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OBJECTIVE: Farmworkers are at increased risk of adverse health outcomes related to occupational heat exposure and inadequate access to water, shade, or rest breaks. Presently, there is a dearth of studies examining the prevalence of dehydration and related factors in U.S. farmworkers. Our objectives were to characterize hydration status during typical workdays and to identify risk factors associated with increased dehydration in migrant farmworkers employed in Florida. METHODS: Urine samples were collected and analyzed for urine specific gravity (USG) 2-3 times per person per day over five days in May 2021 and 2022. Data collection included demographic characteristics, wet-bulb-globe-temperature (WBGT), and information on working conditions (task type, duration, and crop units harvested), fluid intake, clothing worn, and heat safety behaviors. Multivariable mixed regression models were used to evaluate risk factors associated with change in USG levels (continuous) during a work shift. RESULTS: A total of 111 farmworkers participated in this study providing 1020 cumulative USG measurements, of which 96.8% of end-of-shift USG samples were above 1.020 indicating potential dehydration. In multivariable models, dehydration assessed using change in USG levels significantly declined with age (ß = -0.078; 95%CI: 0.150, -0.006) but showed significant increase with body mass index (ß = 0.016; 95%CI: 0.003, 0.028), WGBT (ß = 0.054; 95%CI:0.044, 0.064), mean shift duration, and state of primary residence. We did not find significant associations of dehydration with type of clothing worn, intake of employer-provided water, or crop units harvested during a shift in this sample of farmworkers. CONCLUSION: Our findings underscore the need for additional research to evaluate adverse outcomes related to dehydration and to better understand recovery patterns from chronic dehydration across workweeks and harvest seasons in migrant farmworkers.
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Deshidratación , Agricultores , Migrantes , Humanos , Deshidratación/epidemiología , Migrantes/estadística & datos numéricos , Masculino , Factores de Riesgo , Adulto , Femenino , Agricultores/estadística & datos numéricos , Florida/epidemiología , Persona de Mediana Edad , Adulto Joven , Exposición Profesional/análisis , Gravedad Específica , Calor/efectos adversos , AgriculturaRESUMEN
Efforts to mitigate outdoor water use in Florida's urban landscapes increasingly include promotion of regionally appropriate landscaping based on its documented effectiveness. Targeted initiatives, however, require an understanding of mechanisms underpinning low irrigation use in single-family homes with Florida-Friendly Landscaping (FFL). This paper reports survey research conducted in southwest Florida to identify factors associated with irrigation practices among FFL clients. Results indicate that approximately half of survey participants irrigated less frequently than once per week year-round. Aesthetic considerations, horticultural knowledge, and membership in a homeowner's association (HOA) with rules regarding yard care were key variables underlying landscape characteristics and maintenance, while property values, water conservation attitudes, lawn grass, and in-ground irrigation system use significantly predicted irrigation practices. Homes with in-ground irrigation systems were more than six times more likely to water their landscapes at least once per week during the warm season when residential outdoor water use is at its peak. A $100,000 increase in a home's market value increased the odds of weekly watering by a multiplicative factor of two, whereas a one-point increase in a six-item Likert scale used to measure a homeowner's water conservation attitude decreased the odds by 76%. Homes with no grass in the landscape were 71% less likely to water on a weekly basis. Providing homeowners, and HOAs, with educational resources that build on existing support for water conservation could augment adoption of low maintenance plants and sustainable practices in Florida's urban landscapes.
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Conservación de los Recursos Naturales , Agua , Riego Agrícola , Florida , PlantasRESUMEN
In this study, an alphavirus vector platform was used to deliver replicon particles (RPs) expressing African swine fever virus (ASFV) antigens to swine. Alphavirus RPs expressing ASFV p30 (RP-30), p54 (RP-54) or pHA-72 (RP-sHA-p72) antigens were constructed and tested for expression in Vero cells and for immunogenicity in pigs. RP-30 showed the highest expression in Vero cells and was the most immunogenic in pigs, followed by RP-54 and RP-sHA-p72. Pigs primed with two doses of the RP-30 construct were then boosted with a naturally attenuated ASFV isolate, OURT88/3. Mapping of p30 identified an immunodominant region within the amino acid residues 111-130. However, the principal effect of the prime-boost was enhanced recognition of an epitope covered by the peptide sequence 61-110. The results suggest that a strategy incorporating priming with a vector-expressed antigen followed by boosting with an attenuated live virus may broaden the recognition of ASFV epitopes.
