RESUMEN
Globalization and climate change have contributed to the simultaneous increase and spread of arboviral diseases. Cocirculation of several arboviruses in the same geographic region provides an impetus to study the impacts of multiple concurrent infections within an individual vector mosquito. Here, we describe coinfection and superinfection with the Mayaro virus (Togaviridae, Alphavirus) and Zika virus (Flaviviridae, Flavivirus) in vertebrate and mosquito cells, as well as Aedes aegypti adult mosquitoes, to understand the interaction dynamics of these pathogens and effects on viral infection, dissemination, and transmission. Aedes aegypti mosquitoes were able to be infected with and transmit both pathogens simultaneously. However, whereas Mayaro virus was largely unaffected by coinfection, it had a negative impact on infection and dissemination rates for Zika virus compared to single infection scenarios. Superinfection of Mayaro virus atop a previous Zika virus infection resulted in increased Mayaro virus infection rates. At the cellular level, we found that mosquito and vertebrate cells were also capable of being simultaneously infected with both pathogens. Similar to our findings in vivo, Mayaro virus negatively affected Zika virus replication in vertebrate cells, displaying complete blocking under certain conditions. Viral interference did not occur in mosquito cells. IMPORTANCE Epidemiological and clinical studies indicate that multiple arboviruses are cocirculating in human populations, leading to some individuals carrying more than one arbovirus at the same time. In turn, mosquitoes can become infected with multiple pathogens simultaneously (coinfection) or sequentially (superinfection). Coinfection and superinfection can have synergistic, neutral, or antagonistic effects on viral infection dynamics and ultimately have impacts on human health. Here we investigate the interaction between Zika virus and Mayaro virus, two emerging mosquito-borne pathogens currently circulating together in Latin America and the Caribbean. We find a major mosquito vector of these viruses-Aedes aegypti-can carry and transmit both arboviruses at the same time. Our findings emphasize the importance of considering co- and superinfection dynamics during vector-pathogen interaction studies, surveillance programs, and risk assessment efforts in epidemic areas.
Asunto(s)
Aedes , Infecciones por Alphavirus , Coinfección , Sobreinfección , Infección por el Virus Zika , Animales , Humanos , Aedes/virología , Alphavirus , Infecciones por Alphavirus/complicaciones , Infecciones por Alphavirus/virología , Mosquitos Vectores/virología , Vertebrados/virología , Virus Zika , Infección por el Virus Zika/complicaciones , Infección por el Virus Zika/virologíaRESUMEN
Dengue is the most burdensome vector-borne viral disease in the world. Dengue virus (DENV), the etiological cause of dengue, is transmitted primarily by the Aedes aegypti mosquito. Like any arbovirus, the transmission cycle of dengue involves the complex interactions of a multitude of human and mosquito factors. One point during this transmission cycle that is rich in these interactions is the biting event by the mosquito, upon which its saliva is injected into the host. A number of components in mosquito saliva have been shown to play a pivotal role in the transmission of dengue, however one such component that is not as well characterized is extracellular vesicles. Here, using high-performance liquid chromatography in tandem with mass spectrometry, we show that dengue infection altered the protein cargo of Aedes aegypti extracellular vesicles, resulting in the packaging of proteins with infection-enhancing ability. Our results support the presence of an infection-dependent pro-viral protein packaging strategy that uses the differential packaging of pro-viral proteins in extracellular vesicles of Ae. aegypti saliva to promote transmission. These studies represent the first investigation into the function of Ae. aegypti extracellular vesicle cargo during dengue infection.
