RESUMO
It has been estimated that individuals with COVID-19 can shed replication-competent virus up to a maximum of 20 days after initiation of symptoms. The majority of studies that addressed this situation involved hospitalized individuals and those with severe disease. Studies to address the possible presence of SARS-CoV-2 during the different phases of COVID-19 disease in mildly infected individuals, and utilization of viral culture techniques to identify replication-competent viruses, have been limited. This report describes two patients with mild forms of the disease who shed replication-competent virus for 24 and 37 days, respectively, after symptom onset.
Assuntos
COVID-19/imunologia , COVID-19/virologia , SARS-CoV-2/crescimento & desenvolvimento , Cultura de Vírus , Animais , Chlorocebus aethiops , Feminino , Humanos , Pessoa de Meia-Idade , SARS-CoV-2/patogenicidade , Células Vero/ultraestrutura , Células Vero/virologia , Carga Viral , Eliminação de Partículas ViraisRESUMO
In cell culture, cell structures suffer strong impact due to centrifugation during processing for electron microscope observation. In order to minimise this effect, a new protocol was successfully developed. Using conventional reagents and equipments, it took over one week, but cell compression was reduced to none or the lowest deformation possible.
Assuntos
Aedes/ultraestrutura , Vírus da Dengue/ultraestrutura , Microscopia Eletrônica de Transmissão/métodos , Aedes/virologia , Animais , Técnicas de Cultura de Células , Centrifugação/métodos , Chlorocebus aethiops , Fixadores , Indicadores e Reagentes , Células Vero/ultraestruturaRESUMO
In cell culture, cell structures suffer strong impact due to centrifugation during processing for electron microscope observation. In order to minimise this effect, a new protocol was successfully developed. Using conventional reagents and equipments, it took over one week, but cell compression was reduced to none or the lowest deformation possible.
Assuntos
Animais , Aedes/ultraestrutura , Vírus da Dengue/ultraestrutura , Microscopia Eletrônica de Transmissão/métodos , Aedes/virologia , Técnicas de Cultura de Células , Centrifugação/métodos , Chlorocebus aethiops , Fixadores , Indicadores e Reagentes , Células Vero/ultraestruturaRESUMO
OBJECTIVE: To investigate whether the HlyA-induced vacuolating effect is produced by V. cholerae O1 ElTor strains isolated from different geographic origins, including Mexico. MATERIAL AND METHODS: Supernatant-induced haemolysis, vacuolating activity and cytotoxicity in Vero cells were recorded. PCR, RFLP analysis and molecular cloning were performed. RESULTS: All ElTor strains analyzed induced cellular vacuolation. Ribotype 2 strains isolates from the U.S. gulf coast yielded the highest titer of vacuolating activity. Eight of nine strains were haemolytic, while all strains were PCR positive for the hlyA gene. We cloned the hlyA gene from two ElTor strains, a toxigenic (2514-88, ctxAB+) and a non-toxigenic Mexican strain (CM 91-3, ctxAB-). Supernatant from those recombinant E. coli strains induced haemolysis, cell vacuolation and cytotoxicity. RFLP-PCR analysis revealed similarities in the hlyA gene from all strains tested. CONCLUSION: The HlyA-induced vacuolating effect is a widespread phenotype of epidemic V. cholerae O1 ElTor strains.
OBJETIVO: Analizar el efecto vacuolizante de cepas de V. cholerae O1 ElTor aisladas de diferente origen geográfico, incluyendo México. MATERIAL Y MÉTODOS: Se realizaron pruebas de hemolisis, vacuolización y citotoxicidad en células Vero, así como PCR, análisis por RFLP y clonación molecular. RESULTADOS: Todas las cepas indujeron el efecto vacuolizante. Las cepas del ribotipo 2, aisladas de las costas del Golfo en Estados Unidos, presentaron títulos altos de vacuolización. El gen hlyA fue amplificado en las nueve cepas mediante PCR, aunque sólo ocho fueron hemolíticas. Se clonó el gen hlyA de una cepa toxigénica (2514-88, ctxAB+) y de una cepa no toxigénica aislada en México (CM 91-3, ctxAB-). El sobrenadante de las clonas recombinantes indujo hemólisis, efecto vacuolizante y citotoxicidad. El RFLP mostró alta similitud del gen hlyA de las cepas estudiadas. CONCLUSIÓN: El efecto vacuolizante es un fenotipo ampliamente distribuido en cepas epidémicas de V. cholerae O1 biotipo ElTor.
