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1.
Mem. Inst. Oswaldo Cruz ; 116: e200443, 2021. tab, graf
Article in English | LILACS | ID: biblio-1154874

ABSTRACT

BACKGROUND The coronaviruses (CoVs) called the attention of the world for causing outbreaks of severe acute respiratory syndrome (SARS-CoV), in Asia in 2002-03, and respiratory disease in the Middle East (MERS-CoV), in 2012. In December 2019, yet again a new coronavirus (SARS-CoV-2) first identified in Wuhan, China, was associated with a severe respiratory infection, known today as COVID-19. This new virus quickly spread throughout China and 30 additional countries. As result, the World Health Organization (WHO) elevated the status of the COVID-19 outbreak from emergency of international concern to pandemic on March 11, 2020. The impact of COVID-19 on public health and economy fueled a worldwide race to approve therapeutic and prophylactic agents, but so far, there are no specific antiviral drugs or vaccines available. In current scenario, the development of in vitro systems for viral mass production and for testing antiviral and vaccine candidates proves to be an urgent matter. OBJECTIVE The objective of this paper is study the biology of SARS-CoV-2 in Vero-E6 cells at the ultrastructural level. METHODS In this study, we documented, by transmission electron microscopy and real-time reverse transcription polymerase chain reaction (RT-PCR), the infection of Vero-E6 cells with SARS-CoV-2 samples isolated from Brazilian patients. FINDINGS The infected cells presented cytopathic effects and SARS-CoV-2 particles were observed attached to the cell surface and inside cytoplasmic vesicles. The entry of the virus into cells occurred through the endocytic pathway or by fusion of the viral envelope with the cell membrane. Assembled nucleocapsids were verified inside rough endoplasmic reticulum cisterns (RER). Viral maturation seemed to occur by budding of viral particles from the RER into smooth membrane vesicles. MAIN CONCLUSIONS Therefore, the susceptibility of Vero-E6 cells to SARS-CoV-2 infection and the viral pathway inside the cells were demonstrated by ultrastructural analysis.


Subject(s)
Humans , Animals , Vero Cells/virology , Cytoplasmic Vesicles/virology , Cytopathogenic Effect, Viral , SARS-CoV-2/physiology , Chlorocebus aethiops , Nucleocapsid , Reverse Transcriptase Polymerase Chain Reaction , Microscopy, Electron, Transmission , Endocytosis , Endoplasmic Reticulum/virology , Virus Internalization , Real-Time Polymerase Chain Reaction
2.
Biomédica (Bogotá) ; 38(supl.2): 135-143, ago. 2018. tab, graf
Article in English | LILACS | ID: biblio-1038798

ABSTRACT

ABSTRACT Introduction: Dengue virus replication has been considered mainly cytoplasmic, however, studies indicate that some flaviviruses may use the intranuclear pathway as part of the machinery that the virus uses to increase infection capacity in the host cell. This paper describes alterations at nuclear level in the cell infected with dengue, which are likely involved in the virus replication processes. Objective: This paper addresses the ultrastructural observations of C6/36 cells of the Aedes albopictus mosquito infected with dengue virus type 2. Materials and methods: C6/36 cells were infected in culture medium with the serum of a patient positively diagnosed for dengue 2. Subsequently, the cells were incubated for 10 days and the cytopathic effect was assessed. The cells were processed for immunofluorescence assays and transmission electron microscopy. Results: The immunofluorescence assays confirmed the presence of viral protein E associated with cellular syncytia in the culture. In the ultrastructural study, the infected cells showed vesicular-tubular structures and dilated cisterns of the endoplasmic reticulum at the cytoplasmic level. Viral particles were found exclusively in cytoplasm localized within the vacuoles. Nuclei of cellular syncytia showed membrane structures arranged in a circular shape and, in some cases, these syncytia displayed lysis; in no case viral particles were observed at the nuclear level. Conclusions: The ultrastructural alterations of nuclei in cells infected with the dengue virus using electron microscopy techniques had not been reported before, as far as we know. It is likely that such modifications are associated with replicative processes at an intranuclear level as an alternate replication mechanism.


RESUMEN Introducción. La replicación del virus del dengue se ha considerado principalmente citoplásmica; sin embargo, en diversos estudios se ha informado que algunos flavivirus pueden utilizar factores intranucleares como parte de la maquinaria que utilizan para aumentar la capacidad de infección en la célula huésped. En este trabajo se describen las alteraciones a nivel nuclear en células infectadas con dengue, probablemente involucradas en procesos de replicación viral. Objetivo. Presentar las observaciones ultraestructurales de células C6/36 de Aedes albopictus infectadas con el virus del dengue de tipo 2. Materiales y métodos. Se infectaron células C6/36 con suero de un paciente con diagnóstico de dengue 2; posteriormente, se mantuvieron en medio de cultivo durante 10 días y se evaluó el efecto citopático. Las células se procesaron para los ensayos de inmunofluorescencia y microscopía electrónica de transmisión, con el fin de hacer el estudio ultraestructural. Resultados. Los ensayos de inmunofluorescencia confirmaron la presencia de la proteína E viral asociada con sincitios celulares en el cultivo. En el estudio ultraestructural, las células infectadas tenían estructuras vesiculares y tubulares, y cisternas dilatadas del retículo endoplásmico en el citoplasma. Las partículas virales se encontraron exclusivamente en vacuolas localizadas en el citoplasma. Los núcleos de los sincitios celulares contenían estructuras de membrana dispuestas en forma circular y, en algunos casos, dichos sincitios presentaban lisis. En ningún caso se observaron partículas virales en el núcleo. Conclusiones. No se habían reportado alteraciones ultraestructurales en los núcleos de células infectadas con el virus del dengue detectadas mediante técnicas de microscopia electrónica. Es probable que tales modificaciones estén asociadas con procesos intranucleares de replicación como un mecanismo alternativo.


