RESUMEN
Equine rotavirus species A (ERVA) G3P[12] and G14P[12] are two dominant genotypes that cause foal diarrhoea with a significant economic impact on the global equine industry. ERVA can also serve as a source of novel (equine-like) rotavirus species A (RVA) reassortants with zoonotic potential as those identified previously in 2013-2019 when equine G3-like RVA was responsible for worldwide outbreaks of severe gastroenteritis and hospitalizations in children. One hurdle to ERVA research is that the standard cell culture system optimized for human rotavirus replication is not efficient for isolating ERVA. Here, using an engineered cell line defective in antiviral innate immunity, we showed that both equine G3P[12] and G14P[12] strains can be rapidly isolated from diarrhoeic foals. The genome sequence analysis revealed that both G3P[12] and G14P[12] strains share the identical genotypic constellation except for VP7 and VP6 segments in which G3P[12] possessed VP7 of genotype G3 and VP6 of genotype I6 and G14P[12] had the combination of VP7 of genotype G14 and VP6 of genotype I2. Further characterization demonstrated that two ERVA genotypes have a limited cross-neutralization. The lack of an in vitro broad cross-protection between both genotypes supported the increased recent diarrhoea outbreaks due to equine G14P[12] in foals born to dams immunized with the inactivated monovalent equine G3P[12] vaccine. Finally, using the structural modelling approach, we provided the genetic basis of the antigenic divergence between ERVA G3P[12] and G14P[12] strains. The results of this study will provide a framework for further investigation of infection biology, pathogenesis and cross-protection of equine rotaviruses.
Asunto(s)
Antígenos Virales , Diarrea , Genotipo , Enfermedades de los Caballos , Infecciones por Rotavirus , Rotavirus , Animales , Caballos , Rotavirus/genética , Rotavirus/inmunología , Rotavirus/aislamiento & purificación , Rotavirus/clasificación , Infecciones por Rotavirus/veterinaria , Infecciones por Rotavirus/virología , Infecciones por Rotavirus/inmunología , Enfermedades de los Caballos/virología , Enfermedades de los Caballos/inmunología , Diarrea/virología , Diarrea/veterinaria , Antígenos Virales/genética , Antígenos Virales/inmunología , Genoma Viral/genética , Filogenia , Línea CelularRESUMEN
Introduction: Rotavirus A is a major cause of acute dehydrating diarrhea in neonatal pigs resulting in significant mortality, morbidity, reduced performance and economic loss. Commercially available prebiotic galacto-oligosaccharides are similar to those of mammalian milk and stimulate the development of the microbiota and immune system in neonates. Little is known about the effects of supplementing sows' diets with galacto-oligosaccharides during gestation. This study aimed to determine if dietary galacto-oligosaccharide supplementation during gestation could improve immunity, reduce rotavirus infection and modulate the microbiota in sows and neonates in a commercial farm setting with confirmed natural endemic rotavirus challenge. Methods: In a randomized controlled trial, control sows received lactation diet with no galacto-oligosaccharide supplementation and test sows received lactation diet with 30 g/day galacto-oligosaccharide top-dressed into feed daily, seven days before farrowing. Colostrum was collected from sows 24 hours post-partum and tested for rotavirus specific antibodies. Fecal samples were collected from sows and piglets three days post-partum, tested for rotavirus A by qPCR and the microbiome composition assessed by 16s rRNA gene sequencing. Results: Supplementation with galacto-oligosaccharides during gestation significantly increased rotavirus-specific IgG and IgA in sow colostrum and reduced the number of rotavirus positive piglet fecal samples. Abundance of potential pathogens Treponema and Clostridiales were higher in fecal samples from non-galacto-oligosaccharide fed sows, their piglets and rotavirus positive samples. Discussion: This study demonstrates that galacto-oligosaccharide supplementation during gestation significantly increases rotavirus specific IgG and IgA in sow colostrum thereby reducing neonatal rotavirus infection and suppresses potential pathogenic bacteria in nursing sows and neonatal piglets.