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Virus de la Fiebre Porcina Africana/inmunología , Fiebre Porcina Africana/inmunología , Antígenos Virales/inmunología , Vacunas Virales/inmunología , Fiebre Porcina Africana/prevención & control , Fiebre Porcina Africana/virología , Virus de la Fiebre Porcina Africana/genética , Alphavirus/genética , Alphavirus/metabolismo , Animales , Anticuerpos Antivirales/inmunología , Antígenos Virales/administración & dosificación , Antígenos Virales/genética , Chlorocebus aethiops , Evaluación Preclínica de Medicamentos , Expresión Génica , Inmunización Secundaria , Epítopos Inmunodominantes/administración & dosificación , Epítopos Inmunodominantes/genética , Epítopos Inmunodominantes/inmunología , Porcinos , Células Vero , Vacunas Virales/administración & dosificaciónRESUMEN
Hendra and Nipah viruses (genus Henipavirus, family Paramyxoviridae) are highly pathogenic bat-borne viruses. The need for high biocontainment when studying henipaviruses has hindered the development of therapeutics and knowledge of the viral infection cycle. We have performed a genome-wide siRNA screen at biosafety level 4 that identified 585 human proteins required for henipavirus infection. The host protein with the largest impact was fibrillarin, a nucleolar methyltransferase that was also required by measles, mumps and respiratory syncytial viruses for infection. While not required for cell entry, henipavirus RNA and protein syntheses were greatly impaired in cells lacking fibrillarin, indicating a crucial role in the RNA replication phase of infection. During infection, the Hendra virus matrix protein co-localized with fibrillarin in cell nucleoli, and co-associated as a complex in pulldown studies, while its nuclear import was unaffected in fibrillarin-depleted cells. Mutagenesis studies showed that the methyltransferase activity of fibrillarin was required for henipavirus infection, suggesting that this enzyme could be targeted therapeutically to combat henipavirus infections.
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Proteínas Cromosómicas no Histona/metabolismo , Infecciones por Henipavirus/virología , Virus Nipah/enzimología , Animales , Chlorocebus aethiops , Proteínas Cromosómicas no Histona/genética , Células HeLa , Virus Hendra/metabolismo , Humanos , Mutación , Virus Nipah/genética , Virus Nipah/patogenicidad , ARN Interferente Pequeño , Células Vero , Proteínas de la Matriz Viral/metabolismoRESUMEN
FK506 and rapamycin (Rap) are immunosuppressive drugs that act principally on T-lymphocytes. The receptors for both drugs are FK506-binding proteins (FKBPs), but the molecular mechanisms of immunosuppression differ. An FK506-FKBP complex inhibits the protein phosphatase calcineurin, blocking a key step in T-cell activation, while the Rap -FKBP complex binds to the protein kinase target of rapamycin (TOR), which is involved in a subsequent signalling pathway. Both drugs, and certain non-immunosuppressive compounds related to FK506, have potent antimalarial activity. There is however conflicting evidence on the involvement of Plasmodium calcineurin in the action of FK506, and the parasite lacks an apparent TOR homologue. We therefore set out to establish whether inhibition of the Plasmodium falciparum FKBP PfFKBP35 itself might be responsible for the antimalarial effects of FK506 and Rap. Similarities in the antiparasitic actions of FK506 and Rap would constitute indirect evidence for this hypothesis. FK506 and Rap acted indistinguishably on: (i) specificity for different intra-erythrocytic stages in culture, (ii) kinetics of killing or irreversible growth arrest of parasites and (iii) interactions with other antimalarial agents. Furthermore, PfFKBP35's inhibitory effect on calcineurin was independent of FK506 under a range of conditions, suggesting that calcineurin is unlikely to be involved in the antimalarial action of FK506.