Subject(s)
Animals , Humans , Cell Nucleus/ultrastructure , Cytopathogenic Effect, Viral , Dengue Virus/physiology , Vacuoles/virology , Viremia/virology , Virus Replication , Microscopy, Electron , Giant Cells/virology , Cell Line , Viral Envelope Proteins/analysis , Aedes/cytology , Cytoplasm/virology , Dengue/virology , Dengue Virus/isolation & purification , Microscopy, Fluorescence
3.
Braz. j. microbiol ; 48(1): 125-131, Jan.-Mar. 2017. graf
Article in English | LILACS | ID: biblio-839349

ABSTRACT

Abstract Small ruminant lentiviruses isolated from peripheral blood leukocytes and target organs can be propagated in vitro in fibroblasts derived from goat synovial membrane cells. These cells are obtained from tissues collected from embryos or fetuses and are necessary for the establishment of the fibroblast primary culture. A new alternative type of host cells, derived from goat umbilical cord, was isolated and characterized phenotypically with its main purpose being to obtain cell monolayers that could be used for the diagnosis and isolation of small ruminant lentiviruses in cell culture. To accomplish this goal, cells were isolated from umbilical cords; characterized phenotypically by flow cytometry analysis; differentiate into osteogenic, chondrogenic and adipogenic lineage; and submitted to viral challenge. The proliferation of goat umbilical cord cells was fast and cell monolayers formed after 15 days. These cells exhibited morphology, immunophenotype, growth characteristics, and lineage differentiation potential similar to mesenchymal stem cells of other origins. The goat umbilical cord derived cells stained positive for vimentin and CD90, but negative for cytokeratin, CD34 and CD105 markers. Syncytia and cell lysis were observed in cell monolayers infected by CAEV-Cork and MVV-K1514, showing that the cells are permissive to small ruminant lentivirus infection in vitro. These data demonstrate the proliferative competence of cells derived from goat umbilical cords and provide a sound basis for future research to standardize this cell lineage.


Subject(s)
Animals , Umbilical Cord/cytology , Lentivirus/physiology , Mesenchymal Stem Cells/virology , Osteogenesis , Virus Replication , In Vitro Techniques , Goats , Biomarkers , Cell Differentiation , Cells, Cultured , Immunophenotyping , Cell Culture Techniques , Chondrogenesis , Cytopathogenic Effect, Viral , Adipogenesis , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/pathology
4.
Article in English | WPRIM | ID: wpr-311350

ABSTRACT

Enterovirus 71 is a neuroinvasive virus that is associated with severe neurological complications. We had earlier suggested that the replication capacity of a severe strain was higher than that of a mild strain. The recombinant 3CRV and 3CDRV virus strains were successfully rescued in our previous study. In the present study, we found no difference in virulence between 3CRV and severe strains. However, the capacity of replication and to cause cell injury of 3CDRV strain decreased in vitro, especially at 39.5 °C. Replacement of 3CD region in the severe strain led to milder symptoms, less body weight loss, and lower viral load in ICR mice. Histopathological findings indicated less severe injury in mice infected with 3CDRV strain. This study suggests that the 3CD region contributes to the attenuation of the severe strain, including its replication capacity and temperature sensitivity.


Subject(s)
Animals , Cytopathogenic Effect, Viral , Enterovirus A, Human , Genetics , Virulence , Enterovirus Infections , Pathology , Virology , Gene Expression Regulation, Viral , Mice , Mice, Inbred ICR , Mutation , Viral Load , Viral Proteins , Genetics , Metabolism , Virulence , Virus Replication
5.
Mem. Inst. Oswaldo Cruz ; 111(2): 106-113, Feb. 2016. tab, graf
Article in English | LILACS | ID: lil-772612

ABSTRACT

The influence of different infectious agents and their association with human papillomavirus (HPV) in cervical carcinogenesis have not been completely elucidated. This study describes the association between cytological changes in cervical epithelium and the detection of the most relevant aetiological agents of sexually transmitted diseases. Samples collected from 169 patients were evaluated by conventional cytology followed by molecular analysis to detect HPV DNA, Chlamydia trachomatis, herpes simplex virus 1 and 2,Neisseria gonorrhoeae, Mycoplasma genitalium, Trichomonas vaginalis, andTreponema pallidum, besides genotyping for most common high-risk HPV. An association between cytological lesions and different behavioural habits such as smoking and sedentariness was observed. Intraepithelial lesions were also associated with HPV and C. trachomatis detection. An association was also found between both simple and multiple genotype infection and cytological changes. The investigation of HPV and C. trachomatisproved its importance and may be considered in the future for including in screening programs, since these factors are linked to the early diagnosis of patients with precursor lesions of cervical cancer.