RESUMEN
Poorly performing piglets receiving commercial milk replacers do not benefit from the naturally occurring probiotic galacto-oligosaccharides otherwise found in sow milk. Study objectives were to investigate the effects of complete milk replacer supplemented with galacto-oligosaccharides on the microbiome, gut architecture and immunomodulatory goblet cell expression of poorly performing piglets that could benefit from milk replacement feeding when separated from sows and housed with fit siblings in environmentally controlled pens. The study is novel in that it is one of the first to investigate the effects of supplementing complete milk replacer with galacto-oligosaccharides in poorly performing piglets. Gastrointestinal tract samples were collected from piglets, and the microbiome composition was assessed by 16s ribosomal ribonucleic acid gene sequencing. Gut architectural features, villus/crypt ratio and enumeration of goblet cells in tissues were assessed by histopathological techniques. The most abundant taxa identified at the genus level were Lactobacillus, Streptococcus, Prevotella, Lactococcus and Leuconostoc. Milk replacer plus galacto-oligosaccharides significantly improved gut architectural features and villus/crypt ratio throughout the gastrointestinal tract, increased the number of goblet cells and revealed a differential abundance of beneficial probiotic bacteria, particularly Lactobacillus and Bifidobacterium. In these respects, galacto-oligosaccharide-supplemented milk replacer may be a useful addition to animal husbandry in poorly performing, non-thriving animals when moved to environmentally controlled pens away from sows and fit siblings, thereby modulating the microbiome and gastrointestinal tract performance.
RESUMEN
The primary objective of this study was to investigate if common colonic community indicators could be identified from the microbiota of 22-day-old suckling pigs in repeated small-scale trials. A total of three separate trials were conducted at different times in the same year and facility with genetically similar animals. Colonic samples were collected from four pigs in each trial and the microbiome composition assessed by 16s rRNA gene sequencing. Pig weight, average daily gain (ADG), bacterial diversity, and abundance were not significantly different between repeated trials, except for a significant difference in Jaccard Similarity. At genus level, the most abundant taxa identified were Porphyromonadaceae unclassified (15.81%), Ruminococcaceae unclassified, (12.78%), Prevotella (7.26%), Clostridiales unclassified (6.99%), Lactobacillus (6.58%), Phascolarctobacterium (6.52%), and Firmicutes unclassified (5.69%). The secondary objective was to establish if pooled data in terms of microbial diversity and abundance of the colonic microbiota related to weight and ADG. Pig weight at day 22 and ADG positively correlated with α-diversity. Abundance of potential protein digesting and short-chain fatty acid producing operational taxonomic units ascribed to Terrisporobacter, Ruminococcaceae unclassified, Intestinimonas, and Dorea correlated with weight and ADG, suggesting a nutritional role for these common colonic community microbiota members in suckling pigs.
Asunto(s)
Microbioma Gastrointestinal , Microbiota , Animales , Clostridiales/genética , Colon/microbiología , Microbioma Gastrointestinal/genética , Prevotella , ARN Ribosómico 16S/genética , PorcinosRESUMEN
The struggle to control the COVID-19 pandemic is made challenging by the emergence of virulent SARS-CoV-2 variants. To gain insight into their replication dynamics, emergent Alpha (A), Beta (B) and Delta (D) SARS-CoV-2 variants were assessed for their infection performance in single variant- and co-infections. The effectiveness of thapsigargin (TG), a recently discovered broad-spectrum antiviral, against these variants was also examined. Of the 3 viruses, the D variant exhibited the highest replication rate and was most able to spread to in-contact cells; its replication rate at 24 h post-infection (hpi) based on progeny viral RNA production was over 4 times that of variant A and 9 times more than the B variant. In co-infections, the D variant boosted the replication of its co-infected partners at the expense of its own initial performance. Furthermore, co-infection with AD or AB combination conferred replication synergy where total progeny (RNA) output was greater than the sum of corresponding single-variant infections. All variants were highly sensitive to TG inhibition. A single pre-infection priming dose of TG effectively blocked all single-variant infections and every combination (AB, AD, BD variants) of co-infection at greater than 95% (relative to controls) at 72 hpi. Likewise, TG was effective in inhibiting each variant in active preexisting infection. In conclusion, against the current backdrop of the dominant D variant that could be further complicated by co-infection synergy with new variants, the growing list of viruses susceptible to TG, a promising host-centric antiviral, now includes a spectrum of contemporary SARS-CoV-2 viruses.