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Antimaláricos/farmacología , Plasmodium falciparum/efectos de los fármacos , Proteínas Protozoarias/genética , Sirolimus/farmacología , Proteínas de Unión a Tacrolimus/genética , Tacrolimus/farmacología , Plasmodium falciparum/genética , Proteínas Protozoarias/metabolismo , Proteínas de Unión a Tacrolimus/metabolismoRESUMEN
UNLABELLED: Wongabel virus (WONV) is an arthropod-borne rhabdovirus that infects birds. It is one of the growing array of rhabdoviruses with complex genomes that encode multiple accessory proteins of unknown function. In addition to the five canonical rhabdovirus structural protein genes (N, P, M, G, and L), the 13.2-kb negative-sense single-stranded RNA (ssRNA) WONV genome contains five uncharacterized accessory genes, one overlapping the N gene (Nx or U4), three located between the P and M genes (U1 to U3), and a fifth one overlapping the G gene (Gx or U5). Here we show that WONV U3 is expressed during infection in insect and mammalian cells and is required for efficient viral replication. A yeast two-hybrid screen against a mosquito cell cDNA library identified that WONV U3 interacts with the 83-amino-acid (aa) C-terminal domain of SNF5, a component of the SWI/SNF chromatin remodeling complex. The interaction was confirmed by affinity chromatography, and nuclear colocalization was established by confocal microscopy. Gene expression studies showed that SNF5 transcripts are upregulated during infection of mosquito cells with WONV, as well as West Nile virus (Flaviviridae) and bovine ephemeral fever virus (Rhabdoviridae), and that SNF5 knockdown results in increased WONV replication. WONV U3 also inhibits SNF5-regulated expression of the cytokine gene CSF1. The data suggest that WONV U3 targets the SWI/SNF complex to block the host response to infection. IMPORTANCE: The rhabdoviruses comprise a large family of RNA viruses infecting plants, vertebrates, and invertebrates. In addition to the major structural proteins (N, P, M, G, and L), many rhabdoviruses encode a diverse array of accessory proteins of largely unknown function. Understanding the role of these proteins may reveal much about host-pathogen interactions in infected cells. Here we examine accessory protein U3 of Wongabel virus, an arthropod-borne rhabdovirus that infects birds. We show that U3 enters the nucleus and interacts with SNF5, a component of the chromatin remodeling complex that is upregulated in response to infection and restricts viral replication. We also show that U3 inhibits SNF5-regulated expression of the cytokine colony-stimulating factor 1 (CSF1), suggesting that it targets the chromatin remodeling complex to block the host response to infection. This study appears to provide the first evidence of a virus targeting SNF5 to inhibit host gene expression.
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Ensamble y Desensamble de Cromatina , Proteínas Cromosómicas no Histona/metabolismo , Interacciones Huésped-Patógeno , Rhabdoviridae/inmunología , Rhabdoviridae/fisiología , Factores de Transcripción/metabolismo , Proteínas Virales/metabolismo , Animales , Línea Celular , Núcleo Celular/química , Cromatografía de Afinidad , Insectos , Mamíferos , Microscopía Confocal , Técnicas del Sistema de Dos HíbridosRESUMEN
Stormwater ponds are installed in urban developments to provide the ecosystem services of flood control and water treatment. In coastal areas, these ponds are connected to watersheds that can drain directly into protected estuaries, making their design, function, and maintenance critical to environmental protection. However, stormwater ponds in residential areas are increasingly managed as aesthetic amenities that add value to real estate rather than as engineered devices with special maintenance requirements. To help extend the life of neighborhood stormwater systems and improve ecosystem services, homeowners should follow best management practices for nutrient management and add shoreline plantings and non-invasive, beneficial aquatic plants to their ponds. This study used focus group and survey research to document the knowledge, behaviors, and attitudes of homeowners living near stormwater ponds in a master-planned community in Florida. The study was designed to use a social marketing research approach to promote Extension best practices. Findings indicate that many residents were aware of the functional components of stormwater systems and respondents' receptivity to best management practices was mediated by age, their attitudes about water quality and whether their home was adjacent to a pond. These findings can be used to target Extension audiences and improve adoption of stormwater pond best management practices for increased protection of water quality.
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Conservación de los Recursos Naturales/métodos , Inundaciones , Vivienda , Estanques , Urbanización , Ecosistema , Florida , Calidad del AguaRESUMEN
In recent years, bats have been identified as a natural reservoir for a diverse range of viruses. Nelson Bay orthoreovirus (NBV) was first isolated from the heart blood of a fruit bat (Pteropus poliocephalus) in 1968. While the pathogenesis of NBV remains unknown, other related members of this group have caused acute respiratory disease in humans. Thus the potential for NBV to impact human health appears plausible. Here, to increase our knowledge of NBV, we examined the replication and infectivity of NBV using different mammalian cell lines derived from bat, human, mouse and monkey. All cell lines supported the replication of NBV; however, L929 cells showed a greater than 2 log reduction in virus titre compared with the other cell lines. Furthermore, NBV did not induce major cytopathic effects in the L929 cells, as was observed in other cell lines. Interestingly, the related Pteropine orthoreoviruses, Pulau virus (PulV) and Melaka virus (MelV) were able to replicate to high titres in L929 cells but infection resulted in reduced cytopathic effect. Our study demonstrates a unique virus-host interaction between NBV and L929 cells, where cells effectively control viral infection/replication and limit the formation of syncytia. By elucidating the molecular mechanisms that control this unique relationship, important insights will be made into the biology of this fusogenic virus.
