RESUMEN
SARS-CoV-2 is the virus responsible for causing the global COVID-19 pandemic. Identifying the presence of this virus in the environment could potentially improve the effectiveness of disease control measures. Environmental SARS-CoV-2 monitoring may become increasingly demanded in areas where the available testing methods are ineffective. In this study, we present an electrochemical polymer composites biosensor for measuring SARS-CoV-2 whole-virus particles in the environment. The sensitized layer was prepared from molecularly imprinted polymer (MIP) composites of inactivated SARS-CoV-2. Testing demonstrated increased sensor signaling with SARS-CoV-2 specifically, while lower responses were observed to the negative controls, H5N1 influenza A virus and non-imprinted polymers (NIPs). This sensor detected SARS-CoV-2 at concentrations as low as 0.1 fM in buffer and samples prepared from reservoir water with a 3 log-scale linearity.
RESUMEN
Over the last decade infections with the mosquito transmitted chikungunya virus (CHIKV) have become a major worldwide concern, and considerable efforts have been made in understanding the interaction of this virus with the host cell machinery. Studies have documented the induction of the unfolded protein response (UPR), as well as the induction of apoptosis and autophagy in response to CHIKV infection. This study comparatively analysed these three processes in two cell lines, Hela and HepG2. Infection of Hela cells was characterized by activation of the PERK/eIF2α branch of the UPR, the induction of autophagy and early apoptosis, while infection of HepG2 cells was characterized by activation of the IRE/XBP1 branch of the UPR, limited or no activation of autophagy and comparatively later apoptosis. These results show that the specific cell context is an important mediator of the host cell response to CHIKV infection.
Asunto(s)
Virus Chikungunya/patogenicidad , Estrés del Retículo Endoplásmico , Interacciones Huésped-Patógeno , Apoptosis , Autofagia , Células Epiteliales/fisiología , Células Epiteliales/virología , Células HeLa , Células Hep G2 , Hepatocitos/fisiología , Hepatocitos/virología , Humanos , Respuesta de Proteína DesplegadaRESUMEN
Chikungunya virus (CHIKV), the virus responsible for the disease chikungunya fever in humans, is transmitted by Aedes mosquitoes. While significant progress has been made in understanding the process by which CHIKV enters into mammalian cells, far less progress has been made in understanding the CHIKV entry process in insect cells. This study sought to identify mosquito-cell-expressed CHIKV-binding proteins through a combination of virus overlay protein binding assays (VOPBA) and mass spectroscopy. A 50-kDa CHIKV-binding protein was identified as the ATP synthase ß subunit (ATPSß). Co-immunoprecipitation studies confirmed the interaction, and colocalization analysis showed cell-surface and intracellular co-localization between CHIKV and ATPSß. Both antibody inhibition and siRNA-mediated downregulation experiments targeted to ATPSß showed a significant reduction in viral entry and virus production. These results suggest that ATPSß is a CHIKV-binding protein capable of mediating the entry of CHIKV into insect cells.
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Aedes/virología , Virus Chikungunya/fisiología , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Receptores Virales/metabolismo , Internalización del Virus , Animales , Expresión Génica , Interacciones Huésped-Patógeno , Espectrometría de Masas , Unión Proteica , Acoplamiento ViralRESUMEN
Despite the availability of an effective vaccine, Japanese encephalitis remains a significant cause of morbidity and mortality in many parts of Asia. Japanese encephalitis is caused by the Japanese encephalitis virus (JEV), a mosquito transmitted flavivirus. Many of the details of the virus replication cycle in mosquito cells remain unknown. This study sought to determine whether GRP78, a well-characterized flavivirus E protein interacting protein, interacted with JEV E protein in insect cells, and whether this interaction was mediated at the cell surface. GRP78 was shown to interact with JEV E protein by coimmunoprecipitation, and was additionally shown to interact with voltage dependent anion protein (VDAC) through the same methodology. Antibody inhibition experiments showed that neither GRP78 nor VDAC played a role in JEV internalization to insect cells. Interestingly, VDAC was shown to be significantly relocalized in response to JEV infection, and significant levels of colocalization between VDAC and GRP78 and VDAC and ribosomal L28 protein were seen in JEV infected but not uninfected cells. This is the first report of relocalization of VDAC in response to JEV infection and suggests that this may be a part of the JEV replication strategy in insect cells.