Asunto(s)
Aedes/metabolismo , Dengue/transmisión , Vesículas Extracelulares/metabolismo , Proteínas de Insectos/metabolismo , Mosquitos Vectores/metabolismo , Aedes/virología , Animales , Células Cultivadas , Dengue/virología , Virus del Dengue , Femenino , Humanos , Mosquitos Vectores/virologíaRESUMEN
MicroRNAs (miRNAs) are important regulators of biological processes, including host-virus interaction. This study investigated the involvement of Drosophila melanogaster miR-8-5p in host-virus interaction. Drosophila flies and cells challenged with Drosophila C virus (DCV) were found to have lower miR-8-5p abundance compared to uninfected samples. Lowering miR-8-5p abundance by experimental inhibition of the miRNA led to an increase in viral accumulation, suggesting that the observed decrease in the miR-8-5p abundance during DCV infection enhances viral replication. miR-8-5p putative targets were identified and included dJun, a transcription factor gene whose mammalian homologue cJun is induced by various viruses through kinase activation. Increasing miR-8-5p abundance using miR-8-5p mimics resulted in a decrease in dJun and GFP reporter levels. Furthermore, when the putative target in dJun was mutated, addition of miR-8-5p mimics did not result in the same antagonistic effect on dJun. These results show negative regulation of dJun by miR-8-5p and suggest that an miRNA-mediated pathway is involved in dJun regulation during viral infection. To analyse the role of dJun during DCV infection, dJun was knocked down in cells prior to DCV infection. Knockdown of dJun decreased DCV replication, providing evidence that dJun up-regulation that is concomitant with miR-8-5p down-regulation during DCV infection supports viral replication. These results highlight the role of miRNA in regulating the transcription factor gene dJun and uncover a previously unrecognized mechanism by which dJun is regulated during host-virus interaction.
Asunto(s)
Dicistroviridae/fisiología , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Drosophila melanogaster/virología , MicroARNs/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Replicación Viral , Animales , Dicistroviridae/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Interacciones Huésped-Patógeno , MicroARNs/genética , Proteínas Proto-Oncogénicas c-jun/genéticaRESUMEN
Dengue neurological disease is an uncommon yet severe complication of dengue infection. It can manifest as encephalitis, encephalopathy, neuro-ophthalmic complications, or neuromuscular disorders. Severe infection can result in viral shedding across multiple body sites. We describe a case of severe neuro-ophthalmic dengue infection in an otherwise healthy returned traveller, presenting with prolonged multiple-body-site viral detections by PCR. The dengue virus (DENV) dynamics and serological response support a direct DENV neuropathogenicity. A retrospective review of the laboratory data at the Victorian Infectious Diseases Reference Laboratory (VIDRL) suggests that blood is the most frequent sample type with DENV detection (92% of all DENV-positive samples). Genotype variation is seen across different sample types. The similarity of CSF and nasopharyngeal DENV subtypes (genotype 1 and 3) suggests a possible correlation between nasopharyngeal replication and neurological complications. The case presented highlights the direct neuropathogenicity of DENV early in the course of infection, and a potential correlation between nasopharyngeal replication and neurological disease.
Asunto(s)
Virus del Dengue , Dengue , Humanos , Dengue/virología , Dengue/diagnóstico , Virus del Dengue/genética , Virus del Dengue/aislamiento & purificación , Masculino , Genotipo , Adulto , Estudios RetrospectivosRESUMEN
MicroRNAs (miRNAs) are a group of small noncoding RNAs that regulate gene expression during important biological processes including development and pathogen defense in most living organisms. Presently, no miRNAs have been identified in the mosquito Culex tarsalis (Diptera: Culicidae), one of the most important vectors of West Nile virus (WNV) in North America. We used small RNA sequencing data and in vitro and in vivo experiments to identify and validate a repertoire of miRNAs in Cx. tarsalis mosquitoes. Using bioinformatic approaches we analyzed small RNA sequences from the Cx. tarsalis CT embryonic cell line to discover orthologs for 86 miRNAs. Consistent with other mosquitoes such as Aedes albopictus and Culex quinquefasciatus, miR-184 was found to be the most abundant miRNA in Cx. tarsalis. We also identified 20 novel miRNAs from the recently sequenced Cx. tarsalis genome, for a total of 106 miRNAs identified in this study. The presence of selected miRNAs was biologically validated in both the CT cell line and in adult Cx. tarsalis mosquitoes using RT-qPCR and sequencing. These results will open new avenues of research into the role of miRNAs in Cx. tarsalis biology, including development, metabolism, immunity, and pathogen infection.