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Línea Celular/virología , Fibroblastos/virología , Orthoreovirus/fisiología , Tropismo Viral , Animales , Quirópteros , Haplorrinos , Humanos , Ratones , Orthoreovirus/crecimiento & desarrollo , Carga Viral , Cultivo de Virus , Replicación ViralRESUMEN
Bovine ephemeral fever virus (BEFV) is an arthropod-borne rhabdovirus that is classified as the type species of the genus Ephemerovirus. In addition to the five canonical rhabdovirus structural proteins (N, P, M, G, and L), the large and complex BEFV genome contains several open reading frames (ORFs) between the G and L genes (α1, α2/α3, ß, and γ) encoding proteins of unknown function. We show that the 10.5-kDa BEFV α1 protein is expressed in infected cells and, consistent with previous predictions based on its structure, has the properties of a viroporin. Expression of a BEFV α1-maltose binding protein (MBP) fusion protein in Escherichia coli was observed to inhibit cell growth and increase membrane permeability to hygromycin B. Increased membrane permeability was also observed in BEFV-infected mammalian cells (but not cells infected with an α1-deficient BEFV strain) and in cells expressing a BEFV α1-green fluorescent protein (GFP) fusion protein, which was shown by confocal microscopy to localize to the Golgi complex. Furthermore, the predicted C-terminal cytoplasmic domain of α1, which contains a strong nuclear localization signal (NLS), was translocated to the nucleus when expressed independently, and in an affinity chromatography assay employing a GFP trap, the full-length α1 was observed to interact specifically with importin ß1 and importin 7 but not with importin α3. These data suggest that, in addition to its function as a viroporin, BEFV α1 may modulate components of nuclear trafficking pathways, but the specific role thereof remains unclear. Although rhabdovirus accessory genes occur commonly among arthropod-borne rhabdoviruses, little is known of their functions. Here, we demonstrate that the BEFV α1 ORF encodes a protein which has the structural and functional characteristics of a viroporin. We show that α1 localizes in the Golgi complex and increases cellular permeability. We also show that BEFV α1 binds importin ß1 and importin 7, suggesting that it may have a yet unknown role in modulating nuclear trafficking. This is the first functional analysis of an ephemerovirus accessory protein and of a rhabdovirus viroporin.
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Virus de la Fiebre Efímera Bovina/metabolismo , Fiebre Efímera/metabolismo , Carioferinas/metabolismo , Proteínas Virales/metabolismo , beta Carioferinas/metabolismo , Secuencias de Aminoácidos , Animales , Bovinos , Núcleo Celular/genética , Núcleo Celular/metabolismo , Fiebre Efímera/genética , Fiebre Efímera/virología , Virus de la Fiebre Efímera Bovina/química , Virus de la Fiebre Efímera Bovina/genética , Carioferinas/genética , Señales de Localización Nuclear , Unión Proteica , Transporte de Proteínas , Proteínas Virales/química , Proteínas Virales/genética , beta Carioferinas/genéticaRESUMEN
Infection of cells with African swine fever virus (ASFV) can lead to the formation of zipper-like stacks of structural proteins attached to collapsed endoplasmic reticulum (ER) cisternae. We show that the collapse of ER cisternae observed during ASFV infection is dependent on the viral envelope protein, J13Lp. Expression of J13Lp alone in cells is sufficient to induce collapsed ER cisternae. Collapse was dependent on a cysteine residue in the N-terminal domain of J13Lp exposed to the ER lumen. Luminal collapse was also dependent on the expression of J13Lp within stacks of ER where antiparallel interactions between the cytoplasmic domains of J13Lp orientated N-terminal domains across ER cisternae. Cisternal collapse was then driven by disulphide bonds between N-terminal domains arranged in antiparallel arrays across the ER lumen. This provides a novel mechanism for biogenesis of modified stacks of ER present in cells infected with ASFV, and may also be relevant to cellular processes.