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Aedes/virología , Virus de la Encefalitis Japonesa (Especie)/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Canales Aniónicos Dependientes del Voltaje/metabolismo , Animales , Western Blotting , Línea Celular , Chaperón BiP del Retículo Endoplásmico , Procesamiento de Imagen Asistido por Computador , Inmunoprecipitación , Microscopía ConfocalRESUMEN
Despite the availability of effective vaccines, Japanese encephalitis virus (JEV) infections remain a leading cause of encephalitis in many Asian countries. The virus is transmitted to humans by Culex mosquitoes, and, while the majority of human infections are asymptomatic, up to 30% of JE cases admitted to hospital die and 50% of the survivors suffer from neurological sequelae. Microglia are brain-resident macrophages that play key roles in both the innate and adaptive immune responses in the CNS and are thus of importance in determining the pathology of encephalitis as a result of JEV infection.
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Encefalitis Japonesa/inmunología , Macrófagos/inmunología , Macrófagos/virología , Microglía/inmunología , Animales , Encéfalo/inmunología , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/virología , Culex/virología , Virus de la Encefalitis Japonesa (Especie)/inmunología , Virus de la Encefalitis Japonesa (Especie)/metabolismo , Encefalitis Japonesa/patología , Encefalitis Japonesa/virología , Humanos , Microglía/metabolismo , Microglía/virología , Neuronas/metabolismo , Neuronas/patología , Neuronas/virologíaRESUMEN
An Asian Zika virus (ZIKV) isolated from a Thai patient that was serially passaged in Primary Dog Kidney (PDK) cells for attenuation displayed both big and small plaque-forming viruses by the 7th passage. Two small-plaque isolates were selected and purified for characterization as attenuated ZIKV candidates. In vitro growth kinetics showed significantly reduced titers for small-plaque isolates in Vero cells early post-infection compared to the parental ZIKV and a big-plaque isolate, but no significant difference was observed in C6/36 cells. Viral entry experiments elucidate that titer reduction likely occurred due to the diminished entry capabilities of a small-plaque isolate. Additionally, a small-plaque isolate displayed lowered neurovirulence in newborn mice compared to 100% lethality from infection with the parental ZIKV. Genomic analysis revealed the same three unique non-synonymous mutations for both small-plaque isolates: two on the envelope (E) protein at residues 310, alanine to glutamic acid (A310E), and 393, glutamic acid to lysine (E393K), and one on residue 355 of NS3, histidine to tyrosine (H355Y). Three-dimensional (3D) mapping suggests that the E protein mutations located on the receptor-binding and fusion domain III likely affect cell entry, tropism, and virulence. These ZIKV isolates and genotypic markers will be beneficial for vaccine development.
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Culicidae , Infección por el Virus Zika , Virus Zika , Animales , Chlorocebus aethiops , Perros , Ácido Glutámico/metabolismo , Humanos , Mamíferos , Ratones , Mutación , Células Vero , Internalización del Virus , Replicación ViralRESUMEN
Rickettsia felis is a rickettsial pathogen primarily associated with the cat flea, Ctenocephalides felis. Although laboratory studies have confirmed that R. felis is maintained by transstadial and transovarial transmission in C. felis, distinct mechanisms of horizontal transmission of R. felis among cat fleas are undefined. Based on the inefficient vertical transmission of R. felis by cat fleas and the detection of R. felis in a variety of haematophagous arthropods, we hypothesize that R. felis is horizontally transmitted between cat fleas. Towards testing this hypothesis, flea transmission of R. felis via a bloodmeal was assessed weekly for 4 weeks. Rhodamine B was used to distinguish uninfected recipient and R. felis-infected donor fleas in a rickettsial horizontal transmission bioassay, and quantitative real-time PCR assay was used to measure transmission frequency; immunofluorescence assay also confirmed transmission. Female fleas acquired R. felis infection more readily than male fleas after feeding on a R. felis-infected bloodmeal for 24 h (69.3% and 43.3%, respectively) and both Rickettsia-uninfected recipient male and female fleas became infected with R. felis after cofeeding with R. felis-infected donor fleas (3.3-40.0%). Distinct bioassays were developed to further determine that R. felis was transmitted from R. felis-infected to uninfected fleas during cofeeding and copulation. Vertical transmission of R. felis by infected fleas was not demonstrated in this study. The demonstration of horizontal transmission of R. felis between cat fleas has broad implications for the ecology of R. felis rickettsiosis.
