dystrophies other than Duchenne muscular dystrophy (DMD) are genetic diseases characterized by increasing muscle weakness, loss of ambulation, and ultimately cardiac and respiratory failure. There are currently no effective therapeutics available. Having demonstrated the efficacy of a N-163 strain of Aureobasidium Pullulans (Neu-REFIX) produced B-1, 3-1,6-Glucan in pre-clinical and clinical studies of Duchenne muscular dystrophy (DMD) earlier, we assessed the effectiveness of this novel Beta glucan in the other muscular dystrophies in the present study. Methods: In this 60-day study, six patients with muscular dystrophies other than DMD consumed one 8g gel of Neu-REFIX beta-glucan along with their usual standard of care treatment regimen, and their biomarkers of relevance to muscle function such as serum calcium (SC), creatine phosphokinase (CPK), and alkaline phosphatase (ALP) levels along with functional improvement criteria, which is, Medical research council (MRC) scale and North Star Ambulatory assessment (NSAA), assessed at baseline and following the intervention. Results: After the intervention, the SC levels significantly decreased from a mean baseline value of 9.28 mg/dL to 8.31 mg/dL (p-value = 0.02). With a p-value of 0.29, the mean CPK value dropped from 2192.33 IU/L to 1567.5 IU/L. Following the intervention, the ALP levels dropped from 200.33 to 75.5 U/L (p-value = 0.15). MRC scale improved in three out of six patients. NSAA remained stable. There were no adverse effects. Conclusion: This study has proven the safety of Neu REFIX beta-glucan food supplement and its efficacy in improving both plasma biomarkers and functional parameters of muscle in a short duration of 2 months. Further validation by evaluation of muscle function for a longer duration is recommended to confirm the efficacy of Neu-REFIX food supplement as a potential adjuvant DMT in muscular dystrophies.
Assuntos
Distrofia Muscular de Duchenne , beta-Glucanas , Humanos , Distrofia Muscular de Duchenne/genética , Biomarcadores , Músculos , Debilidade MuscularRESUMO
We developed an efficient cell culture system for genotype 4 hepatitis E virus using the HE-JF5/15F strain recovered from a fulminant hepatitis patient. The sixth-passage virus in the culture supernatant reached 1.5 x 10(8) copies/ml at 10 days postinoculation and possessed 10 nucleotide mutations with four amino acid changes.
Assuntos
Vírus da Hepatite E/crescimento & desenvolvimento , Vírus da Hepatite E/isolamento & purificação , Hepatite E/virologia , Substituição de Aminoácidos/genética , Técnicas de Cultura de Células/métodos , Linhagem Celular , Genótipo , Vírus da Hepatite E/genética , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutação de Sentido Incorreto , RNA Viral/genética , Análise de Sequência de DNARESUMO
Using RNA preparations extracted from PLC/PRF/5 cells transfected with infectious genotype 3 hepatitis E virus (HEV) cDNA clones or inoculated with a fecal suspension containing a genotype 4 HEV, the 5'-terminal sequence of a 2.2-kb subgenomic RNA of genotype 3 and 4 HEVs was determined. Despite an insertion of T after nucleotide 5116 or an ORF3-null mutation in genotype 4 HEV and/or one of the genotype 3 variants, it was found that the subgenomic RNA of genotype 3 and 4 HEVs initiates exclusively at nucleotide 5122 with the common sequence of 5'-GC, which is identical to that of the prototype genotype 1 HEV.
Assuntos
Regiões 5' não Traduzidas/genética , Genoma Viral , Vírus da Hepatite E/crescimento & desenvolvimento , Vírus da Hepatite E/genética , RNA Viral/genética , Análise de Sequência de DNA , Sequência de Bases , Linhagem Celular , Meios de Cultura , Meios de Cultivo Condicionados , Fezes/virologia , Genótipo , Hepatite E/virologia , Vírus da Hepatite E/patogenicidade , Humanos , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Capuzes de RNA/genética , RNA Viral/química , Transfecção , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Upon phylogenetic analysis of a partial S gene sequence [396 nucleotides (nt)], 928 hepatitis B virus (HBV) strains obtained from 899 viremic subjects in 28 major cities on 15 islands of Indonesia in 1989-2007 segregated into four HBV genotypes. Genotype B was predominant (66%), followed by genotype C (26%), genotype D (7%), and genotype A (0.8%). Comparative and phylogenetic analyses of the 396-nt S gene sequence of 928 HBV isolates and whole genomic sequences of 25 selected HBV isolates revealed a total of 14 subgenotypes within genotypes A-D: two (A1 and A2) in genotype A (HBV/A), five (B2, B3, B5, B7, and a novel subgenotype, tentatively designated B8) in HBV/B, five (C1, C2, C5, C6, and another novel subgenotype, C7) in HBV/C, and two (D1 and D3) in HBV/D. The distribution of HBV genotypes/subgenotypes, including B8 and C7, seems to be associated with ethnological origins in Indonesia.