Subject(s)
Adolescent , Adult , Female , Humans , Middle Aged , Young Adult , Cervix Uteri/microbiology , Chlamydia trachomatis/isolation & purification , DNA, Viral/isolation & purification , Papillomaviridae/isolation & purification , Squamous Intraepithelial Lesions of the Cervix/microbiology , Uterine Cervical Neoplasms/prevention & control , Carcinogenesis , Coinfection , Cross-Sectional Studies , Cytopathogenic Effect, Viral , Cervix Uteri/pathology , Chlamydia Infections/complications , Chlamydia Infections/epidemiology , Early Detection of Cancer/methods , Epithelium/virology , Genotype , Genotyping Techniques , Herpesvirus 1, Human/isolation & purification , /isolation & purification , Molecular Typing , Mycoplasma genitalium/isolation & purification , Neisseria gonorrhoeae/isolation & purification , Papillomaviridae/classification , Papillomavirus Infections/complications , Papillomavirus Infections/epidemiology , Risk Factors , Treponema pallidum/isolation & purification , Trichomonas vaginalis/isolation & purification , Uterine Cervical Neoplasms/microbiology
6.
Mem. Inst. Oswaldo Cruz ; 111(2): 120-127, Feb. 2016. tab
Article in English | LILACS | ID: lil-772616

ABSTRACT

This study investigated the rate of human papillomavirus (HPV) persistence, associated risk factors, and predictors of cytological alteration outcomes in a cohort of human immunodeficiency virus-infected pregnant women over an 18-month period. HPV was typed through L1 gene sequencing in cervical smears collected during gestation and at 12 months after delivery. Outcomes were defined as nonpersistence (clearance of the HPV in the 2nd sample), re-infection (detection of different types of HPV in the 2 samples), and type-specific HPV persistence (the same HPV type found in both samples). An unfavourable cytological outcome was considered when the second exam showed progression to squamous intraepithelial lesion or high squamous intraepithelial lesion. Ninety patients were studied. HPV DNA persistence occurred in 50% of the cases composed of type-specific persistence (30%) or re-infection (20%). A low CD4+T-cell count at entry was a risk factor for type-specific, re-infection, or HPV DNA persistence. The odds ratio (OR) was almost three times higher in the type-specific group when compared with the re-infection group (OR = 2.8; 95% confidence interval: 0.43-22.79). Our findings show that bonafide (type-specific) HPV persistence is a stronger predictor for the development of cytological abnormalities, highlighting the need for HPV typing as opposed to HPV DNA testing in the clinical setting.


Subject(s)
Adult , Female , Humans , Pregnancy , Young Adult , DNA, Viral/classification , HIV , HIV Seropositivity/virology , Papillomaviridae/isolation & purification , Papillomavirus Infections/diagnosis , Pregnancy Complications, Infectious/virology , Squamous Intraepithelial Lesions of the Cervix/diagnosis , Chronic Disease , Coinfection , Cytopathogenic Effect, Viral , DNA, Viral/isolation & purification , HIV , Longitudinal Studies , Molecular Typing/methods , Phylogeny , Predictive Value of Tests , Prospective Studies , Papillomaviridae/classification , Papillomavirus Infections/virology , Recurrence , Risk Factors , Reproductive Tract Infections/virology , Socioeconomic Factors
7.
Braz. j. microbiol ; 46(1): 279-283, 05/2015. graf
Article in English | LILACS | ID: lil-748263

ABSTRACT

Bovine Herpesvirus 4 (BoHV-4) is a member of Gammaherpesvirinae sub-family and belongs to genus Rhadinovirus. This virus has been associated with different clinical manifestations and research activity has put forward a strong correlation among virus infection, postpartum metritis, and abortion. The goal of this work was to characterize a virus strain isolate from a cow’s uterine outflow. From swabs drawn of uterine secretion, a virus strain was isolated and characterized by its cytopathology, morphology, and molecular biology approaches. In culture there was CPE development, characterized mainly by long strands with several small balloons along them, radiated from infected cells. Electron microscopy analysis revealed virus particles that had icosahedrical capsid symmetry surrounded by a loose envelope, typical of a herpesvirus. A 2,571 bp PCR product after HindIII digestion generated four fragments, whose base pair composition were 403, 420, 535, and 1,125 bp. Restriction enzymes HindIII and BamHI generated the expected diagnostic bands as well as a 2,350 bp hypermolar fragment as a result of BamHI treatment to demonstrate that agent was a bovine herpesvirus 4, appertaining to DN-599 group.