Asunto(s)
Aedes , Culex , Culicidae , MicroARNs , Fiebre del Nilo Occidental , Virus del Nilo Occidental , Animales , Virus del Nilo Occidental/genética , Culex/genética , Culicidae/genética , MicroARNs/genética , Mosquitos Vectores/genética , Aedes/genéticaRESUMEN
BACKGROUND: Hepatitis C virus (HCV) is one of the major health concerns globally, with genotype 3a as the most prevalent in Pakistan. Lack of efficient HCV genotype 3a small animal models as well as genomic replicons has hampered the complete understanding of its life cycle, pathogenesis and therapeutic options. In this study we aimed to develop a persistent HCV genotype 3a infectious cell culture model. METHODS: We inoculated Huh-7 cells with HCV genotype 3a serum. Cells and media supernatant were collected at different time periods up to 40th day post infection. Culture media supernatant was also collected to find out its ability to infect naive Huh-7 cells. RESULTS: HCV replication was confirmed at both RNA and protein level through Real Time RCR and western blot using HCV core as marker. In order to validate the persistence of our model for HCV genotype 3a replication we inhibited the HCV replication through core specific siRNAs. The HCV RNA was detected intracellularly from the day one post infection up till 40th day, while HCV core protein was detected from the second day up to 40th day consistently. In culture media supernatant HCV RNA was also actively detected conferring its ability to infect the naive Huh-7 cells. Furthermore, core specific siRNA showed significant inhibition at 24th hour post transfection both at RNA and protein level with progressive increase in the expression of core gene after 3rd day. It clearly depicts that the Huh-7 successfully retained the HCV replication after degradation of siRNA. CONCLUSION: Finally, we report that our persistent infection cell culture model consistently replicate HCV genotype 3a for more than 1 month.
Asunto(s)
Hepacivirus/crecimiento & desarrollo , Hepacivirus/patogenicidad , Hepatocitos/fisiología , Hepatocitos/virología , Western Blotting , Técnicas de Cultivo de Célula/métodos , Genotipo , Hepacivirus/genética , Humanos , ARN Viral/biosíntesis , ARN Viral/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Virales/biosíntesisRESUMEN
HIV (Human immunodeficiency virus) transmission has been reduced by protected sex and screening of blood products and other body fluids in the developed countries. It has been reported that Pakistan is at high risk of HIV/AIDS infection but presently the prevalence rate is considerably low. The number of reported cases of HIV/AIDS in Pakistan has been continuously increasing since 1987. By 2010 the total number of registered cases has reached to 6000 and this figure is on the rise with the passage of time. Some serious strategies must be implemented to control this deadly disease.
Asunto(s)
Infecciones por VIH/epidemiología , Humanos , Incidencia , Pakistán/epidemiologíaRESUMEN
BACKGROUND: Several factors have been proposed to assess the clinical outcome of HCV infection. The correlation of HCV genotypes to possible serum markers in clinical prediction is still controversial. The main objective of this study was to determine the existence of any correlation between HCV genotypes to viral load and different clinical serum markers. METHODS: We performed a prospective cross-sectional and observational study. About 3160 serum HCV RNA positive patients were chosen from 4020 randomly selected anti-HCV positive patients. Statistical analysis was performed using the SPSS 16 software package. ROC (receiver operating characteristics) curves were used to compare diagnostic values of serum markers to predict genotypes. RESULTS: The most prevalent genotype was 3a (73.9%) followed by 1a (10.7%), 4a (6.4%) and 3b (6.1%) in Pakistani population. No correlation was found between viral load and serum markers for genotype 3a in a large no. of sample (n = 2336). While significant correlation was observed between viral load and AST in genotype 3b, ALP with viral load and ALT for genotype 1a. Patients with genotype 4a showed a significant inverse correlation with viral load and Hb level and AST with ALP. For genotype 4a, AUC (area under the curve) of ALT, ALP, AST, bilirubin, Hb level and viral load was 0.790, 0.763, 0.454, 0.664, 0.458 and 0.872 respectively. CONCLUSIONS: In conclusion, there was a significant variable response of HCV genotypes with serum markers. Severity of disease is independent of serum marker level in genotype 3a, while the liver damage in genotype 4a may associate with viral cytopathic effect as well as the immune-mediated process. An index using six serum markers may correctly predict genotype 4a in patients with ≥ 75% accuracy.