Asunto(s)
Tratamiento Farmacológico de COVID-19 , Coinfección , SARS-CoV-2 , Tapsigargina , Antivirales/farmacología , Antivirales/uso terapéutico , Humanos , Pandemias , SARS-CoV-2/efectos de los fármacos , Tapsigargina/farmacología , Tapsigargina/uso terapéuticoRESUMEN
The long-term control strategy of SARS-CoV-2 and other major respiratory viruses needs to include antivirals to treat acute infections, in addition to the judicious use of effective vaccines. Whilst COVID-19 vaccines are being rolled out for mass vaccination, the modest number of antivirals in use or development for any disease bears testament to the challenges of antiviral development. We recently showed that non-cytotoxic levels of thapsigargin (TG), an inhibitor of the sarcoplasmic/endoplasmic reticulum (ER) Ca2+ ATPase pump, induces a potent host innate immune antiviral response that blocks influenza A virus replication. Here we show that TG is also highly effective in blocking the replication of respiratory syncytial virus (RSV), common cold coronavirus OC43, SARS-CoV-2 and influenza A virus in immortalized or primary human cells. TG's antiviral performance was significantly better than remdesivir and ribavirin in their respective inhibition of OC43 and RSV. Notably, TG was just as inhibitory to coronaviruses (OC43 and SARS-CoV-2) and influenza viruses (USSR H1N1 and pdm 2009 H1N1) in separate infections as in co-infections. Post-infection oral gavage of acid-stable TG protected mice against a lethal influenza virus challenge. Together with its ability to inhibit the different viruses before or during active infection, and with an antiviral duration of at least 48 h post-TG exposure, we propose that TG (or its derivatives) is a promising broad-spectrum inhibitor against SARS-CoV-2, OC43, RSV and influenza virus.
Asunto(s)
Antivirales/farmacología , Betacoronavirus/efectos de los fármacos , Coronavirus Humano OC43/efectos de los fármacos , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Virus Sincitial Respiratorio Humano/efectos de los fármacos , SARS-CoV-2/efectos de los fármacos , Tapsigargina/farmacología , Animales , Antivirales/uso terapéutico , Betacoronavirus/fisiología , Línea Celular , Línea Celular Tumoral , Células Cultivadas , Coronavirus Humano OC43/fisiología , Estrés del Retículo Endoplásmico , Humanos , Subtipo H1N1 del Virus de la Influenza A/fisiología , Ratones , Pruebas de Sensibilidad Microbiana , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Infecciones por Orthomyxoviridae/virología , Virus Sincitial Respiratorio Humano/fisiología , Ribavirina/farmacología , SARS-CoV-2/fisiología , Tapsigargina/uso terapéutico , Replicación Viral/efectos de los fármacosRESUMEN
An entirely plasmid-based reverse genetics (RG) system was recently developed for rotavirus (RV), opening new avenues for in-depth molecular dissection of RV biology, immunology, and pathogenesis. Several improvements to further optimize the RG efficiency have now been described. However, only a small number of individual RV strains have been recovered to date. None of the current methods have supported the recovery of murine RV, impeding the study of RV replication and pathogenesis in an in vivo suckling mouse model. Here, we describe useful modifications to the RG system that significantly improve rescue efficiency of multiple RV strains. In addition to the 11 group A RV segment-specific (+)RNAs [(+)ssRNAs], a chimeric plasmid was transfected, from which the capping enzyme NP868R of African swine fever virus (ASFV) and the T7 RNA polymerase were expressed. Second, a genetically modified MA104 cell line was used in which several components of the innate immunity were degraded. Using this RG system, we successfully recovered the simian RV RRV strain, the human RV CDC-9 strain, a reassortant between murine RV D6/2 and simian RV SA11 strains, and several reassortants and reporter RVs. All these recombinant RVs were rescued at a high efficiency (≥80% success rate) and could not be reliably rescued using several recently published RG strategies (<20%). This improved system represents an important tool and great potential for the rescue of other hard-to-recover RV strains such as low-replicating attenuated vaccine candidates or low-cell culture passage clinical isolates from humans or animals.IMPORTANCE Group A rotavirus (RV) remains as the single most important cause of severe acute gastroenteritis among infants and young children worldwide. An entirely plasmid-based reverse genetics (RG) system was recently developed, opening new ways for in-depth molecular study of RV. Despite several improvements to further optimize the RG efficiency, it has been reported that current strategies do not enable the rescue of all cultivatable RV strains. Here, we described a helpful modification to the current strategies and established a tractable RG system for the rescue of the simian RRV strain, the human CDC-9 strain, and a murine-like RV strain, which is suitable for both in vitro and in vivo studies. This improved RV reverse genetics system will facilitate study of RV biology in both in vitro and in vivo systems that will facilitate the improved design of RV vaccines, better antiviral therapies, and expression vectors.