Assuntos
Vírus da Hepatite B/genética , Hepatite B/epidemiologia , Anticorpos Antivirais/sangue , Genótipo , Geografia , Hepatite B/sangue , Hepatite B/genética , Hepatite B/imunologia , Vírus da Hepatite B/classificação , Hepatite D/epidemiologia , Vírus Delta da Hepatite/genética , Vírus Delta da Hepatite/isolamento & purificação , Humanos , Indonésia/epidemiologia , Epidemiologia Molecular/métodos , Dados de Sequência Molecular , Ilhas do Pacífico/epidemiologia , Filogenia , RNA Viral/genéticaRESUMO
We recently developed a cell culture system for hepatitis E virus (HEV) in PLC/PRF/5 cells, using a genotype 3 HEV (JE03-1760F strain). Thirteen generations of consecutive passages of culture supernatant were successfully carried out in PLC/PRF/5 cells, with the highest HEV load reaching 10(8) copies/ml in the culture medium. Based on continuous release of progenies into culture medium, 50% tissue culture infectivity doses were estimated to be 2.0 x 10(3) copies for wild-type JE03-1760F and 1.4 x 10(2) copies for p13 (progeny in the thirteenth passage). Earlier appearance and greater increase in the yield of progenies in the culture supernatant were evident in p13 compared with wild-type. The cell culture-produced variants in primary propagation (p0) and consecutive passages (p5 [fifth passage], p10 [tenth], and p13) differed from the wild-type virus by 1, 9, 18, and 19 nucleotides (nt), respectively, over the entire genome of 7226nt, excluding the poly(A) tail. Three of five non-synonymous mutations in p13 were shared by a variant (fifth passage) in another series of passages of JE03-1760F. These results suggest that adaptation of HEV variants to growth in vitro is associated with a limited number of mutations similar to hepatitis A virus.
Assuntos
Vírus da Hepatite E/crescimento & desenvolvimento , Vírus da Hepatite E/genética , Hepatite E/virologia , Mutação , RNA Viral/genética , Idoso , Substituição de Aminoácidos , Linhagem Celular Tumoral , Fezes/virologia , Vírus da Hepatite E/classificação , Vírus da Hepatite E/patogenicidade , Humanos , Dados de Sequência Molecular , Filogenia , RNA Viral/metabolismo , Análise de Sequência de RNA , Inoculações Seriadas , Cultura de VírusRESUMO
Naturally occurring galactomannans were sulfated to give sulfated galactomannans with degrees of substitution of 0.7-1.4 per sugar unit and molecular weights of M¯n=0.6×10(4)-2.4×10(4). Sulfated galactomannans were found to have specific biological activities in vitro such as anticoagulant, anti-HIV and anti-Dengue virus activities. The biological activities were compared with those of standard dextran and curdlan sulfates, which are polysaccharides with potent antiviral activity and low cytotoxicity. It was found that sulfated galactomannans had moderate to high anticoagulant activity, 13.4-36.6unit/mg, compared to that of dextran and curdlan sulfates, 22.7 and 10.0unit/mg, and high anti-HIV and anti-Dengue virus activities, 0.04-0.8µg/mL and 0.2-1.1µg/mL, compared to those curdlan sulfates, 0.1µg/mL, respectively. The cytotoxicity on MT-4 and LCC-MK2 cells was low. Surface plasmon resonance (SPR) of sulfated galactomannans revealed strong interaction with poly-l-lysine as a model compound of virus proteins, and suggested that the specific biological activities might originate in the electrostatic interaction of negatively charged sulfate groups of sulfated galactomannans and positively charged amino groups of surface proteins of viruses. These results suggest that sulfated galactomannans effectively prevented the infection of cells by viruses and the degree of substitution and molecular weights played important roles in the biological activities.