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Biomarcadores/análisis , Hepacivirus/genética , Hepacivirus/patogenicidad , Hepatitis C/diagnóstico , Hepatitis C/patología , Carga Viral , Adolescente , Adulto , Anciano , Estudios Transversales , Femenino , Genotipo , Hepacivirus/clasificación , Hepatitis C/virología , Humanos , Hígado/patología , Masculino , Persona de Mediana Edad , Pronóstico , Estudios Prospectivos , Índice de Severidad de la Enfermedad , Resultado del Tratamiento , Adulto JovenRESUMEN
HCV is a leading cause of hepatocellular carcinoma and cirrhosis all over the world. Claudins belong to family of tight junction's proteins that are responsible for establishing barriers for controlling the flow of molecules around cells. For therapeutic strategies, regulation of viral entry into the host cells holds a lot of promise. During HCV infection claudin-1 is highly expressed in liver and believed to be associated with HCV virus entry after HCV binding with or without co-receptor CD81. The claudin-1 assembly with tight junctions is regulated by post translational modifications. During claudins assembly and disassembly with tight junctions, phosphorylation is required at C-terminal tail. In cellular proteins, interplay between phosphorylation and O-ß-GlcNAc modification is believed to be functional switch, but it is very difficult to monitor these functional and vibrant changes in vivo. Netphos 2.0 and Disphos 1.3 programs were used for potential phosphorylation; NetPhosK 1.0 and KinasePhos for kinase prediction; and YinOYang 1.2 and OGPET to predict possible O-glycosylation sites. We also identified Yin Yang sites that may have potential for O-ß-GlcNAc and phosphorylation interplay at same Ser/Thr residues. We for the first time proposed that alternate phosphorylation and O-ß-GlcNAc modification on Ser 192, Ser 205, Ser 206; and Thr 191 may provide an on/off switch to regulate assembly of claudin-1 at tight junctions. In addition these phosphorylation sites may be targeted by novel chemotherapeutic agents to prevent phosphorylation lead by HCV viral entry complex.
Asunto(s)
Hepacivirus/fisiología , Proteínas de la Membrana/metabolismo , Procesamiento Proteico-Postraduccional , Internalización del Virus , Secuencia de Aminoácidos , Claudina-1 , Biología Computacional/métodos , Glicosilación , Humanos , Datos de Secuencia Molecular , Fosforilación , Alineación de SecuenciaRESUMEN
BACKGROUND: Chronic HCV is one of the major causes of morbidity and mortality in the present day world. The assessment of disease progression not only provides useful information for diagnosis and therapeutic supervision judgment but also for monitoring disease. Different invasive and non invasive methods are applied to diagnose the disease from initial to end stage (mild fibrosis to cirrhosis). Although, liver biopsy is still considered as gold standard to identify liver histological stages, an assessment of the disease development based on non-invasive clinical findings is also emerging and this may replace the need of biopsy in near future. This review gives brief insight on non-invasive methods currently available for predicting liver fibrosis in HCV with their current pros and cons to make easier for a clinician to choose better marker to assess liver fibrosis in HCV infected patients. METHODS: More than 200 studies regarding invasive and noninvasive markers available for HCV liver disease diagnosis were thoroughly reviewed. We examined year wise results of these markers based on their sensitivity, specificity, PPV, NPV and AUROCs. RESULTS: We found that in all non-invasive serum markers for HCV, FibroTest, Forn's Index, Fibrometer and HepaScore have high five-year predictive value but with low AUROCs (0.60~0.85) and are not comparable to liver biopsy (AUROC = 0.97). Even though from its beginning, Fibroscan is proved to be best with high AUROCs (> 0.90) in all studies, no single noninvasive marker is able to differentiate all fibrosis stages from end stage cirrhosis. Meanwhile, specific genetic markers may not only discriminate fibrotic and cirrhotic liver but also differentiate individual fibrosis stages. CONCLUSIONS: There is a need of marker which accurately determines the stage based on simplest routine laboratory test. Genetic marker in combination of imaging technique may be the better non invasive diagnostic method in future.