Assuntos
Animais , Proteínas de Bactérias/toxicidade , Cólera/virologia , Meios de Cultivo Condicionados/toxicidade , Proteínas Hemolisinas/toxicidade , Células Vero/microbiologia , Vibrio cholerae O1/patogenicidade , Austrália/epidemiologia , Proteínas de Bactérias/genética , Chlorocebus aethiops , Cólera/epidemiologia , DNA Bacteriano/genética , Proteínas Hemolisinas/genética , Hemólise , América Latina/epidemiologia , Fenótipo , Ribotipagem , Romênia/epidemiologia , Estados Unidos/epidemiologia , Vacúolos , Células Vero/ultraestrutura , Vibrio cholerae O1/classificação , Vibrio cholerae O1/genética , Vibrio cholerae O1/isolamento & purificação , Virulência/genéticaRESUMO
OBJECTIVE: To investigate whether the HlyA-induced vacuolating effect is produced by V. cholerae O1 ElTor strains isolated from different geographic origins, including Mexico. MATERIAL AND METHODS: Supernatant-induced haemolysis, vacuolating activity and cytotoxicity in Vero cells were recorded. PCR, RFLP analysis and molecular cloning were performed. RESULTS: All ElTor strains analyzed induced cellular vacuolation. Ribotype 2 strains isolates from the U.S. gulf coast yielded the highest titer of vacuolating activity. Eight of nine strains were haemolytic, while all strains were PCR positive for the hlyA gene. We cloned the hlyA gene from two ElTor strains, a toxigenic (2514-88, ctxAB+) and a non-toxigenic Mexican strain (CM 91-3, ctxAB-). Supernatant from those recombinant E. coli strains induced haemolysis, cell vacuolation and cytotoxicity. RFLP-PCR analysis revealed similarities in the hlyA gene from all strains tested. CONCLUSION: The HlyA-induced vacuolating effect is a widespread phenotype of epidemic V. cholerae O1 ElTor strains.
Assuntos
Proteínas de Bactérias/toxicidade , Cólera/virologia , Meios de Cultivo Condicionados/toxicidade , Proteínas Hemolisinas/toxicidade , Células Vero/microbiologia , Vibrio cholerae O1/patogenicidade , Animais , Austrália/epidemiologia , Proteínas de Bactérias/genética , Chlorocebus aethiops , Cólera/epidemiologia , DNA Bacteriano/genética , Proteínas Hemolisinas/genética , Hemólise , América Latina/epidemiologia , Fenótipo , Ribotipagem , Romênia/epidemiologia , Estados Unidos/epidemiologia , Vacúolos , Células Vero/ultraestrutura , Vibrio cholerae O1/classificação , Vibrio cholerae O1/genética , Vibrio cholerae O1/isolamento & purificação , Virulência/genéticaRESUMO
Vero cells, a cell line established from the kidney of the African green monkey (Cercopithecus aethiops), were cultured in F-10 Ham medium supplemented with 10% fetal calf serum at 37 degrees C on membranes of poly(L-lactic acid) (PLLA), poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and their blends in different proportions (100/0, 60/40, 50/50, 40/60, and 0/100). The present study evaluated morphology of cells grown on different polymeric substrates after 24 h of culture by scanning electron microscopy. Cell adhesion was also analyzed after 2 h of inoculation. For cell growth evaluation, the cells were maintained in culture for 48, 120, 240, and 360 h. For cytochemical study, the cells were cultured for 120 or 240 h, fixed, processed for histological analysis, and stained with Toluidine blue, pH 4.0, and Xylidine ponceau, pH 2.5. Our results showed that cell adhesion was better when 60/40 and 50/50 blends were used although cells were able to grow and proliferate on all blends tested. When using PLLA/PHBV (50/50) slightly flattened cells were observed on porous and smooth areas. PLLA/PHBV (40/60) blends presented flattened cells on smooth areas. PLLA/PHBV (0/100), which presented no pores, also supported spreading cells interconnected by thin filaments. Histological sections showed that cells grew as a confluent monolayer on different substrates. Cytochemical analysis showed basophilic cells, indicating a large amount of RNA and proteins. Hence, we detected changes in cell morphology induced by alterations in blend proportions. This suggests that the cells changed their differentiation pattern when on various PLLA/PHBV blend surfaces.
Assuntos
Materiais Biocompatíveis , Técnicas de Cultura de Células/métodos , Hidroxibutiratos , Ácido Láctico , Membranas Artificiais , Polímeros , Células Vero/citologia , Animais , Adesão Celular/fisiologia , Chlorocebus aethiops , Histocitoquímica , Microscopia Eletrônica de Varredura , Poliésteres , Porosidade , Células Vero/ultraestruturaRESUMO
Vero cells, a cell line established from the kidney of the African green monkey (Cercopithecus aethiops), were cultured in F-10 Ham medium supplemented with 10 percent fetal calf serum at 37°C on membranes of poly(L-lactic acid) (PLLA), poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and their blends in different proportions (100/0, 60/40, 50/50, 40/60, and 0/100). The present study evaluated morphology of cells grown on different polymeric substrates after 24 h of culture by scanning electron microscopy. Cell adhesion was also analyzed after 2 h of inoculation. For cell growth evaluation, the cells were maintained in culture for 48, 120, 240, and 360 h. For cytochemical study, the cells were cultured for 120 or 240 h, fixed, processed for histological analysis, and stained with Toluidine blue, pH 4.0, and Xylidine ponceau, pH 2.5. Our results showed that cell adhesion was better when 60/40 and 50/50 blends were used although cells were able to grow and proliferate on all blends tested. When using PLLA/PHBV (50/50) slightly flattened cells were observed on porous and smooth areas. PLLA/PHBV (40/60) blends presented flattened cells on smooth areas. PLLA/PHBV (0/100), which presented no pores, also supported spreading cells interconnected by thin filaments. Histological sections showed that cells grew as a confluent monolayer on different substrates. Cytochemical analysis showed basophilic cells, indicating a large amount of RNA and proteins. Hence, we detected changes in cell morphology induced by alterations in blend proportions. This suggests that the cells changed their differentiation pattern when on various PLLA/PHBV blend surfaces.