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Virus de la Fiebre Porcina Africana/fisiología , Retículo Endoplásmico/ultraestructura , Proteínas Virales/biosíntesis , Virus de la Fiebre Porcina Africana/ultraestructura , Animales , Técnicas de Cultivo de Célula , Chlorocebus aethiops , Técnica del Anticuerpo Fluorescente , Membranas Intracelulares/ultraestructura , Microscopía Electrónica de Transmisión , Plásmidos , Estructura Terciaria de Proteína , Transfección , Células Vero , Proteínas del Envoltorio Viral/biosíntesis , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/ultraestructura , Proteínas Virales/química , Proteínas Virales/ultraestructura , Ensamble de VirusRESUMEN
BACKGROUND: Hendra virus (HeV) is a pleomorphic virus belonging to the Paramyxovirus family. Our long-term aim is to understand the process of assembly of HeV virions. As a first step, we sought to determine the most appropriate cell culture system with which to study this process, and then to use this model to define the morphology of the virus and identify the site of assembly by imaging key virus encoded proteins in infected cells. METHODS: A range of primary cells and immortalised cell lines were infected with HeV, fixed at various time points post-infection, labelled for HeV proteins and imaged by confocal, super-resolution and transmission electron microscopy. RESULTS: Significant differences were noted in viral protein distribution depending on the infected cell type. At 8 hpi HeV G protein was detected in the endoplasmic reticulum and M protein was seen predominantly in the nucleus in all cells tested. At 18 hpi, HeV-infected Vero cells showed M and G proteins throughout the cell and in transmission electron microscope (TEM) sections, in pleomorphic virus-like structures. In HeV infected MDBK, A549 and HeLa cells, HeV M protein was seen predominantly in the nucleus with G protein at the membrane. In HeV-infected primary bovine and porcine aortic endothelial cells and two bat-derived cell lines, HeV M protein was not seen at such high levels in the nucleus at any time point tested (8,12, 18, 24, 48 hpi) but was observed predominantly at the cell surface in a punctate pattern co-localised with G protein. These HeV M and G positive structures were confirmed as round HeV virions by TEM and super-resolution (SR) microscopy. SR imaging demonstrated for the first time sub-virion imaging of paramyxovirus proteins and the respective localisation of HeV G, M and N proteins within virions. CONCLUSION: These findings provide novel insights into the structure of HeV and show that for HeV imaging studies the choice of tissue culture cells may affect the experimental results. The results also indicate that HeV should be considered a predominantly round virus with a mean diameter of approximately 280 nm by TEM and 310 nm by SR imaging.
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Virus Hendra/fisiología , Virus Hendra/ultraestructura , Ensamble de Virus , Animales , Línea Celular , Humanos , Microscopía , Imagen ÓpticaRESUMEN
OBJECTIVES: This study aimed to explore farmworkers' experiences of social vulnerability during the peak period of the COVID-19 pandemic. METHODS: Telephone surveys of 63 migrant and seasonal farmworkers across three major agricultural areas in Florida were conducted. The survey, designed and disseminated with critical support from a statewide farmworker membership and advocacy organization, included items related to social and occupational precarity and a suite of demographic conditions, including specific employment-based indicators and categories. Data were analyzed with SPSS using a series of statistical significance tests including Pearson's Chi-Square, Fisher's Exact, T-tests, and Mann-Whitney U. An open-ended question regarding employment precarity was also analyzed for frequencies of responses pertaining to a set of descriptive categories. RESULTS: Survey findings demonstrated a high degree of social vulnerability among the farmworker sample, with notable variation in the type and severity of vulnerability and risk exposure across employment-based indicators and occupational categories. For example, a cross-industry comparison between vegetable field workers and greenhouse nursery workers revealed a disparity in COVID-exposure risk through commuting characteristics, as 43% of vegetable field workers used shared, employer-provided transport, while 68% of nursery workers used personal vehicles. CONCLUSION: While previous research has broadly established the extreme precarity of migrant and seasonal farmworkers during the peak COVID-19 period, the variability of experience, exposure to risk, and social vulnerability between farmworkers representing distinct employment-based indicators and occupational categories demonstrated in this study contributes to widening awareness of the importance of assessing farmworker experiences at a more granular level. In addition to delineating social vulnerability across key demographic categories, cross-industry comparisons between farmworkers revealed significant discrepancies in risk and vulnerability to COVID-19. Future research that further explores this variability may reveal opportunities to improve disaster-relief planning and mitigate social vulnerability in future disaster scenarios. The importance of surveying the vulnerability of worker populations, aside from geographic communities, is highlighted.