Asunto(s)
Diagnóstico por Imagen/métodos , Hepatitis C Crónica/diagnóstico , Hepatitis C Crónica/genética , Cirrosis Hepática/diagnóstico , Cirrosis Hepática/genética , Animales , Marcadores Genéticos , Hepatitis C Crónica/patología , Hepatitis C Crónica/virología , Humanos , Cirrosis Hepática/patologíaRESUMEN
Hepatocellular carcinoma is mainly associated with viral hepatitis B and C. Activation of cell growth stimulator IGF-II gene is observed in tumor formation especially in viral associated hepatocellular carcinoma. Elevated IGF-II levels are indicator of increased risk for cholangiocellular and hepatocellular carcinomas through over saturation of IGF-II binding capacities with IGF receptors leading to cellular dedifferentiation. In HCV, core protein is believed to trans-activate host IGF-II receptor through PKC pathway and the inhibition of tumor cell growth can be achieved by blocking IGF-II pathway either at transcriptional level or increasing its binding with IGFBPs (Insulin like growth factor proteins) at C-terminal, so that it is not available in free form. IGFBP-6 is a specific inhibitor of IGF-II actions. Affinity of IGFBPs with IGFs is controlled by post-translational modifications. Phosphorylation of IGFBPs inhibits IGFs action on target cells while O-glycosylation prevents binding of IGFBP-6 to glycosaminoglycans and cell membranes and resulting in a 10-fold higher affinity for IGF-II. O-glycosylation and phosphorylation operate the functional expression of cellular proteins, this switching on and off the protein expression is difficult to monitor in vivo. By using neural network based prediction methods, we propose that alternate O-ß-GlcNAc modification and phosphorylation on Ser 204 control the binding of IGFBP-6 with IGF-II. This information may be used for developing new therapies by regulating IGFBP-6 assembly with IGF-II to minimize the risk of viral associated hepatocellular carcinoma. We can conclude that during HCV/HBV infection, O-ß-GlcNAc of IGFBP-6 at Ser 204 diminish their binding with IGF-II, increase IGF-II cellular expression and promote cancer progression which can lead to hepatocellular carcinoma. Furthermore, this site can be used for developing new therapies to control the IGF-II actions during viral infection to minimize the risk of hepatocellular carcinoma.
Asunto(s)
Acetilglucosamina/metabolismo , Carcinoma Hepatocelular/virología , Hepatitis B/complicaciones , Hepatitis C/complicaciones , Proteína 6 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Factor II del Crecimiento Similar a la Insulina/metabolismo , Serina/metabolismo , Secuencia de Aminoácidos , Glicosilación , Hepacivirus/patogenicidad , Hepatitis B/virología , Virus de la Hepatitis B/patogenicidad , Hepatitis C/virología , Humanos , Datos de Secuencia Molecular , Fosforilación , Unión Proteica , Alineación de SecuenciaRESUMEN
BACKGROUND AND AIMS: HCV infection may lead to hepatic fibrosis. In this study, we tried to determine whether there is any correlation of HCV genotypes and viral load to the clinical parameters such as ALT, AST, ALP, bilirubin, Hb level, patient's age and gender; and then correlated this association with disease progression in liver biopsy samples. METHODS: In cross-sectional and observational study, 6048 serum HCV RNA positive patients were chosen. The study consists of 53 months from March 2006 to September 2010. Patients were divided into three cohorts to validate our data. Statistical analysis and correlation of lab parameters with viral factors was determined by using SPSS version 16. RESULTS: The most prevalent genotype was 3 (70.9%) followed by 1 (13.3%) and 4 (7.4%), collectively. During Univariate analysis, in all cohorts; serum bilirubin, ALP, ALT and AAR showed significant correlation with genotypes, however multivariate analysis showed that all genotypes except 4a have no association with host biochemical markers. Disease progression was also independent of all genotypes. Serum ALP, ALT, bilirubin and viremea levels were significantly elevated in patients with genotype 4a. Viral load showed negative association with serum bilirubin (r = -0.112, P = 0.000) and ALP levels (r = -0.098, P = 0.000). We observed positive correlation of ALP and bilirubin levels, while negative associations of viral load with HCV liver disease progression. CONCLUSION: Disease progression seems independent of the genotypes. Relationship between ALP and bilirubin with viral load may be an attractive marker to guess disease progression in patients with hepatitis C.