Subject(s)
Animals , Cattle , Female , Cattle Diseases/virology , Herpesviridae Infections/veterinary , /classification , /isolation & purification , Tumor Virus Infections/veterinary , Brazil , Cytopathogenic Effect, Viral , DNA, Viral/genetics , DNA, Viral/metabolism , Exudates and Transudates/virology , Herpesviridae Infections/virology , /genetics , Microscopy, Electron, Transmission , Polymorphism, Restriction Fragment Length , Tumor Virus Infections/virology , Uterus/pathology , Uterus/virology , Virus Cultivation , Virion/ultrastructure
8.
Mycobiology ; : 189-192, 2014.
Article in English | WPRIM | ID: wpr-729225

ABSTRACT

During a search for neuraminidase inhibitors derived from medicinal fungi, we found that the fermentation broth of Phellinus linteus exhibited potent neuraminidase inhibitory activity. Through bioassay-guided fractionation, two active compounds were purified from the ethyl acetate-soluble portion of the fermentation broth of P. linteus. These structures were identified as inotilone (1) and 4-(3,4-dihydroxyphenyl)-3-buten-2-one (2) by spectroscopic methods. Compounds 1 and 2 inhibited H1N1 neuraminidase activity with IC50 values of 29.1 and 125.6 microM, respectively, in a dose-dependent manner. They also exhibited an antiviral effect in a viral cytopathic effect reduction assay using MDCK cells. These results suggest that compounds 1 and 2 from the culture broth of P. linteus would be good candidates for the prevention and therapeutic strategies towards viral infections.


Subject(s)
Cytopathogenic Effect, Viral , Fermentation , Fungi , Inhibitory Concentration 50 , Madin Darby Canine Kidney Cells , Neuraminidase
9.
Chinese Journal of Virology ; (6): 357-363, 2013.
Article in Chinese | WPRIM | ID: wpr-339945

ABSTRACT

To study the proliferation characteristics of PPV in differently infected way and the variance of concentrations in different cells. A strain of porcine parvovirus(PPV) was adapted to PK-15 cells, and a Real-time fluorescent quantitative PCR (FQ-PCR) assay was developed based on the specific region of the NS1 gene of PPV to quantify the PPV. The FQ-PCR was used to measure the viral concentration of virus-infected cells by simultaneous or step by step inoculation and plot one-step growth curves. The proliferation characteristics of PPV strain in different cells lines (HeLa, MDBK, PK-15 ,ST, F81, BHK-21 and Marc-145) was also compared. The results showed the PK-15 cell -adapted strain of PPV produced CPE after 12 passages, and maintained stable CPE at the following 10 messages. The one-step growth curve showed that the virus concentration of simultaneous inoculation was higher than that of the step-by-step inoculation, and the proliferation cycle of step-by-step inoculation was shorter. The proliferation ability of PPV strain in different cells showed that CPE appeared first inPK-15, followed by ST, HeLa and MDBK, and the virus concentration was highest in ST, followed byPK-15, MDBK and HeLa. NO proliferation was observed in F81, BHK-21 and Marc-145 cells. These findings lay a material foundation for the basic researches on PPV and the development of vaccine.


Subject(s)
Animals , Cell Line , Cricetinae , Cytopathogenic Effect, Viral , DNA, Viral , Genetics , Female , Haplorhini , Humans , Male , Parvoviridae Infections , Virology , Parvovirus, Porcine , Genetics , Physiology , Real-Time Polymerase Chain Reaction , Reproducibility of Results , Sensitivity and Specificity , Swine , Viral Proteins , Genetics , Virus Replication
10.
Chinese Journal of Virology ; (6): 392-397, 2013.
Article in Chinese | WPRIM | ID: wpr-339939

ABSTRACT

This research aims to evaluate the application of Real - time cell assay (RTCA) based on microelectronics sensor technology in the detection of HEV71 induced cell lesion. Growth indexes of RD cells at different stages were observed dynamically, appropriate cell concentration was selected to test HEV71 infectivity and to determine the HEV71 neutralizing antibody titer in serum. The traditional microplate test was used as methodology comparison and results validation at the same time. Cell impedance was transformed to cell index (CI) value and visual dynamic curve through software, and the result showed that the observation of HEV71 infectivity was more than 5d when the RD cells concentration was 1. 5 X 10(4) hole on the 96 electronic orifice plate. Compared with the traditional cytopathic effect (CPE) through microscope observation method, the end point judgment results were consistent between these two methods at 132h (about 5. 5d) post virus inoculation. In the neutralization tests, three CI values of neutralizing antibody titers against HEV71 in human serum were correspond to those obtained from traditional 96 microplate microscopy. RTCA also suggested that the presentation time of CPE induced by the i virus could be different even the end point judgment was the same with the same neutralization antibody titer. Compared with the traditional microplate monitoring method, RTCA can save labor and eliminate the hands-on error in the monitoring HEV71 infectivity and antibody titer detection in serum. RTCA can be served as one of the supplementary methods of traditional detection method, with the advantages of dynamically observing the occurrence and development of cell pathological changes, and the variation of virus infectivity and serum neutralizing antibodies.


