RESUMO
Papaya sticky disease, or "meleira", is one of the major diseases of papaya in Brazil and Mexico, capable of causing complete crop loss. The causal agent of sticky disease was identified as an isometric virus with a double stranded RNA (dsRNA) genome, named papaya meleira virus (PMeV). In the present study, PMeV dsRNA and a second RNA band of approximately 4.5 kb, both isolated from latex of papaya plants with severe symptoms of sticky disease, were deep-sequenced. The nearly complete sequence obtained for PMeV dsRNA is 8,814 nucleotides long and contains two putative ORFs; the predicted ORF1 and ORF2 display similarity to capsid proteins and RdRp's, respectively, from mycoviruses tentatively classified in the family Totiviridae. The sequence obtained for the second RNA is 4,515 nucleotides long and contains two putative ORFs. The predicted ORFs 1 and 2 display 48% and 73% sequence identity, respectively, with the corresponding proteins of papaya virus Q, an umbravirus recently described infecting papaya in Ecuador. Viral purification in a sucrose gradient allowed separation of particles containing each RNA. Mass spectrometry analysis indicated that both PMeV and the second RNA virus (named papaya meleira virus 2, PMeV2) were encapsidated in particles formed by the protein encoded by PMeV ORF1. The presence of both PMeV and PMeV2 was confirmed in field plants showing typical symptoms of sticky disease. Interestingly, PMeV was detected alone in asymptomatic plants. Together, our results indicate that sticky disease is associated with double infection by PMeV and PMeV2.
Assuntos
Carica/virologia , Genoma Viral , Filogenia , Doenças das Plantas/virologia , Vírus de Plantas/genética , Vírus de RNA/genética , Totiviridae/genética , Sequência de Aminoácidos , Sequência de Bases , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Conformação de Ácido Nucleico , Fases de Leitura Aberta , Vírus de Plantas/classificação , Vírus de Plantas/ultraestrutura , Vírus de RNA/classificação , Vírus de RNA/ultraestrutura , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Totiviridae/classificação , Totiviridae/ultraestrutura , Vírion/genética , Vírion/ultraestruturaRESUMO
Citrus leprosis is one of the most destructive diseases of Citrus spp. and is associated with two unrelated virus groups that produce particles primarily in either the cytoplasm or nucleus of infected plant cells. Symptoms of leprosis, including chlorotic spots surrounded by yellow haloes on leaves and necrotic spots on twigs and fruit, were observed on leprosis-affected mandarin and navel sweet orange trees in the state of Querétaro, Mexico. Serological and molecular assays showed that the cytoplasmic types of Citrus leprosis virus (CiLV-C) often associated with leprosis symptomatic tissues were absent. However, using transmission electron microscopy, bullet-shaped rhabdovirus-like virions were observed in the nuclei and cytoplasm of the citrus leprosis-infected leaf tissues. An analysis of small RNA populations from symptomatic tissue was carried out to determine the genome sequence of the rhabdovirus-like particles observed in the citrus leprosis samples. The complete genome sequence showed that the nuclear type of CiLV (CiLV-N) present in the samples consisted of two negative-sense RNAs: 6,268-nucleotide (nt)-long RNA1 and 5,847-nt-long RNA2, excluding the poly(A) tails. CiLV-N had a genome organization identical to that of Orchid fleck virus (OFV), with the exception of shorter 5' untranslated regions in RNA1 (53 versus 205 nt) and RNA2 (34 versus 182 nt). Phylogenetic trees constructed with the amino acid sequences of the nucleocapsid (N) and glycoproteins (G) and the RNA polymerase (L protein) showed that CiLV-N clusters with OFV. Furthermore, phylogenetic analyses of N protein established CiLV-N as a member of the proposed genus Dichorhavirus. Reverse-transcription polymerase chain reaction primers for the detection of CiLV-N were designed based on the sequence of the N gene and the assay was optimized and tested to detect the presence of CiLV-N in both diseased and symptom-free plants.
Assuntos
Citrus/virologia , Doenças das Plantas/virologia , Vírus de Plantas/classificação , Vírus de RNA/classificação , Sequência de Aminoácidos , DNA Complementar/química , DNA Complementar/genética , Frutas/virologia , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , México , Dados de Sequência Molecular , Nucleocapsídeo/genética , Filogenia , Folhas de Planta/virologia , Vírus de Plantas/genética , Vírus de Plantas/ultraestrutura , Vírus de RNA/genética , Vírus de RNA/ultraestrutura , RNA Viral/genética , Alinhamento de Sequência , Análise de Sequência de DNA , VírionRESUMO
The causal agents of Citrus leprosis are viruses; however, extant diagnostic methods to identify them have failed to detect known viruses in orange, mandarin, lime and bitter orange trees with severe leprosis symptoms in Mexico, an important citrus producer. Using high throughput sequencing, a virus associated with citrus leprosis was identified, belonging to the proposed Dichorhavirus genus. The virus was termed Citrus Necrotic Spot Virus (CNSV) and contains two negative-strand RNA components; virions accumulate in the cytoplasm and are associated with plasmodesmata-channels interconnecting neighboring cells-suggesting a mode of spread within the plant. The present study provides insights into the nature of this pathogen and the corresponding plant response, which is likely similar to other pathogens that do not spread systemically in plants.