Asunto(s)
Enzimas/sangre , Hepacivirus/aislamiento & purificación , Hepacivirus/patogenicidad , Hepatitis C/patología , Hepatitis C/virología , Pruebas de Función Hepática , Carga Viral , Adolescente , Adulto , Anciano , Bilirrubina/sangre , Biomarcadores , Biopsia , Niño , Estudios Transversales , Progresión de la Enfermedad , Femenino , Hemoglobinas/análisis , Hepatitis C/diagnóstico , Humanos , Hígado/patología , Masculino , Persona de Mediana Edad , Pronóstico , Adulto JovenRESUMEN
BACKGROUND: The 9.6 kb long RNA genome of Hepatitis C virus (HCV) is under the control of RNA dependent RNA polymerase, an error-prone enzyme, for its transcription and replication. A high rate of mutation has been found to be associated with RNA viruses like HCV. Based on genetic variability, HCV has been classified into 6 different major genotypes and 11 different subtypes. However this classification system does not provide significant information about the origin of the virus, primarily due to high mutation rate at nucleotide level. HCV genome codes for a single polyprotein of about 3011 amino acids which is processed into structural and non-structural proteins inside host cell by viral and cellular proteases. RESULTS: We have identified a conserved NS4A protein sequence for HCV genotype 3a reported from four different continents of the world i.e. Europe, America, Australia and Asia. We investigated 346 sequences and compared amino acid composition of NS4A protein of different HCV genotypes through Multiple Sequence Alignment and observed amino acid substitutions C22, V29, V30, V38, Q46 and Q47 in NS4A protein of genotype 1b. Furthermore, we observed C22 and V30 as more consistent members of NS4A protein of genotype 1a. Similarly Q46 and Q47 in genotype 5, V29, V30, Q46 and Q47 in genotype 4, C22, Q46 and Q47 in genotype 6, C22, V38, Q46 and Q47 in genotype 3 and C22 in genotype 2 as more consistent members of NS4A protein of these genotypes. So the different amino acids that were introduced as substitutions in NS4A protein of genotype 1 subtype 1b have been retained as consistent members of the NS4A protein of other known genotypes. CONCLUSION: These observations indicate that NS4A protein of different HCV genotypes originally evolved from NS4A protein of genotype 1 subtype 1b, which in turn indicate that HCV genotype 1 subtype 1b established itself earlier in human population and all other known genotypes evolved later as a result of mutations in HCV genotype 1b. These results were further confirmed through phylogenetic analysis by constructing phylogenetic tree using NS4A protein as a phylogenetic marker.
Asunto(s)
Proteínas Portadoras/genética , Evolución Molecular , Hepacivirus/clasificación , Hepacivirus/genética , Proteínas no Estructurales Virales/genética , Américas , Asia , Australia , Análisis por Conglomerados , Biología Computacional/métodos , Europa (Continente) , Genotipo , Hepacivirus/aislamiento & purificación , Hepatitis C/virología , Humanos , Péptidos y Proteínas de Señalización Intracelular , Datos de Secuencia Molecular , Filogenia , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
Hepatitis C virus (HCV) genotype 3a is considered a significant risk factor for the development of liver diseases and hepatocellular carcinoma for most of the cases in Pakistan. Because of the limited efficiency of the current therapy, RNA interference (RNAi), which results in sequence-specific degradation of HCV RNA, has potential as a powerful alternative molecular therapeutic approach. The envelope genes (E1 and E2) of HCV come in immediate contact with cells during infection and therefore might be a relevant target for new drug development. In the present study, the expression of E1 and E2 genes of HCV genotype 3a was dramatically reduced at both the mRNA and protein level using gene-specific small interfering RNAs (siRNA) when compared to mock-transfected and cells treated with control siRNAs. The potential of siRNAs to inhibit HCV-3a replication in serum-infected Huh-7 cells was also demonstrated by combined treatment of siRNAs against the E1 and E2 genes, which resulted in a significant decrease in HCV viral copy number. This clearly demonstrates that the RNAi-mediated silencing of HCV E1 and E2 is among the first of its type for the development of an effective siRNA-based therapeutic option against HCV-3a.