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Infecciones por Rickettsia/transmisión , Rickettsia felis/patogenicidad , Siphonaptera/microbiología , Animales , Gatos , Femenino , MasculinoRESUMEN
Haematophagous arthropods are the primary vectors in the transmission of Rickettsia, yet the molecular mechanisms mediating the rickettsial infection of arthropods remain elusive. This study utilized a biotinylated protein pull-down assay together with LC-MS/MS to identify interaction between Ixodes scapularis histone H2B and Rickettsia felis. Co-immunoprecipitation of histone with rickettsial cell lysate demonstrated the association of H2B with R. felis proteins, including outer-membrane protein B (OmpB), a major rickettsial adhesin molecule. The rickettsial infection of tick ISE6 cells was reduced by approximately 25 % via RNA-mediated H2B-depletion or enzymic treatment of histones. The interaction of H2B with the rickettsial adhesin OmpB suggests a role for H2B in mediating R. felis internalization into ISE6 cells.
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Vectores Arácnidos/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Histonas/metabolismo , Ixodes/metabolismo , Rickettsia felis/metabolismo , Animales , Vectores Arácnidos/microbiología , Proteínas de la Membrana Bacteriana Externa/genética , Línea Celular , Histonas/genética , Ixodes/microbiología , Unión Proteica , Rickettsia felis/genéticaRESUMEN
Zika virus (ZIKV) is a flavivirus that was first identified in 1947. Initially, the virus was of little concern for health authorities given there were very few casualties among those suffering an infection. As such, only limited studies were performed on ZIKV. Recently, the viral infection has been linked to microcephaly in infants, which has prompted a dramatic increase in scientific interest in ZIKV research, including methods to allow for rapid virus identification. In this work we report the development of a new type of ZIKV electrochemical biosensor based on surface imprinted polymers and graphene oxide composites. The biosensor was used to detect ZIKV by measuring changes in the electrical signal with changing virus concentrations in buffer and serum using standard electrochemical techniques. The detection limit of our method is similar to the detection limit of the real-time quantitative reverse transcription PCR method.
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Técnicas Biosensibles/métodos , Sangre/virología , Técnicas Electroquímicas/métodos , Virus Zika/aislamiento & purificación , Aedes/virología , Animales , Técnicas Biosensibles/instrumentación , Línea Celular , Técnicas Electroquímicas/instrumentación , Electrodos , Oro/química , Grafito/química , Humanos , Límite de Detección , Nanocompuestos/química , Polímeros/química , Propiedades de SuperficieRESUMEN
The endosomal sorting complexes required for transport (ESCRT) pathway accessory protein apoptosis linked gene-2-interacting protein X (ALIX) has been shown to be upregulated during dengue virus (DENV) replication. Yeast-two-hybrid screens have additionally shown that ALIX interacts with DENV NS3 protein, but evaluation of the interaction through a replicon assay failed to show a functional significance to the interaction. In this study the interaction between DENV NS3 and ALIX was investigated by co-immunoprecipitation, and functional significance assessed by investigation of DENV production in ALIX expression regulated cells. The results showed that ALIX both interacted and co-localized with DENV NS3 protein and that upregulation of ALIX resulted in a significantly increased viral titer, while either siRNA or CRISPR-Cas9 mediated down regulation of ALIX significantly reduced viral production, without affecting relative DENV genome levels. These results are consistent with ALIX playing a significant role in the DENV replication cycle either during late infection or at viral egress.