Subject(s)
Antibodies, Neutralizing , Blood , Antibodies, Viral , Blood , Cell Line, Tumor , Cytopathogenic Effect, Viral , Electric Impedance , Enterovirus A, Human , Allergy and Immunology , Virulence , Enterovirus Infections , Virology , Humans , Microelectrodes , Neutralization Tests , Methods
11.
Pesqui. vet. bras ; 32(7): 653-657, jul. 2012. tab
Article in English | LILACS | ID: lil-644572

ABSTRACT

A retrospective study of 24 cases of papillomas in dogs was performed from January 2001 to March 2011. Additionally, immunohistochemistry (IHC) was used to characterize and evaluate the samples. We found that disease was observed more in mixed breed dogs, ages ranging from 6 months to 10 years (mean 3.1 years), and there was no gender predilection. The main lesion sites were the skin (75%), lips (16.7%), and eyelids (8.3%). Upon histological evaluation, we observed papillary exophytic proliferation of squamous epithelium and papillary endophytic proliferation (inverted) in 87.5% and 12.5% of cases, respectively. The tumors were characterized by spinous layer hyperplasia (87.5%) with koilocytes (70.8%) and intranuclear pale basophilic inclusions bodies (8.3%), prominent granular layer with large amounts of keratohyalin granules (95.8%), and hyperkeratosis in the stratum corneum (100%). Positive immunostaining for Papillomavirus was found in 83.3% of cases, which were distributed between the granular layer and the stratum corneum. These findings indicate the following: that papillomas in dogs are caused by Papillomavirus, the viral cytopathic effect induces epithelial lesions, viral particles are found inside the cell nuclei, and inclusions bodies are rare.


Foi realizado um estudo retrospectivo de 24 casos de papilomas em cães diagnosticados no período de janeiro 2001 a março de 2011, bem como a sua caracterização imuno-histoquímica (IHQ). Cães sem raça definida foram os mais afetados, a idade média foi de 3,1 anos, com variação de 6 meses a 10 anos e não houve predileção sexual. Quanto à localização das lesões, 75,0% estavam na pele, 16,7% no lábio e 8,3% em pálpebra. Na avaliação histológica havia proliferação papilar exofítica do epitélio escamoso em 87,5% e papilar endofítica (invertido) em 12,5%. O tumor era caracterizado por hiperplasia do estrato espinhoso (87,5%) com coilócitos (70,8%) e inclusões intranucleares basofílicas pálidas (8,3%); o estrato granular estava proeminente com grande quantidade de grânulos de querato-hialina (95,8%); e havia hiperqueratose do estrato córneo (100%). Na avaliação IHQ para Papillomavirus houve marcação nos estratos granuloso e córneo em 83,3%. Estes achados indicam que os papilomas em cães são causados por Papillomavirus, as lesões epiteliais são decorrentes do efeito citopático viral, as partículas virais estão no núcleo das células e corpúsculos de inclusão são raros.


Subject(s)
Animals , Dogs , Immunohistochemistry , Papillomavirus Infections/veterinary , Lambdapapillomavirus/isolation & purification , Papilloma/diagnosis , Cytopathogenic Effect, Viral
12.
Braz. j. infect. dis ; 16(1): 19-26, Jan.-Feb. 2012. ilus
Article in English | LILACS | ID: lil-614545

ABSTRACT

OBJECTIVE: Epstein-Barr virus (EBV) is a ubiquitous human γ-herpes virus, which can adapt and evade host immune defense. Dendritic cells (DCs) play a pivotal role in the initiation and maintenance of immune responses. This study investigated the effects of EBV on cord blood monocytes derived DCs (CBDC). METHODS: Monocytes were isolated from cord blood and cultured in medium containing recombinant IL-4 and GM-CSF to induce DCs development. B95-8 supernatant was added in monocytes culture medium for EBV infection at day 0. Phenotypic characterization of DCs, apoptotic cells, and mitochondrial membrane potential (MMP) were detected by flow cytometry. The morphology was observed by Hoechst 33258 staining and TUNEL staining, the expression of X-linked inhibitor of apoptosis protein (XIAP) was detected by Western blotting assay and caspase 3, 8 and 9 activity was measured. RESULTS: Phenotypic characterization of DCs was changed in EBV-treated group. Chromatin condensation and DNA fragmentation were observed in EBV induced CBDC apoptosis. In addition, caspase 3, caspase 8, and caspase 9 activation were enhanced in the EBV-treated group. This was accompanied by the loss of MMP. Furthermore, XIAP expression was down-regulated in the EBV-treated group and compared to mock-infected group. CONCLUSION: These results suggested that EBV could inhibit CBDC phenotypic differentiation, and induce CBDC apoptosis in caspase-dependent manner with involvement of the mitochondrial pathway. This might help EBV to evade host immune responses to establish persistent infection.