Asunto(s)
Hepacivirus/crecimiento & desarrollo , ARN Interferente Pequeño/metabolismo , Proteínas del Envoltorio Viral/antagonistas & inhibidores , Línea Celular , Expresión Génica , Genotipo , Hepacivirus/clasificación , Hepacivirus/genética , Humanos , Datos de Secuencia Molecular , Pakistán , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Interferente Pequeño/genética , ARN Viral/antagonistas & inhibidores , ARN Viral/biosíntesis , ARN Viral/genética , Análisis de Secuencia de ADN , Proteínas del Envoltorio Viral/biosíntesis , Proteínas del Envoltorio Viral/genéticaRESUMEN
BACKGROUND: Hepatitis C can lead to liver fibrosis and cirrhosis. We compared readily available non-invasive fibrosis indexes for the fibrosis progression discrimination to find a better combination of existing non-invasive markers. METHODS: We studied 157 HCV infected patients who underwent liver biopsy. In order to differentiate HCV fibrosis progression, readily available AAR, APRI, FI and FIB-4 serum indexes were tested in the patients. We derived a new fibrosis-cirrhosis index (FCI) comprised of ALP, bilirubin, serum albumin and platelet count. FCI = [(ALP × Bilirubin) / (Albumin × Platelet count)]. RESULTS: Already established serum indexes AAR, APRI, FI and FIB-4 were able to stage liver fibrosis with correlation coefficient indexes 0.130, 0.444, 0.578 and 0.494, respectively. Our new fibrosis cirrhosis index FCI significantly correlated with the histological fibrosis stages F0-F1, F2-F3 and F4 (r = 0.818, p < 0.05) with AUROCs 0.932 and 0.996, respectively. The sensitivity and PPV of FCI at a cutoff value < 0.130 for predicting fibrosis stage F0-F1 was 81% and 82%, respectively with AUROC 0.932. Corresponding value of FCI at a cutoff value ≥1.25 for the prediction of cirrhosis was 86% and 100%. CONCLUSIONS: The fibrosis-cirrhosis index (FCI) accurately predicted fibrosis stages in HCV infected patients and seems more efficient than frequently used serum indexes.
Asunto(s)
Hepatitis C Crónica/sangre , Hepatitis C Crónica/patología , Cirrosis Hepática/sangre , Cirrosis Hepática/patología , Índice de Severidad de la Enfermedad , Adulto , Alanina Transaminasa/sangre , Área Bajo la Curva , Aspartato Aminotransferasas/sangre , Bilirrubina/sangre , Biopsia , Distribución de Chi-Cuadrado , Femenino , Humanos , Masculino , Persona de Mediana Edad , Recuento de Plaquetas , Valor Predictivo de las Pruebas , Curva ROC , Albúmina Sérica/metabolismo , Carga Viral , Adulto JovenRESUMEN
We have developed an in situ bioprinting method that allows the printing of cells under true physiological conditions by applying self-assembling ultrashort peptides as bioinks. This method avoids cell stressing methods, such as UV-treatment, chemical crosslinking and viscous bioink printing methods. We further demonstrate that different nanomaterials can easily be synthesized or incorporated in the 3D bioprinted peptide scaffolds which opens up the possibility of functionalized 3D scaffolds.
Asunto(s)
Materiales Biocompatibles/química , Bioimpresión , Hidrogeles/química , Péptidos/química , Impresión Tridimensional , Andamios del Tejido/química , Materiales Biocompatibles/síntesis química , Células Cultivadas , Humanos , Hidrogeles/síntesis química , Conformación Molecular , Simulación de Dinámica Molecular , Tamaño de la Partícula , Péptidos/síntesis química , ARN/análisis , ARN/genética , RNA-Seq , Propiedades de SuperficieRESUMEN
BACKGROUND: Hepatitis C virus (HCV) is a major causative agent of liver associated diseases throughout the world, with genotype 3a responsible for most of the cases in Pakistan. Due to the limited efficiency of current therapy, RNA interference (RNAi) a novel regulatory and powerful silencing approach for molecular therapeutics through a sequence-specific RNA degradation process represents an alternative option. RESULTS: The current study was purposed to assess and explore the possibility of RNAi to silence the HCV-3a Core gene expression, which play complex role in regulation of cell growth and host genes expression essential for infectivity and disease progression. To identify the potent siRNA target sites, 5 small interfering RNAs (siRNAs) against Core gene were designed and in vitro transcribed after consensus sequence analysis of different HCV-3a isolates. Antiviral effects of siRNAs showed upto 80% inhibition of Core gene expression by different siRNAs into Huh-7 cells as compared with Mock transfected and control siRNAs treated cells. For long lasting effect of siRNAs, vector based short hairpin siRNAs (shRNAs) were designed and tested against HCV-3a Core which resulted in a similar pattern of inhibition on RNA and protein expression of HCV Core as synthetic siRNAs. Furthermore, the efficacy of cell culture tested siRNA and shRNA, were evaluated for inhibition of HCV replication in HCV infected serum inoculated Huh-7 cells and a significant decrease in HCV viral copy number was observed. CONCLUSIONS: Our results support the possibility of using consensus siRNA and shRNA-based molecular therapy as a promising strategy in effective inhibition of HCV-3a genotype.
