RESUMO
BACKGROUND: Occurrence of extrachromosomal dsRNA elements has been described in the red-yeast Xanthophyllomyces dendrorhous, with numbers and sizes that are highly variable among strains with different geographical origin. The studies concerning to the encapsidation in viral-like particles and dsRNA-curing have suggested that some dsRNAs are helper viruses, while others are satellite viruses. However, the nucleotide sequences and functions of these dsRNAs are still unknown. In this work, the nucleotide sequences of four dsRNAs of the strain UCD 67-385 of X. dendrorhous were determined, and their identities and genome structures are proposed. Based on this molecular data, the dsRNAs of different strains of X. dendrorhous were analyzed. RESULTS: The complete sequences of L1, L2, S1 and S2 dsRNAs of X. dendrorhous UCD 67-385 were determined, finding two sequences for L1 dsRNA (L1A and L1B). Several ORFs were uncovered in both S1 and S2 dsRNAs, but no homologies were found for any of them when compared to the database. Instead, two ORFs were identified in each L1A, L1B and L2 dsRNAs, whose deduced amino acid sequences were homologous with a major capsid protein (5'-ORF) and a RNA-dependent RNA polymerase (3'-ORF) belonging to the Totiviridae family. The genome structures of these dsRNAs are characteristic of Totiviruses, with two overlapped ORFs (the 3'-ORF in the -1 frame with respect to the 5'-ORF), with a slippery site and a pseudoknot in the overlapped regions. These structures are essential for the synthesis of the viral polymerase as a fusion protein with the viral capsid protein through -1 ribosomal frameshifting. In the RNase protection analysis, all the dsRNAs in the four analyzed X. dendrorhous strains were protected from enzymatic digestion. The RT-PCR analysis revealed that, similar to strain UCD 67-385, the L1A and L1B dsRNAs coexist in the strains VKM Y-2059, UCD 67-202 and VKM Y-2786. Furthermore, determinations of the relative amounts of L1 dsRNAs using two-step RT-qPCR revealed a 40-fold increment of the ratio L1A/L1B in the S2 dsRNA-cured strain compared to its parental strain. CONCLUSIONS: Three totiviruses, named as XdV-L1A, XdV-L1B and XdV-L2, were identified in the strain UCD 67-385 of X. dendrorhous. The viruses XdV-L1A and XdV-L1B were also found in other three X. dendrorhous strains. Our results suggest that the smaller dsRNAs (named XdRm-S1 and XdRm-S2) of strain UCD 67-385 are satellite viruses, and particularly that XdRm-S2 is a satellite of XdV-L1A.
Assuntos
Basidiomycota/virologia , RNA Viral/genética , RNA Viral/isolamento & purificação , Totivirus/classificação , Totivirus/isolamento & purificação , Sequência de Aminoácidos , Sequência de Bases , Análise por Conglomerados , Dados de Sequência Molecular , Fases de Leitura Aberta , Filogenia , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/isolamento & purificação , Vírus Satélites/classificação , Vírus Satélites/genética , Vírus Satélites/isolamento & purificação , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Totivirus/genéticaRESUMO
Double-stranded RNA (dsRNA) molecules are widely found in yeasts and filamentous fungi. It has been suggested that may play important roles in the evolution of eukaryote genomes and may be a valuable tool in yeast typing. The characterization of these extrachromosomal genetic elements is usually a laborious process, especially when trying to analyze a large number of samples. In this chapter, we describe a simple method to isolate dsRNA elements from yeasts using low amounts of starting material, and their application to different Xanthophyllomyces dendrorhous strains. Furthermore, the methodologies for enzymatic and hybridization characterizations, and quantification of relative dsRNA abundance are detailed.