Subject(s)
Humans , Apoptosis/physiology , Cytopathogenic Effect, Viral/physiology , Dendritic Cells/pathology , Fetal Blood/cytology , /physiology , Monocytes/pathology , Blotting, Western , Cell Differentiation , Caspases/immunology , Dendritic Cells/virology , Flow Cytometry , /immunology , /immunology , Monocytes/cytology , Monocytes/virology , Phenotype , X-Linked Inhibitor of Apoptosis Protein/immunology
13.
Acta Pharmaceutica Sinica ; (12): 116-123, 2012.
Article in Chinese | WPRIM | ID: wpr-323070

ABSTRACT

This study is to report the preparation of complexes of Ad5 and anionic liposomes (AL-Ad5), the amplification of adenoviruses with enhanced green fluorescent protein (eGFP) reporter gene performed by HEK 293 cells, the adenoviral vectors purified by cesium chloride gradient centrifugation, and the titer of adenovirus determined by cytopathic effect (CPE) method, hexon capsid immunoassay and quantitative-PCR (Q-PCR), separately. The prescription and experiment conditions were optimized by central composite design (CCD). The complexes of Ad5 and AL-Ad5 were formulated by the calcium-induced phase change method. The morpholopy, particle size and zeta potential were detected by dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively. Additionally, the bicolourable fluoresce-labeled complexes (F(labeled)-AL-Ad5) were prepared and their intracellular location in MDCK cells was detected by confocal laser scanning microscopy (CLSM). The results indicate that the complexes of AL-Ad5 exhibited a uniform distribution with a particle size of 211 +/- 10 nm and a zeta potential of -41.2 +/- 2.2 mV. The result of CLSM demonstrates that the intracellular location of red fluoresce-labeled adenovirus was consistent with that of green fluoresce-labeled liposomes suggesting that the naked adenovirus was well encapsulated by the anionic liposomes in complexes of AL-Ad5.


Subject(s)
Adenoviridae , Genetics , Animals , Anions , Cytopathogenic Effect, Viral , Dogs , Drug Compounding , Methods , Genetic Vectors , Green Fluorescent Proteins , Chemistry , HEK293 Cells , Humans , Liposomes , Chemistry , Pharmacokinetics , Madin Darby Canine Kidney Cells , Microscopy, Confocal , Microscopy, Electron, Transmission , Particle Size , Polymerase Chain Reaction , Methods , Recombinant Fusion Proteins
14.
Chinese Journal of Virology ; (6): 326-330, 2011.
Article in Chinese | WPRIM | ID: wpr-286034

ABSTRACT

To investigate biological characteristics of the IVpi-189 progeny virus derived from the culture of influenza A virus as a live-attenuated vaccine candidate. Persistent infection of a cultured cell line with influenza A virus (MDCK-IVpi) was established by incubating continuously influenza virus-infected cells at a lower temperature. The infectious progeny virus derived from MDCK-IVpi cells at the 189rd subculture was designated as the IVpi-189 strain of influenza virus. The cytopathic effect induced by IVpi-189 virus was observed under different temperature conditions. The production of infectious progeny virus was examined at 38 and 32 degrees C by plaque titration of cell-associated and released virus. IVpi-189 virus showed cytopathic effect as strong as that of IVwt in infected cell line of MDCK at 32 degrees C. However, when culture temperature was raised to 38 degrees C, the cytopathic effect induced by IVpi-189 virus was delayed and less pronounced. Virus growth in IVpi-189 virus-infected cells at 38 degrees C was significantly reduced as compared with that of IVwt virus, although both viruses yielded nearly equivalent high titers of cell-associated and released virus at 32 degrees C. The reasons of the decreased proliferative ability of IVpi-189 virus at high culture temperature were unrelated with virus inactivation or the release of progeny virus, but associated with the decreased replication of infectious progeny virus in the infected cells. IVpi-189 virus derived from MDCK cells infected persistently with influenza A virus showed biological characteristics as a potential live-attenuated vaccine candidate.


Subject(s)
Animals , Cell Line , Cytopathogenic Effect, Viral , Dogs , Humans , Influenza A virus , Genetics , Physiology , Temperature , Virus Cultivation , Methods , Virus Replication
15.
Biomédica (Bogotá) ; 30(2): 276-282, jun. 2010. ilus, tab
Article in Spanish | LILACS | ID: lil-560970