Asunto(s)
Antivirales/farmacología , Productos Biológicos/farmacología , Expresión Génica/efectos de los fármacos , ARN Interferente Pequeño/farmacología , Proteínas del Núcleo Viral/antagonistas & inhibidores , Proteínas del Núcleo Viral/genética , Antivirales/síntesis química , Productos Biológicos/síntesis química , Productos Biológicos/genética , Línea Celular , Hepatocitos/virología , Humanos , Datos de Secuencia Molecular , ARN Interferente Pequeño/síntesis química , ARN Interferente Pequeño/genética , ARN Viral/genética , Análisis de Secuencia de ADNRESUMEN
RNA activation (RNAa) is a newly emerging area of research in which dsRNA targeting promoter regions can induce the expression of the target gene. Although still in its infancy, it is already having significant impacts in several research areas in particular as cancer therapeutics. So far, the scope of RNAa has been limited to mammals and Caenorhabditis elegans with no indication of its prevalence in insects. In this study, we aimed to demonstrate the presence of RNAa in the insect dengue vector Aedes aegypti. Furthermore, we looked to uncover some details surrounding the involvement of host factors in order to present this as a new technique for insect research. The outcomes of this study provide new opportunities to further research into arthropod-borne diseases and insect biology in the same way as RNA interference.
Asunto(s)
Aedes/genética , Mosquitos Vectores/genética , ARN Bicatenario/genética , Activación Transcripcional , Aedes/metabolismo , Animales , Animales Modificados Genéticamente/genética , Animales Modificados Genéticamente/metabolismo , Mosquitos Vectores/metabolismo , ARN Bicatenario/metabolismoRESUMEN
Aedes mosquitoes vector many viruses with divergent characteristics, yet the criteria needed for a virus to be vectored by an arthropod remain unknown. The intracellular cholesterol transporter protein Niemann-Pick C1 (NPC1) has been identified as the necessary entry receptor for filoviruses such as Ebola and Marburg viruses. While homologues of NPC1 are observed in mosquitoes, currently no filovirus has been identified as circulating in mosquitoes. This work aimed at increasing the understanding of the mosquito vector by examining the capability of a virus to gain the ability to enter mosquito cells. We developed a model system of Aedes cells expressing human NPC1 (hNPC1) and attempted to infect these cells with recombinant vesicular stomatitis virus expressing the Ebola virus glycoprotein. As compared to the control cells, no significant increase in infection was observed in cells expressing hNPC1, demonstrating that the expression of human NPC1 alone is not sufficient to support filovirus infection, and that host factors other than NPC1 determine filovirus susceptibility of mosquito cells.
Asunto(s)
Aedes , Proteína Niemann-Pick C1 , Animales , Línea Celular , Humanos , Péptidos y Proteínas de Señalización Intracelular , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Mosquitos Vectores , Tolerancia , Receptores Virales/metabolismo , Internalización del VirusRESUMEN
The recent emergence of the flavivirus Zika and neurological complications, such as Guillain-Barré syndrome and microcephaly in infants, has brought serious public safety concerns. Among the risk factors, antibody-dependent enhancement (ADE) poses the most significant threat, as the recent re-emergence of the Zika virus (ZIKV) is primarily in areas where the population has been exposed and is in a state of pre-immunity to other closely related flaviviruses, especially dengue virus (DENV). Here, we describe a protocol for quantifying the effect of human serum antibodies against DENV on ZIKV infection in primary human cells or cell lines.