Asunto(s)
Regulación Viral de la Expresión Génica , Interacciones Huésped-Patógeno/genética , Virus Reordenados/genética , Genética Inversa/métodos , Rotavirus/genética , Proteínas Virales/genética , Virus de la Fiebre Porcina Africana/genética , Virus de la Fiebre Porcina Africana/inmunología , Animales , Chlorocebus aethiops , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/inmunología , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunidad Innata , Factores Reguladores del Interferón/deficiencia , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/inmunología , Ratones , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/inmunología , Plásmidos/química , Plásmidos/metabolismo , Caperuzas de ARN , Virus Reordenados/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Rotavirus/inmunología , Factor de Transcripción STAT1/deficiencia , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/inmunología , Transfección , Células Vero , Proteínas Virales/inmunología , Replicación ViralRESUMEN
Pigs are evidently more resistant to avian than swine influenza A viruses, mediated in part through frontline epithelial cells and alveolar macrophages (AM). Although porcine AM (PAM) are crucial in influenza virus control, their mode of control is unclear. To gain insight into the possible role of PAM in the mediation of avian influenza virus resistance, we compared the host effects and replication of two avian (H2N3 and H6N1) and three mammalian (swine H1N1, human H1N1 and pandemic H1N1) influenza viruses in PAM. We found that PAM were readily susceptible to initial infection with all five avian and mammalian influenza viruses but only avian viruses caused early and extensive apoptosis (by 6 h of infection) resulting in reduced virus progeny and moderated pro-inflammation. Full length viral PB1-F2 present only in avian influenza viruses is a virulence factor that targets AM for mitochondrial-associated apoptotic cell death. With the use of reverse genetics on an avian H5N1 virus, we found that full length PB1-F2 contributed to increased apoptosis and pro-inflammation but not to reduced virus replication. Taken together, we propose that early apoptosis of PAM limits the spread of avian influenza viruses and that PB1-F2 could play a contributory role in the process.
Asunto(s)
Apoptosis , Virus de la Influenza A/fisiología , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/virología , Infecciones por Orthomyxoviridae/metabolismo , Infecciones por Orthomyxoviridae/virología , Replicación Viral , Animales , Humanos , Virus de la Influenza A/clasificación , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/patología , Mutación , Infecciones por Orthomyxoviridae/inmunología , Polimorfismo Genético , PorcinosRESUMEN
Group A rotaviruses (GARV) are a significant cause of enteritis in young pigs. The aim of this study was to extend our understanding of the molecular epidemiology of porcine GARV in the UK by investigating the genetic diversity of GARV on a conventional farrow-to-finish farm. Faecal samples were obtained from six batches of pigs in 2009 and 8 batches in 2010, when the pigs were 2, 3 (time point omitted in 2009), 4, 5, 6 and 8 weeks of age. Presence of rotavirus was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in 89% and 80% of samples from 2009 and 2010, respectively. A combination of multiplex PCRs and sequencing identified four VP7 genotypes (G2, G3, G4 and G5) and three VP4 genotypes (P[6], P[7] and P[32]) present in almost every combination over the 2 years. The predominant genotype combination was G5P[32] in 2009 and G4P[32] in 2010. Conservation among the P[32] sequences between 2009 and 2010 suggests that reassortment may have led to the different genotype combinations. There were significant changes in the predominant VP7 genotype prior to weaning at 4 weeks, and post weaning when pigs were moved to a different building. Phylogenetic analysis indicated that introduction of new viruses onto the farm was limited. Taken together, these findings suggest that genetically diverse GARV strains persist within the farm environment.