ABSTRACT

Introducción. Los virus entéricos se han visto implicados en brotes de enfermedad diarreica aguda, enfermedades transmitidas por alimentos, hepatitis A y meningitis aséptica, en los que el vehículo de transmisión del agente ha sido el agua.Objetivo. Estandarizar un método de concentración para la detección de virus entéricos en aguas de consumo.Materiales y métodos. Se concentraron 20 litros de agua a un volumen de 6 ml mediante filtración y ultrafiltración tangencial. Como controles positivos se prepararon soluciones de 20 litros a concentraciones virales de 1, 10, 50 y 100 TCID50 (Tissue Culture Infectious Dose 50%) de Poliovirus Sabin de tipo 1. Las partículas virales fueron recuperadas por cultivo en células sensibles a la infección e identificadas por amplificación del genoma viral mediante reacción en cadena de la polimerasa, siguiendo los estándares internacionales de los Centers for Disease Control and Prevention (CDC) de Atlanta. Resultados. Todos los controles positivos causaron efecto citopático en células de rabdomiosarcoma y L20B y fueron detectados por RT- PCR (Real Time- PCR) convencional, directamente de las muestras. Los controles negativos no mostraron efecto citopático ni amplificación viral por RT-PCR.Conclusiones. La ultrafiltración tangencial mostró ser un método rápido y eficaz al recuperar virus desde una TCID50, además de ser reproducible y sencillo. Tiene la ventaja de permitir la detección de su capacidad de contagiosidad viral por el cultivo celular, y la identificación por RT-PCR.


Introduction. Enteric viruses have been implicated in acute diarrheal disease, food-borne disease, hepatitis A and meningitis outbreaks, in which water was the vehicle of transmission.Objective. A concentration method was standardized for the detection of enteric viruses in drinking water. Materials and methods. Twenty liters of water were concentrated to 6 ml by filtration and tangential ultrafiltration. Viral solutions of 20 L each were prepared at 1, 10, 50 and 100 TCID50 of Sabin poliovirus type 1 as positive controls. Viral particles were recovered by tissue culture and detected by conventional polymerase chain reaction (PCR), according to the international standards recommended by the Enterovirus Laboratory at the Centers for Disease Control and Prevention, Atlanta, GA. Results. All positive controls showed cytopathic effect on L20B and RD cells and were amplified by conventional PCR directly from samples. Negative controls did not show any amplification or viral cytopathic effect. Conclusions. Tangential ultrafiltration for concentrating viruses proved to be a fast, efficient recovery and reproducible. It has the advantage of allowing the detection (at the 1 TCID50 level) and identification of viruses by RT-PCR and the demonstration of viral infectivity by tissue culture.


Subject(s)
Cytopathogenic Effect, Viral , Enterovirus , Poliovirus , Reverse Transcriptase Polymerase Chain Reaction , Water Quality
16.
Rev. bras. anal. clin ; 42(4): 265-267, 2010. ilus, tab
Article in Portuguese | LILACS | ID: lil-579393

ABSTRACT

O vírus, contrariamente a outras classes de agentes que afetam o organismo, não são observáveis em microscopia óptica, refletindo-se apenas microscopicamente em efeitos virais (ECPV), que permitem identificá-los por constituírem um conjunto de alterações patognomônicas a nível celular. O aumento nuclear, a variação da morfologia celular, a perda do padrão da cromatina, as inclusões intranucleares ou intracitoplasmáticas ou a multinucleação, são alguns dos ECPV mais comuns. Entre os vírus que afetam a mucosa genital na família Herpesviridae, inserem-se Herpes Simplex 2 e o Citomegalovirus, cujo diagnóstico deverá ter em conta a complexidade dos mecanismos de replicação. Assim, e apesar dos vários métodos para a detecção do vírus, a citologia continua a ser um método viável para o diagnóstico destas infecções virais, sendo que a compreensão do processo de infecção contribui para a correta avaliação microscópica. Este trabalho tem como finalidade efetuar uma revisão bibliográfica sobre o grupo Herpes, focando o Herpes Simplex 2 e o Citomegalovirus, assim como, explicar e relacionar os ECPV, associados ao grupo Herpes , tendo por base os mecanismos de infecção.


Subject(s)
Cell Biology , Cytomegalovirus , Cytopathogenic Effect, Viral , Herpesviridae , Simplexvirus
17.
Article in English | WPRIM | ID: wpr-360594

ABSTRACT

<p><b>OBJECTIVE</b>To perform gene expression profiles comparison so that to identify and understand the potential differences in pathogenesis between the pandemic and seasonal A (H1N1) influenza viruses.</p><p><b>METHODS</b>A549 cells were infected with A/California/07/09 (H1N1) and A/GuangdongBaoan/51/08 (H1N1) respectively at the same MOI of 2 and collected at 2, 4, 8, and 24 h post infection (p.i.). Gene expression profiles of A549 cells were obtained using the 22 K Human Genome Oligo Array, and differentially expressed genes were analyzed at selected time points.</p><p><b>RESULTS</b>Microarrays results indicated that both of the viruses suppressed host immune response related pathways including cytokine production while pandemic H1N1 virus displayed weaker suppression of host immune response than seasonal H1N1 virus. Observation on similar anti-apoptotic events such as activation of apoptosis inhibitor and down-regulation of key genes of apoptosis pathways in both infections showed that activities of promoting apoptosis were different in later stage of infection.</p><p><b>CONCLUSIONS</b>The immuno-suppression and anti-apoptosis events of pandemic H1N1 virus were similar to those seen by seasonal H1N1 virus. The pandemic H1N1 virus had an ability to inhibit biological pathways associated with cytokine responses, NK activation and macrophage recognition.</p>


