RESUMO
Chronic bee paralysis virus (CBPV) is responsible for chronic paralysis, an infectious and contagious disease of adult honey bees (Apis mellifera L.). The full-length nucleotide sequences of the two major RNAs of CBPV have previously been characterized. The Orf3 of RNA1 has shown significant similarities to the RNA-dependent RNA polymerase (RdRp) of positive single-stranded RNA viruses, whereas the Orf3 of RNA2 encodes a putative structural protein (pSP). In the present study, honey bees originating from 9 different countries (Austria, Poland, Hungary, Spain, Belgium, Denmark, Switzerland, Uruguay and France) were analysed for the presence of CBPV genome. The complete genomic nucleotide sequence of the RdRp (1947bp) and of the pSP (543bp) from 24 honey bee positive samples was determined and the phylogenetic relationship among isolates was investigated. Four distinct genotypes of CBPV were observed.
Assuntos
Abelhas/virologia , Filogenia , RNA Polimerase Dependente de RNA/genética , Proteínas Virais/genética , Proteínas Estruturais Virais/genética , Vírus não Classificados/classificação , Vírus não Classificados/genética , Animais , Áustria , Bélgica , Análise por Conglomerados , Dinamarca , França , Geografia , Hungria , Epidemiologia Molecular , Dados de Sequência Molecular , Polônia , Análise de Sequência de DNA , Homologia de Sequência , Espanha , Suíça , Uruguai , Vírus não Classificados/isolamento & purificaçãoRESUMO
Bee samples were collected in French apiaries that displayed severe losses and mortality during the winter (from November 2007 to March 2008). They were screened for the presence of Israeli acute paralysis virus (IAPV) by using RT-PCR. Five out of 35 surveyed apiaries, located in two different geographical areas, were found positive. This represents the first reported detection of IAPV in France. The specificity of the PCR products was checked by sequencing. The phylogenetic analysis showed that French isolates of IAPV were closely related to a cluster including American and Australian isolates. Nevertheless, most of American isolates previously reported to be associated to Colony Collapse Disorder (CCD) and an Israeli isolate first isolated in 2004 from dead bees were included in another cluster. Since IAPV was detected in only 14% of the affected apiaries, it was not possible to establish a causal link between IAPV and the severe winter losses that occurred.
Assuntos
Abelhas/virologia , Vírus de Insetos/isolamento & purificação , Infecções por Picornaviridae/veterinária , Picornaviridae/isolamento & purificação , Animais , Sequência de Bases , França , Interações Hospedeiro-Patógeno , Vírus de Insetos/classificação , Vírus de Insetos/genética , Israel , Dados de Sequência Molecular , Filogenia , Picornaviridae/classificação , Picornaviridae/genética , Infecções por Picornaviridae/virologia , RNA Viral/química , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Chronic bee paralysis virus (CBPV) is a common single-stranded RNA virus which may cause significant losses in honey bee colonies. As this virus seems to exhibit neurotropism, an in situ hybridization based method was developed to localize the genomic and antigenomic CBPV RNAs in infected honey bee brains. Double-stranded cDNA probes as well as genomic and antigenomic-specific single-stranded cDNA probes were prepared, using the polymerase chain reaction in presence of labelled d-UTP with non-radioactive digoxigenin. Both genomic and antigenomic RNAs were detected the brain of honey bee infected naturally or artificially. Hybridization signals were obtained in some somata and neuropile regions of the brain. In particular, high signals were observed at the level of the mushroom bodies and central complex, regions that are known to be engaged in higher neuronal functions and in the optic and antennal lobes that are sensorial neuropiles. Thus, the presence of virus at these levels may explain the nervous symptoms observed in infected bees. The in situ hybridization procedure proved to be a useful tool to localize specifically CBPV and may be helpful for understanding the observed symptoms.
Assuntos
Abelhas/virologia , Encéfalo/virologia , Vírus de RNA/isolamento & purificação , RNA Viral/análise , Vírus não Classificados/isolamento & purificação , Animais , Abelhas/anatomia & histologia , DNA Complementar , Genoma Viral , Hibridização de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase , Vírus de RNA/genética , RNA Viral/isolamento & purificação , Vírus não Classificados/genéticaRESUMO
Detection of Chronic bee paralysis virus (CBPV) is reported for the first time in two species of ants (Camponotus vagus and Formica rufa) and in Varroa destructor. A quantitative real-time PCR (qPCR) method was used to detect and quantify CBPV in infected bees, ants and mites. A minus-strand-specific RT-PCR was used to assess viral replication. These results suggest a new way by which the infection may be spread and other sites of viral persistence in the close apiary environment.
