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Molecular and biological characterization of deformed wing virus of honeybees (Apis mellifera L.).

ABSTRACT: Deformed wing virus (DWV) of honeybees (Apis mellifera) is closely associated with characteristic wing deformities, abdominal bloating, paralysis, and rapid mortality of emerging adult bees. The virus was purified from diseased insects, and its genome was cloned and sequenced. The genomic RNA of DWV is 10,140 nucleotides in length and contains a single large open reading frame encoding a 328-kDa polyprotein. The coding sequence is flanked by a 1,144-nucleotide 5' nontranslated leader sequence and a 317-nucleotide 3' nontranslated region, followed by a poly(A) tail. The three major structural proteins, VP1 (44 kDa), VP2 (32 kDa), and VP3 (28 kDa), were identified, and their genes were mapped to the N-terminal section of the polyprotein. The C-terminal part of the polyprotein contains sequence motifs typical of well-characterized picornavirus nonstructural proteins: an RNA helicase, a chymotrypsin-like 3C protease, and an RNA-dependent RNA polymerase. The genome organization, capsid morphology, and sequence comparison data indicate that DWV is a member of the recently established genus Iflavirus.

SUBMITTER: Lanzi G

PROVIDER: S-EPMC1472076 | biostudies-literature | 2006 May

REPOSITORIES: biostudies-literature

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Molecular and biological characterization of deformed wing virus of honeybees (Apis mellifera L.).

Lanzi Gaetana G de Miranda Joachim R JR Boniotti Maria Beatrice MB Cameron Craig E CE Lavazza Antonio A Capucci Lorenzo L Camazine Scott M SM Rossi Cesare C

Journal of virology 20060501 10

Deformed wing virus (DWV) of honeybees (Apis mellifera) is closely associated with characteristic wing deformities, abdominal bloating, paralysis, and rapid mortality of emerging adult bees. The virus was purified from diseased insects, and its genome was cloned and sequenced. The genomic RNA of DWV is 10,140 nucleotides in length and contains a single large open reading frame encoding a 328-kDa polyprotein. The coding sequence is flanked by a 1,144-nucleotide 5' nontranslated leader sequence an ...[more]

PMID: 16641291

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Project description:Honeybees are globally threatened by several pathogens, especially deformed wing virus (DWV), as the presence of DWV in western honeybees is indicative of colony loss. The high mortality rate is further exacerbated by the lack of effective treatment, and therefore understanding the immune and apoptosis responses could pave an avenue for the treatment method. In this study, DWV was directly injected into the white-eyed pupae stage of western honeybees (Apis mellifera). The DWV loads and selected gene responses were monitored using the real-time PCR technique. The results showed that honeybee pupae that were injected with the highest concentration of viral loads showed a significantly higher mortality rate than the control groups. Deformed wings could be observed in newly emerged adult bees when the infected bees harbored high levels of viral loads. However, the numbers of viral loads in both normal and crippled wing groups were not significantly different. DWV-injected honeybee pupae with 104 and 107 copy numbers per bee groups showed similar viral loads after 48 h until newly emerged adult bees. Levels of gene expression including immune genes (defensin, abaecin, and hymenoptaecin) and apoptosis genes (buffy, p53, Apaf1, caspase3-like, caspase8-like, and caspase9-like) were analyzed after DWV infection. The expressions of immune and apoptosis genes were significantly different in infected bees compared to those of the control groups. In the pupae stage, the immune genes were activated by injecting DWV (defensin and hymenoptaecin) or Escherichia coli (defensin, abaecin, and hymenoptaecin), a positive control. On the contrary, the expression of apoptosis-related genes (buffy, caspase3-like, caspase8-like, and caspase9-like genes) was suppressed at 96 h post-infection. In DWV-infected newly emerged adult bees, abaecin, hymenoptaecin, Apaf1, and caspase8-like genes were upregulated. However, these genes were not significantly different between the normal and crippled wing bees. Our results suggested that DWV could activate the humoral immunity in honeybees and that honeybee hosts may be able to protect themselves from the virus infection through immune responses. Apoptosis gene expressions were upregulated in newly emerged adult bees by the virus, however, they were downregulated during the initial phase of viral infection.

