RESUMO
Total nucleic acids from sporulated oocysts of Eimeria tenella isolated from Changchun in China were found to contain three extrachromosomal double-stranded RNA segments (dsRNAs) of 1.4, 2.4 and 3.6 kb in sizes. These RNAs were resistant to RNase A digestion under high salt concentration (0.3 M NaCl). RNA-dependent RNA polymerase (RDRP) activity was detected in crude extracts of E. tenella sporulated oocysts containing these nucleic acid species. Virus-like particles (VLPs) were shown to have a diameter of approximately 38 nm under Electron Microscopy (EM) after purification by sucrose density gradient centrifugation. In keeping with the nomenclature generally adopted for protozoan viruses, we have named this isolate as E. tenella virus (ETV) which is the first virus isolated from E. tenella.
Assuntos
Eimeria tenella/virologia , Vírus de RNA/genética , RNA de Cadeia Dupla/análise , RNA Viral/análise , Vírion/genética , Animais , Galinhas , Coccidiose/parasitologia , Coccidiose/veterinária , Eimeria tenella/genética , Eletroforese em Gel de Ágar , Microscopia Eletrônica , Oocistos/virologia , Doenças das Aves Domésticas/parasitologia , Vírus de RNA/enzimologia , Vírus de RNA/isolamento & purificação , Vírus de RNA/ultraestrutura , RNA de Cadeia Dupla/isolamento & purificação , RNA Viral/isolamento & purificação , RNA Polimerase Dependente de RNA/metabolismo , Organismos Livres de Patógenos Específicos , Vírion/enzimologia , Vírion/isolamento & purificação , Vírion/ultraestruturaRESUMO
Monoclonal antibodies (MAb) were prepared against the 40-kDa capsid protein of Cryptosporidium parvum virus (CPV) by immunizing mice with purified recombinant CPV40 protein. In immunoblotting analysis, MAbCPV40-1 bound to a 40-kDa protein in extracts of C. parvum oocysts. This 40-kDa protein was localized in the sporozoite cytoplasm by immunofluorescence (IFA) staining with MAbCPV40-1. In a dot-blot assay, MAbCPV40-1 was capable of detecting 10(2) non-bleach-treated and 10(2)-10(3) bleach-treated C. parvum oocysts. MAbCPV40-1 was capable of detecting CPV40 antigen in both soluble and total C. parvum oocyst protein extracts, indicating a potential use for detecting this parasite in environmental samples.
Assuntos
Anticorpos Monoclonais/imunologia , Proteínas do Capsídeo/imunologia , Cryptosporidium parvum/isolamento & purificação , Immunoblotting/métodos , Vírus de RNA , Animais , Bovinos , Cryptosporidium parvum/virologia , Eletroforese em Gel de Poliacrilamida , Imunofluorescência , Hibridomas , Camundongos , Camundongos Endogâmicos BALB C , Oocistos/virologia , Vírus de RNA/química , Vírus de RNA/imunologia , Proteínas Recombinantes/imunologia , Sensibilidade e Especificidade , SimbioseRESUMO
Study of the mechanism of transmission of hepatitis B virus is important for public health. An improved experimental model is described for studying vertical transmission of hepatitis B virus (HBV) via human spermatozoa. Recombinant plasmid pIRES2-EGFP-HBx which would express enhanced green fluorescent protein (EGFP) used as a marker for the expression of hepatitis B virus X (HBx) gene was constructed successfully and confirmed by PCR, EcoR I and Sal I digestion, and DNA sequencing. After exposure to the plasmid, human spermatozoa were used to fertilize zona-free hamster ova in vitro. Two-cell embryos were collected and classified into group A with green fluorescence and group B without green fluorescence under fluorescence microscope. The results showed that HBx DNA positive bands were detected in the embryos with green fluorescence (PCR and RT-PCR) and positive controls (PCR) indicating presence and expression of HBx gene. In contrast, HBx gene expression was not detected in the embryos without green fluorescence and negative controls (PCR and RT-PCR). This improved experimental model is more efficient, accurate and reliable for studying further perinatal transmission of HBV or other viruses causing chronic human disease possibly via the male germ line.