Assuntos
Antivirais/química , Vírus da Dengue/efeitos dos fármacos , HIV/efeitos dos fármacos , Mananas/química , Anticoagulantes/química , Anticoagulantes/uso terapêutico , Antivirais/uso terapêutico , Dengue/tratamento farmacológico , Dengue/virologia , Galactose/análogos & derivados , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Humanos , Mananas/uso terapêutico , Polilisina/química , Sulfatos/química , beta-Glucanas/químicaRESUMO
Dengue virus (DENV) infectious disease is a major public health problem worldwide; however, licensed vaccines or specific antiviral drugs against this infection are not available. To identify novel anti-DENV compounds, we screened 1280 pharmacologically active compounds using focus reduction assay. Bromocriptine (BRC) was found to have potent anti-DENV activity and low cytotoxicity (half maximal effective concentration [EC50], 0.8-1.6 µM; and half maximal cytotoxicity concentration [CC50], 53.6 µM). Time-of-drug-addition and time-of-drug-elimination assays suggested that BRC inhibits translation and/or replication steps in the DENV life cycle. A subgenomic replicon system was used to verify that BRC restricts RNA replication step. Furthermore, a single amino acid substitution (N374H) was detected in the NS3 protein that conferred resistance to BRC. In summary, BRC was found to be a novel DENV inhibitor and a potential candidate for the treatment of DENV infectious disease.
Assuntos
Antivirais/farmacologia , Bromocriptina/farmacologia , Vírus da Dengue/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Dengue/tratamento farmacológico , Vírus da Dengue/fisiologia , Farmacorresistência Viral , Humanos , Replicon/efeitos dos fármacos , Ensaio de Placa ViralRESUMO
High prevalence of infection with high-risk human papilloma virus (HPV) ranging from 25 to 100% (average 31%) was observed in breast cancer (BC) patients in Singapore using novel DNA chip technology. Early stage of BC demonstrated higher HPV positivity, and BC positive for estrogen receptor (ER) showed significantly higher HPV infection rate. This unique association of HPV with BC in vivo prompted us to investigate a possible involvement of HPV in early stages of breast carcinogenesis. Using normal breast epithelial cells stably transfected with HPV-18, we showed apparent upregulation of mRNA for the cytidine deaminase, APOBEC3B (A3B) which is reported to be a source of mutations in BC. HPV-induced A3B overexpression caused significant γH2AX focus formation, and DNA breaks which were cancelled by shRNA to HPV18 E6, E7 and A3B. These results strongly suggest an active involvement of HPV in the early stage of BC carcinogenesis via A3B induction.
Assuntos
Neoplasias da Mama/patologia , Neoplasias da Mama/virologia , Carcinogênese , Citidina Desaminase/metabolismo , Papillomaviridae/fisiologia , Adulto , Idoso , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/metabolismo , Transformação Celular Viral , Citidina Desaminase/deficiência , Citidina Desaminase/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Instabilidade Genômica , Células HEK293 , Humanos , Glândulas Mamárias Humanas/metabolismo , Glândulas Mamárias Humanas/patologia , Pessoa de Meia-Idade , Antígenos de Histocompatibilidade Menor , Prognóstico , Receptores de Estrogênio/metabolismo , Fatores de TempoRESUMO
The roles of Type I interferon (IFN) in human immunodeficiency virus Type 1 (HIV-1) neuropathogenesis are poorly understood; both protective and deleterious effects of IFN signaling have been described. We used genetically modified mice deficient in the Type I IFN receptor (IFNRKO) to analyze the progress of HIV-1 brain infection and neuropathogenesis in the absence of IFN signaling. IFNRKO and wild-type (WT) mice on the 129xSv/Ev or C57BL/6 strain backgrounds were infected systemically with EcoHIV, a chimeric HIV-1 that productively infects mice. IFNRKO mice showed higher HIV-1 expression in spleen and peritoneal macrophages and greater virus infiltration into the brain compared to WT mice. Neuropathogenesis was studied by histopathological, immunohistochemical, immunofluorescence, and polymerase chain reaction analyses of brain tissues after the virus was inoculated into the brain by stereotaxic intracerebral injection. Both IFNRKO and WT mice showed readily detectable HIV-1 and brain lesions, including microglial activation, astrocytosis, and increased expression of genes coding for inflammatory cytokines and chemokines typical of human HIV-1 brain disease. Parameters of HIV-1 neuropathogenesis, including HIV-1 expression in microglia/macrophages, were significantly greater in IFNRKO than in WT mice. Our results show unequivocally that Type I IFN signaling and responses limit HIV-1 infection and pathogenesis in the brains of mice.