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BACKGROUND: The pigeon crop is specially adapted to produce milk that is fed to newly hatched young. The process of pigeon milk production begins when the germinal cell layer of the crop rapidly proliferates in response to prolactin, which results in a mass of epithelial cells that are sloughed from the crop and regurgitated to the young. We proposed that the evolution of pigeon milk built upon the ability of avian keratinocytes to accumulate intracellular neutral lipids during the cornification of the epidermis. However, this cornification process in the pigeon crop has not been characterised. RESULTS: We identified the epidermal differentiation complex in the draft pigeon genome scaffold and found that, like the chicken, it contained beta-keratin genes. These beta-keratin genes can be classified, based on sequence similarity, into several clusters including feather, scale and claw keratins. The cornified cells of the pigeon crop express several cornification-associated genes including cornulin, S100-A9 and A16-like, transglutaminase 6-like and the pigeon 'lactating' crop-specific annexin cp35. Beta-keratins play an important role in 'lactating' crop, with several claw and scale keratins up-regulated. Additionally, transglutaminase 5 and differential splice variants of transglutaminase 4 are up-regulated along with S100-A10. CONCLUSIONS: This study of global gene expression in the crop has expanded our knowledge of pigeon milk production, in particular, the mechanism of cornification and lipid production. It is a highly specialised process that utilises the normal keratinocyte cellular processes to produce a targeted nutrient solution for the young at a very high turnover.
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Columbidae/genética , Perfilación de la Expresión Génica , Leche/fisiología , Triglicéridos/genética , Animales , Apoptosis , Evolución Biológica , Diferenciación Celular , Columbidae/crecimiento & desarrollo , Células Epidérmicas , Epidermis/metabolismo , Queratinocitos/citología , Queratinocitos/metabolismo , Transglutaminasas/genética , Triglicéridos/biosíntesis , beta-Queratinas/genéticaRESUMEN
BACKGROUND: The scarcity of certain nucleic acid species and the small size of target sequences such as miRNA, impose a significant barrier to subcellular visualization and present a major challenge to cell biologists. Here, we offer a generic and highly sensitive visualization approach (oligo fluorescent in situ hybridization, O-FISH) that can be used to detect such nucleic acids using a single-oligonucleotide probe of 19-26 nucleotides in length. RESULTS: We used O-FISH to visualize miR146a in human and avian cells. Furthermore, we reveal the sensitivity of O-FISH detection by using a HIV-1 model system to show that as little as 1-2 copies of nucleic acids can be detected in a single cell. We were able to discern newly synthesized viral cDNA and, moreover, observed that certain HIV RNA sequences are only transiently available for O-FISH detection. CONCLUSIONS: Taken together, these results suggest that the O-FISH method can potentially be used for in situ probing of, as few as, 1-2 copies of nucleic acid and, additionally, to visualize small RNA such as miRNA. We further propose that the O-FISH method could be extended to understand viral function by probing newly transcribed viral intermediates; and discern the localisation of nucleic acids of interest. Additionally, interrogating the conformation and structure of a particular nucleic acid in situ might also be possible, based on the accessibility of a target sequence.
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ADN Complementario/ultraestructura , ADN Viral/ultraestructura , VIH-1/ultraestructura , Hibridación Fluorescente in Situ/métodos , MicroARNs/ultraestructura , ARN Viral/ultraestructura , Animales , Anticuerpos Monoclonales/inmunología , Biotina/inmunología , Línea Celular , Pollos , ADN Complementario/genética , ADN Viral/genética , Dosificación de Gen/genética , Células HEK293 , VIH-1/genética , Células HeLa , Humanos , Células Jurkat , MicroARNs/genética , Microscopía/métodos , Sondas de Oligonucleótidos , ARN Viral/genéticaRESUMEN
Autophagy is an intracellular pathway that can contribute to innate antiviral immunity by delivering viruses to lysosomes for degradation or can be beneficial for viruses by providing specialized membranes for virus replication. Here, we show that the picornavirus foot-and-mouth disease virus (FMDV) induces the formation of autophagosomes. Induction was dependent on Atg5, involved processing of LC3 to LC3II, and led to a redistribution of LC3 from the cytosol to punctate vesicles indicative of authentic autophagosomes. Furthermore, FMDV yields were reduced in cells lacking Atg5, suggesting that autophagy may facilitate FMDV infection. However, induction of autophagosomes by FMDV appeared to differ from starvation, as the generation of LC3 punctae was not inhibited by wortmannin, implying that FMDV-induced autophagosome formation does not require the class III phosphatidylinositol 3-kinase (PI3-kinase) activity of vps34. Unlike other picornaviruses, for which there is strong evidence that autophagosome formation is linked to expression of viral nonstructural proteins, FMDV induced autophagosomes very early during infection. Furthermore, autophagosomes could be triggered by either UV-inactivated virus or empty FMDV capsids, suggesting that autophagosome formation was activated during cell entry. Unlike other picornaviruses, FMDV-induced autophagosomes did not colocalize with the viral 3A or 3D protein. In contrast, â¼50% of the autophagosomes induced by FMDV colocalized with VP1. LC3 and VP1 also colocalized with the cellular adaptor protein p62, which normally targets ubiquitinated proteins to autophagosomes. These results suggest that FMDV induces autophagosomes during cell entry to facilitate infection, but not to provide membranes for replication.