Project description:The globally distributed ectoparasite Varroa destructor is a vector for viral pathogens of the Western honeybee (Apis mellifera), in particular the Iflavirus Deformed Wing Virus (DWV). In the absence of Varroa low levels DWV occur, generally causing asymptomatic infections. Conversely, Varroa-infested colonies show markedly elevated virus levels, increased overwintering colony losses, with impairment of pupal development and symptomatic workers. To determine whether changes in the virus population were due Varroa amplifying and introducing virulent virus strains and/or suppressing the host immune responses, we exposed Varroa-naïve larvae to oral and Varroa-transmitted DWV. We monitored virus levels and diversity in developing pupae and associated Varroa, the resulting RNAi response and transcriptome changes in the host. Exposed pupae were stratified by Varroa association (presence/absence) and virus levels (low/high) into three groups. Varroa-free pupae all exhibited low levels of a highly diverse DWV population, with those exposed per os (group NV) exhibiting changes in the population composition. Varroa-associated pupae exhibited either low levels of a diverse DWV population (group VL) or high levels of a near-clonal virulent variant of DWV (group VH). These groups and unexposed controls (C) could be also discriminated by principal component analysis of the transcriptome changes observed, which included several genes involved in development and the immune response. All Varroa tested contained a diverse replicating DWV population implying the virulent variant present in group VH, and predominating in RNA-seq analysis of temporally and geographically separate Varroa-infested colonies, was selected upon transmission from Varroa, a conclusion supported by direct injection of pupae in vitro with mixed virus populations. Identification of a virulent variant of DWV, the role of Varroa in its transmission and the resulting host transcriptome changes furthers our understanding of this important viral pathogen of honeybees.

Project description:Honey bees (Apis mellifera) play a crucial role in agriculture through their pollination activities. However, they have faced significant health challenges over the past decades that can limit colony performance and even lead to collapse. A primary culprit is the parasitic mite Varroa destructor, known for transmitting harmful bee viruses. Among these viruses is deformed wing virus (DWV), which impacts bee pupae during their development, resulting in either pupal demise or in the emergence of crippled adult bees. In this study, we focused on DWV master variant B. DWV-B prevalence has risen sharply in recent decades and appears to be outcompeting variant A of DWV. We generated a molecular clone of a typical DWV-B strain to compare it with our established DWV-A clone, examining RNA replication, protein expression, and virulence. Initially, we analyzed the genome using RACE-PCR and RT-PCR techniques. Subsequently, we conducted full-genome RT-PCR and inserted the complete viral cDNA into a bacterial plasmid backbone. Phylogenetic comparisons with available full-length sequences were performed, followed by functional analyses using a live bee pupae model. Upon the transfection of in vitro-transcribed RNA, bee pupae exhibited symptoms of DWV infection, with detectable viral protein expression and stable RNA replication observed in subsequent virus passages. The DWV-B clone displayed a lower virulence compared to the DWV-A clone after the transfection of synthetic RNA, as evidenced by a reduced pupal mortality rate of only 20% compared to 80% in the case of DWV-A and a lack of malformations in 50% of the emerging bees. Comparable results were observed in experiments with low infection doses of the passaged virus clones. In these tests, 90% of bees infected with DWV-B showed no clinical symptoms, while 100% of pupae infected with DWV-A died. However, at high infection doses, both DWV-A and DWV-B caused mortality rates exceeding 90%. Taken together, we have generated an authentic virus clone of DWV-B and characterized it in animal experiments.

Dataset Information

Molecular and biological characterization of deformed wing virus of honeybees (Apis mellifera L.).

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Molecular and biological characterization of deformed wing virus of honeybees (Apis mellifera L.).

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