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Proteínas de Unión al Calcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Virus del Dengue/crecimiento & desarrollo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Interacciones Huésped-Patógeno , Proteínas no Estructurales Virales/metabolismo , Replicación Viral , Expresión Génica , Técnicas de Silenciamiento del Gen , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Inmunoprecipitación , Mapeo de Interacción de Proteínas , ARN Helicasas/metabolismo , Serina Endopeptidasas/metabolismo , Carga ViralRESUMEN
Infection with the mosquito transmitted dengue virus (DENV) remains a significant worldwide public health problem. While the majority of infections are asymptomatic, infection can result in a range of symptoms. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression through repression or degradation of mRNAs. To understand the contribution of miRNAs to DENV 2 replication, we screened a number of candidate miRNAs for variations in expression levels during DENV 2 infection of HepG2 (liver) cells. Seven miRNAs were identified as differentially expressed, and one, miR-21, was differentially expressed at all time points examined. Interestingly, miR-21 was also differentially regulated in DENV 2 infection under conditions of antibody dependent enhancement of infection, and in direct Zika virus infection, but not in DENV 4 infection. The role of miR-21 during DENV infection was further examined by treating HepG2 cells with an anti-miR-21 (AMO-21) before DENV infection. The results showed a significant reduction in DENV 2 production, clearly suggesting that miR-21 plays a key role in DENV 2 replication. To further confirm the role of miR-21 in DENV infection, a peptide nucleic acid-21 (PNA-21) construct with a nucleotide sequence complementary to AMO-21, was co-administered with AMO-21 as an AMO-21/PNA-21 complex followed by DENV 2 infection. The results showed that AMO-21 significantly reduced DENV 2 titer, PNA-21 significantly increased DENV 2 titer and the combined AMO-21/PNA-21 showed no difference from non-treated infection controls. Taken together, the results show that miR-21 promotes DENV 2 replication, and this mechanism could serve as a possible therapeutic intervention point.
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Virus del Dengue/efectos de los fármacos , Células Hep G2/efectos de los fármacos , Células Hep G2/virología , MicroARNs/farmacología , Replicación Viral/efectos de los fármacos , Células A549 , Antagomirs/farmacología , Proteínas Portadoras , Replicación del ADN/efectos de los fármacos , Dengue/virología , Virus del Dengue/genética , Regulación de la Expresión Génica/efectos de los fármacos , Interacciones Huésped-Patógeno , Humanos , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Factor 88 de Diferenciación Mieloide/metabolismo , Ácidos Nucleicos de Péptidos/farmacología , Serogrupo , Células U937RESUMEN
Dengue virus (DENV) remains a significant public health problem in many tropical and sub-tropical countries worldwide. The DENV envelope (E) protein is the major antigenic determinant and the protein that mediates receptor binding and endosomal fusion. In contrast to some other DENV proteins, relatively few cellular interacting proteins have been identified. To address this issue a co-immuoprecipitation strategy was employed. The predominant co-immunoprecipitating proteins identified were actin and actin related proteins, however the results suggested that actin was the only bona fide interacting partner. Actin was shown to interact with the E protein of DENV 2 and 4, and the interaction between actin and DENV E protein was shown to occur in a truncated DENV consisting of only domains I and II. Actin was shown to decrease during infection, but this was not associated with a decrease in gene transcription. Actin-related proteins also showed a decrease in expression during infection that was not transcriptionally regulated. Cytoskeletal reorganization was not observed during infection, suggesting that the interaction between actin and E protein has a cell type specific component.