Assuntos
Formigas/virologia , Abelhas/virologia , Vírus de Insetos/isolamento & purificação , Ácaros/virologia , Vírus de RNA/isolamento & purificação , RNA Viral/biossíntese , Animais , Doença Crônica , Genoma Viral , Vírus de Insetos/genética , Vírus de Insetos/fisiologia , Paralisia/virologia , Reação em Cadeia da Polimerase/métodos , Vírus de RNA/genética , Vírus de RNA/fisiologia , RNA Viral/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Especificidade da Espécie , Replicação ViralRESUMO
The complete sequences of the two major RNAs of Chronic bee paralysis virus (CBPV) have been determined. RNA 1 (3674nt long) and RNA 2 (2305nt long) are positive single-stranded RNAs that are capped but not polyadenylated. The 3' ends of both RNAs are unreactive to polymerisation or ligation even in denaturing conditions, a feature already observed in alphanodavirus RNAs. The three previously described smaller RNAs [Overton, H.A., Buck, K.W., Bailey, L., et al., 1982. Relationships between the RNA components of Chronic bee-paralysis virus and those of chronic bee-paralysis virus associate. J. Gen. Virol. 63, 171-179], were not detected in this study, supporting the hypothesis that they would correspond to the three RNAs of the Chronic bee paralysis satellite virus (CBPSV). RNA 1 and RNA 2 encoded three and four overlapping open reading frames (ORFs), respectively. The amino acid sequences deduced from the ORF 3 on RNA 1 shared the conserved motifs of the RNA-dependent RNA polymerase (RdRp) sequence and presented similarities with members of the Nodaviridae and Tombusviridae families. However, no similarities were found between the other CBPV deduced amino acid sequences and sequences in the NCBI databases, suggesting that CBPV is the prototype of a new family of positive single-stranded RNA viruses.
Assuntos
Abelhas/virologia , Vírus de Insetos/genética , Filogenia , Vírus de RNA/genética , Vírus não Classificados/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Genoma Viral , Vírus de Insetos/classificação , Vírus de Insetos/isolamento & purificação , Vírus de Insetos/fisiologia , Dados de Sequência Molecular , Nodaviridae/genética , Fases de Leitura Aberta , Estrutura Terciária de Proteína , Vírus de RNA/classificação , Vírus de RNA/isolamento & purificação , Vírus de RNA/fisiologia , RNA Viral/genética , RNA Polimerase Dependente de RNA/genética , Alinhamento de Sequência , Análise de Sequência de RNA , Tombusviridae/genética , Proteínas Virais/química , Proteínas Virais/genética , Vírus não Classificados/classificação , Vírus não Classificados/isolamento & purificação , Vírus não Classificados/fisiologiaRESUMO
A new RT-PCR test has been developed to diagnose Chronic bee paralysis virus (CBPV) that is able to detect genetically variable viral isolates. In fact, up to 8.7% divergence between partial nucleotide sequences from viral isolates from French honey bees was highlighted in a preliminary variability study. The previously-described RT-PCR was unable to detect all these viral isolates and RT-PCR diagnosis needed improvement. The new RT-PCR test can detect up to 40% more CBPV isolates.
Assuntos
Abelhas/virologia , DNA Viral/genética , Vírus de Insetos/genética , Vírus de Insetos/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Sequência de Bases , França , Variação Genética , Vírus de Insetos/patogenicidade , Dados de Sequência MolecularRESUMO
A two-step real-time RT-PCR assay, based on TaqMan technology using a fluorescent probe (FAM-TAMRA) was developed to quantify Chronic bee paralysis virus (CBPV) genome in bee samples. Standard curves obtained from a CBPV control RNA and from a plasmid containing a partial sequence of CBPV showed that this assay provided linear detection over a 7-log range (R(2)>0.99) with a limit of detection of 100 copies, and reliable inter-assay and intra-assay reproducibility. Standardisation including RNA purification and cDNAs synthesis was also validated. The CBPV TaqMan methodology was first evaluated by quantifying the CBPV genomic load in bee samples from an experimental infection obtained by topical application. Up to 1.9 x 10(10) CBPV copies per segment of insect body (head, thorax and abdomen) were revealed whereas a lower CBPV genomic load was detected in dissected organs such as mandibular and hypopharyngeal glands, brain and alimentary canal (up to 7.2 x 10(6) CBPV copies). The CBPV genomic loads in different categories of bees from a hive presenting the trembling symptoms typical of Chronic paralysis were then quantified. Significantly higher CBPV loads were found in guard, symptomatic and dead bees (up to 1.9 x 10(13) CBPV copies) than in forager, drones and house bees (up to 3.4 x 10(6) CBPV copies). The results obtained for symptomatic or dead bees support the correlation between high CBPV genomic load and pathology expression. Moreover, the high CBPV genomic load revealed in guard bees highlights the possible pivotal role played by this category of bees in CBPV infection.