Assuntos
Citrus/virologia , Genoma Viral , Nucleocapsídeo/genética , Doenças das Plantas/virologia , Vírus de Plantas/genética , Vírus de RNA/genética , Citrus/imunologia , Frutas/imunologia , Frutas/virologia , Regulação Viral da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Interações Hospedeiro-Patógeno , México , Nucleocapsídeo/ultraestrutura , Filogenia , Células Vegetais/imunologia , Células Vegetais/virologia , Doenças das Plantas/imunologia , Imunidade Vegetal , Folhas de Planta/imunologia , Folhas de Planta/virologia , Vírus de Plantas/classificação , Vírus de Plantas/isolamento & purificação , Vírus de Plantas/ultraestrutura , Plasmodesmos/imunologia , Plasmodesmos/virologia , Vírus de RNA/classificação , Vírus de RNA/isolamento & purificação , Vírus de RNA/ultraestruturaRESUMO
Pseudocercospora griseola (Sacc.) Crous & Braun is a widespread fungal phytopathogen that is responsible for angular leaf spot in the common bean (Phaseolus vulgaris L.). A number of fungal phytopathogens have been shown to harbour mycoviruses, and this possibility was investigated in populations of Pseudocercospora griseola. The total nucleic acid extracts of 61 fungal isolates were subjected to agarose gel electrophoresis. Small fragments (800-4800 bp) could be identified in 42 of the samples. The presence of dsRNA in isolate Ig838 was confirmed by treatment of total nucleic acid with DNase, RNase A, and nuclease S1. Transmission electron microscopy revealed the presence of viral-like particles 40 nm in diameter in the mycelia of 2 fungal isolates, namely 29-3 and Ig838. The transmission of dsRNA by means of conidia was 100% for isolate 29-3, but there was loss of 1-6 fragments of dsRNA in monosporic colonies of isolate Ig848. Cycloheximide treatment failed to inhibit the mycovirus in isolate 29-3, but proved efficient in the elimination of the 2.2, 2.0, 1.8, 1.2 and 1.0 kb fragments in 2 colonies of isolate Ig848. The occurrence of a mycovirus in Pseudocercospora griseola was demonstrated for the first time in the present study.
Assuntos
Ascomicetos/virologia , Phaseolus/microbiologia , Doenças das Plantas/microbiologia , Folhas de Planta/microbiologia , Vírus de RNA/genética , Antifúngicos/farmacologia , Ascomicetos/efeitos dos fármacos , Cicloeximida/farmacologia , Microscopia Eletrônica de Transmissão , Folhas de Planta/ultraestrutura , Vírus de RNA/ultraestrutura , RNA de Cadeia Dupla/genética , RNA Viral/genética , RNA Viral/ultraestruturaRESUMO
The barn-owl (Tyto Alba) and striped-owl (Rhinoptynx clamator) belong respectively to the families Tytonidae and Strigidae. Avian paramyxoviruses have been isolated from a variety of species of wild and domestic birds wordlwide causing diverse clinical symptoms and signs. Paramyxoviruses belong to the family Paramyxoviridae and Avulovirus genus, including nine serotypes (APMV 1 to 9). The lymphoid leukosis is a retrovirus-induced neoplasia. The avian retroviruses belong to the Retroviridae family and to the Alpharetrovirus genus. Coronaviruses can cause respiratory and enteric disease in several species of birds. They belong to the Coronaviridae family and to the groups 3a e 3c. In this study, we describe the presence of viruses in four owls, two barn owls (Tyto alba) and two striped owls (Rhinoptynx clamator), rescued from tree-lined streets of Sao Paulo, Brazil and sent to the Recovery Center of Wild Animals of the Tietê Ecological Park, where the animals died. Fragments of lung, liver and small intestine of these birds were processed for transmission electron microscopy utilizing negative staining (rapid preparation), immunoelectron microscopy and immunocitochemistry techniques. Under the transmission electron microscopy paramyxovirus particles, pleomorphic, roughly spherical or filamentous, measuring 100 to 500 nm of diameter containing an envelope covered by spikes, an herring-bone helical nucleocapsid-like structure, measuring 15 to 20 nm in diameter, were visualized in the samples of lung, liver and small intestine of all owls. In small intestine samples of the two striped-owl (owls 3 and 4) it was detected pleomorphic coronavirus particles with a diameter of 75-160 nm containing a solar corona-shaped envelope, with projections of approximately 20 nm of diameter. In liver fragments of one striped-owl (owl 4) pleomorphic particles of retrovirus with a diameter of 80-145 nm containing an envelope with short projections and diameter of 9 nm were....