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Actinas/metabolismo , Virus del Dengue/fisiología , Dengue/metabolismo , Interacciones Huésped-Patógeno , Proteínas del Envoltorio Viral/metabolismo , Actinas/análisis , Aedes , Animales , Anticuerpos Antivirales , Línea Celular , Células HEK293 , Proteínas de Choque Térmico HSP27/análisis , Proteínas de Choque Térmico HSP27/metabolismo , Humanos , Miosinas/análisis , Miosinas/metabolismo , Mapas de Interacción de Proteínas , Proteínas del Envoltorio Viral/análisisRESUMEN
OBJECTIVE: To generate insights into the mechanism of NVP induced hepatotoxicity. METHODS: Liver (HepG2) cells were cultured with various concentrations of NVP. This cell line was chosen because it has low expression of cytochrome P450, allowing evaluation of the effects of NVP rather than specific metabolites. Cytotoxicity was determined using a proliferation assay and cell numbers were monitored using trypan blue exclusion assay for long term culture experiments and apoptosis induction was determined by morphological and biochemical investigation. RESULTS: HepG2 cells treated with the highest concentration of NVP tested (819 µM) initially showed a rounded morphology and all cells had died by week three of exposure. Nuclear condensation and fragmentation, increased Annexin V/propidium iodide staining and caspase 9 activation all supported the induction of apoptosis in HepG2 cells in response to NVP treatment. CONCLUSIONS: There is a clear induction of apoptosis in response to NVP which suggests that NVP has significant cytotoxicity, over and above any cytotoxicity of metabolites and may contribute directly to patient hepatotoxicity.
RESUMEN
The avian influenza polymerase protein PB2 subunit is an important mediator of cross species adaptation and adaptation to mammalian cells is strongly but not exclusively associated with an adaptive mutation of the codon at position 627 of the PB2 protein which alters the glutamate normally found at this position to a lysine. This study sought to identify host cell factors in both mammalian and avian cells that interacted in a species specific or species independent manner. Two PB2 fusion proteins differing only in codon 627 were generated and transfected into mammalian and avian cells and interacting proteins identified through co-immunoprecipitation. A number of proteins including Hsp90 were identified and further investigation showed that Hsp90 interacted with both isoforms of PB2 in both mammalian and avian cells. Hsp90 is thus identified as a species independent interacting protein, further confirming that this protein may be a suitable target for anti-influenza drug development.
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Proteínas HSP90 de Choque Térmico/metabolismo , Subtipo H5N1 del Virus de la Influenza A/enzimología , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas Virales/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Núcleo Celular/enzimología , Citoplasma/enzimología , Humanos , Inmunoprecipitación , Subtipo H5N1 del Virus de la Influenza A/genética , Mutación , Subunidades de Proteína/metabolismo , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/genética , Proteínas Recombinantes de Fusión/metabolismo , Especificidad de la Especie , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
BACKGROUND: Cat fleas, Ctenocephalides felis, are known biological vectors for Rickettsia felis. Rickettsial transmission can be vertical via transovarial transmission within a flea population, as well as horizontal between fleas through a bloodmeal. The previously undescribed infection kinetics of bloodmeal-acquired R. felis in cat fleas provides insight into the R. felis-flea interaction. FINDINGS: In the present study, dissemination of R. felis in previously uninfected cat fleas fed an R. felis-infected bloodmeal was investigated. At weekly intervals for 28 days, rickettsial propagation, accumulation, and dissemination in gut epithelial cells, specifically in the hindgut and the specialized cells in the neck region of midgut, were observed on paraffin sections of infected cat fleas by immunofluorescence assay (IFA) and confirmed by PCR detection of R. felis 17-kDa antigen gene. IFA results demonstrate ingested rickettsiae in vacuoles during early infection of the gut; lysosomal activity, indicated by lysosome marker staining of freshly-dissected gut, suggests the presence of phagolysosome-associated vacuoles. Subsequent to infection in the gut, rickettsiae spread to the hemocoel and other tissues including reproductive organs. Densely-packed rickettsiae forming mycetome-like structures were observed in the abdomen of infected male cat fleas during late infection. Ultrastructural analysis by transmission electron microscopy (TEM) confirmed the presence and infection characteristics of Rickettsia including rickettsial destruction in the phagolysosome, rickettsial division, and accumulation in the flea gut. CONCLUSIONS: This study intimately profiles R. felis dissemination in cat fleas and further illuminates the mechanisms of rickettsial transmission in nature.