Asunto(s)
Antígenos Virales/genética , Proteínas de la Cápside/genética , Genotipo , Infecciones por Rotavirus/veterinaria , Rotavirus/genética , Sus scrofa/virología , Enfermedades de los Porcinos/virología , Animales , Heces/virología , Variación Genética , Epidemiología Molecular , Filogenia , ARN Viral/genética , Análisis de Secuencia de ARN , Porcinos , Reino UnidoRESUMEN
UNLABELLED: Rotavirus is an enteric pathogen that causes morbidity and mortality in young mammals, including pigs. Outbreaks of rotavirus on commercial farms have a significant economic impact in terms of losses in production. Effective cleaning and disinfection along with good farm management can reduce rotavirus contamination in the environment, and decrease the chance of outbreaks of disease. This study investigated the efficacy of six commercial disinfectants against MS2 bacteriophage and Group A porcine rotavirus, in the presence of high and low levels of organic matter to simulate the farm environment. A phenolic-based disinfectant (Bi-OO-cyst) was effective at all levels of organic matter concentrations. Iodophore-based disinfectants did not have a significant virucidal effect against rotavirus under any conditions. For peroxygen compound-based disinfectants and glutaraldehyde-based disinfectants, organic matter load made a significant difference in reducing efficacy. This highlights the importance of thorough cleaning with detergent before disinfection to reduce viral contamination on the farm and decrease rotavirus disease incidence in pigs. SIGNIFICANCE AND IMPACT OF THE STUDY: Infection of rotavirus has a negative impact on the health and growth of pigs in production. Given that the virus is transmitted faecal-orally, use of an effective disinfectant on farm, which works even in high organic matter, has the potential to save costs in terms of outbreaks of disease and viral contamination. Here, we test a number of commercial disinfectants of which one a phenolic compound, Bio-OO-cyst, shows effectivity even in high organic matter, implying its use could have a huge impact in reducing viral contamination and preventing losses in production.
Asunto(s)
Desinfectantes/farmacología , Infecciones por Rotavirus/prevención & control , Infecciones por Rotavirus/veterinaria , Rotavirus/efectos de los fármacos , Enfermedades de los Porcinos/prevención & control , Animales , Brotes de Enfermedades , Desinfección , Porcinos , Enfermedades de los Porcinos/virologíaRESUMEN
Rotavirus is endemic in pig farms where it causes a loss in production. This study is the first to characterise porcine rotavirus circulating in UK pigs. Samples from diarrheic pigs with rotavirus enteritis obtained between 2010 and 2012 were genotyped in order to determine the diversity of group A rotavirus (GARV) in UK pigs. A wide range of rotavirus genotypes were identified in UK pigs: six G types (VP7); G2, G3, G4, G5, G9 and G11 and six P types (VP4); P[6], P[7], P[8], P[13], P[23], and P[32]. With the exception of a single P[8] isolate, there was less than 95% nucleotide identity between sequences from this study and any available rotavirus sequences. The G9 and P[6] genotypes are capable of infecting both humans and pigs, but showed no species cross-over within the UK as they were shown to be genetically distinct, which suggested zoonotic transmission is rare within the UK. We identified the P[8] genotype in one isolate, this genotype is almost exclusively found in humans. The P[8] was linked to a human Irish rotavirus isolate in the same year. The discovery of human genotype P[8] rotavirus in a UK pig confirms this common human genotype can infect pigs and also highlights the necessity of surveillance of porcine rotavirus genotypes to safeguard human as well as porcine health.
Asunto(s)
Variación Genética , Filogenia , Infecciones por Rotavirus/veterinaria , Rotavirus/genética , Enfermedades de los Porcinos/epidemiología , Proteínas Virales/genética , Animales , Secuencia de Bases , Genotipo , Humanos , Datos de Secuencia Molecular , Estudios Retrospectivos , Rotavirus/clasificación , Infecciones por Rotavirus/epidemiología , Infecciones por Rotavirus/transmisión , Infecciones por Rotavirus/virología , Porcinos , Enfermedades de los Porcinos/transmisión , Enfermedades de los Porcinos/virología , Reino Unido/epidemiologíaRESUMEN
Exposure to interferon results in the rapid transcriptional induction of genes, many of which function to create an antiviral environment in potential host cells. For the majority of adenoviruses, replication is unaffected by the actions of interferon. It has previously been shown, using non-gastrointestinal cells, that the species F human adenoviruses are sensitive to the action of interferon. Here, we have developed an enterocyte-like cell-culture model to re-evaluate this question, and determined the effects of interferon on species F adenovirus during infection of gastrointestinal cells. We show that species F adenovirus type 40 is sensitive to the effects of interferon in gastrointestinal-like cells, which may help to explain its fastidious growth in culture.