Assuntos
Basidiomycota/citologia , Fracionamento Químico/métodos , Cromossomos Fúngicos/química , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/isolamento & purificação , RNA Fúngico/química , RNA Fúngico/isolamento & purificação , Basidiomycota/genética , DNA Complementar/biossíntese , Membranas Artificiais , Hibridização de Ácido NucleicoRESUMO
BACKGROUND: In most of the infected fungi, the mycoviruses are latent or cryptic, the infected fungus does not show disease symptoms, and it is phenotypically identical to a non-infected strain of the same species. Because of these properties, the initial stage in the search for fungi infected with mycoviruses is the detection of their viral genome, which in most of the described cases corresponds to double-stranded RNA (dsRNA). So to analyze a large number of fungal isolates it is necessary to have a simple and rapid method to detect dsRNA. RESULTS: A rapid method to isolate dsRNA from a virus-infected filamentous fungus, Botrytis cinerea, and from a killer strain of Saccharomyces cerevisiae using commercial minicolumns packed with CF11 cellulose was developed. In addition to being a rapid method, it allows to use small quantities of yeasts or mycelium as starting material, being obtained sufficient dsRNA quantity that can later be analyzed by agarose gel electrophoresis, treated with enzymes for its partial characterization, amplified by RT-PCR and cloned in appropriate vectors for further sequencing. CONCLUSIONS: The method yields high quality dsRNA, free from DNA and ssRNA. The use of nucleases to degrade the DNA or the ssRNA is not required, and it can be used to isolate dsRNA from any type of fungi or any biological sample that contains dsRNA.
Assuntos
Botrytis/virologia , RNA de Cadeia Dupla/isolamento & purificação , RNA Viral/isolamento & purificação , Saccharomyces cerevisiae/virologia , Cromatografia Líquida/métodos , Virologia/métodosRESUMO
Beauveria bassiana strains from different hosts and geographic origins were assayed for the presence of double-stranded RNA (dsRNA). Two of them (15.4%) showed extra bands, with approximately 4.0-3.5 kb and 2-0.7 kb, respectively, after electrophoretic separation of undigested nucleic acids. Virus-like particles were approximately 28-30 nm diam. The dsRNA was maintained after conidiogenesis (vertical transmission) and was transmitted horizontally by hyphal anastomosis. Strains purged of dsRNA obtained after cycloheximide treatment showed increased conidial production when compared with strains carrying dsRNA particles. Bioassays demonstrated hypovirulence associated with dsRNA. The mean mortality against the insect Euschistus heros was reduced in strains containing dsRNA when compared with the isogenic dsRNA-free ones.
Assuntos
Beauveria/patogenicidade , Beauveria/virologia , Infecções por Vírus de RNA/virologia , Vírus de RNA/isolamento & purificação , RNA de Cadeia Dupla/isolamento & purificação , Animais , Beauveria/ultraestrutura , Desoxirribonuclease I/metabolismo , Insetos , Microscopia Eletrônica de Transmissão , Vírus de RNA/genética , Vírus de RNA/metabolismo , Vírus de RNA/ultraestrutura , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA de Cadeia Dupla/ultraestrutura , RNA Viral/química , RNA Viral/genética , Técnica de Amplificação ao Acaso de DNA Polimórfico , Ribonuclease Pancreático/metabolismo , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , VirulênciaRESUMO
Trichomonas vaginalis can be infected with double-stranded RNA (dsRNA) viruses designated T. vaginalis virus (TVV), which may have important implications for trichomonal virulence and disease pathogenesis. We tested for TVV in 40 fresh T. vaginalis isolates from Cuban patients by total extraction of nucleic acids (DNA and RNA). TVV was detected in 22 (55 percent) of the 40 T. vaginalis isolates. This gives an estimate of the infection rate of Cuban T. vaginalis isolates by the dsRNA virus. Future research should focus on the association between trichomonosis symptoms and the presence of TVV.