Subject(s)
Apoptosis , Genetics , Cell Line, Tumor , Cytopathogenic Effect, Viral , Disease Outbreaks , Down-Regulation , Epithelial Cells , Metabolism , Virology , Gene Expression , Gene Expression Profiling , Humans , Immunity, Innate , Genetics , Influenza A Virus, H1N1 Subtype , Classification , Virulence , Influenza, Human , Epidemiology , Genetics , Allergy and Immunology , Virology , Oligonucleotide Array Sequence Analysis , Pandemics , Seasons , Up-Regulation , Virulence
18.
Acta Pharmaceutica Sinica ; (12): 436-441, 2010.
Article in Chinese | WPRIM | ID: wpr-353378

ABSTRACT

Human rhinoviruses (HRVs) are the causative pathogens in more than half of viral upper respiratory tract infections. Currently, no antiviral agents that are active against HRVs are available for clinical use. Because only higher primates are susceptible to HRVs, the screening of new drug is most commonly based on the cell line model. In this study, isolated rabbit airway smooth muscles (ASM) tissue model has been established, and the airway responsiveness with different treatment has been examined. Relative to control tissues, the maximal constrictor (Tmax) response to ACh increased significantly 150% in ASM inoculated with HRV, and relaxation to isoproterenol has been attenuated to 63%. And the abnormal responsiveness can be inhibited in presence of pretreatment with several new compounds which have been exhibited effective anti-HRV activity on cell lines. The results demonstrate that the established ASM model will be applied to screening the anti-HRVs drugs.


Subject(s)
Acetylcholine , Pharmacology , Animals , Antiviral Agents , Pharmacology , Cytopathogenic Effect, Viral , HeLa Cells , Humans , Isoproterenol , Pharmacology , Muscle Contraction , Muscle Relaxation , Muscle, Smooth , Virology , Piperidines , Pharmacology , Pyridazines , Pharmacology , Rabbits , Rhinovirus , Trachea , Virology
19.
Acta Pharmaceutica Sinica ; (12): 395-398, 2010.
Article in Chinese | WPRIM | ID: wpr-250573

ABSTRACT

Influenza virus is a virus causing upper respiratory tract infection disease with high morbidity and mortality. China is considered as an area with high rate of influenza morbidity. Prevention and treatment of influenza currently rely on vaccines and antiviral agents in the world. In addition, traditional Chinese medicines also have been used in clinical for influenza therapy. In vitro anti-influenza virus activities of 10 traditional Chinese medicines were studied by cytopathic effect (CPE). Qingre Jiedu oral liquid (factory H) had strong antiviral activity against influenza virus A/Guangdong Luohu/219/2006 (H1N1); Yinhuang oral liquid had strong antiviral activity against influenza virus A/Hanfang/359/95 and A/Yuefang/243/72 (H3N2). Qingkailing oral liquid (factory G) had strong antiviral activity against influenza virus A/Jifang/15/90 (H3N2). Qingre Jiedu oral liquid (factory H) had strong antiviral activity against influenza virus A/Jifang/15/90, A/Yuefang/243/72 (H3N2) and virus B.


Subject(s)
Administration, Oral , Animals , Antiviral Agents , Pharmacology , Bridged Bicyclo Compounds, Heterocyclic , Pharmacology , Cell Line , Chlorogenic Acid , Pharmacology , Cytopathogenic Effect, Viral , Dogs , Drugs, Chinese Herbal , Pharmacology , Flavonoids , Pharmacology , Indoles , Pharmacology , Influenza A Virus, H1N1 Subtype , Influenza A Virus, H3N2 Subtype , Influenza B virus , Iridoids , Pharmacology
20.
Acta Pharmaceutica Sinica ; (12): 403-407, 2010.
Article in Chinese | WPRIM | ID: wpr-250571

ABSTRACT

Ribavirin is a broad-spectrum inhibitor against several unrelated DNA or RNA viruses in vitro and in vivo. In this paper the in vitro and in vivo study of anti-influenza virus activity of ribavirin (RBV) injection had been reported. The in vitro antiviral activity of ribavirin injection against influenza virus A and B was studied by CPE. The in vivo protective action of ribavirin injection against influenza A/FM/1/47(H1N1) mouse adapted strain infected mouse was studied with mouse model. The results showed ribavirin injection has strong inhibitory activity against 7 virus strains tested in vitro. Ribavirin injection could significantly increase virus infected mouse survival rate and survival days and improve lung pathogen and lung index.


Subject(s)
Animals , Antiviral Agents , Pharmacology , Therapeutic Uses , Cell Line , Cytopathogenic Effect, Viral , Dogs , Female , Influenza A Virus, H1N1 Subtype , Influenza A Virus, H3N2 Subtype , Influenza B virus , Injections , Lung , Pathology , Mice , Orthomyxoviridae Infections , Drug Therapy , Pathology , Ribavirin , Pharmacology , Therapeutic Uses
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