Asunto(s)
Adenoviridae/efectos de los fármacos , Adenoviridae/patogenicidad , Infecciones por Adenovirus Humanos/prevención & control , Antivirales/farmacología , Tracto Gastrointestinal/virología , Interferones/farmacología , Adenoviridae/crecimiento & desarrollo , Técnicas de Cultivo de Célula , Quimioprevención/métodos , Humanos , Modelos TeóricosRESUMEN
Campylobacter jejuni activates the host transcription factor NF-kappaB that regulates the expression of a number of genes involved in the inflammatory response to bacterial infection. Signaling pathways leading to NF-kappaB by pathogens and/or their products include transmembrane Toll-like receptors (TLRs) and intracellular receptors nucleotide-binding oligomerization domain proteins (Nods). This study was carried out to investigate the role of TLRs (TLR2 and TLR4) and Nods (Nod1 and Nod2) receptors in mediating NF-kappaB activation by C. jejuni. By means of transfecting receptors/molecules under study and measuring reporter gene activity, NF-kappaB activation and subsequent cytokine production by live, heat-killed C. jejuni, or boiled cell extract (BCE) were observed in a range of tissue culture cell lines. This activation is reduced upon transfection of cells with the dominant negative versions (DNV) of TLR-adaptor molecule MyD88. NF-kappaB activation was observed to be augmented in cell lines transfected with TLR2, Nod1, and Nod2 but not with TLR4. Additionally, NF-kappaB activation by C. jejuni was observed to be independent of Nod1 and Nod2 in cells transfected with DNV of these receptors. NF-kappaB activation pathway by C. jejuni may represent a novel mechanism utilising unknown receptors up-regulated by yet to be characterized active component(s). To our knowledge, such observations have not been previously reported for C. jejuni or any other food-borne pathogen.
Asunto(s)
Campylobacter jejuni/inmunología , Campylobacter jejuni/patogenicidad , FN-kappa B/inmunología , FN-kappa B/metabolismo , Células Cultivadas , Citocinas/biosíntesis , Humanos , Proteína Adaptadora de Señalización NOD1/metabolismo , Proteína Adaptadora de Señalización NOD2/metabolismo , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/metabolismoRESUMEN
BACKGROUND: Campylobacter jejuni, the commonest cause of bacterial diarrhoea worldwide, can also induce colonic inflammation. To understand how a previously identified heat stable component contributes to pro-inflammatory responses we used microarray and real-time quantitative PCR to investigate the transcriptional response to a boiled cell extract of Campylobacter jejuni NCTC 11168. RESULTS: RNA was extracted from the human colonocyte line HCA-7 (clone 29) after incubation for 6 hours with Campylobacter jejuni boiled cell extract and was used to probe the Affymetrix Human Genome U133A array. Genes differentially affected by Campylobacter jejuni boiled cell extract were identified using the Significance Score algorithm of the Bioconductor software suite and further analyzed using the Ingenuity Pathway Analysis program. The chemokines CCL20, CXCL3, CXCL2, Interleukin 8, CXCL1 and CXCL6 comprised 6 of the 10 most highly up-regulated genes, all with Significance Scores > or = 10. Members of the Tumor Necrosis Factor alpha/Nuclear Factor-kappaB super-family were also significantly up-regulated and involved in the most significantly regulated signalling pathways (Death receptor, Interleukin 6, Interleukin 10, Toll like receptor, Peroxisome Proliferator Activated Receptor-gamma and apoptosis). Ingenuity Pathway Analysis also identified the most affected functional gene networks such as cell movement, gene expression and cell death. In contrast, down-regulated genes were predominantly concerned with structural and metabolic functions. CONCLUSION: A boiled cell extract of Campylobacter jejuni has components that can directly switch the phenotype of colonic epithelial cells from one of resting metabolism to a pro-inflammatory one, particularly characterized by increased expression of genes for leukocyte chemoattractant molecules.