Assuntos
Modelos Animais de Doenças , Vírus da Hepatite B/fisiologia , Hepatite B/transmissão , Transmissão Vertical de Doenças Infecciosas , Espermatozoides/virologia , Animais , Cricetinae , Embrião de Mamíferos/virologia , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hepatite B/virologia , Vírus da Hepatite B/genética , Vírus da Hepatite B/metabolismo , Humanos , Masculino , Mesocricetus , Oocistos/virologia , Plasmídeos , Recombinação Genética , Transativadores/genética , Transativadores/metabolismo , Proteínas Virais Reguladoras e AcessóriasRESUMO
Differences in the virulence and fecundity of Cryptosporidium parvum isolates have been observed by several researchers studying cryptosporidiosis. The purpose of the present study was to determine if there was a correlation between intracellular levels of the viral symbiont CPV in C. parvum and fecundity of two isolates of the parasite, namely C. parvum Beltsville (B) and C. parvum Iowa (I). Dairy calves infected with 10(6)C. parvum-B excreted 5-fold more oocysts compared with calves infected with the same number of C. parvum-I oocysts. The increased fecundity of the former strain was corroborated by semi-quantitative PCR assay of DNA isolated from cell cultures infected with either C. parvum-B or C. parvum-I. Quantitative reverse transcriptase-PCR analysis of viral RNA revealed a 3-fold greater number of CPV in C. parvum-B compared with C. parvum-I oocysts. These findings may indicate a role for CPV in fecundity and possibly virulence of C. parvum.
Assuntos
Doenças dos Bovinos/parasitologia , Criptosporidiose/veterinária , Cryptosporidium parvum/fisiologia , Cryptosporidium parvum/virologia , Fezes/parasitologia , Fertilidade , Animais , Sequência de Bases , Bovinos , Sobrevivência Celular , Criptosporidiose/parasitologia , Cryptosporidium parvum/isolamento & purificação , DNA de Protozoário/isolamento & purificação , Dados de Sequência Molecular , Oocistos/virologia , Contagem de Ovos de Parasitas , RNA Mensageiro/isolamento & purificação , RNA de Protozoário/isolamento & purificação , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SimbioseRESUMO
The purpose of this study was to determine if the viral symbiont of Cryptosporidium parvum (CPV) sporozoites could be used as a target for sensitive detection of the parasite in food samples. Polyclonal sera specific to the recombinant viral capsid protein (rCPV40) was used in a dot blot hybridization assay to detect oocysts recovered from green onions and cilantro. Small batches of chopped green onions and cilantro leaves were artificially contaminated with three different concentrations of oocysts: 10(6), 10(2), and 10(1). rCPV40 was superior in detecting oocysts compared with other antibodies directed toward total oocyst protein and oocyst surface antigens. This study provides evidence that CPV is an excellent target for sensitive detection of C. parvum oocysts in foods.
Assuntos
Antígenos Virais/genética , Coriandrum/parasitologia , Cryptosporidium parvum/isolamento & purificação , Cryptosporidium parvum/virologia , Parasitologia de Alimentos , Cebolas/parasitologia , Oocistos/isolamento & purificação , Animais , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Contaminação de Alimentos/análise , Oocistos/virologia , Contagem de Ovos de Parasitas , Sensibilidade e EspecificidadeRESUMO
The purpose of this study was to characterize the viral symbiont (CPV) of Cryptosporidium parvum sporozoites and evaluate the CPV capsid protein (CPV40) as a target for sensitive detection of the parasite. Recombinant CPV40 was produced in Escherichia coli, purified by affinity chromatography, and used to prepare polyclonal rabbit sera specific for the viral capsid protein. Anti-rCPV40 recognized a 40 kDa and a 30 kDa protein in C. parvum oocysts and appeared to localize to the apical end of the parasite. Anti-rCPV40 serum was capable of detecting as few as 1 C. parvum oocyst in a dot blot assay, the sensitivity being at least 1000-fold greater than sera reactive with total native C. parvum oocyst protein or specific for the 41 kDa oocyst surface antigen. Water samples were seeded with C. parvum oocysts and incubated at 4, 20, or 25 degrees C for greater than 3 months to determine if CPV levels were correlated with oocyst infectivity. Samples were removed monthly and subjected to mouse and cell culture infectivity, as well as PCR analysis for infectivity and viral particle presence. While sporozoite infectivity declined by more than 75% after 1 month at 25 degrees C, the CPV signal was similar to that of control samples at 4 degrees C. By 3 months at 20 degrees C, the C. parvum oocysts were found to be non-infectious, but retained a high CPV signal. This study indicates that CPV is an excellent target for sensitive detection of C. parvum oocysts in water, but may persist for an indefinite time after oocysts become non-infectious.