Assuntos
Adolescente , Animais , Feminino , Humanos , Vírus de RNA/isolamento & purificação , RNA de Cadeia Dupla/isolamento & purificação , Vaginite por Trichomonas/virologia , Trichomonas vaginalis/virologia , Cuba , DNA Viral/análise , Eletroforese em Gel de Ágar , Vírus de RNA/genética , RNA de Cadeia Dupla/genética , RNA Viral/análise , Trichomonas vaginalis/isolamento & purificação , Trichomonas vaginalis/patogenicidadeRESUMO
Trichomonas vaginalis can be infected with double-stranded RNA (dsRNA) viruses designated T. vaginalis virus (TVV), which may have important implications for trichomonal virulence and disease pathogenesis. We tested for TVV in 40 fresh T. vaginalis isolates from Cuban patients by total extraction of nucleic acids (DNA and RNA). TVV was detected in 22 (55%) of the 40 T. vaginalis isolates. This gives an estimate of the infection rate of Cuban T. vaginalis isolates by the dsRNA virus. Future research should focus on the association between trichomonosis symptoms and the presence of TVV.
Assuntos
Vírus de RNA/isolamento & purificação , RNA de Cadeia Dupla/isolamento & purificação , Vaginite por Trichomonas/virologia , Trichomonas vaginalis/virologia , Adolescente , Animais , Cuba , DNA Viral/análise , Eletroforese em Gel de Ágar , Feminino , Humanos , Vírus de RNA/genética , RNA de Cadeia Dupla/genética , RNA Viral/análise , Trichomonas vaginalis/isolamento & purificação , Trichomonas vaginalis/patogenicidadeRESUMO
To determine the incidence of rotavirus infection among dairy herds in the State of São Paulo, Brazil, 576 faecal samples obtained from calves aged 1-45 days with and without diarrhoea, reared on 63 dairy cattle farms, were analyzed. Polyacrylamide gel electrophoresis (PAGE) identified 28 samples positive for group A rotavirus, while four samples, two diarrhoeic and two non-diarrhoeic, showed a bisegmented genome with a typical picobirnavirus pattern. Electron microscopy revealed spherical virus particles with a diameter of 37 nm and without a defined surface structure. The present study is the first report of a bisegmented virus identified in cattle in Brazil.
Assuntos
Doenças dos Bovinos/virologia , Picobirnavirus/isolamento & purificação , RNA de Cadeia Dupla/isolamento & purificação , Animais , Brasil , Bovinos , Diarreia/veterinária , Diarreia/virologia , Eletroforese em Gel de Poliacrilamida/veterinária , Fezes/virologia , Microscopia EletrônicaRESUMO
A simple double-stranded RNA mycovirus was detected in a wild-type Botrytis cinerea 55k strain. The virus was located in the fungus cytoplasm as free particles of approximately 28 nm in diameter. The mycovirus possesses a single double-stranded genome segment of 1.8 kilobase pairs (kbp) encapsidated within an isometric protein coat whose main structural component is a polypeptide of 68 kDa. Cells infected with this virus showed an important degree of cellular degeneration.
Assuntos
Botrytis/virologia , Vírus de RNA/isolamento & purificação , RNA de Cadeia Dupla/genética , Botrytis/ultraestrutura , Citoplasma/virologia , Eletroforese em Gel de Poliacrilamida , Microscopia Eletrônica , Vírus de RNA/genética , RNA de Cadeia Dupla/isolamento & purificação , Vírion/isolamento & purificaçãoRESUMO
In wild-type Botrytis cinerea CVg25 strain we have detected the presence of extrachromosomal genetic elements corresponding to double-stranded RNA molecules. These genetic elements have been designated L, M1 and M2 with molecular sizes of 8.3, 2.0 and 1.4 kb, respectively. The visualization by electron microscopy of mycelium ultrathin sections from B. cinerea CVg25 showed the presence of isometric virus-like particles of about 40 nm in diameter. Linear sucrose gradient centrifugation of mycelium-free extracts was done to determine if the double-stranded RNAs were associated with virus-like particles. The gradient profile obtained at 260 and 280 nm revealed a major peak that was analyzed by both agarose-gel electrophoresis and electron microscopy. It was observed that only the L-double-stranded RNA molecule copurified with isometric virus-like particles. These virus-like particles had a similar morphology and size as those detected by electron microscopy in the mycelium sections. These results suggest that only the L-double-stranded RNA would be encapsidated.