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Enfermedades de los Gatos/transmisión , Ctenocephalides/microbiología , Infecciones por Rickettsia/veterinaria , Rickettsia felis/fisiología , Animales , Proteínas Bacterianas/genética , Enfermedades de los Gatos/microbiología , Enfermedades de los Gatos/parasitología , Gatos , ADN Bacteriano/genética , Femenino , Masculino , Fagosomas/microbiología , Fagosomas/ultraestructura , Reacción en Cadena de la Polimerasa/veterinaria , Infecciones por Rickettsia/transmisión , Rickettsia felis/genética , Rickettsia felis/aislamiento & purificación , Vacuolas/microbiología , Vacuolas/ultraestructuraRESUMEN
BACKGROUND: A number of studies have implicated the direct involvement of the liver in dengue virus (DENV) infection, and it has been widely shown that liver cells subsequently undergo apoptosis. The mechanism by which liver cells undergo apoptosis in response to DENV infection remains unclear. To provide further information on the mechanism of apoptosis in DENV infected liver cells, HepG2 cells were infected with DENV 2 and analyzed for the induction of ER stress, apoptosis and autophagy. RESULTS: In response to DENV infection, HepG2 cells showed the induction of both the ER resident unfolded protein response as well as the Noxa/PUMA stress response pathways. Proteolytic activation of caspases 4, 7, 8 and 9 was observed as well as changes in mitochondrial transmembrane potential. Increased monodansylcadaverine staining was observed in DENV infected cells, consistent with the previously reported induction of autophagy. CONCLUSIONS: These results are consistent with a model in which the induction of multiple ER stress pathways is coupled with the induction of multiple cell death pathways as a mechanism to ensure the removal of infected liver cells from the system.
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Virus del Dengue/fisiología , Dengue/patología , Dengue/virología , Estrés del Retículo Endoplásmico , Hígado/patología , Hígado/virología , Transducción de Señal , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Autofagia/genética , Caspasas/metabolismo , Muerte Celular , Supervivencia Celular/genética , Activación Enzimática , Regulación Neoplásica de la Expresión Génica , Células Hep G2 , Humanos , Hígado/enzimología , Potencial de la Membrana Mitocondrial , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Factor de Transcripción CHOP/metabolismo , Respuesta de Proteína DesplegadaRESUMEN
Rickettsial diversity is intriguing in that some species are transmissible to vertebrates, while others appear exclusive to invertebrate hosts. Of particular interest is Rickettsia felis, identifiable in both stored product insect pests and hematophagous disease vectors. To understand rickettsial survival tactics in, and probable movement between, both insect systems will explicate the determinants of rickettsial pathogenicity. Towards this objective, a population of Liposcelis bostrychophila, common booklice, was successfully used for rickettsial isolation in ISE6 (tick-derived cells). Rickettsiae were also observed in L. bostrychophila by electron microscopy and in paraffin sections of booklice by immunofluorescence assay using anti-R. felis polyclonal antibody. The isolate, designated R. felis strain LSU-Lb, resembles typical rickettsiae when examined by microscopy. Sequence analysis of portions of the Rickettsia specific 17-kDa antigen gene, citrate synthase (gltA) gene, rickettsial outer membrane protein A (ompA) gene, and the presence of the R. felis plasmid in the cell culture isolate confirmed the isolate as R. felis. Variable nucleotide sequences from the isolate were obtained for R. felis-specific pRF-associated putative tldD/pmbA. Expression of rickettsial outer membrane protein B (OmpB) was verified in R. felis (LSU-Lb) using a monoclonal antibody. Additionally, a quantitative real-time PCR assay was used to identify a significantly greater median rickettsial load in the booklice, compared to cat flea hosts. With the potential to manipulate arthropod host biology and infect vertebrate hosts, the dual nature of R. felis provides an excellent model for the study of rickettsial pathogenesis and transmission. In addition, this study is the first isolation of a rickettsial pathogen from a non-hematophagous arthropod.