La lechuza (Tyto Alba) y el búho de orejas (Rhinoptynx clamator) pertenecen respectivamente a las familias Strigidae y Tytonidae. El paramixovirus aviario se ha aislado de especies de vida silveste como las aves domésticas por todo el mundo, causando diversos síntomas clínicos. El paramixovirus pertenece a la familia Paramyxoviridae y al Avulovirus genus que incluye nueve serotipos (APMV 1 a 9). La leucosis linfoide es una neoplasia inducida por retrovirus. Los retrovirus aviarios pertenecen a la familia Retroviridae y el género Alpharetrovirus. Los coronavirus pueden causar enfermedades respiratorias y entéricas en varias especies de aves. Ellos pertenecen a la familia Coronaviridae y a los grupos 3a y 3c. En este estudio, se describe la presencia del virus en cuatro búhos, dos lechuzas (Tyto alba) y dos búhos de orejas (Rhinoptynx clamator), rescatados de las calles arboladas de São Paulo, Brasil y enviados al Centro de Recuperación de Animales Silvestres del Parque Ecológico de Tietê, donde hubo murieron los animales. Fragmentos de pulmón, delhígado y del intestino delgado de estas aves fueron procesados para microscopía electrónica de transmisión utilizando tinción negativa (preparación rápida), inmunomicroscopía y técnicas de inmunocitoquímica. Bajo microscopía electrónica de transmisión, partículas de paramixovirus, pleomórficas, aproximadamente esféricas o filamentosas, de 100 a 500 nm de diámetro con un sobre cubierto por espigas, y nucleocápside helicoidal con características de espiga, midiendo 15 a 20 nm de diámetro, fueron visualizadas en las muestras de pulmón, hígado e intestino delgado de todos los búhos. En muestras de intestino delgado de dos búho de orejas (búhos 3 y 4) se detectaron partículas pleomórficas con coronavirus de un diámetro de 75-160 nm con un sobre con forma de corona solar, con proyecciones de aproximadamente 20 nm de diámetro. En el hígado de un búho de orejas (búho 4) se observaron partículas pleomórficas de retrovirus con ...
Assuntos
Animais , Estrigiformes/anatomia & histologia , Estrigiformes/virologia , Vírus de RNA/imunologia , Vírus de RNA/ultraestrutura , Brasil , Coronavirus/imunologia , Coronavirus/ultraestrutura , Imuno-Histoquímica , Microscopia Eletrônica de Transmissão , Paramyxoviridae/imunologia , Paramyxoviridae/ultraestrutura , Retroviridae/imunologia , Retroviridae/ultraestruturaRESUMO
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 is a flagellated, parasitic protozoan that inhabits the urogenital tract of humans. Some isolates of T. vaginalis are infected with a double-stranded RNA (dsRNA) virus, which was described in the literature as homogeneous icosahedral viral particles with an isometric symmetry and 33 nm in diameter. This study examined in detail the viral particles in T. vaginalis isolate 347 and describes a heterogeneous population of viral particles. The different dsRNA viruses were only observed after a change in the technique. The sample was prepared by the negative staining carbon-film method directly onto freshly cleft mica. The detected viruses ranged in size from 33 to 200 nm. Among the shapes observed were filamentous, cylindrical, and spherical particles. These results show that T. vaginalis may be a reservoir for several different dsRNA viruses simultaneously.
Assuntos
Coloração Negativa/métodos , Vírus de RNA/ultraestrutura , Trichomonas vaginalis/virologia , Animais , Centrifugação com Gradiente de Concentração/métodos , Microscopia Eletrônica , RNA de Cadeia Dupla/ultraestrutura , Trichomonas vaginalis/crescimento & desenvolvimento , Vírion/ultraestruturaRESUMO
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
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
Examination of the intestinal contents of free-living Oryzomys nigripes rats by PAGE revealed two sharply defined bands that could be stained by ethidium bromide or by silver nitrate with comparable intensities. The molecules forming these bands were susceptible to digestion by pancreatic RNase A but not by RNase T1 or by DNase I. Their lengths were estimated to be about 2.6 and 1.5 kbp, respectively, by comparison with rotavirus SA11 genome segments. They cosedimented in CsCl gradients at a density of 1.39 to 1.40 g/ml, together with uniform particles approximately 35 nm in diameter with indistinct surface structure. It is suggested that these particles represent an as yet undescribed virus with a bisegmented double-stranded RNA genome, for which the name 'picobirnavirus' is proposed.