Assuntos
Encéfalo/metabolismo , Encéfalo/patologia , Infecções por HIV/metabolismo , Infecções por HIV/patologia , HIV-1/metabolismo , Interferon Tipo I/deficiência , Animais , Regulação Viral da Expressão Gênica , Infecções por HIV/genética , HIV-1/genética , Interferon Tipo I/genética , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Through combining vaccine-derived measles and mumps viruses (MM), we efficiently targeted a wide range of hematopoietic cancer cell lines. MM synergistically killed many cell lines including acute myeloid leukemia (AML) cell lines. Further investigation suggested that enhanced oncolytic effect of MM was due to increased apoptosis induction. In an U937 xenograft AML mouse model, MM displayed greater tumor suppression and prolonged survival. Furthermore, MM efficiently killed blasts from 16 out of 20 AML patients and elicited more efficient killing effect on 11 patients when co-administered with Ara-C. Our results demonstrate that MM is a promising therapeutic candidate for hematological malignancies.
Assuntos
Leucemia Mieloide Aguda/terapia , Leucemia Mieloide Aguda/virologia , Vírus do Sarampo/fisiologia , Vírus da Caxumba/fisiologia , Terapia Viral Oncolítica/métodos , Adulto , Animais , Chlorocebus aethiops , Efeito Citopatogênico Viral , Humanos , Células Jurkat , Masculino , Vírus do Sarampo/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Vírus da Caxumba/imunologia , Células U937 , Células Vero , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Curdlan sulfate (CRDS), a sulfated 1â3-ß-D glucan, previously shown to be a potent HIV entry inhibitor, is characterized in this study as a potent inhibitor of the Dengue virus (DENV). CRDS was identified by in silico blind docking studies to exhibit binding potential to the envelope (E) protein of the DENV. CRDS was shown to inhibit the DENV replication very efficiently in different cells in vitro. Minimal effective concentration of CRDS was as low as 0.1 µg/mL in LLC-MK2 cells, and toxicity was observed only at concentrations over 10 mg/mL. CRDS can also inhibit DENV-1, 3, and 4 efficiently. CRDS did not inhibit the replication of DENV subgenomic replicon. Time of addition experiments demonstrated that the compound not only inhibited viral infection at the host cell binding step, but also at an early post-attachment step of entry (membrane fusion). The direct binding of CRDS to DENV was suggested by an evident reduction in the viral titers after interaction of the virus with CRDS following an ultrafiltration device separation, as well as after virus adsorption to an alkyl CRDS-coated membrane filter. The electron microscopic features also showed that CRDS interacted directly with the viral envelope, and caused changes to the viral surface. CRDS also potently inhibited DENV infection in DC-SIGN expressing cells as well as the antibody-dependent enhancement of DENV-2 infection. Based on these data, a probable binding model of CRDS to DENV E protein was constructed by a flexible receptor and ligand docking study. The binding site of CRDS was predicted to be at the interface between domains II and III of E protein dimer, which is unique to this compound, and is apparently different from the ß-OG binding site. Since CRDS has already been tested in humans without serious side effects, its clinical application can be considered.