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Autofagia/fisiología , Virus de la Fiebre Aftosa/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Fagosomas/virología , Internalización del Virus , Androstadienos , Animales , Proteína 5 Relacionada con la Autofagia , Western Blotting , Células CHO , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Cricetinae , Cricetulus , Proteínas Fluorescentes Verdes , Ratones , Microscopía Fluorescente , Proteínas Asociadas a Microtúbulos/deficiencia , WortmaninaRESUMEN
BACKGROUND: Bluetongue virus (BTV) is an arbovirus that is responsible for 'bluetongue', an economically important disease of livestock. Although BTV is well characterised at the protein level, less is known regarding its interaction with host cells. During studies of virus inclusion body formation we observed what appeared to be a large proportion of cells in mitosis. Although the modulation of the cell cycle is well established for many viruses, this was a novel observation for BTV. We therefore undertook a study to reveal in more depth the impact of BTV upon cell division. METHODS: We used a confocal microscopy approach to investigate the localisation of BTV proteins in a cellular context with their respective position relative to cellular proteins. In addition, to quantitatively assess the frequency of aberrant mitosis induction by the viral non-structural protein (NS) 2 we utilised live cell imaging to monitor HeLa-mCherry tubulin cells transfected with a plasmid expressing NS2. RESULTS: Our data showed that these 'aberrant mitoses' can be induced in multiple cell types and by different strains of BTV. Further study confirmed multiplication of the centrosomes, each resulting in a separate mitotic spindle during mitosis. Interestingly, the BTV NS1 protein was strongly localised to the centrosomal regions. In a separate, yet related observation, the BTV NS2 protein was co-localised with the condensed chromosomes to a region suggestive of the kinetochore. Live cell imaging revealed that expression of an EGFP-NS2 fusion protein in HeLa-mCherry tubulin cells also results in mitotic defects. CONCLUSIONS: We hypothesise that NS2 is a microtubule cargo protein that may inadvertently disrupt the interaction of microtubule tips with the kinetochores during mitosis. Furthermore, the BTV NS1 protein was distinctly localised to a region encompassing the centrosome and may therefore be, at least in part, responsible for the disruption of the centrosome as observed in BTV infected mammalian cells.
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Virus de la Lengua Azul/fisiología , Interacciones Huésped-Patógeno , Mitosis , Animales , Línea Celular , Citosol/química , Citosol/virología , Microscopía Confocal , Proteínas Virales/análisisRESUMEN
BACKGROUND: Hendra virus (HeV) is an Australian bat-borne zoonotic paramyxovirus that repeatedly spills-over to horses causing fatal disease. Human cases have all been associated with close contact with infected horses. METHODS: A full-length antigenome clone of HeV was assembled, a reporter gene (GFP or luciferase) inserted between the P and M genes and transfected to 293T cells to generate infectious reporter gene-encoding recombinant viruses. These viruses were then assessed in vitro for expression of the reporter genes. The GFP expressing recombinant HeV was used to challenge ferrets to assess the virulence and tissue distribution by monitoring GFP expression in infected cells. RESULTS: Three recombinant HeV constructs were successfully cloned and rescued; a wild-type virus, a GFP-expressing virus and a firefly luciferase-expressing virus. In vitro characterisation demonstrated expression of the reporter genes, with levels proportional to the initial inoculum levels. Challenge of ferrets with the GFP virus demonstrated maintenance of the fatal phenotype with disease progressing to death consistent with that observed previously with the parental wild-type isolate of HeV. GFP expression could be observed in infected tissues collected from animals at euthanasia. CONCLUSIONS: Here, we report on the first successful rescue of recombinant HeV, including wild-type virus and viruses expressing two different reporter genes encoded as an additional gene cassette inserted between the P and M genes. We further demonstrate that the GFP virus retained the ability to cause fatal disease in a well-characterized ferret model of henipavirus infection despite the genome being an extra 1290 nucleotides in length.
