RESUMEN
BACKGROUND: Human enterovirus 71 (EV71) was previously known to enter cells through clathrin or caveolar mediated endocytic pathways. However, we observed chlorpromazine (CPZ) or dynasore (DNS), which inhibit clathrin and dynamin mediated endocytosis, did not suppress EV71 cell entry in particular cell types. So the current knowledge of entry mechanisms by EV71 is not complete. METHODS: Viral infection was examined by flow cytometry or end-point dilution assays. Viral entry was monitored by immunofluorescence or pseudoviral infections. Various inhibitors were utilized for manipulating endocytic pathways. Cellular proteins were knockdown by siRNA. RESULTS: CPZ and DNS did not inhibit but rather enhance viral infection in A549 cells, while they inhibited infections in other cells tested. We further found CPZ did not affect EV71 binding to target cells and failed to affect viral translation and replication, but enhanced viral entry in A549 cells. Immunofluorescence microscopy further confirmed this increased entry. Using siRNA experiment, we found that the enhancement of EV71 infection by CPZ did not require the components of clathrin mediated endocytosis. Finally, CPZ also enhanced infection by Coxackivirus A16 in A549 cells. CONCLUSIONS: CPZ and DNS, previously reported as EV71 entry inhibitors, may rather lead to increased viral infection in particular cell types. CPZ and DNS increased viral entry and not other steps of viral life cycles. Therefore, our study indicated an unknown dynamin-independent entry pathway utilized by enteroviruses that cause Hand-Foot-and-Mouth Diseases.
Asunto(s)
Endocitosis/efectos de los fármacos , Enterovirus Humano A/efectos de los fármacos , Enterovirus Humano A/fisiología , Infecciones por Enterovirus/virología , Enfermedad de Boca, Mano y Pie/virología , Internalización del Virus/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular , Clorpromazina/farmacología , Clatrina/metabolismo , Dinaminas/metabolismo , Infecciones por Enterovirus/metabolismo , Regulación Viral de la Expresión Génica/efectos de los fármacos , Enfermedad de Boca, Mano y Pie/metabolismo , Humanos , Hidrazonas/farmacología , Acoplamiento Viral/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
In this study, chitosan (CH) coating with different number-average molecular weight (MW, ca. 5, 19 and 61 kDa) was electrostatic sprayed on strawberry. The effects of MW on strawberry quality changes were evaluated during 15 days of storage at 4 °C. The qualities of strawberry included mold growth, weight loss, firmness, total soluble solids (TSS), pH, flavonoids content, superoxide dismutase (SOD) activity and malondialdehyde (MDA) content. Results showed that CH coating could significantly maintain the strawberry qualities during storage compared to uncoated treatment. CH coating with 61 kDa was more effective in retarding the increases of pH and MDA, and could better maintain flavonoids content. However, MW had no significant impact on mold growth, weight loss, firmness, SOD activity of coated strawberry. According to evaluation criteria, CH coating with 61 kDa had better performance on strawberry preservation with the highest synthetic value (6.93), and could be used to maintain quality and prolong the shelf life of strawberry during cold storage.
Asunto(s)
Quitosano/química , Materiales Biocompatibles Revestidos/química , Conservación de Alimentos , Fragaria , Flavonoides/química , Fragaria/microbiología , Concentración de Iones de Hidrógeno , Malondialdehído , Peso Molecular , Solubilidad , Superóxido Dismutasa/metabolismoRESUMEN
Enterovirus 71 (EV71) is a major pathogen that causes hand, foot and mouth disease (HFMD), which is a life threatening disease in certain children. The pathogenesis of EV71-caused HFMD is poorly defined due to the lack of simple and robust animal models with severe phenotypes that recapitulate symptoms observed in humans. Here, we generated the infectious clone of a clinical isolate from a severe HFMD patient. Virus rescued from the cDNA clone was infectious in cell lines. When administrated intraperitoneally to neonatal ICR, BALB/c and C57 immune competent mice at a dosage of1.4 × 104â pfu per mouse, the virus caused weight loss, paralysis and death in the infected mice after 4-5 days of infection. In the infected mice, detectable viral replication was detected in various tissues such as heart, liver, brain, lung, kidney, small intestine, leg skeletal muscle and medulla oblongata. The histology of the infected mice included massive myolysis, glomerular atrophy, villous blunting in small intestine, widened alveolar septum, diminished alveolar spaces and lymphocytes infiltration into the lung. By using the UV-inactivated virus as a control, we elucidated that the virus first amplified in the leg skeletal muscle tissue and the muscle tissue served as a primary viral replication site. In summary, we generated a stable EV71 infectious clone that is capable of infecting neonatal immune competent mice without adaptive mutations and provide a simple, valuable animal model for the studies of EV71pathogenesis and therapy.