Assuntos
Anticorpos Facilitadores/efeitos dos fármacos , Vírus da Dengue/efeitos dos fármacos , Dengue/imunologia , Replicação Viral/efeitos dos fármacos , beta-Glucanas/farmacologia , Animais , Linhagem Celular , Vírus da Dengue/imunologia , Vírus da Dengue/fisiologia , Macaca mulatta , Microscopia EletrônicaRESUMO
Humans are frequently infected with three anelloviruses which have circular DNA genomes of 3.6-3.9 kb [Torque teno virus (TTV)], 2.8-2.9 kb [Torque teno mini virus (TTMV)] and 3.2 kb [a recently discovered anellovirus named Torque teno midi virus (TTMDV)]. Unexpectedly, human TTMDV DNA was not detectable in any of 74 chimpanzees tested, although all but one tested positive for both human TTV and TTMV DNA. Using universal primers for anelloviruses, novel variants of TTMDV that are phylogenetically clearly separate from human TTMDV were identified from chimpanzees, and over the entire genome, three chimpanzee TTMDV variants differed by 17.9-20.3 % from each other and by 40.4-43.6 % from all 18 reported human TTMDVs. A newly developed PCR assay that uses chimpanzee TTMDV-specific primers revealed the high prevalence of chimpanzee TTMDV in chimpanzees (63/74, 85 %) but low prevalence in humans (1/100). While variants of TTV and TTMV from chimpanzees and humans were phylogenetically interspersed, those of TTMDV were monophyletic for each species, with sequence diversity of <33 and <20 % within the 18 human and three chimpanzee TTMDV variants, respectively. Maximum within-group divergence values for TTV and TTMV were 51 and 57 %, respectively; both of these values were substantially greater than the maximum divergence among TTMDV variants (44 %), consistent with a later evolutionary emergence of TTMDV. However, substantiation of this hypothesis will require further analysis of genetic diversity using an expanded dataset of TTMDV variants in humans and chimpanzees. Similarly, the underlying mechanism of observed infrequent cross-species infection of TTMDV between humans and chimpanzees deserves further analysis.
Assuntos
Infecções por Vírus de DNA/transmissão , Genoma Viral , Torque teno virus/genética , Animais , Sequência de Bases , Clonagem Molecular , Infecções por Vírus de DNA/sangue , Infecções por Vírus de DNA/epidemiologia , Infecções por Vírus de DNA/virologia , DNA Viral/sangue , DNA Viral/genética , Transmissão de Doença Infecciosa , Variação Genética , Humanos , Dados de Sequência Molecular , Pan troglodytes/virologia , Filogenia , Prevalência , Torque teno virus/classificação , Torque teno virus/patogenicidade , Proteínas Virais/genéticaRESUMO
The function of the hepatitis E virus (HEV) open reading frame 3 (ORF3) protein remains unclear. To elucidate the role of the ORF3 protein in the virus life cycle, an infectious cDNA clone (pJE03-1760F/wt) that can replicate efficiently in PLC/PRF/5 and A549 cells and release progeny into the culture medium was used to generate a derivative ORF3-deficient (DeltaORF3) mutant whose third in-frame AUG codon of ORF3 was mutated to GCA. The DeltaORF3 mutant in the culture medium of mutant RNA-transfected PLC/PRF/5 cells was able to infect and replicate within PLC/PRF/5 and A549 cells as efficiently as the wild-type pJE03-1760F/wt virus. However, less than 1/100 of the number of progeny was detectable in the culture medium of DeltaORF3 mutant-infected PLC/PRF/5 cells compared with wild-type-infected PLC/PRF/5 cells, and the HEV RNA level in the culture medium of DeltaORF3 mutant-infected A549 cells was below or near the limit of detection. An immunocapture PCR assay revealed that the ORF3 protein is present on the surface of cell-culture-generated wild-type HEV but not on the DeltaORF3 mutant. Wild-type HEV in the culture supernatant peaked at a sucrose density of 1.15-1.16 g ml(-1), in contrast with the DeltaORF3 mutant in culture supernatant, which banded at 1.27-1.28 g ml(-1), similar to HEV in cell lysate and faecal HEV. These results suggest that the ORF3 protein is responsible for virion egress from infected cells and is present on the surface of released HEV particles, which may be associated with lipids.