Assuntos
Fungos Mitospóricos/virologia , Vírus de RNA/ultraestrutura , Herança Extracromossômica , Genoma Viral , Corpos de Inclusão Viral/ultraestrutura , Microscopia Eletrônica , Vírus de RNA/genética , Vírus de RNA/isolamento & purificação , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/isolamento & purificação , RNA Viral/genética , RNA Viral/isolamento & purificaçãoRESUMO
A method is described for rapid extraction of double-stranded RNAs from entomopathogenic fungi. Lyophilised and ground mycelium is incubated with 6 M guanidine thiocyanate, centrifuged, and the cleared lysate applied to a QIAGEN silica-based mini-spin column. Following washing with 70% isopropanol, bound nucleic acids are eluted under low salt conditions and treated with DNAse I prior to analysis by non-denaturing agarose gel electrophoresis.
Assuntos
Ascomicetos/química , RNA de Cadeia Dupla/isolamento & purificação , RNA Fúngico/isolamento & purificação , Animais , Insetos/microbiologiaRESUMO
Central neurons in culture represent a limitless substratum for research in neurobiology and experimental neurology. Primary cultures of NIH mouse neurons have shown that about 83% of total cells in the cultures are neuron clumps, detected by their reaction with the neuron specific-enolase (NSE) marker. Herpes Simplex Virus type 1 (HSV-1) can grow efficiently in these cultures, as it does in nonneuronal cultures usually used for antiviral drugs testing. For that reason, the primary neuronal cultures were used for testing antiviral activity against HSV-1, after an overnight treatment with different concentrations of dsRNA from phi 6 bacteriophage. The dsRNA started to be toxic for the cells at concentrations of 4 micrograms/ml, but it was found that 1 microgram/ml of this dsRNA protected all the neuronal cultures from HSV-1 infection. The dsRNA value for effective dose (ED50) was 0.27 microgram/ml.
Assuntos
Antivirais/farmacologia , Neurônios/microbiologia , RNA de Cadeia Dupla/farmacologia , Simplexvirus/fisiologia , Animais , Bacteriófagos/química , Células Cultivadas , Camundongos , RNA de Cadeia Dupla/isolamento & purificação , RNA Viral/isolamento & purificação , RNA Viral/farmacologia , Simplexvirus/efeitos dos fármacosRESUMO
Polyacrylamide gel electrophoresis of nucleic acids extracted from porcine faecal samples revealed in several samples the presence of two discrete bands. The bands were resistant to digestion with of DNase I and RNase T1, but not with RNase A in low salt conditions, indicating that they consisted of double stranded (ds) RNA. The two bands from different samples varied in sizes, in a range between 2.4-2.6 kbp and 1.7-1.9 kbp for the slow and fast moving band respectively. The bands cosedimented in CsCl gradients at an average density of 1.415 g/ml with icosahedral virus particles of a diameter of 34 nm and a triangulation number equal to 3. Aggregates of virus, which appeared to be immunocomplexes, were seen in one sample. From 244 faecal samples collected in one farm, 27 (11.1%) were found to contain the characteristic dsRNA pattern, with a higher prevalence in samples from animals 15 to 35 days old. The agent was equally distributed among samples from diarrhoeic or non-diarrhoeic animals. These results confirm the circulation among pigs of a novel virus, possibly of vertebrates, with a bisegmented double stranded RNA genome, similar to viruses previously described in humans, wild rats, guinea pigs, pigs, and chickens, for which the name "picobirnavirus" has been proposed.