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Genes Reporteros , Virus Hendra/genética , Virus Hendra/patogenicidad , Infecciones por Henipavirus/virología , Animales , Línea Celular , Modelos Animales de Enfermedad , Hurones , Proteínas Fluorescentes Verdes/genética , Humanos , Luciferasas/genética , Masculino , Coloración y Etiquetado/métodos , Análisis de Supervivencia , VirulenciaRESUMEN
BACKGROUND: There is widespread agreement that work organization is an important element of occupational safety and health, but the health effects of many aspects of work organization are likely to vary considerably across different sectors of work and geographies. METHODS: We examined existing employment policies and work organization-related research relevant specifically to immigrant workers in the Agriculture, Forestry, and Fishing (AgFF) Sector of the US workforce focusing, when possible, on the southeastern US. RESULTS: A number of specific aspects of work organization within AgFF subsectors have been described, but most of this literature exists outside the purview of occupational health. There are few studies that directly examine how attributes of work organization relevant to the AgFF Sector affect workers', much less immigrant workers', occupational health exposures and outcomes. CONCLUSIONS: In contrast to the broader literature, research linking occupational health outcomes to work organization in the AgFF Sector is limited and weak. A systematic program of research and intervention is needed to develop strategies that eliminate or substantially mitigate the deleterious health effects of occupational exposures whose origins likely lie in the organization of AgFF work.
Asunto(s)
Agricultura/organización & administración , Emigrantes e Inmigrantes , Agricultura Forestal/organización & administración , Enfermedades Profesionales/prevención & control , Salud Laboral/etnología , Traumatismos Ocupacionales/prevención & control , Política Organizacional , Comercio/legislación & jurisprudencia , Comercio/organización & administración , Explotaciones Pesqueras , Regulación Gubernamental , Humanos , Sindicatos , Enfermedades Profesionales/etnología , Exposición Profesional/prevención & control , Salud Laboral/legislación & jurisprudencia , Traumatismos Ocupacionales/etnología , Política Pública , Sudeste de Estados UnidosRESUMEN
Crystal structure analysis of Flavivirus methyltransferases uncovered a flavivirus-conserved cavity located next to the binding site for its cofactor, S-adenosyl-methionine (SAM). Chemical derivatization of S-adenosyl-homocysteine (SAH), the product inhibitor of the methylation reaction, with substituents that extend into the identified cavity, generated inhibitors that showed improved and selective activity against dengue virus methyltransferase (MTase), but not related human enzymes. Crystal structure of dengue virus MTase with a bound SAH derivative revealed that its N6-substituent bound in this cavity and induced conformation changes in residues lining the pocket. These findings demonstrate that one of the major hurdles for the development of methyltransferase-based therapeutics, namely selectivity for disease-related methyltransferases, can be overcome.
Asunto(s)
Antivirales/química , Virus del Dengue/enzimología , Inhibidores Enzimáticos/química , Metiltransferasas/antagonistas & inhibidores , Metiltransferasas/química , S-Adenosilmetionina/análogos & derivados , S-Adenosilmetionina/química , Proteínas Virales/antagonistas & inhibidores , Proteínas Virales/química , Antivirales/farmacología , Sitios de Unión , Cristalografía por Rayos X , Dengue/tratamiento farmacológico , Dengue/enzimología , Dengue/genética , Virus del Dengue/genética , Inhibidores Enzimáticos/farmacología , Humanos , Metiltransferasas/genética , Metiltransferasas/metabolismo , S-Adenosilmetionina/farmacología , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Bluetongue virus (BTV) is a double stranded (ds) RNA virus (genus Orbivirus; family Reoviridae), which is considered capable of infecting all species of domestic and wild ruminants, although clinical signs are seen mostly in sheep. BTV is arthropod-borne ("arbovirus") and able to productively infect and replicate in many different cell types of both insects and mammalian hosts. Although the organ and cellular tropism of BTV in ruminants has been the subject of several studies, many aspects of its pathogenesis are still poorly understood, partly because of inherent problems in distinguishing between "virus replication" and "virus presence".BTV replication and organ tropism were studied in a wide range of infected sheep tissues, by immuno-fluorescence-labeling of non-structural or structural proteins (NS2 or VP7 and core proteins, respectively) using confocal microscopy to distinguish between virus presence and replication. These results are compared to gross and microscopic pathological findings in selected organs from infected sheep. Replication was demonstrated in two major cell types: vascular endothelial cells, and agranular leukocytes which morphologically resemble lymphocytes, monocytes/macrophages and/or dendritic cells. Two organs (the skin and tonsils) were shown to support relatively high levels of BTV replication, although they have not previously been proposed as important replication sites during BTV infection. The high level of BTV replication in the skin is thought to be of major significance for the pathogenesis and transmission of BTV (via biting insects) and a refinement of our current model of BTV pathogenesis is discussed.