Assuntos
Genes Essenciais , Vírus da Hepatite E/fisiologia , Proteínas Virais/fisiologia , Replicação Viral , Linhagem Celular , Códon de Iniciação/genética , Técnicas de Inativação de Genes , Genes Virais , Vírus da Hepatite E/genética , Humanos , Dados de Sequência Molecular , Análise de Sequência de DNA , Proteínas Virais/análise , Proteínas Virais/genética , Vírion/químicaRESUMO
A full-length infectious cDNA clone (pJE03-1760F/wt) of a genotype 3 hepatitis E virus (HEV) (strain JE03-1760F) obtained from a faecal specimen was constructed in this study. Upon transfection of the capped in vitro transcripts of pJE03-1760F/wt into PLC/PRF/5 cells, the viral RNA levels in the culture supernatant started to increase on day 6 post-transfection (p.t.) and reached 10(7) copies ml(-1) on day 28 p.t. Detection of increasing numbers of cells with ORF2 protein expression by immunofluorescence assay at 5, 7, 11 and 15 days p.t. indicated the spread of HEV infection in cell culture. When the cDNA-derived virus in culture supernatant was inoculated into PLC/PRF/5 or A549 cells, it grew as efficiently as the faeces-derived virus in both cells, reaching 10(6) copies ml(-1) at 30 days post-inoculation. Our reverse genetics system for HEV that is usable in a robust cell-culture system will be useful for elucidation of the mechanism of HEV replication and functional roles of HEV proteins.
Assuntos
DNA Complementar/genética , DNA Viral/genética , Vírus da Hepatite E/genética , Vírus da Hepatite E/fisiologia , RNA Viral/genética , Animais , Células Cultivadas , Clonagem Molecular , Surtos de Doenças , Genótipo , Hepatite E/epidemiologia , Hepatite E/virologia , Vírus da Hepatite E/patogenicidade , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Mapeamento por Restrição , Transfecção , ZoonosesRESUMO
Ten murine monoclonal antibodies (MAbs) against a synthetic peptide corresponding to the well-conserved, C-terminal 24-amino acid portion of ORF3 protein of hepatitis E virus (HEV) were produced and characterized. Immunofluorescent assays using the anti-ORF3 MAbs revealed accumulation of ORF3 protein in the cytoplasm of PLC/PRF/5 cells transfected with ORF3-expressing plasmid or inoculated with cell-culture-generated HEV. The anti-ORF3 MAbs could capture HEV particles in culture medium and serum at variable efficiency of up to 61 and 49%, respectively, but not those in feces. By sandwiching between immobilized and enzyme-labeled anti-ORF3 MAbs in ELISA, ORF3 antigen was detected in the culture media with an HEV RNA titer of >10(6) copies/ml and increased in parallel with the increase in HEV load. HEV progenies in the culture supernatant, with ORF3 protein on the surface, banded at a low buoyant density of 1.15 g/cm(3) in sucrose. A representative anti-ORF3 MAb (TA0536) could partially neutralize the infection of cell-culture-generated HEV in a cell culture system. These results indicate that ORF3 protein, at least its C-terminal portion, is present on the surface of HEV virions released from infected cells and support a previously proposed assumption that ORF3 protein is associated with virus release from infected cells.
Assuntos
Anticorpos Monoclonais/imunologia , Fezes/virologia , Vírus da Hepatite E/imunologia , Hepatite E/imunologia , Fases de Leitura Aberta , Soro/virologia , Proteínas Virais/imunologia , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Meios de Cultura/química , Hepatite E/diagnóstico , Vírus da Hepatite E/genética , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Testes de Neutralização , Alinhamento de Sequência , Soro/imunologia , Proteínas Virais/síntese química , Proteínas Virais/genéticaRESUMO
BACKGROUND: One of the remaining challenges in the prevention of mother-to-child transmission of HIV is to reduce the risk of the transmission during pregnancy. It remains to be investigated which factors affect intrauterine HIV transmission and how they can be identified and addressed during pregnancy. METHODS: Granulocyte elastase in the endocervical mucus of HIV-positive pregnant women in Zambia was measured, and its association with intrauterine transmission of HIV-1 from the mother to the fetus was investigated. RESULTS: The intrauterine transmission rate determined by polymerase chain reaction assay of DNA from neonates at birth was 15.3%. The risk for intrauterine transmission was 8.65-fold higher in women who were positive for granulocyte elastase than in those who were negative. CONCLUSION: We suggest that the women showing positive granulocyte elastase at delivery be strongly suspected of having and if having had chorioamnionitis during pregnancy, which could affect the intrauterine transmission of HIV.