Assuntos
Animais , Materiais Biocompatíveis , Células Vero/citologia , Hidroxibutiratos , Ácido Láctico , Membranas Artificiais , Polímeros , Técnicas de Cultura de Células/métodos , Adesão Celular/fisiologia , Chlorocebus aethiops , Células Vero/ultraestrutura , Histocitoquímica , Microscopia Eletrônica de Varredura , PorosidadeRESUMO
The Vero cell line, a non-phagocytic cell, has supported the intracellular mechanism of Leishmania (L.) chagasi. This strain (MHOM/BR/501/MS00) was isolated from a human case of visceral leishmaniasis in Mato Grosso do Sul, Brazil and cultivated in Schneider's Drosophila medium with 20% of heat inactivated fetal calf serum. It was allowed to infect the Vero cells at a ratio of 10 to 20 promastigotes per cell. Within six hours of incubation, promastigote forms were found attached to Vero cells without any particular orientation. The number of amastigotes per cell increased during the incubation period. Results showed that promastigotes of L. (L.) chagasi could interact, transform to amastigote forms and multiply in non-phagocytic cells, demonstrating a new model to study the intracellular cycle of this protozoan.
Assuntos
Leishmania infantum/fisiologia , Células Vero/parasitologia , Animais , Chlorocebus aethiops , Meios de Cultura , Leishmania infantum/crescimento & desenvolvimento , Microscopia Eletrônica/veterinária , Células Vero/ultraestruturaRESUMO
We studied the fate of different Trypanosoma cruzi trypomastigote forms after they invade Vero cells persistently colonised with Coxiella burnetii. When the invasion step was examined we found that persistent C. burnetii infection per se reduced only tissue-culture trypomastigote invasion, whereas raising vacuolar pH with Bafilomycin A1 and related drugs, increased invasion of both metacyclic and tissue-culture trypomastigotes when compared with control Vero cells. Kinetic studies of trypomastigote transfer indicated that metacyclic trypomastigotes parasitophorous vacuoles are more efficiently fused to C. burnetii vacuoles. The higher tissue-culture trypomastigote hemolysin and transialidase activities appear to facilitate their faster escape from the parasitophorous vacuole. Sialic acid deficient Lec-2 cells facilitate the escape of both forms. Endosomal-lysosomal sequential labelling with EEA1, LAMP-1, and Rab7 of the parasitophorous vacuoles formed during the entry of each infective form revealed that the phagosome maturation processes are also distinct. Measurements of C. burnetii vacuolar pH disclosed a marked preference for trypomastigote fusion with more acidic rickettsia vacuoles. Our results thus suggest that intravacuolar pH modulates the traffic of trypomastigote parasitophorous vacuoles in these doubly infected cells.
Assuntos
Doença de Chagas/complicações , Coxiella burnetii , Febre Q/complicações , Trypanosoma cruzi , Células Vero/microbiologia , Animais , Doença de Chagas/transmissão , Chlorocebus aethiops , Progressão da Doença , Concentração de Íons de Hidrogênio , Microscopia Confocal , Vacúolos/metabolismo , Células Vero/parasitologia , Células Vero/ultraestruturaRESUMO
The Mojuí dos Campos virus (MDCV) was isolated from the blood of an unidentified bat (Chiroptera) captured in Mojuí dos Campos, Santarém, State of Pará, Brazil, in 1975 and considerated to be antigenically different from other 102 arboviruses belonging to several antigenic groups isolated in the Amazon region or another region by complement fixation tests. The objective of this work was to develop a morphologic, an antigenic and physicochemical characterization of this virus. MDCV produces cytopathic effect in Vero cells, 24 h post-infection (p.i), and the degree of cellular destruction increases after a few hours. Negative staining electron microscopy of the supernatant of Vero cell cultures showed the presence of coated viral particles with a diameter of around 98 nm. Ultrathin sections of Vero cells, and brain and liver of newborn mice infected with MDCV showed an assembly of the viral particles into the Golgi vesicles. The synthesis kinetics of the proteins for MDCV were similar to that observed for other bunyaviruses, and viral proteins could be detected as early as 6 h p.i. Our results reinforce the original studies which had classified MDCV in the family Bunyaviridae, genus Bunyavirus as an ungrouped virus, and it may represent the prototype of a new serogroup.