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Artrópodos/microbiología , Rickettsia felis/aislamiento & purificación , Animales , Insectos/microbiología , Modelos Animales , Reacción en Cadena de la Polimerasa , Infecciones por Rickettsia , Análisis de Secuencia de ADNRESUMEN
Recently, several stress-related proteins including GRP78, hsp70, and hsp90 have been implicated as dengue virus receptors in various cell types, with hsp90/70 being implicated as a receptor complex in monocytes and macrophages, while GRP78 has been implicated as a liver cell expressed dengue virus receptor. To assess whether the hsp90/70 complex plays a role in the internalization of the dengue viruses into liver cells, we undertook infection inhibition studies with lipopolysaccharide and antibodies directed against both hsp70 and hsp90, individually and in combination. No inhibition of any dengue serotype was seen in the presence of lipopolysaccharide or antibodies directed against either hsp70 or hsp90 either singly or in combination. A moderate inhibition of dengue virus serotype 2 entry into liver cells was observed in the presence of antibodies directed against GRP78. These results confirm a proposed role for GRP78 as a dengue virus serotype 2 receptor protein and suggest that the recently identified hsp90/70 complex does not play a role in dengue virus internalization into liver cells.
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Virus del Dengue/fisiología , Dengue/virología , Proteínas HSP70 de Choque Térmico/fisiología , Proteínas HSP90 de Choque Térmico/fisiología , Receptores Virales/fisiología , Línea Celular Tumoral , Chaperón BiP del Retículo Endoplásmico , Proteínas de Choque Térmico/fisiología , Hepatocitos/virología , Humanos , Chaperonas Moleculares/fisiología , Replicación ViralRESUMEN
Dengue virus, the causative agent of dengue fever, dengue shock syndrome, and dengue hemorrhagic fever, infects susceptible cells by initially binding to a receptor(s) located on the host cell surface. Evidence to date suggests that receptor usage may be cell and serotype specific, and this study sought to identify dengue virus serotype 1 binding proteins on the surface of liver cells, a known target organ. By using a virus overlay protein binding assay (VOPBA), in both nondenaturing and denaturing gel systems, a putative dengue virus serotype 1 binding protein of approximately 37 kDa expressed on the surface of liver (HepG2) cells was identified. Mass spectrometry analysis identified a candidate protein, the 37/67-kDa high-affinity laminin receptor. Entry of the dengue virus serotype 1 was significantly inhibited in a dose-dependent manner by both antibodies directed against the 37/67-kDa high-affinity laminin receptor and soluble laminin. No inhibition of virus entry was seen with dengue virus serotypes 2, 3, or 4, demonstrating that the 37/67-kDa high-affinity laminin receptor is a serotype-specific receptor for dengue virus entry into liver cells.
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Virus del Dengue/fisiología , Hígado/virología , Receptores de Laminina/análisis , Receptores Virales/análisis , Animales , Chlorocebus aethiops , Virus del Dengue/clasificación , Peso Molecular , Serotipificación , Células VeroRESUMEN
OBJECTIVES: This study sought to undertake a comparative analysis of the internalization and propagation of all four dengue serotypes in a single cell line of human liver origin, HepG2. METHODS: Virus production after infection was determined by the plaque assay technique. Internalization profiles were determined by incubating virus and cells on ice and then raising the temperature for various times. The contribution of extracellular matrix components to internalization was determined by pretreatment of cells with either trypsin or heparinase III. RESULTS: HepG2 cells were able to support the propagation of all four serotypes with mature viruses being produced by 12 h for dengue serotype 4 and by 17-18 h for the remaining serotypes. Virus internalization showed a plateau for serotypes 1, 2 and 4 entry while serotype 3 showed a constant increase in internalization for up to 5 h. Pretreatment of HepG2 cells with heparinase III or trypsin both resulted in a reduction in viral production, with the smallest effect being noted for dengue serotype 3. CONCLUSION: These results suggest that the interaction between the dengue virus and liver cells is a complex one that requires both protein and nonprotein elements, and has a significant serotype/strain element.