Asunto(s)
Enterovirus Humano A/genética , Enterovirus Humano A/patogenicidad , Enfermedad de Boca, Mano y Pie/virología , Animales , Animales Recién Nacidos , Línea Celular Tumoral , Chlorocebus aethiops , ADN Complementario , Modelos Animales de Enfermedad , Humanos , Lactante , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Mutación , ARN Viral , Organismos Libres de Patógenos Específicos , Células Vero , Virulencia , Replicación ViralRESUMEN
BACKGROUND: Enteroviruses cause hand, foot and mouth disease (HFMD). The host B-cells recognize the viral proteins and provoke humoral responses. Deciphering the B-cell responses to the viral epitopes helps diagnosis and vaccine development. OBJECTIVES: The objective of the present study was to investigate for the first time the landscape of genome-wide linear B-cell epitopes of enterovirus 71 in HFMD population. STUDY DESIGN: The peptides encompassing the entire coding region of EV71 were chemically synthesized and displayed on a microarray. The peptide microarray was used to screen serum samples from an HFMD population, including EV71-, CAV10-, CAV16- and CAV6-infected patients. We identified the dominant epitope-containing-peptides (DECPs) that react with the sera of more than 20% of the HFMD population and the common DECPs that cross-react with the sera from other enteroviruses-infected population. RESULTS: Ten DECPs reacting with IgM and 9 DECPs reacting with IgG antibodies were identified, of which, 6 IgM and 5 IgG common DECPs cross-reacted with the sera from other enteroviruses. Some DECPs preferentially reacted with IgG or IgM antibodies and some epitope-antibody interactions correlated with the severity of HFMD. CONCLUSIONS: We uncovered the DECPs and the common DECPs among a group of enteroviruses in HFMD population and found that some epitope-antibody reactions were associated with the outcome of HFMD. These data may guide developing vaccines against the enteroviruses and help the diagnosis and prognosis of HFMD.
Asunto(s)
Anticuerpos Antivirales/sangre , Enterovirus Humano A/inmunología , Epítopos de Linfocito B/genética , Enfermedad de Boca, Mano y Pie/inmunología , Antígenos Virales/inmunología , Niño , Preescolar , China/epidemiología , Reacciones Cruzadas , Enterovirus Humano A/genética , Femenino , Enfermedad de Boca, Mano y Pie/sangre , Enfermedad de Boca, Mano y Pie/epidemiología , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Lactante , Masculino , Tamizaje Masivo , Péptidos/inmunología , Análisis por Matrices de ProteínasRESUMEN
Since late 2012, coxsackievirus A6 (CVA6) has gradually become the predominant pathogen responsible for hand-foot-mouth disease (HFMD) in several provinces of China. A total of 626 patients diagnosed with HFMD in Shanghai, China from January 2012 to September 2013 were enrolled in this study. Of these, 292 CVA6 infected cases were subjected to clinical analyses. Whole-genome sequencing, recombination and phylogenetic analyses were also performed. A recombinant CVA6 monophyletic lineage was found during an outbreak of CVA6-associated HFMDs in Shanghai, China in November 2012, and accounted for 21.9% (64/292) of the CVA6 strains during the study period. Recombination analyses showed that the 2C gene of the novel CVA6 virus was probably derived from a coxsackievirus A4 (CVA4) strain circulating in the population. Clinical observation showed that this recombinant CVA6 virus led to a more generalized rash than did the non-recombinant CVA6 virus. This newly emerged CVA6 lineage was associated with a considerable proportion of HFMD cases from 2012 to 2013 in Shanghai, and poses a potential threat to public health.