Asunto(s)
Campylobacter jejuni/química , Campylobacter jejuni/inmunología , Factores Quimiotácticos/inmunología , Colon/inmunología , Células Epiteliales/inmunología , Perfilación de la Expresión Génica , Línea Celular , Quimiocinas/biosíntesis , Factores Quimiotácticos/aislamiento & purificación , Colon/citología , Regulación hacia Abajo , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/inmunología , Regulación hacia ArribaRESUMEN
Human enteric adenoviruses propagate poorly in conventional human cell lines used to grow other adenovirus serotypes. As human enteric adenoviruses have a defect in counteracting the cellular interferon (IFN) response in cell culture, to aid in growth of the virus, a 293-based cell line defective in its ability to respond to IFN was constructed. This cell line (293-SV5/V) constitutively expresses V-protein of the paramyxovirus Simian virus 5, which degrades the signal transducer and activator of transcription 1 (STAT1) and thereby prevents the STAT1-mediated IFN response. Analysis of human enteric adenovirus type 40 (HAdV-40)-infected 293-SV5/V cells compared with parental 293 cells shows that the recombinant line allows more rapid production of virus and results in higher titres. These results suggest that the defect in HAdV-40 in counteracting the IFN response can be overcome at least partially through the use of 293-SV5/V cell lines.
Asunto(s)
Adenovirus Humanos/crecimiento & desarrollo , Interferones/farmacología , Factor de Transcripción STAT1/fisiología , Replicación Viral/efectos de los fármacos , Adenovirus Humanos/fisiología , Línea Celular , Humanos , Factor de Transcripción STAT1/biosíntesis , Factor de Transcripción STAT1/genéticaRESUMEN
Enteropathogenic Escherichia coli (EPEC) cause infantile diarrhoea and are characterized by their ability to produce attaching and effacing lesions on the surface of intestinal epithelial cells. EPEC employ a filamentous type III secretion system to deliver effector molecules that subvert mammalian cell function to generate actin- and cytokeratin-rich pedestals beneath adherent bacteria. Tir is a major effector protein that is delivered to the plasma membrane of the eukaryotic cell where it acts as the receptor for the bacterial adhesin intimin. Host cell proteins that are recruited to the site of intimate attachment include focal adhesion and cytoskeletal proteins that contribute to pedestal formation. We have used Tir as bait in a yeast two-hybrid screen to identify the protein 14-3-3tau as a binding partner. 14-3-3 proteins are a family of adaptor proteins that modulate protein function in all eukaryotic cells. Here we demonstrate that the tau isoform (also known as theta) of 14-3-3 can bind specifically to Tir in a phosphorylation-independent manner, and that the interaction occurs during the infection process by co-immunoprecipitation of the partners from infected HeLa cell extracts. 14-3-3tau is recruited to the site of the pedestal (3 h after infection) and can decorate attached EPEC in the later stages of the infection process (5-7 h). Pedestal formation can be impaired by depletion of cellular 14-3-3tau using small interfering RNAs. This study indicates a direct functional role for the 14-3-3tau:Tir interaction and is the first to demonstrate the association of a host protein with the surface of EPEC.
Asunto(s)
Proteínas 14-3-3/metabolismo , Actinas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiología , Receptores de Superficie Celular/metabolismo , Adhesión Bacteriana , Biopolímeros , Extractos Celulares , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Células HeLa , Humanos , Fosforilación , Unión Proteica , Transporte de Proteínas , ARN Interferente Pequeño/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
Campylobacter jejuni is a food-borne pathogen responsible for infectious enterocolitis. The early-response transcription factor NF-kappa B triggers the expression of genes associated with cellular immune and inflammatory responses. Co-incubation of HeLa cells with viable C. jejuni leads to the activation of the transcription factor NF-kappa B as determined by specific induction of a cellular luciferase-based reporter. Boiled cell-free extracts of C. jejuni are also potent dose-dependent stimulators of NF-kappa B-dependent transcription, the levels of which can reach up to 1000-fold as compared with independent controls. Using both cultured HeLa cells and human colonic epithelial (HCA-7) cells, the activation of NF-kappa B by C. jejuni boiled extract has been monitored through the degradation of IKB alpha and DNA binding of the nuclear translocated p50/p65 heterodimer of NF-kappa B. These events are co-ordinated with elaboration of the pro-inflammatory cytokine interleukin-8. Fractionation of the boiled C. jejuni extract suggests that the majority of the bioactive component has a molecular mass of 3 kDa or less, which is insensitive to proteinase K treatment.