Assuntos
Vírus de RNA/isolamento & purificação , RNA de Cadeia Dupla/isolamento & purificação , RNA Viral/isolamento & purificação , Suínos/microbiologia , Animais , Diarreia/microbiologia , Diarreia/veterinária , Eletroforese em Gel de Poliacrilamida , Fezes/microbiologia , Prevalência , Vírus de RNA/classificação , Vírus de RNA/genética , Vírus de RNA/ultraestrutura , RibonucleasesRESUMO
Viral particles purified from species of the protozoan parasite Leishmania braziliensis subsp. guyanensis by centrifugation in CsCl gradients were examined for the presence of viral polymerase. We demonstrated that RNA-dependent RNA polymerase is associated with viral particles. Viral transcription was studied in vitro with pulse-chase experiments and by assaying the RNase sensitivity of the viral transcripts. Viral polymerase synthesized full-length transcripts within 1 h. Double-strained, genome-length, and single-stranded RNAs were produced in this system. The nature of the RNA extracted from virions was also tested by RNase protection assays; both single-stranded and double-stranded RNAs were found.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Leishmania braziliensis/microbiologia , Leishmania/microbiologia , Vírus/enzimologia , Animais , Centrifugação com Gradiente de Concentração , Leishmania braziliensis/enzimologia , RNA de Cadeia Dupla/biossíntese , RNA de Cadeia Dupla/isolamento & purificação , RNA Viral/biossíntese , RNA Viral/isolamento & purificação , Transcrição Gênica , Vírus/genética , Vírus/isolamento & purificaçãoRESUMO
Human rotaviruses are the major, recognized cause of infantile diarrhea worldwide. Characterization of naturally occurring human isolates indicates that there are six human rotavirus serotypes, four of which (serotype 1 to 4) are widespread. We utilized monoclonal antibodies specific for the VP of serotypes 1, 2, 3, and 4 as capture antibody in a sandwich enzyme-linked immunosorbent to serotype rotaviruses directly in stool samples. The stool samples were collected from 1983 through 1986, from two epidemiologic studies in the area of Buenos Aires, Argentina. All four serotypes assayed were found Serotype 2 and 3 viruses, which were detected most frequently in 1983 and 1984, were virtually undetected in 1985 and 1986 (chi 2 = 23, P = less than 0.001 for this difference). No significant difference was noted among the three collection sites for serotype prevalence. These results indicate that the changing predominance of rotavirus serotype in a given region can involve multiple serotypes at the same time. Analysis of an outbreak of diarrhea in two neighboring families which occurred during a prospective study of community diarrhea documented inter- and intra-family spread of one serotype of virus.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Rotavirus/classificação , Argentina/epidemiologia , Diarreia/epidemiologia , Diarreia/microbiologia , Surtos de Doenças , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Fezes/microbiologia , Humanos , RNA de Cadeia Dupla/isolamento & purificação , RNA Viral/isolamento & purificação , SorotipagemRESUMO
Eight viruses of the Corriparta serogroup (Reoviridae: Orbivirus) that were known to be heterogeneous on the basis of serology and polyacrylamide gel electrophoresis were examined by reciprocal RNA-RNA blot hybridization of genomic RNA. Conserved and variant genes were identified by the degree of hybridization between cognate genes of different isolates. The eight viruses were divided into three subsets on the basis of the number of shared genes. Four of the viruses, isolated in Australia, formed one subset of related isolates and shared five conserved genes. Another isolate, Acado, was variant in all 10 genes and was considered to be a second subset. The remaining three isolates formed a third subset and shared four conserved genes. Genes 1, 3 and 10 were the most variable among the Corriparta serogroup isolates. Subsets of isolates within a serogroup which are highly related in the majority of the 10 genes and less related to serogroup viruses in another subset have not been reported previously. The phylogenetic relationship of Corriparta serogroup members suggested by the blot hybridization data is not apparent in the current taxonomic classification of these viruses which is based primarily upon serological data. The hybridization data on the Corriparta serogroup viruses are discussed and contrasted with other Orbivirus serogroups which have been examined similarly.